Soil Biology
Neeraj Shrivastava
Shubhangi Mahajan
Ajit Varma Editors
Symbiotic Soil
Microorganisms
Biology and Applications
Soil Biology
Volume 60
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Ajit Varma, Amity Institute of Microbial Technology,
Amity University, Noida, Uttar Pradesh, India
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Neeraj Shrivastava • Shubhangi Mahajan •
Ajit Varma
Editors
Symbiotic Soil
Microorganisms
Biology and Applications
Editors
Neeraj Shrivastava
Amity Institute of Microbial Technology
Amity University Uttar Pradesh (AUUP)
Noida, Uttar Pradesh, India
Shubhangi Mahajan
Amity Institute of Microbial Technology
Amity University Uttar Pradesh (AUUP)
Noida, Uttar Pradesh, India
Ajit Varma
Amity Institute of Microbial Technology
Amity University Uttar Pradesh (AUUP)
Noida, Uttar Pradesh, India
ISSN 1613-3382
ISSN 2196-4831 (electronic)
Soil Biology
ISBN 978-3-030-51915-5
ISBN 978-3-030-51916-2 (eBook)
https://doi.org/10.1007/978-3-030-51916-2
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to Springer Nature Switzerland
AG 2021
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Contents
Part I
1
2
3
4
Fungal Symbiosis
Current Status–Enlightens in Its Biology and Omics Approach on
Arbuscular Mycorrhizal Community . . . . . . . . . . . . . . . . . . . . . . .
Tulasikorra, O. Siva Devika, K. Mounika, I. Sudhir Kumar, Suman
Kumar, G. Sabina Mary, Uday Kumar, and Manoj Kumar
3
An Insight through Root-Endophytic-Mutualistic Association in
Improving Crop Productivity and Sustainability . . . . . . . . . . . . . . .
Nitika Thakur
31
Interaction Between Root Endophytes and Plants: Their Bioactive
Products and Significant Functions . . . . . . . . . . . . . . . . . . . . . . . . .
Dhriti Kapoor and Nitika Kapoor
45
Unravelling the Role of Endophytes in Micronutrient Uptake and
Enhanced Crop Productivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Kanchan Vishwakarma, Nitin Kumar, Chitrakshi Shandilya, and Ajit
Varma
63
5
Dual and Tripartite Symbiosis of Invasive Woody Plants . . . . . . . .
Robin Wilgan
87
6
Eco-friendly Association of Plants and Actinomycetes . . . . . . . . . . .
Saraswathy Nagendran, Surendra S. Agrawal, and Aryaman Girish
Patwardhan
99
7
The Arbuscular Mycorrhizal Symbiosis of Trees: Structure,
Function, and Regulating Factors . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Leszek Karliński
v
vi
Contents
8
Effectiveness of Arbuscular Mycorrhizas in Improving Carob
Culture in the Mediterranean Regions . . . . . . . . . . . . . . . . . . . . . . 129
Abdellatif Essahibi, Laila Benhiba, Cherki Ghoulam, and Ahmed
Qaddoury
9
Leaf Endophytes and Their Bioactive Compounds . . . . . . . . . . . . . 147
Parikshana Mathur, Payal Mehtani, and Charu Sharma
10
Role of Endophytic Fungus Piriformospora indica in Nutrient
Acquisition and Plant Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Neha Sharma and Ajit Varma
11
The Role of Symbiotic Fungi in Nutri-Farms . . . . . . . . . . . . . . . . . . 171
Saumya Singh and Ajit Varma
Part II
Bacterial Symbiosis
12
Understanding the Evolution of Plant Growth-Promoting
Rhizobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Pratyusha Sambangi, Vadlamudi Srinivas, and Subramaniam
Gopalakrishnan
13
Rhizobia–Legume Symbiosis During Environmental Stress . . . . . . . 201
Sriram Shankar, Ekramul Haque, Tanveer Ahmed,
George Seghal Kiran, Saqib Hassan, and Joseph Selvin
14
Archaeal Symbiosis for Plant Health and Soil Fertility . . . . . . . . . . 221
Ranjith Sellappan, Senthamilselvi Dhandapani,
Anandakumar Selvaraj, and Kalaiselvi Thangavel
15
Microbial Symbionts of Aquatic Plants . . . . . . . . . . . . . . . . . . . . . . 229
Tejaswini Dash, Klaus-J. Appenroth, and K. Sowjanya Sree
16
Rhizobium Presence and Functions in Microbiomes of
Non-leguminous Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Alexandra Díez-Méndez and Esther Menéndez
Part III
Insect–Fungus Mutualism
17
Symbiotic Harmony Between Insects and Fungi: A Mutualistic
Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Saraswathy Nagendran, Surendra S. Agrawal, and Sheba Abraham
18
Panorama of Metarhizium: Host Interaction and Its Uses in
Biocontrol and Plant Growth Promotion . . . . . . . . . . . . . . . . . . . . . 289
Srinivas Patil, Gargi Sarraf, and Amit C. Kharkwal
Contents
vii
19
Arbuscular Mycorrhizal Fungi: Potential Plant Protective Agent
Against Herbivorous Insect and Its Importance in Sustainable
Agriculture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
Anandakumar Selvaraj and Kalaiselvi Thangavel
20
Eradication of Malaria by the Mutualistic Interaction Between
Wickerhamomyces anomalus and Anopheles sp. . . . . . . . . . . . . . . . . 339
Arpit Gupta, Arpita Balakrishnan, and Amit C. Kharkwal
Part IV
Microbial Symbiosis in Disease and Stress Management
21
Halophyte–Endophyte Interactions: Linking Microbiome
Community Distribution and Functionality to Salinity . . . . . . . . . . 363
Bliss Ursula Furtado and Katarzyna Hrynkiewicz
22
Root Endophytic Microbes and Their Potential Applications
in Crop Disease Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
Alka Tripathi, Ajit Varma, and Swati Tripathi
23
Do Mycorrhizal Fungi Enable Plants to Cope with Abiotic
Stresses by Overcoming the Detrimental Effects of Salinity
and Improving Drought Tolerance? . . . . . . . . . . . . . . . . . . . . . . . . 391
I. Ortas, M. Rafique, and F. Ö. Çekiç
24
Combined Use of Beneficial Bacteria and Arbuscular Mycorrhizal
Fungi for the Biocontrol of Plant Cryptogamic Diseases: Evidence,
Methodology, and Limits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
Yuko Krzyzaniak, Maryline Magnin-Robert, Béatrice Randoux,
Joël Fontaine, and Anissa Lounès-Hadj Sahraoui
25
Remediation of Toxic Metal-Contaminated Soil and
Its Revitalisation with Arbuscular Mycorrhizal Fungi . . . . . . . . . . . 469
Irena Maček
Part I
Fungal Symbiosis
Chapter 1
Current Status–Enlightens in Its Biology
and Omics Approach on Arbuscular
Mycorrhizal Community
Tulasikorra, O. Siva Devika, K. Mounika, I. Sudhir Kumar, Suman Kumar,
G. Sabina Mary, Uday Kumar, and Manoj Kumar
Abstract Symbiotic association has been subject to great heed in part because of the
prime of the disease to world agriculture, but also because both host and arbuscular
mycorrhizal fungi are persuadable to proceed investigational approaches. The goal
of the review is to furnish an overview of the microbial association system and
designate concurrent remarkable studies that amend our comprehension of the
biology and Omics of VAM fungi. The genomic studies have been organized to
long-term well-established areas of investigation, including disease development
and the characterization of proteins in relation to host. VAM fungi act as biological
control in global sustainable development under varied agroecological regions.
VAM fungi illustrate the plant–root/microbial interactions which serves gaining
knowledge and a wide out-look of issues in crop production/protection.
Keywords AMF · Genes · Omic approach · Spores · Enzyme activity · Biocontrol ·
Symbiosis · Ecology
Tulasikorra · I. S. Kumar · S. Kumar · U. Kumar
Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras
Hindu University, Varanasi, Uttar Pradesh, India
O. Siva Devika
Department of Soil Science and Agricultural Chemistry, Institute of Agricultural Sciences,
Banaras Hindu University, Varanasi, Uttar Pradesh, India
K. Mounika
Department of Genetics and Plant Breeding, Institute of Agricultural Sciences, Banaras Hindu
University, Varanasi, Uttar Pradesh, India
G. Sabina Mary
Department of Plant Pathology, Odisha University of Agriculture & Technology, Bhubaneswar,
India
M. Kumar (*)
Department of Plant Pathology, Acharya N.G. Ranga Agricultural University, Agricultural
College, Bapatla, Andhra Pradesh, India
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_1
3
4
1.1
Tulasikorra et al.
Introduction
Arbuscular mycorrhizal fungi (AMF) is one of the most widely circulated, ecologically and economically significant fungal groups and are ubiquitously associated
with the vast majority of plant families in numerous habitats around the world,
extending from the tropics (Zhao et al. 2001) or arctic-alpine habitats (Haselwandter
1987) to mesic (Muthukumar and Udaiyan 2000) and arid habitats (O’Connor et al.
2002). AMF is included in the order of Glomales (Morton and Benny 1990)
classified in the phylum of the Zygomycota, defined as compulsively symbiotic,
asexual organisms, and form symbioses with most land plants in almost any terrestrial environment (Smith and Read 2008). AMF uplifts crop development through
enhancement of growth, intake of nutrients and water, confronts the plant under
diverse stress situations, and also releases antioxidants. Omic approach is a crucial
aspect to perceive a deep insight into appropriate mechanisms of the mentioned
activities of AMF. Several SSU-targeting PCR primers (Lee et al. 2008) that amplify
fragments of 500–800 bp have been widely applied in ecological studies (Zhang
et al. 2010). AMF encompasses a wide range of species that performs variety of
functions. Phylogenetic analyzes, based on three rDNA markers, offer accurate and
stable resolution from phylum to species level. In comparison, 109 known species
and 27 populations comprising as yet unknown organisms have been studied
(Kruger et al. 2012). Besides these, AMF release several proteins and enzymes.
Proteins implicate disparate signaling pathways and also plays crucial role in
arbuscule formation. Enzymes catalyze in the decomposition of complex unavailable
organic substrates to simple utilizable forms through which plant uptake can be
enhanced. However, by keeping in view regarding change in climate and varied
ecology, current study emphasized the role of AMF in modern agriculture and
sustainability.
1.2
Goal for Studying Its Biology and Omics Approaches
Mycorrhizal fungi have a broad range of roles and can also lead as nectrotroph
lifestyle, endophytic, antagonists among host and nonhost plants, with roles which
vary in association of life. It is a long-term goal to unravel and cope mycorrhizal
functioning in our world. We must review full spectrum of varied factors, i.e.,
deciphering the intricacies of plant–microbe interactions, gene function, trade-off
in mutualistic symbiosis, capacity to adapt the changing soils or environment or how
to described genus and species.
1 Current Status–Enlightens in Its Biology and Omics Approach on Arbuscular. . .
5
Aim to Study
• How the AMF symbiosis form and arise?
• Broaden our perspective to include all functional and phylogeny studies.
• The most enigmatic groups are Glomales.
• Furthermore, characterization studies on wide range of plant and fungal species
could provide hints for understanding the unexplained relationship mutualistic
association in VAM fungi.
• Owing to their role, VAM can be classified as essential linkage species of plant
community.
• Elaborating research network pools that span from genetics to ecosystem levels.
• Essential tools for phytoremediation practices.
• It shed light on various pathways regulating the development of VAM fungi.
• In addition, the potential role of AM as biocontrol agents needs to be considered
and utilized in plant breeding programs for the selection of pathogen-resistant
cultivars.
• AMF as potential bicontrol agent due to low cost, wide availability, and environmental friendliness.
• Applications in the field of agroecology.
1.3
Phylogeny of New Speices of AMF
Diversispora Diversisporaceae family (order Diversisporales, subphylum
Glomeromycotina) are described by species producing spores at the tip of a
funnel-shaped hypha or cylindrical hyphal structure (Błaszkowski and Chwat
2013). Diversisporales comprises five families of the Diversisporaceae that has
five genera (Spatafora et al. 2016a, b). Symanczik et al. (2018) reconstructed the
true phylogeny of the “omaniana” fungus and to describe a new species in the genus
Diversispora.
Glomus Phylogenetic analyzes of rDNA sequences were performed in the Glomus
group A sensu (Schwarzott et al. 2001) which verified their uniqueness relative to
other recognized Glomus species. Thus the fungi are described as G. africanum
sp. nov. and G. iranicum sp. nov. Błaszkowski (2010) was the first person to report a
phylogenetic sequencing of the two new species Glomus africanum and
G. iranicum.
Dominikia and Kamienskia All Dominikia and Kamienskia spp. form colorless or
very pale colored, small glomoid hypogeous spores in more or less compact clusters.
Błaszkowski et al. (2015) included seven group of fungi G. achrum, G. bistratum,
G. iranicum, G. minutum, G. perpusillum, and two undescribed fungi with glomoid.
Lactarius It is vital species of ectomycorrhizal communities in different habitat
types ranging from low elevation to higher elevation rural and urban areas. Twenty
genus identified with three different species along with one unresolved species
(Barge and Cripps 2016). Eight taxa Lactarius alpinus v. mitis with Alnus incana,
L. barrowsii with Pinus flexilis, L. aff. brunneoviolaceus with Salix reticulata,
6
Tulasikorra et al.
L. luculentus v. laetus under mixed conifers, L. aff. Olivinus with Picea engelmannii,
L. pseudodelicatus with Populus tremuloides, and L. aff. tuomikoskii with Picea
engelmannii were identified and, respectively.
Laccaria Laccaria species has long been recognized as important ectomycorrhizal
associates of ectotrophic plants worldwide. They are known to form associations
with members of the Pinaceae, Dipterocarpaceae, Fagaceae, Myrtaceae, Betulaceae,
Tiliaceae and Salicaceae (Wilson et al. 2013). Ramos et al. (2017) during their study,
they found two species Fagus grandifolia var. mexicana and Laccaria
trichodermophora.
Acaulospora Acaulospora ignota and Claroideoglomus hanlinii, two new species
of arbuscular mycorrhizal fungi (Glomeromycota) from Brazil and Cuba. On basis of
morphological studies A. ignota, is distinguished by yellow colored, relatively small
(65–80 μm diam when globose) spores, whose upper surface of the structural
laminate spore wall layer is ornamented with flattened elevations or inconspicuous
warts these are usually deteriorating and disappearing with age. The most
distinguishing features of the second species, C. hanlinii, are its relatively small
(45–90 μm) and dark-colored (olive-yellow) spores, with a simple two-layered spore
wall. Acaulospora koreana isolated from forest soils in South Korea (Lee et al.
2018). New species listed of VAM fungi (Table 1.1).
1.4
Genomics
Genomics plays an great role to investigate genes in association with plant root and
VAM fungi Early decades scientists used limited technologies about genetically to
ecology level. We recently focus on AM fungus interaction and physiology of the
AM symbiosis and also is turning point to study their biology that yet remain to be
elucidated would now became accessible for investigation. Genomic sequencing
would provide information on genetic variants that may occur disease or reduction of
threat of disease development, even in asymptomatic plant.
To date, full genome sequences are available for only two AM fungi from a single
genus, Rhizophagus (Chen et al. 2018; Kobayashi et al. 2018). The isolate
DAOM197198 of Rhizophagus irregularis was used for genome sequencing as
(1) it is widely used for laboratory studies (2) it is highly sporulating, providing
large quantities of biological material and (3) a smaller genome size than many other
AM fungal species. The sequencing program was announced in 2004 and was
supported by the Joint Genome Initiative and founded by the US Department of
Energy. Glomus Genome Consortium was formed with several contributors, participated to provide biological material and sequences. Furthermore, the genome size
was estimated to be 153 Mb, in accordance with the value of 154.8_6.2 Mb
measured by flow cytometry. The first AM fungal genome was released in May
2013 (http://genome.jgi.doe.gov/Gloin1/Gloin1.home.html) before publication
(Tisserant et al. 2013). It was found that the genome of R. irregularis is haploid
1 Current Status–Enlightens in Its Biology and Omics Approach on Arbuscular. . .
7
Table 1.1 New species listed of VAM fungi
VAM fungi
Acaulospora
koreana
Host
Lindera obtusiloba and
Styrax obassia
Acaulospora
saccata
A. fragilissima
Scutellospora ovalis
Rhizophagus
neocaledonicus
Acaulospora ignota
Sorghum vulgare
Claroideoglomus
hanlinii
Septoglomus
mexicanum
Rhizosphere soils of
plant species of the
Poaceae
Roots of Phoenix
dactylifera
Rhizospheric soil of
two endemic Mexican
legumes: Prosopis
laevigata and Mimosa
luisana
Zea mays.
Location
Forest soils
in South
Korea
Soils in New
Caledonia
(South
Pacific)
Accession no.
MN792884.1
(SSU)
References
Lee et al.
(2018)
KY362430
(ITS)
Crossay et al.
(2018)
Natal, Brazil
KP191472
(ITS)
Błaszkowski
et al. (2015)
Cuba
KP191482.1
(its)
MK570915.1
(its)
Błaszkowski
et al. (2015)
ChimalSánchez
et al. (2020)
Pereira et al.
(2015)
Błaszkowski
(2017), Zu
et al. (2019)
Mexico
Rhizoglomus
dalpeae
R. Silesianum.
Savanna zone
R. maiae
Shrubs growing in a
tropical humid reserve.
Date palm plantation.
Brazil
Oman
LN881565.1
(its)
KF060313.1
(its)
MG183939.1
(its)
MN130954.1
(its)
MN130954.1
(its)
MN130957.1
(its)
MG459210.1,
Lolium perenne, Trifolium pratense, Plantago
lanceolata and
Hieracium pilosella.
Inka nut (Plukenetia
volubilis)
France, Germany and
Switzerland
LR723644.1
(its)
LR723643.1
Oehl et al.
(2019)
Peru
MN081581.1
CorazonGuivin et al.
(2020)
Acaulospora
papillosa
Septoglomus fuscum
and S. furcatum
Diversispora
omaniana,
Septoglomus
nakheelum, and
Rhizophagus
arabicus spp
Septoglomus nigrum
Paraglomus
occidentale
Roots of Arctotheca
populifolia and Cordia
oncocalyx in dry forest
Coal mine spoil heap in
Northeastern
Brazil
South Africa
and Brazil
Benin, West
Africa
Poland
Błaszkowski
et al. (2019)
Błaszkowski
et al. (2019)
Błaszkowski
et al. (2019)
Symanczik
et al. (2018)
8
Tulasikorra et al.
and does not show any evidence of recent duplication. Transposable elements
(TE) are strongly represented (up to 55 Mb of the genome is formed by repeated
TE) as expected from a large fungal genome. The mitochondrial (mt) genome of the
AM fungi is also considered during the sequencing, because it played a great role to
investigate the fungal ecology unlike normal fungi Malbreil et al., 2014 .
These genomic data has opened a new era in the study of AM fungi. The advent of
large scale sequencing approaches the studies on AM fungal taxonomy and systematics had risen to a new level (Spatafora et al. 2016a, b). New operational taxonomic
units (OTUs) at the species, genus, and higher taxonomic levels were identified with
the help of metagenomic approaches based on next-generation sequencing methodologies (Opik et al. 2013). Analysis of the sequenced genomes revealed that the
capacity for plant cell wall degradation and secondary metabolite production was
less even in AM fungi like many other biotrophic and some ectomycorrhizal fungi.
But they contain a large repertoire of small secreted proteins of unknown functions,
potentially candidate effectors for modulating the interaction with their host (Lin
et al. 2014; Zeng et al. 2018). The first indication of the AM fungi being fatty acid
auxotrophs was provided by genome sequence (Wewer et al. 2014). However, the
symbiotic physiology of AM fungi has been widely investigated, and it shows that
phosphorus is not only a nutrient recruited by extraradical hyphae in soil and
translocated to the host plant (Graham et al. 1981), but it is also a major regulator
for the establishment and function of AM symbiosis (Javot et al. 2007). A large
number of transcriptomic studies have given a good knowledge of physiology,
metabolism, and cell regulation modified in the host plants after the symbiotic
relationship.
1.5
Fungal Metabolism During Symbiotic Life
The role of AM fungi in mineral nutrient supply for the plants is the most widely
studied aspect. This supply of nutrients requires different metabolic machinery and
the coenocytic nature of AM fungal hyphae allows easier, faster, and low-energydemanding transport. Genomics has helped in the identification of a number of genes
and transcripts responsible for the effective symbiotic relationship and are explained
below.
• Most importantly for Phosphate transport and metabolism, R. irregularis contains
at least four different kinds of putative phosphatases that are expressed in
intraradical mycelium (IRM) that are able to cleave a broad range of substrates
to release Pi (Tisserant et al. 2012). Alkaline phosphatase activity and candidate
genes have been identified in AM fungi (Liu et al. 2013).
• Coming to the Nitrogen transport and metabolism, which is often underestimated,
AM fungi supply significant amounts of the total N taken up by plants
(Govindarajulu et al. 2005). Two high-affinity N transporters have been partially
characterized in R. irregularis namely, GinAMT1 and GinAMT2 (Pérez-Tienda
1 Current Status–Enlightens in Its Biology and Omics Approach on Arbuscular. . .
9
et al. 2011). Both the transporters are expressed in extraradical mycelium (ERM),
but GinAMT2 transcript levels are higher in intraradical mycelium (IRM) and
GinAMT1 is induced in ERM at low N concentrations. Tian et al 2010 has
identified a putative high-affinity nitrate transporter and a transcriptomic
approach showed that this gene was expressed more in IRM (Tisserant
et al. 2012).
• Total 445 gene models involved in carbohydrate metabolism including transport
of sugars from the plant to the fungus have been identified in R. irregularis and
AM fungal monosaccharide transporters (MST) and sucrose transporters were
also isolated further (Helber et al. 2011).
• A set of 432 lipid-related genes were identified for either transport or metabolism
in Gloin1 with respect to lipid metabolism. An unexpected and specific regulation
mechanism has been observed for lipid metabolism, as shown by numerous
experiments (Trépanier et al. 2005). All the genes involved in fatty acid synthesis
are present in R. irregularis has given by the biochemical studies showing that the
fungus did not obtain its fatty acid from the plant but was able to synthesize them
(Tisserant et al. 2012).
1.6
Proteomics
Limited research work has been reported in the field of protein biology. Scientists
had a hard task to differentiate the proteins of AM Fungi from plants. Moreover,
Post-translation modification assigns the covalent and usually enzymatic protein
modification after the biosynthesis of proteins. Moreover, proteins were controlled
by post-translation mechanism more than 200 different types which have been
reported it includes phosphorylation, ubiquitination, and proteolysis (Packer et al.
1997). Furthermore, polyprotiens separated by 2DE Electrophoresis on the basis of
isoelectric points and analysis of polypeptides by mass spectrometry methods either
de novo sequencing or peptide mass fingerprinting might outcome in identification
of fungal proteins. Antibodies corresponding to fungal proteins were used to identify
specific fungal species (Göbel et al. 1995). Early proteomics work on AM interactions made it clear that proteins aware of AM symbiosis in the early stages of
colonization are not quite the same caused in the late stages when the symbiosis is
formed and functional (Bestel-Corre et al. 2004). Della proteins are core for several
signaling pathways of gibberellic acid (GA), are required for arbuscule formation.
GA signaling also influences the arbuscule formation in monocots. (Floss et al.
2013). Glomalin Soil Protein (GRSP), a widespread glycoprotein developed by
arbuscular mycorrhizal fungi (AMF), plays a critical role in the functioning of the
ecological regeneration and the environment (Jiang et al. 2017). The subsequent
proteomics revealed that E.augustifolia seedlings inoculated with AMF improved
the secondary metabolism level of the phenylpropane pathway, increased the signal
transduction capacity of Ca2+ and ROS scavenging, encouraged protein biosynthesis, acceleration protein folding and prevented protein degradation under salt stress
10
Tulasikorra et al.
(Jia-Dong et al. 2019). We listed few concurrent proteomic studies on rhziobial
symbioses (Table 1.2).
1.7
Symbiotic Root–Microbe Interactions
The Symbiotic Plant-Mycorrhizal Symbioses
Earlier decades, two model legumes were used as forefont of proteomic research into
root association symbiosis, Medicago truncatula and Lotus japonicas (Cook 1999).
Proteins secreted by the general use of secretion systems. Primarily, Gram-negative
bacteria classifies four types of secretion systems (Types I, II, III, IV, V, VI (T1SS or
TOSS, T2SS, T3SS or TTSS, T4SS or TFSS, T5SS, and T6SS, respectively). Type
V to Type VI Animal secretion system and remaining plant secretion system. Nodo
is the first secreted rhizobial protein for which symbiosis played a role could be seen
in Rhizobium leguminosarum (de Maagd et al. 1989). The secretion pathways
discussed so far are also important for processes that are not related to host
interactions. Several secretion systems, by contrast, appear to have specialized in
mediating such interactions, with ability to translocate effector proteins into the
cytoplasm of the host cell as a defining element. These include the T3SS, T4SS,
T6SS Frame works. Viprey et al. (1998) provided the first definite evidence of
involvement of T3SS in the rhizobium-legume symbiosis. T3SS substrates come
in two different forms: effectors and proteins for helpers. Effectors are translocated
into eukaryotic host cells whereas helpers proteins help to translocate. Proteins
secreted by rhizobial T3SS are called outer proteins or Nops nodulations. So far
three proteins have been identified as helpers viz., NopA, NopB, and NopX (Saad
et al. 2008). HrpW has recently been shown to promote translocation of the T3SS
effector into plant leaves, making it an aid protein rather than a true effector (Kvitko
et al. 2007). HopG1 cuts down basal tobacco resistance (Oh and Collmer 2005).
These homology-based findings expand significantly the pool of potential T3SS
rhizobial effectors.
The Symbiotic Plant-Mycorrhizal Symbioses
In AM symbioses, which are the most common associations and have existed for
about the last 450 M years (Remy et al. 1994). Arbuscular mycorrhiza
(AM) originated from the Latin word “arbusculum” and the Greek words “mycos”
and “rhiza,” respectively, meaning small tree, fungus, and root. Predominantly two
types classified into ectomycorrhiza and endomycorrhiza. Ectomycorrhiza tends to
form symbiotic mutual relationships with woody plants, including birch, beech,
willow, pine, oak, spruce, and fir. An intracellular surface, known as the Hartig
Net is distinguished by ectomycorrhizal connections. The Hartig net is a highly
branched hyphae which connect the epidermal and cortical root cells. In addition,
ectomycorrhiza can be identified by forming a dense hyphal sheath that surrounds
the surface of the root so-called mantle. In other words, ectomycorrhiza has just
survived off the root. Together, ectomycorrhiza has only 5–10% of terrestrial plant
1 Current Status–Enlightens in Its Biology and Omics Approach on Arbuscular. . .
11
Table 1.2 Proteomic studies on rhziobial symbioses
Sl.
No
1
3
Rhizobium
leguminosarum
bv. trifolii
Frankia alni
4
B. japonicum
Identified proteins
60 deficient strain altered
proteins in QSS,
52 identified
Identification of two inducible flavonoids and 10 constituent proteins
126 proteins identified as
N-replete, relative to
N-limiting circumstances
17 proteins identified
5
M. truncatula
7 proteins identified
2DE
6
Rhizobium
leguminosarum
bv. trifolii
Funneliformis
mosseae and
Rhizophagus
irregularis
10 proteins identified in
association with
subterranean clover cultivar
3 genes (Fm201, Ri14–3-3
and RiBMH2) encoding
14–3-3-like proteins for abiotic stress responses and
arbuscule formation during
AM
Symbiosis
Identification of protein
abundance variations as part
of the local reactions of pea
nodules grown under splitroot conditions and
subjected to water stress.
377 plant protein identification in nodules,
SNARE proteins
(LjVAMP72a and
LjVAMP72b) for root symbiosis and root growth in
Lotus
Japonicas seeds
VAPYRIN protein interacts
with a symbiotic R-SNARE
of the VAMP721 family
A. thaliana as model plant
450 proteins in perennial
plants.
111 differentially expressed
proteins seed profiling I pea
seed
2DE
2
7
Species
S. meliloti
8
R. Leguminosarumbv.
viciae
9
S. meliloti
10
Mesorhizobium loti
11
P.
hybrida,
N. benthamiana
12
Glomus intraradices
13
R. Leguminosarum
Methods
MALDI-TOF 2DE
References
Gao et al.
(2007)
2DE, N-terminal
Sequencing
Guerreiro
et al. (1997)
MALDITOF,2DE
Alloisio
et al. (2007)
2DE
Panter et al.
(2000)
Bestel-Corre
et al. (2004)
Morris and
Djordjevic
(2001)
Sun et al.
(2018)
Starch
gel-electrophoresis
2DE
Irar et al.
(2014)
2DE
Larrainzar
et al. (2007)
Sogawa
et al. (2019)
SDS-PAGE
Semiautomated
image analysis
pipeline
Bapaume
et al. (2019)
2DE
Lingua et al.
(2002)
Mamontova
et al. (2019)
SDS-PAGE
(continued)
12
Tulasikorra et al.
Table 1.2 (continued)
Sl.
No
14
15
16
17
18
19
20
Species
Rhizophagus
irregularis
R. irregularis
Pinus pinaster–
Hebeloma
cylindrosporum
M. truncatula–R.
irregularis,
G. intraradices
Tree root–Laccaria
bicolor
Glomus mosseae
(GM) and
G. intraradices,
Funneliformis
mosseae
Identified proteins
75 proteins identified in
E. angustifolia seedlings
529 different peptides that
were confidently mapped to
474 R. irregularis proteins
in chicory roots
869 proteins constituting the
exoproteome of Hebeloma
cylindrosporum.
Root membrane proteome,
cadmium tolerance
Methods
LC-MS/MS
analysis
LC-MS/MS
analysis
References
Jia et al.
(2019)
Murphy
et al. (2019)
LC-MS/MS
analysis
Doré et al.
(2015)
2-DE, 2D-DIGE,
LC–MS/MS
Zhan et al.
(2018)
224 proteins identified in
involved in the cell wall
remodeling linked to hyphal
growth
3473 proteins were identified in the roots of Amorpha
fruticosa
131 differentially expressed
proteins (DEPs) were identified in F. mosseae treated
samples of soyabean.
2-DELC-MS/MS
Vincent
et al. (2012)
LC-MS/MS
Song et al.
(2015)
iTRAQ, LC-MS/
MS
Bai et al.
(2019)
species. On the other hand, endomycorrhizae are present in more than 80% of
established plant species, including grains and greenhouse plants such as most fruits,
grasses, flowers, and fruit trees. Endomycorrhizal relationships are characterized by
fungal penetration of the cortical cells, and the formation of abscesses and vesicles
by the fungi. Endomycorrhiza, in other words, has an attachment process inside the
root, with the hyphae of the fungus spreading outside the root (Fig. 1.1). Compared
with the ectomycorrhiza it is a more invasive relationship. Endomycorrhiza is further
subdivided into specific types: Arbuscular Mycorrhizae, Ericaceous, Arbutoid, and
Orchidaceous. Bestel and Co-workers (2004) characterized on Medicago truncatula
infected with Glomus mosseae find 14 protein changes in the preinfection stage and
23 protein changes in 14 days of arbuscular development. Analysis of proteins
shows that mycorrhizal fungi induced improvements in the redox or stress response
(peroxides and glutathione-transferases), respiration, and alteration of the cell wall.
Velvet and colleagues identified that membrane-associated M. truncatula proteins
with the fungus G. intraradices. Due to a lack of sequence knowledge and difficulties in growing AM fungi in pure culture, the detection of proteins from the fungal
partners of AM symbioses has been much more challenging. A proteome map of
mycorrhizal fungal proteins from Glomus intraradices has been founded and eight
1 Current Status–Enlightens in Its Biology and Omics Approach on Arbuscular. . .
13
Fig. 1.1 A schematic representation diagram on Ectomycorrhiza and Endomycorrhiza
of the 438 solved proteins were detected using tandem mass spectrometry (DumasGaudot et al. 2004).
Other Beneficial Interactions
Mostly grasses may enter into symbiotic relationships with nitrogen-fixing bacteria
promoting plant growth, which does not form a particular root structure like a nodule
(Reinhold-Hurek and Hurek 1998). The Trichoderma genus “free-living” fungi are
considered to be beneficial to plants, mainly through their defense against pathogenic soil fungi, but also through systemic resistance caused in plant roots and shoots
(Harman et al. 2004). A study of the T.harzianum secretome identified an aspartic
protease that could target fungal cell walls containing parasitized fungus proteins.
(Suarez et al. 2005). Gomes et al., 2017 analyzed gene regulation changes in
T. harzianum Epl-1 protein involved botrydial biosynthesis (BcBOT genes) in the
course of mycoparasitism association. Even, Brassicaceae includes plants that are
not hosted by arbuscular mycorrhizal fungi (AMF) such as used plant model
Arabidopsis thaliana (Poveda et al. 2019). However, one of the main challenges
to be addressed is how Trichoderma modulates the plant’s immune response to
establish beneficial interactions (Ramírez-Valdespino et al. 2019). In building a
beneficial relationship between Trichoderma and plants, the effectors can play key
roles, as illustrated in mycorrhizal systems like those of Laccaria bicolor and
Glomus intraradices. (Ramírez-Valdespino et al. 2019). Exploring these protein
functions, our study provides a detailed understanding of the architectural alteration
of the root, cell remodeling, and upkeep of cellular homeostasis during the mutualistic and development stage of plant life. Hopefully, our review provided valuable
14
Tulasikorra et al.
candidate proteins for further study of this beneficial combination of plant–fungal
interactions. Such results will further allow researchers to fully understand the
molecular networks and the regulation of biological pathways underlying plant–
fungal interactions.
1.8
Enzymes
In soil system, unavailable complex organic substrates can be decomposed into
utilizable forms for plants and microbes by the activity of enzymes which govern
nutrient cycling. Enzymes in soil commence through soil flora and fauna and are
substrate specific, also enhances rate of reaction at which organic residues decompose. Soil enzymes are bioindicators for soil quality and soil health (Killham 2002),
its activity relates microbial activity and soil properties. Soil enzymes associate with
the degradation of organic substrates (e.g., hydrolase, glucosidase), in nutrient
mineralization (e.g., amidase, urease, phosphatase, sulfates) besides further activities
such as pesticide degradation. In practice, enzymes have ample utilization in agriculture and bio-degradation which is eco-friendly. In the attempt to enhance soil
enzyme activity, pertinent research reveals the notably varying effect of AMF on
enzyme activity.
AMF forms mutual association with the roots of majority of land plant species
(Wang and Qiu 2006) and are mostly familiar to help phosphorus uptake by host
plant (Smith and Read 2008). Various soil enzymes and its activity alteration as a
result of AMF application is shown (Table 1.3.) Favorable growth conditions for
AMF i.e., generally neutral pH enhance the enzyme activity. Various enzymes were
identified and classified into different releasing groups based on nutrient transformation (Table.1.3) indicates that AMF positively affects the enzyme activity. In
wide range of crops great increase in the enzyme activity was observed by the
application of AMF.
1.9
Biology of AMF on Different Crops: Insight and Impact
Biology Literal meaning of the Latin word Arbusculum, mycos, and rhiza-meaning
tree, fungus, and root, respectively. The primary characteristic feature of arbuscular
mycorrhizal, the evolutionary precursor of most other mutualistic root–microbe
associations (belonging to phylum—Glomeromycota) fungi being the growth of
arbuscles and vesicles in the cortical cells of plant roots. AMF consists of 9–55% of
soil microbes and benefits around 90% of the total agricultural plants.
Insights of the Mycorrhizal Biology
The plants predominantly used to study the plant–fungal partnership are Medicago
truncatula and Lotus japonicus (Hause & Schaarschmidt (2009) Fukai et al. 2012).
1 Current Status–Enlightens in Its Biology and Omics Approach on Arbuscular. . .
15
Table 1.3 Response of soil enzymes activity to the application of AMF
S. No.
1
2
3
4
5
6
7
8
9
Enzyme
Esterase
Chitinase
Trehalase
Phosphatase
Endo-xyloglucanase
Endoglucanase
Endopolymethylgalacturonase
Endopolygalacturonase
Alkaline phosphatase
10
11
12
13
Phosphatase
Urease
Alkaline phosphatase
Acid phosphatase
P-releasing
% increase
in enzyme
activity
256%
197%
444%
166%
78–320%
33–145%
73–266%
148–416%
8–51.6%
P-releasing
N-releasing
P-releasing
P-releasing
39–58%
34–123%
193%
143%
Releasing
type
C-releasing
C-releasing
C-releasing
P-releasing
Plant
enzymes
Crop
Maize
References
Vázquez
et al. (2000)
Soybean
GarciaGarrido
et al. (2000)
Cucumber
(soil incorporated with
clover
leaves)
Red clover
Joner and
Jakobsen
(1995)
Berseem
clover
Ming-Yuan
et al. (2007)
Raiesi and
Ghollarata
(2006)
The development of AM fungi is based on the induction of specific gene
recognition cites and the elicitors such as strigolactones (Akiyama et al. 2010).
The first interaction results into reciprocation of diffusible signals and once the
fungi senses the vicinity of plant root system the signals are triggered toward the
development of host fungal symbiotic relationship (Bonfante and Requena 2011;
Gough and Cullimore 2011). The root released phytohormones belonging to the
class Strigolactones plays the next major role and the highly sensitive (up to a
minimal concentration of 10 nM) relevance in the establishment of symbiosis
(Besserer et al. 2006).
The Strigolactone (SL) biosynthesis pathway and its regulation by the NSP1 and
NSP2 transcription factors. NSP1 and NSP2 (yellow) promote expression of
DWARF27 (D27) (Liu et al. 2003). nsp2 mutants additionally accumulate
orobanchol, but the misregulated gene responsible for this accumulation is unknown.
All compounds with SL activity on plants and fungi are marked in orange. PDR1 is
required for SL transport within the plant tissue and for root exudation. There is
evidence for plant SL perception via a complex between the α/β-fold hydrolase
D14/DAD2 and the F-box leucine-rich repeat (LRR) E3 ligase D3/RMS4/MAX2
(Hamiaux et al. 2012). The mechanism of SL perception by AM fungi is elusive.
Products of deviating biosynthesis pathways (green) also play a role in AM (HerreraMedina et al. 2007). (Fig. 1.2).
The hypopodium of the fungi develops at the root surface based on the physicochemical features of root cell walls. The hyphopodia were noticed by G. gigantea on
16
Tulasikorra et al.
Fig. 1.2 The SL biosynthesis pathway and its regulation
isolated cell wall fragments of roots from the host plant carrot but not on the nonhost
Beta vulgaris. Further studies were performed based on the M. truncatula mutants
(ram) 1 and 2 (Gobbato et al. 2012). The fungal hyphopodium once attached releases
plant inducing signals, which induce expression of a suite of plant genes, calcium
spiking in rhizodermal cells, starch accumulation in roots, and lateral root formation
prior to colonization (Chabaud et al. 2011) Table 1.4.
1 Current Status–Enlightens in Its Biology and Omics Approach on Arbuscular. . .
17
Table 1.4 Host plants for various arbuscular mycorrhizal fungi
Fungus
Glomus mosseae, G. intraradices
G. Mosseae
Glomus mosseae
G. Mosseae, G. versiforme
Glomus etunicatum, G. mosseae, G. intraradices
G. Intraradices
G. Mosseae
Glomus intraradices, G. aggregatum Glomus sp.
Glomus mosseae
G. clarum
Acaulospora, Glomus
Acaulospora, Glomus
Glomus
Acaulospora
Acaulospora
A. scrobiculata, G. aggregatum
A. laevis, G. dimorphicum
A. scrobiculata, G. fasciculatum, Gi. albida,
S. calospora
G. multicaule, G. clarum, G. fasciculatum,
A. delicata, S. scutata
G. maculosum, G. glomerulatum, A. scrobiculata
1.10
Host
Pepper
Tomato (Lycopersicon esculentum cv
Zhongzha105)
Peanut
Trifoliate orange
Tabasi (mutated)
Zucchini squash (Cucurbita pepo L.)
Cotton
Onion (Allium cepa L) lettuce (Lactuca
sativa L.)
Maize, alfa
Mungbean (V. radiata)
Parthenium hysterophorus (L.)
Cynodon dactylon (L.) Pers
Lantana camara (L.)
Digitaria sanguinalis (L.) Scop
Cassia tora (L.)
Andrographis paniculata
Catharanthus roseus
Azadirachta indica
Centella asiatica
Hibiscus rosa- sinensis
AMF Applications in Different Crops Under Varied
Agro-Ecology
Agroecology can be described as the practice of applying ecological concepts and
principles to maintain agriculture sustainable (Gliessman 1992). India encompasses
huge diversity in landforms with differing environmental conditions produced wide
range of soils. Under these fluctuating conditions to obtain ensured yield, application
of chemical fertilizers, herbicides, and pesticides became mandatory which encumber the farmer economically. Because of changing climate and stress conditions,
benefit from the products shall be decreased. Promptly increasing population places
heavy strain on the natural resources for fulfilling requirements. These critical
situations lead the researchers toward inventing environmental friendly, economical,
and efficient techniques like adoption of microorganisms which can be beneficial in
varying environmental conditions to maintain sustainable agriculture. In this study,
we focused on the vesicular arbuscular mycorrhiza fungi and its role in agriculture
(Table 1.5) which can form association with plant roots, enhances nutrient uptake
especially phosphorus, thus helps in better growth of the crop and also resist the
plants toward biotic and abiotic stress conditions.
18
Tulasikorra et al.
Table 1.5 Role of AMF in crop performance under varied agroecology
S. No.
1
Agroecology
Hot summer
Mediterranean
to cold
semiarid
Crop
Persea
Americana
(avocado)
AMF
Glomus
fasciculatum
2
Humid
subtropical
Transgenic
tobacco
hairy roots
Glomus
intraradices
3
Humid
subtropical
Solanum
tuberosum
(potato)
Glomus
intraradices
Glomus
mosseae
4
Humid
subtropical
Cowpea,
Pigeonpea,
groundnut
Glomus
etunicatum,
Gigaspora
margarita
5
Hot desert
climate
Panicum
turgidum
Glomus
etunicatum
Glomus
intraradices
Glomus
mosseae
Impact
Improved the development of root system,
shoot: Root ratio in
plants inoculated with
AMF. Inoculation also
enhanced the uptake
of N, P, and K by plant
tissue as compared to
the rest of the
treatments.
Results reported that
anti-oxidation enzyme
activities were higher
in the plants inoculated
with AMF, through
which plants showed
tolerance against toxic
oxygen species and
phenol level up to the
concentration
25 mg L 1
Chlorophyll content,
fresh and dry weight of
plants at harvest,
growth of above and
below ground parts
were enhanced and
maximum in the
mycorrhizal infested
potato plants
Growth responses of
these three legume
plants inoculated with
mycorrhiza were significant. Shoot dry
matter production and
shoot phosphorus content were enhanced.
Anti-oxidation enzyme
activities viz., superoxide dismutase, peroxidase, glutathione
reductase, and compatible solutes were
enhanced in AMF
inoculated plants under
salt stress and tolerance
to salt stress was
increased significantly.
Reference
Vidal et al.
(1992)
Ibáñez
et al.
(2011)
Lone et al.
(2015)
Ahiabor
and Hirata
(1994)
Hashem
et al.
(2015)
(continued)
1 Current Status–Enlightens in Its Biology and Omics Approach on Arbuscular. . .
19
Table 1.5 (continued)
S. No.
6
Agroecology
Cold semiarid
Crop
Solanum
lycopersicum
AMF
Glomus
intraradices
7
Humid
continental
Zea mays
Glomus
intraradices
8
Semiarid
Triticum
aestivum
Glomus
mosseae
Glomus
fasciculatum
9
Humid
subtropical
Scutellaria
integrifolia
Gigaspora
margarita
Glomus
etunicatum
Glomus
intradicas
10
Hot and dry
climate
Zea mays
Glomus
constrictum
11
Island
Malus
pumila
Allium
porrum
Tagetes
patula
Glornus
epigaeum
Glomus
monosporum
Impact
In salt stress conditions
less H2O2, lipid peroxidation, and higher
proline content were
noticed in AMF inoculated plants. AMF also
reduced the impact of
salt stress on P, Ca, and
K uptake by plants.
Plants cultivated in
clay brick granules
with the colonization
of Glomus showed
higher colonization,
spore production and
root length as compared to rest of the
treatments
Growth parameters
such as plant height,
root length, shoot, and
root biomass production, grain size, and
photosynthetic pigment were found maximum in Glomus
inoculated treatments.
AMF inoculation to
plant roots resulted in
positive effect on
growth parameters
especially plant height,
fresh weights of root,
shoot, and seed.
Plant biomass production on dry weight
basis was significantly
superior in plants inoculated with AMF to
that of the treatments
of non-inoculated.
Positively influenced
the growth of the
plants.
Endomycorrhizal colonization decreased
with increase in phosphorus levels.
Reference
Hajiboland
et al.
(2010)
Gaur and
Adholeya
(2000)
Pal and
Pandey
(2017)
Joshee
et al.
(2007)
Omar
(1995)
Plenchette
et al.
(1983)
(continued)
20
Tulasikorra et al.
Table 1.5 (continued)
S. No.
12
Agroecology
Semiarid
Crop
Sorghum
bicolor
AMF
Glomus spp.
13
Humid
Maize, Sorghum,
chickpea
Glomus
clarum
14
Hot summer
Mediterranean
to cold
semiarid
Trifolium
repens
Glomus
mosseae
15
Humid
subtropical
Poncirus
trifoliata
(citrus)
Glomus
mosseae
Glomus
versiforme
Glomus
diaphanum
1.11
Impact
In response to CO2
hyphal length of AMF,
protein globulin and
aggregate water stability were increased.
Hyphal length, globulin were positively
correlated with aggregate stability
Enhancement of plant
growth, dry weight,
and root length were
recorded in the treatments inoculated with
AMF
Root growth, nodule
formation, dehydrogenase activity, phosphatase, β-glucosidase
activities, and soil fertility were improved
with inoculation in cd
polluted soils.
Mycorrhizal fungus
improved plant biomass, soil protein concentration, soil hyphal
length densities, and
peroxidase activities. It
enhanced water stable
aggregates and also
plant growth under
drought stress conditions. Reduced the hot
water extractable and
hydrolyzable carbohydrates concentrations
in soil.
Reference
Rillig et al.
(2001)
Simpson
and Daft
(1990)
Vivas et al.
(2005)
Wu et al.
(2008)
Opportunities and Challenges: AMF
We believe that significant progress will be made through the development and use
of genetic model systems in the early diverging host and nonhost lineages. Powerful
genetic tools are available already in Physcomitrella patens (moss) and Marchantia
(liverwort) (Zimmer et al. 2013; Hiss et al. 2014). Use of these and the Charophyte
green algae Penium margaritaceum as research models will lead to greater
1 Current Status–Enlightens in Its Biology and Omics Approach on Arbuscular. . .
21
understanding of the host mechanisms that gave rise to efficient AM symbioses
(Sørensen et al. 2014).
Significant progress in the analysis of plant systems modulating symbiotic interactions in AM has been made through the application of genetic, ultra-cytological,
biochemical, and molecular biology techniques. Mycorrhiza-defective legume
mutants provide direct evidence for specific symbiosis-related gene control and
afford the potential for analyzing some host genes and gene functions involved in
early and late events. However, the range of mutant phenotypes available is still
extremely limited. Expansion of the genetic approach to additional mutations is
essential to understand more fully the molecular mechanisms leading to morphological integration and reciprocal functional compatibility between AM symbionts.
Moreover, mutagenesis programs, including mycorrhiza-forming non-legumes such
as maize or tomato, should indicate to what extent AM development is determined
by processes other than those common to nodulation. Despite obvious similarities
between the infection processes of AM fungi and rhizobia in legumes, important
differences also exist. A number of non-nodulating legume genotypes are able to
form mycorrhiza (Duc et al. 1989; Wyss et al. 1990; Gianinazzi-Pearson et al.
1991b), and some nodule infection thread components are not synthesized around
the fungal symbionts in mycorrhizal roots (Gianinazzi-Pearson et al. 1990; Perotto
et al. 1994). Evidence for AM symbioses appears in the fossil record before the
evolution of legume species. This raises the interesting possibility that during
evolution, rhizobia may have exploited plant processes involved in symbiotic
interactions with arbuscular mycorrhizal fungi and modified them to a completely
different purpose, that of nodule development (Gollotte et al. 1995b). Use of
heterologous probes for genes that mediate other plant–microbe interactions has
provided much information on cell-cell interactions in AM. This approach has been
particularly effective in evaluating the restricted expression of defense responses that
is essential for compatibility between plant and fungal symbionts. Clearly, plant–
fungal compatibility is an active phenomenon in AM, because in activation of a
single plant gene, as in the case of the Myc-1 pea mutants, results in resistance.
Moreover, the fact that AM symbioses are widespread in the plant kingdom makes
this symbiosis an interesting model system for dissecting the genetic and molecular
bases of biotrophic plant–microbe interactions in general. Although transport processes between plant and fungal cells are crucial for the proper functioning of AM,
they represent one of the most poorly understood aspects of the symbiosis. The use
of tools such as antibody and nucleic acid probes and promoter-GUS fusions in
transgenic plants will accelerate the investigation of the ill-defined area of interface
function. The complex cellular relationship between roots and AM fungi necessitates
continuous recognition and signal exchange between both partners. These exchanges
affect the regulation of genes whose products participate in the metabolic and
structural changes leading to the symbiosis (Gianinazzi et al. 1995). Signal or
receptor molecules involved in this dialog have not been identified. Polysaccharides,
hormones, and polyamines are among the candidates for signaling, because they are
important components of various developmental processes in plants and are associated with AM development (Dannenberg et al. 1992; E1 Ghachtouli 1995). The
22
Tulasikorra et al.
search for plant genes mediating symbiotic events in defined root tissues at specific
times has begun. The next few years should see exciting advances toward identifying
these plant determinants, the molecules they encode, and the ways they are regulated
in AM, the most common root symbiosis in the plant kingdom.
The idea of mycorrhizae and their use in the agronomic sector has taken hold in
many scientific experiments around the world. Most of the research has focused on
the host plant benefits attributed to arbuscular fungi from the points of view of yield
and resistance to biotic and abiotic stresses. Great efforts have been made in order to
study the processes and metabolic pathways involved in the fungus, aimed at the
greater absorption of nutrients and water and greater resistance to pathogens,
salinity, and heavy metals. Despite the numerous studies on horticultural and
forestry plants, wheat, which is one of the most important food crops worldwide,
has been the subject of many tests on mycorrhizal inoculation. With a view to greater
environmental sustainability, the selection and cultivation of cereals in agricultural
systems with a low environmental impact could be based on the selection of wheat
varieties with highly effective mycorrhizal symbiosis. In the last few years, the
research has recognized notable differences in plant susceptibility and/or responsiveness to AMF among wheat genotypes that differ in ploidy number or geographic
origin (DeVita et al. 2018). Significant genotypic differences were detected in the
ability to form mycorrhizal symbiosis, and some significant markers, representing a
Quantitative Trait Locus (QTL), were detected on wheat chromosomes (Lehnert
et al. 2017).
Future research should therefore not focus only on the AM fungus colonization
capacity, but it could take into account the ability of single grain accessions to form a
mycorrhiza, based on the results obtained by previous genetic characterization. The
identification of molecular markers closely associated with a mycorrhiza could be a
very effective tool for selecting highly effective plants for symbiosis and developing
wheat varieties suitable for low-environmental impact agricultural systems. At the
same time, the identification and selection of the most infectious and efficient
mycorrhizal fungi in combination with wheat will facilitate their use as biofertilizers
to overcome the loss of soil biological fertility, reduce chemical inputs, and alleviate
the effects of biotic and abiotic stress.
1.12
Conclusion
VAM fungi studies offer shot-term snapshots of inner depth behavior on physiological changes. However, it is to be expected that the rapid technological advances in
the field of molecular biology would fill the still current scientific and functional
differences between arbuscular mycorrhizas and other more symbiotic species
whatever experimentally high tractable. Thus, it was concluded that the discovery
of new genera and species provides a means of recognizing and investigating the
practical importance of AM and provides a new potential for habitat protection,
revegetation, and sustainable growth. Mycorrhizal fungus could demonstrate its
1 Current Status–Enlightens in Its Biology and Omics Approach on Arbuscular. . .
23
beneficial effect in a diverse range of environmental conditions, be aggressive
against pathogens, and be able to colonize roots of even disease-resistant varieties.
Genomic sequence databases responsible for the symbiosis for estimation of fungal
taxa number and its ecology. Unpublished work on additional plant mutants in the
mycorrhizal colonization pathway in several laboratories and studies to achieve the
transformation of mycorrhizal fungi would provide innovative input into process
control mechanisms in the near future. Therefore, we encourage future studies to
investigate both root and fungal characteristics as separate entities concurrently.
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Chapter 2
An Insight through
Root-Endophytic-Mutualistic Association
in Improving Crop Productivity
and Sustainability
Nitika Thakur
Abstract The Endophytic Symbiotic Association (ESA) has proven as an important
tool in uplifting various areas related to crop productivity, nutritional profile, issues
related to sustainability, food quality, and security. These beneficial associations
have highlighted the burning scenario that is generally equipped with the conventional and chemical cultivation patterns, but moving toward a safe area which
includes the use of EPHs (Enhanced Plant Holobionts) consortiums is a best choice
toward a sustainable agriculture. The use of these types of associates with mutualistic associations have benefited the host in terms of increased uptake of nitrogen,
efficient utilization of nutrients, increased photosynthetic activity and levels, introducing a systemic resistance toward various plant pathogens, pests, and diseases.
Secretion of osmoprotectant, decreasing the availability of ROS (Reactive Oxygen
Species) and finally leading to enhanced plant growth, crop productivity, and yields.
Further, in this direction many integrated systems (SRI-System of Rice Intensification) have been incorporated with these endophytic symbionts, which have yielded
spectacular results in terms of pest resistance and crop yields. Thus, it can be
concluded that integrating the endophytic symbiotic associations with the different
cropping patterns can easily solve the issues related to crop quality, yields, and
sustainability.
Keywords Endophytes · Enhanced Plant Holobionts (EPHs) · Agricultural
Sustainability · System of Rice Cultivation (SRI) · Reactive Oxygen Species · Crop
quality and productivity
N. Thakur (*)
Department of Biotechnology, Shoolini University of Biotechnology and Management
Sciences, Solan, Himachal Pradesh, India
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_2
31
32
2.1
N. Thakur
Introduction
The endophytes have been known to colonize the plant roots for decades, but the fact
that these mutualistic associations can be directed toward crop sustainability and
improvement came into limelight since recent years. Initial researches took a new
turn when the symbiotic associations between plants and microorganisms were
identified in the form of root galls. Furthermore, when these studies were expanded
these root galls were identified as “Nodules” composed of both bacterial and plant
proteins, which can help in fixing nitrogen (Hirsch 2009) and other essential
nutrients to the leguminous plants. Similarly, these symbiotic associators which
helped in increasing plant productivity were recognized as “Mutualistic fungi’s” or
the arbuscular mycorrhizal fungi (AMF) (Harrison 1997). In the 1920s and 1930s
some strains of fungi “Trichoderma” were reported with an antifungal activity which
was working against the pathogenic fungi (Weindling 1932, 1934) rendering multiple benefits and enhanced productivity to plants (Berch et al. 2005). A similar
group of fungi “Piriformospora” with multiple benefits (Harman 2000; Harman
et al. 2004; Varma et al. 1999) was observed which working efficiently against
various plant diseases. So, the endophytes can be broadly defined as the beneficial
microorganisms that can be extracted or isolated from the surface disinfected plant
tissues and do not visibly harm the plant (Hallmann et al. 1997). Through penetration
and colonization process, these microorganisms become part of regular symbiotic
plant–microbe system representing a composite entity (Shrivastava et al. 2018).
Plants existing in association with the microbial entities constitutes the
“Holobionts” which refers to the combination or assemblage of different ecological
units which symbiotically works together (Margulis 1990; Rogers 2016; Malley
2017) not only restricting itself to some microorganisms but covering a wide era of
all microbial symbiont communities.
The growth curve and performance rate of holobionts can be successfully altered
positively by introducing screened microbial stains of specified microbial communities that basically enhances or modifies the root microflora. These types of
alterations which are intervened purposely result in modification of simple
holobionts to EPH’s (Enhanced Plant Holobionts). The EPH’s can be induced
more numerously for agricultural purposes by inculcating the selected microbial
strains which when given the opportunity to colonize the root zone, can raise crop
yields by root colonization resulting in growth enhancement of both roots and
shoots, enhancing efficient nutritional uptake, inducing resistance to pests, diseases,
and developing tolerance toward biotic/abiotic stress. The EPHs have an altered root
zone having larger and deeper root system, enhanced assured crop yields, efficiency
in nitrogen supply and its utilization, enhanced photosynthesis levels, etc.
2 An Insight through Root-Endophytic-Mutualistic Association in Improving Crop. . .
2.2
33
Endophyte Distribution Pattern, Grouping,
and Transmission Mode
A diverse microbial community (Archaeal, fungal, bacterial, and parodistic communities) are widely distributed within plant groups (Hardoim et al. 2015a, b).
The identification and estimation of these fungal associates range from traditional
1.5 million species (Hawksworth 2001) to the updated newer data by sequence
analysis which highlights 5.1 million fungal species, with one million involved in
mutualistic associations (Strobel and Daisy 2003). These fungal associates predominately belong to class “Ascomycetes” that have been recovered ubiquitously from
regions ranging from extreme hot temperatures to the extreme cold one’s (Arnold
2007, 2008). Similarly, these also colonize different regions within a plant, that may
vary from leaves, stems to the extreme roots, playing an active role in synthesizing
the secondary metabolites (Porras-Alfaro and Bayman 2011; Thakur 2018a). However, the positive increase in the use of endophytes suggests an expanding recognition by the scientific communities to exploit various potential benefits of these
endophytes for crop improvement and to attain sustainability in yields (Hardoim
et al. 2015a, b; Card et al. 2016).
The classification criteria to differentiate various fungal endophytes were based
on unique symbiotic and ecological framework, phylogeny, and life history traits.
The endophytes are classified into Class 1 Endophytes (Clavicipitaceous), which are
known to form (Rodriguez et al. 2009) most economic associations with the aboveground tissues (Johnson et al. 2013) of grasses. The non-clavicipitaceous are further
categorized into 2, 3, and 4 subclasses. The Class 2 endophytes can effectively colonize
roots, stems, and leaves, generally belonging to ascomycota and few basidiomycota.
The plant-endophyte association in case of tropical, non-vascular, and vascular
plants can be observed in case of Endophytes-3. However, Class 4 endophytes or
DSE (Dark Septate Endophytes) are biotrophic facultative fungi which has a special
feature of producing dark melanized septate hyphae while colonizing plant roots
(Vidal and Jaber 2015).
The Endophytic-transmission routes can vary from Horizontal to vertical transmission. A typical example that satisfies both transmission routes is the Epichloe
species. The horizontal transmission depends on the reproductive structures of
endophytes which include the spores that can be either (Gao and Mendgen 2006)
dispersed by wind or by a vector from plant to plant transmission. While the vertical
transmission can be easily mobilized in laboratories enabling the artificial inoculation of grass-endophytic association for commercial purposes (Johnson et al. 2013).
2.3
The Beneficial Endophytic Secretions: Bioactive
Natural Products
The endophytes have been bestowed with beneficial secretions (Fig. 2.1) which
includes bioactive natural products that can work as antimicrobials, anti-insects, and
as plant beneficial addictive.
34
2.3.1
N. Thakur
The Antimicrobial Bioactive Metabolites
There are many promising secondary metabolites which have been identified as
potential antimicrobials (230 secondary metabolites) against pathogens and pests in
various horticultural and agricultural operations. For example, ambuic acid (Li et al.
2001) which is found to be an antifungal and anti-oomycete agent isolated from
Pestalotiopsis microspora which is found to be active against fusarium and Pythium
species. Similarly, a compound showing antifungal, antibacterial, and antialgal
properties known as “Colletonoic acid” in Colletotrichum sp. isolated from (Hussain
et al. 2014) Artemisia annua. A strong bioactive compound called as “Scandenin”
isolated from Derris scandens has a potential antibacterial activity against Bacillus
megaterium. These types of bioactive metabolites are known to be produced by the
fungal associates and none have been reported to be produced by bacterial strains.
Twenty-eight volatile compounds secreted by an endophytic fungus (Muscodor
albus) have been used for inhibiting and killing certain fungi and bacteria. Interestingly, 50 different volatiles have been reported to inhibit the growth of Grampositive and Gram-negative bacteria, and some plant pathogenic fungi. Furthermore,
artificial volatile mixtures are being processed and hold great potential and
Fig. 2.1 Potential beneficial effects of plant-associated symbionts on plant growth and health
(Mitter et al. 2013)
2 An Insight through Root-Endophytic-Mutualistic Association in Improving Crop. . .
35
application in food industry, household applications, industrial uses, and agricultural
implementation.
2.3.2
Anti-Insect Bioactive Potential
The drastic equation of chemical insecticides has led to devastating decrease in crop
yields and crop quality. The use of safe alternatives like biopesticides is a boon to
agricultural sustainability. Additionally, the secretion of these types of anti-insect
metabolites by the endophytes has added an organic texture to the agricultural
practices. The Nodulisporic acids (Indole diterpenes), displays anti-insecticidal
activity against blowfly by activating the insect’s glutamate-induced chloride pathways. Similarly, an insect repellent “naphthalene” produced by Muscodor vitigenus
which is effective against mothballs (Daisy et al. 2002).
The endophytic secondary metabolites (Ola et al. 2013) that may work efficiently
as antimicrobials, anti-insect, etc., may get triggered by the limitation of food
supplies, presence of different cell components, activation and deactivation procedures of cell signaling and increasing competition rates between the different
organisms (Thakur 2017b, c).
2.4
The Quadra-Brigade of Endophytic Microbes: The
Components of EPH’s
The most important symbiotic microbes which have been recognized as backbone in
terms of multiple benefit potential that may range from deriving nutrients and
enhancing plant–endophyte association.
The four important categories have been designed which serves as components of
EPHs:
2.4.1
Rhizobiaceae
This class generally introduces a wide variety of bacteria belonging to family
Rhizobiaceae, which shows (Jones et al. 2007; Boogerd and Van Rossum 1997)
complex interactions in the form of root nodules with the leguminous plants. An
infection thread gets initiated which confronts the bacterial path toward plant root
cells, where final structures known as “bacteroid” can be formed. The complex
nodulated structure is hence initiated by the leguminous plants which contains
leghemoglobin (Iron containing protein), thus proving a hypoxic environment
(Schmidt et al. 1994; Chi et al. 2005) for nitrogen fixation and finally the free
36
N. Thakur
nitrogen gets reduced to ammonium ions that can be easily utilized by the plant. This
class has highly specific interactions that generated ammonium pool for efficient
plant growth (Thakur 2017a, b).
2.4.2
Piriformaspora indica
This class generally penetrates the root cortical cells and colonize the area by
establishing root interactions accompanying programmed plant cell death (Varma
et al. 1999; Samuels and Hebbar 2015).
2.4.3
Trichodermal strains
Trichoderma has been recognized as the most widespread species (Saprophytic
associations) which is probably found in abundance in soil, tree stems, and shoots.
Their association can be short lived via direct penetration into the plant cell walls or
can become perfect endophytes persisting throughout the lifetime (Klein
and Eveleigh 1998; Neumann and Laing 2006; Gill et al. 2016).
2.4.4
AMF: Arbuscular Mycorrhizal Fungi
The obligate association is required as they cannot be cultured or grown unless or
until they get a plant host. The process includes a chemical signaling pathway which
initiates the infection establishment and colonization process, followed by
the formation of “Arbuscules” that are located between the host cell wall and plant
cell membranes (Strack et al. 2003a, b; Parniske 2008a, b).
2.5
EPHs: A Mutualistic Boon for Sustainable Agriculture
The successful development and spread of EPHs highlight a strong potential
(Table 2.1) for increasing the crop productivity, promising better crop yields, and
ensuring agricultural sustainability in terms of cost to benefit ratio. The following
parameters are listed which focusses on EPHs role in various plant protection
mechanisms and enhancing crop productivity.
2 An Insight through Root-Endophytic-Mutualistic Association in Improving Crop. . .
37
Table 2.1 Examples of the abilities of endophytic symbiotic microorganisms to increase plants’
productivity and yield (Harnam and Uphoff 2019)
Interactions of symbiotic
microbes
Rhizobiaceae
Associated
crop
Soyabean
Rhizobium
R. leguminosarum bv. trifolii
Common bean
Rice, wheat,
and corn
AMF (Glomus versiforme)
All types of
crops
Piriformaspora indica
Over 150 plant
species
P. indica
Barley
Trichoderma afroharzianum,
T. virens, T. viride, and other
species
Numerous
crops
T. afroharzianum
Tomato, corn
Effects of endophytic association with the
host plant
Meta-analysis showed 6–176% increase in
soybean yields across 28 studies. On farmer
fields in Michigan, yields were increased by
23–45% where inoculants had not been used
previously. Average yield increased 2–3%
where inoculants had previously been used.
In Indiana, yield, increases were 1.5–2%.
Increases of 2–3.5 t/ha under dry conditions
Increases in yield were seen under field conditions. With corn, not all plant genotypemicrobial combinations increased yield
Across numerous studies in the literature,
AMF inoculation has resulted in increases in
yield but not statistically different from zero.
In grasses, the combination of aerially
applied endophytic fungi and AMF gave
greater than expected results than from either
alone.
Increased shoot and root growth is seen
compared to untreated controls in drought but
not well-watered conditions. Inactivation of
reactive oxygen species (ROS) by gene
expression change was required. Various
studies have identified plant growthpromoting activities of plants whose roots
were colonized by P. indica, as reviewed.
Improvements in plant performance include
better seed germination under temperature,
improved resistance of plantlets during
micropropagation, and stress resistance.
P. indica reduced effects of stresses and
pathogens, inducing reprogramming of plant
gene expression, which resulted in increased
plant biomass and resistance to abiotic
stresses. Include the upregulation of enzymes
that inactivate toxic levels of reactive oxygen
species (ROS) that are formed in plants under
stress.
Inoculation with the organism induced
increased growth responses in numerous
vegetable species, greenhouse ornamental
plants, and cereal crops
Seed treatments applied to corn or tomato
resulted in endophytic colonization of plant
roots. Colonization is associated with
increased resistance to stresses and is causally associated with higher levels of expression of enzymes that inactivate ROS
(continued)
38
N. Thakur
Table 2.1 (continued)
Interactions of symbiotic
microbes
Chitooligosaccharides,
lipochitooligosaccharides
Associated
crop
Rhizobiaceae
and AMF
6-Pentyl-α-pyrone (6PP),
1-Octen-3-ol (1o3),
Harzianic acid (HzA)
T.
afroharzianum
Hydrophobins and other
hydrophobin-like proteins
(Hp)
Trichoderma
spp.
2.5.1
Effects of endophytic association with the
host plant
Application, even to the soil, increased fruit
yield, and increased total amount of polyphenols. Increased seedling growth of roots;
increased yields of corn and other crops
including leaf area, shoot mass, and root
mass; root branching; increased photosynthesis; changes in plant gene expression;
induced resistance to plant diseases. LCOs
are produced by the bacteria, but COs may
elicit similar plant responses. Compounds
added to plants of many kinds result in
season-long disease resistance and plant yield
increases.
Application of this volatile unsaturated lactone molecule, even to the soil, increased
fruit yield and increased the total amount of
polyphenols as effectively as did treatments
with the organism.
Seed treatments with picoliter quantities of
this volatile metabolite resulted in seasonlong improvements to shoot and root growth
in corn as effectively as did treatments with
the fungus itself.
Induced plant Défense responses and are
inhibitory to soil microflora. Hydrophobic
proteins induce plant resistance and increase
plant growth is great variability between
these proteins, and only a few have beneficial
activity.
Induces immunity to a virus, a fungus, a
bacterium, and an oomycete plant pathogen
Promoting Plant Growth and Increasing Market Value
The mutualistic associations have led to an increase in nitrogen-fixing and nitrogen
utilization abilities, which affects the overall plant growth. The various seed treatments and colonization of legumes with various strains of Rhizobiaceae family have
successfully led to increase in crop yields (Thilakarathna and Raizada 2017). The
current market value of such products corresponds to $ 230 million respectively
(Mordor 2018). The proliferation strategies in the roots of rice, wheat, and other
cereals have led to effective colonization and increasing growth and crop yields.
The need of the hour is to increase the photosynthetic efficiencies, which can be
easily met by “reprogramming of plant genes.” This technique includes reframing or
redesigning chloroplasts (For example, redesigning tobacco chloroplasts) which has
shown to enhance crop yield by increasing the photosynthetic abilities (Chi et al.
2010; Wu et al. 2018a, b; Long et al. 2018).
2 An Insight through Root-Endophytic-Mutualistic Association in Improving Crop. . .
39
AMF stands as strong foundation for many plant species for survival purposes,
especially those living under adverse conditions. It has been observed that AMF has
been found to offer limited benefits in terms of crop productivity. (Markmann and
Parniske 2009), but these organisms can increase the uptake of nutrients from the
soil especially the uptake of phosphorus (Strack et al. 2003a, b). Additionally, they
are beneficial in terms of inducing resistance (Tahat et al. 2014; Akhtar et al. 2015;
Alaux et al. 2018) against plant pathogens, reducing abiotic stresses, and providing
maximum benefit in agriculture where the soil disruption is minimum and carbonfarming systems are incorporated. On the other hand, Trichodermal strains have
been known to set up long associations with the host enhancing the root and shoot
growth. The selected known strains (P. indica) of Trichoderma induce crops resistant to pests and pathogens, reduce abiotic stress, and finally activate the internal
systems which refill the plant operating system with more photosynthetic capabilities
and adding on more chlorophyll for efficient plant growth (Thakur 2018a). The
Trichodermal strains and AMF can be easily propagated for successful implementation in agricultural practices (Woo et al. 2014).
2.5.2
Integrated Pest and Disease Management
The endophytes possess an ability to systemically induce the potential to reduce or to
completely eliminate the plant diseases and pests by introducing “systemic resistance.” The best example of endophytic association is seen in Rhizobia-legume
interaction through mycoparasitic colonization, production of bacteriocins which
can fight against number of plant pathogens, production of siderophores which can
lead to sequestration of metal ions preferably iron ions required by pathogens for
their growth. AMF are also categorized in terms of producing an internal systemic
resistance which enables the host to fight against multiple diseases (Mustafa et al.
2017; Kashyap and Thakur 2017). The Solanaceae family when colonized by
endophyte Rhizophagus, is observed with lesser incidence of Phytopthora. The
plant resistance against viruses can also be induced where the symptoms can be
found to a lesser degree than usual cases (Example: tomato leaf curl virus) (Maffei
et al. 2014).
The root colonization process through various endophytic associations has
led to successful reduction in diseases, including root rot pathogens (Verticillium,
Rhizoctonia, etc.). It has also led to reduction in various types of mycotoxins, as in
case of P. indica colonization with wheat roots for the reduction of wheat head
blight, and activation of ROS (Reactive Oxygen Species), thus surviving the biotic
and abiotic stresses (Narayan et al. 2017).
Trichoderma possesses various abilities to control the harmful organisms
(Fusarium, Colletotrichum, Phytopthora, and nematodes) (Thakur 2018a, b).
Various strains like T. harzianum have been successfully employed to control
infections initiated by Pythium (Harman et al. 2018).
40
2.6
2.6.1
N. Thakur
Successful Trials: Integrating Crop Management
Practices with Endophytic Symbionts
The SRI Trial: System of Rice Intensification
The effects of integrating fungal endophyte (Trichoderma asperellum) with the crop
management systems recommended for rice cultivation, were found to be beneficial
and had more impact when grown in SRI, when compared to the conventional
cultivation systems (Uphoff et al. 2010). It was found that the SRI Systems could
modify the environments for rice cultivation and resulted in increased microbial
activities around the root zone, decreased susceptibility to sheath blight, reduction in
methane emissions, and net reduction in greenhouse gases, increased biomass,
increased water use efficiency and higher rates of photosynthesis. This system also
promotes the utilization of organic biofertilizers and amendments in preference to
chemical pesticides and fertilizers.
2.7
Improved Agricultural Prospective of EPHs
EPHs are the promising consortium for improving different aspects of cultivation
systems, providing different cellular and molecular benefits
2.7.1
Developing Tolerance to Stress by Optimizing the Redox
Environment
The symbiotic association of endophytes with their respective hosts results in
altering the genetic makeup by changing the gene expression that ultimately leads
to scavenging or detoxifying the active ROS (Reactive oxygen species), which may
lead to development of toxins. The enzymes involved in detoxification of ROS
include APX (Ascorbate Peroxidases) and CAT (Catalase) etc. (Zuccaro 2002). The
antioxidants which are released by the symbionts may result in lowering the ROS
stress and keeping the internal environment at low ROS levels, so that the internal
system can work efficiently and neutralize the adverse effects. Another advantage is
that these associates can secrete osmoprotectants (proline, betaines, and sugars)
which can help the host partner to survive under drought conditions (Amato et al.
2008; Thakur 2018a, b). This in turn could help in raising photosynthetic levels and
result in better plant growth and productivity.
2 An Insight through Root-Endophytic-Mutualistic Association in Improving Crop. . .
2.7.2
41
Sequestering the Future Agricultural Stress
• Developing an efficient mechanism through symbiotic association to reduce
harmful forms of nitrates and to efficiently increase the nitrogen efficiency rates.
• To reduce the stress (abiotic/biotic) to be faced in near future due to climatic
variation and global warming.
• Enhancing the carbon sequestration so that it can be fixed during plant growth and
reducing methane emissions.
• Increasing soil productivity through the use of organic amendments and symbiotic associations, so that food quality and security issues do not intervene in
agricultural sustainability.
• Creating awareness among the farmer folk to successfully implement the organic
strategies and make more use of these symbiotic associates rather than moving
toward a chemical cultivation mode (Thakur 2018a, b).
2.8
Conclusion
The use of symbiotic endophytes (EPHs) in agricultural applications can enhance
nutritional quality, increased resistance to pest and diseases, increased uptake of
nutrients and water utilization, thus introducing systemic resistance for scavenging
the ROS (Reactive Oxygen Species) which can finally detach or decrease the levels
of toxin production. The utilization of such symbiotic associates can increase soil
productivity by increasing the microbial communities (PGPR’s) around the
rhizospheric area, making it healthier and more fertile for plant growth and finally
boosting up the crop productivity and agricultural sustainability. Therefore, it is
recommended in near future to utilize more EPHs consortium to solve issues related
to carbon sequestering and micro-farming situations.
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Chapter 3
Interaction Between Root Endophytes
and Plants: Their Bioactive Products
and Significant Functions
Dhriti Kapoor and Nitika Kapoor
Abstract Root endophytes exist in almost every identified plant species. Their
capability of entering and flourishing in the plant tissues make them distinctive,
which indicates their multidimensional connections within the host plants. Root
endophytes influence various significant processes of the host. They trigger the
growth of the plants, stimulate defensive response against pathogens, and mitigate
abiotic stresses. Several studies have confirmed that endophytes interaction with the
host plant is the same as that of the plant growth-promoting (PGP) microbes reside in
the rhizosphere. Besides, these endophytes play crucial role in stimulating the
productivity of plants and in boosting up their resistance against temperature,
salinity, heavy metals, drought, and biotic stresses. Furthermore, root endophytes
have potential to synthesize myriad of bioactive compounds including antioxidants,
antibiotics, anticancer and antimicrobial agents, immune-suppressive compounds,
and insecticides. Such compounds comprise a broad range of organic molecules such
as peptides, carbohydrates, aromatics hydrocarbons, etc. and these compounds play
vital role in host–microbe relationship. This chapter aimed to give background
knowledge of the developments in endophyte biology, functions of root endophytes,
and bioactive compounds synthesized by them.
Keywords Root endophytes · Bioactive products · Genotypic and phenotypic
expression · Stress resistance
D. Kapoor
Department of Botany, School of Bioengineering and Biosciences, Lovely Professional
University, Phagwara, Punjab, India
N. Kapoor (*)
PG Department of Botany, Hans Raj Mahila Maha Vidyalaya, Jalandhar, Punjab, India
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_3
45
46
3.1
D. Kapoor and N. Kapoor
Introduction
The term endophyte, coined by De Bary (1866) is defined as any micro-organism
colonizes healthy plant tissues without causing any symptoms and noticeable injury
to the host plant. Most common microbes existing as endophytes in plants are fungi
and bacteria (Padhi et al. 2013; Khare et al. 2018). Endophytes are microorganisms
that live in plant tissue partly or in all of their life cycle. They can be beneficial,
neutral, or disadvantageous depending on their interaction with host plant. For
example, rhizobia and mycorrhizal fungi are regarded as beneficial whereas Fusarium sp. that causes wilt disease in many economically important food crops is taken
as disadvantageous (Anyasi and Atagana 2019; Rodriguez et al. 2009). An array of
symbiotic lifestyle has been defined depending on fitness benefits or impacts to host
and symbionts. Plant ecology, fitness level, and biological activities of plants are
profoundly affected by microbial symbionts. The association of plants with microbial symbionts was found to more than 400 million years old. This relationship starts
the moment a land is inhabited by plants (Kozyrovska 2013; Anyasi and Atagana
2016). Endophytes behave the same way in their colonization ability as phytopathogens. So they can be used as biocontrol agents for various pathogens of plants.
Some of the microorganisms help to assist the plant growth under adverse conditions, whereas other endophytes have been involved in the synthesis of some novel
compounds and antifungal metabolites (Kumar et al. 2014; Afzal et al. 2014).
After colonizing the plant roots, these microorganisms become part of a symbiotic plant-microbe system. Now, these plants should not be regarded as independent
entities instead they should be regarded as composite organisms. Such plants that
exist in association with their microbial colonizers constitute holobionts. The term
holobionts was originally proposed by Margulis (1990) to describe diverse microorganisms that combine asexually to create new integrated hereditary symbionts. But
this concept was later expanded to describe more generally host organisms and all of
their microbial symbionts (Rogers 2016).
The performance and growth of plant holobionts can be improved by purposely
altering the root’s microflora. Such plant holobionts show better growth and performance and they are called as EPHs, i.e., enhanced plant holobionts (EPHs). EPHs
can be induced within agricultural systems by the selective addition of microbes.
EPHs are better than conventional plants as associated microorganisms can be
capitalized on both for the human race and for the sustainability of our environment.
Several benefits associated with EPHs include enhanced plant growth, larger and
deeper root system, higher yield, efficient uptake and use of nutrients, enhanced rate
of photosynthesis and enhanced immunity against various biotic and abiotic stresses
(Harman and Uphoff 2019).
The present chapter focuses on the concept of root endophytes, their lifestyle and
relationship with plants, and beneficial interactions with host plant particularly in
relation to bioactive products synthesized by endophytes.
3 Interaction Between Root Endophytes and Plants: Their Bioactive Products and. . .
3.2
47
Lifestyle of Root Endophytes
An inclusive definition of endophytes does not specify their functional relationship
with host plant. Apart from commensalistic symbionts, they can exist from dormant
pathogens or saprotrophs to mutualistic associates. The mutualistic association by
colonizing plant tissues both intercellularly or intracellularly is a well-versed component of their lifestyle. Most of the recent studies clearly show that survival and
health of plants are very much dependent upon these microorganisms (Fesel and
Zuccaro 2016; Potshangbam et al. 2017). This concept is well explained with
rhizobia-legume symbiosis, which is also considered as one of the best-described
endophytic relationships (Santoyo et al. 2016).
It has been suggested that endophytes have instigated from the rhizosphere
microbes or seed-borne microbial communities, but genomic studies and their
correlation with them show that these microbes are more versatile and may contain
genes for novel traits which are beneficial to the host plant (Ali et al. 2014).
Endophytes also manufacture or induce the host plant to produce metabolites that
improve plant growth and assist them to adapt the different environmental conditions
(Vega et al. 2008; Lugtenberg et al. 2016; Lata et al. 2018; Khare et al. 2018).
There is another concept of “plant microbiome” which explains the coevolution
of plants and their symbionts and tracked the benefits out of the relationship (Turner
et al. 2013). The expression of plant genes in the presence of the endophytes
provides evidences about their effects on the host plant (Berendsen et al. 2015).
The contemporary “omics” based approaches like genome sequencing, comparative
genomics, next-generation sequencing (NGS), microarray, and metagenomics may
provide an in-depth detail on endophytic lifestyle (Kaul et al. 2016). Endophytes
play an imperious role to maintain the health of plants. They help in enhancing
growth and yields of plants by protecting or preparing them against various abiotic
and biotic stresses (Khare et al. 2018).
Harman and Uphoff (2019) divided the plant root endophytes into four major
groups viz. (a) bacteria of the family Rhizobiaceae; (b) arbuscular mycorrhizal fungi
(AMF) of the phylum Glomeromycota; (c) certain strains of fungi of Ascomycetous
genus Trichoderma; and (d) fungi of the order Sebicales, Piriformaspora indica.
Although these organisms are phylogenetically distinct, each group has independently evolved and colonizes the plant roots, thus becoming resident plant root
endophytes. These four groups are regarded as true plant symbionts as they confer
advantages to host plant and at same time they drive nutrients, shelter, and other
benefits from the host plant. Although their mode of infection and lifestyle within
plant roots differ they provide similar advantages to plants (O’Malley 2017).
Bacteria of family Rhizobiaceae infect the leguminous plants through plant root
hairs in conjunction with complex plant-microbe chemical signaling. The host plant
produces infection threads that guide the bacteria to cortical cells of root where they
morph into nitrogen-fixing bacteroids. Later on, the host plant produces a complex
structure called nodule for nitrogen-fixing bacteroids. The cells of nodule contain a
specific protein called leghemoglobin which provide anaerobic environment to
48
D. Kapoor and N. Kapoor
bacteroids necessary for nitrogen fixation. (Andrews and Andrews 2017; Jones et al.
2007). Some of the members of family Rhizobiaceae are also colonize the roots of
nonleguminous plants like cereals and potatoes (Schmidt et al. 1994; Chi et al.
2005). The second group of symbionts, arbuscular mycorrhizal fungi (AMF) are
obligate plant symbionts. AMF form association with roots of terrestrial plant
species which involve complex process of chemical signaling between fungus and
host plant (Markmann and Parniske 2009). After the penetration of fungi in host
plant, a prepenetration apparatus (PPA) analogous to infection thread of rhizobium
bacteria helps the fungi to reach a specific cell of host. After penetration AMF forms
large lobed structures called arbuscules that are located between cell wall and cell
membrane of host plant cell. Large surface area of arbuscules provides greater area
for exchange of nutrients and other metabolites between fungus and host plant cells
(Parniske 2008).
Trichoderma species have many diverse lifestyles including saprophytic growth
in soils where they degrade complex substrates like cellulose, lignin, chitin, etc. In
addition to saprophytic lifestyle they also exhibit symbiotic behavior by colonizing
tree’s branches and roots. Trichoderma species directly penetrate the plant cell wall
and persist as endophyte for entire life of the plant (Harman et al. 2004; Zachow et al.
2016). Piriformaspora indica, the fourth group of symbionts also directly penetrate
the root cells and establish colonies within them. The penetrated fungus further
strengthens their colonies by programmed death of root’s cortical cells (Samuels and
Hebbar 2015).
All the four groups of symbionts produce signaling molecules called SAMPs
(symbiont-associated molecular patterns) which were previously designated as
MAMPs (microbial-associated molecular patterns). SAMPs interact with receptors
located on plasmalemma of plant cell. Such interactions are essential to permit
infection and plant receptivity leading to nodulation in case of rhizobium-legume
interactions and to root infections with AMF (Jones et al. 2007; Parniske 2008).
SAMPs include diverse lipochitooligosaccharides (also called as Nod factors) that
are produced by Rhizobiaceae and AMF; cellotriose produced by P. indica; and
peptides and proteins produced by Trichoderma strains (Johnson et al. 2018; Das
et al. 2015; Ruocco et al. 2015).
SAMP transduced MAPK (mitogen-activated protein kinases) mediated signals
in host plants which in consequence reprogrammed the gene expression of plants.
Reprogramming occurs by alterations in the cell’s chromatin; by DNA methylation
in the upstream regulatory portions of genes or by modification of the histone
proteins. These changes result in “gene priming,” a process where gene products
are not expressed until these products are needed. In a “primed” state, genes are
expressed more rapidly and more fully in response to various abiotic and biotic
stimulating factors (Ramirez-Prado et al. 2018; Conrath et al. 2015). Various
SAMPS produced by different symbionts and their role in plant productivity and
yield are summed up in Table 3.1.
3 Interaction Between Root Endophytes and Plants: Their Bioactive Products and. . .
49
Table 3.1 Role of symbiont produced SAMPs in plant productivity and yield
Symbionts
Rhizobiaceae and
AMF
SAMPs
Chitooligosaccharides
and
lipochitooligosaccharides
Trichoderma
spp.
1-Octen-3-ol
Trichoderma
spp.
Harzianic acid
Trichoderma
spp.
T. afroharzianum
Hydrophobins and other
hydrophobin like
proteins
6-phenyl-α-pyrone
Trichoderma
formosa
Plant response like
proteins
3.3
Effect on plants
Improved seedling growth,
enhanced yield of corn and other
crops, and boosted photosynthetic efficiency.
Seed treatment with this metabolite results in season-long
improvement in shoot and root
growth in corn
Show antifungal and growthpromoting activities; help to
chelate iron.
Increased plant growth and
induce resistance against various
plant diseases.
Increased fruit yield and the total
amount of polyphenols.
Improve yield of plants by
inducing immunity against a
wide spectrum of pathogens
References
Janczarek
et al. (2015),
Das et al.
(2015)
Harman
et al. (2018)
Vinale et al.
(2013)
Ruocco
et al. (2015)
Pascale et al.
(2017)
Cheng et al.
(2018)
Plant–Endophyte Relationship
There are multidimensional interactions between endophytes and their plant hosts,
particularly, in relation to maintaining the health of the plant (Fig. 3.1). Fungal
endophytes change chemical and physical characteristics of the leaf such as highcellulose content and lamina density, which provide toughness resulting in reduced
herbivory, specifically by leaf-cutting ants. Endophytes also prime the host plant’s
defensive responses against phytopathogens (Table 3.2). Early detection of the
phytopathogen is possible by cell surface receptor kinases (RK) and cytoplasmic
kinases (CK) mediated activation of ethylene/jasmonic acid transduction pathway.
Endophytes have the ability to control many plant diseases and pests through
induction of systematic resistance (Harman and Uphoff 2019; Khare et al. 2018;
Chadha et al. 2014). Endophytes also help to remediate the several abiotic and biotic
stresses by enhancing the expression of various stress-responsive genes and by
regulating the level of certain phytohormones like abscisic acids and gibberellins.
Plants colonized by endophytes show enhanced expression of genes that detoxify
reactive oxygen species (ROS). ROS are neutralized by antioxidants and
antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT) ascorbate peroxidase (APOX), glutathione reductase (GR), dehydroascorbate reductase
(DHAR) and monodehydroascorbate reductase (MDHAR), etc. Antioxidants like
ascorbic acid and glutathione are only effective if they are present in reduced form.
Endophytes enhanced the expression of genes encoding for antioxidative enzymes
that maintain the level of reduced forms of antioxidants and detoxify the ROS
(Moore et al. 2016; Tyagi et al. 2017). Endophytic associations also alleviate
50
D. Kapoor and N. Kapoor
Fig. 3.1 Interaction of host plant with endophytes
metal phytotoxicity by various methods which include sequestration, extracellular
precipitation, intracellular accumulation, and biotransformation of toxic metals to
less toxic or nontoxic forms (Mishra et al. 2017).
3.4
Bioactive Product Synthesized by Endophytes
Isolation of the secondary products from the endophytes started in 1980s, where the
main concern was the toxins which are associated with the disease outbreak in
livestock. The information regarding their beneficial effects for human beings and
plants were not apparent till far long. Endophytes can be used as a novel and
beneficial source of drug specifically used as taxol, an anticancer drug, which
came into existence with the recent discovery of Taxomyces andreanae in Montana
in a yew tree (Strobel et al. 1993). Therefore, extraction of the endophytes and their
bioactive products can be useful for the living organisms including human beings as
it further leads to improved vitality in plants and further contributes to the product
formation in plants. According to the reports, taxol acts as a persuasive compound
which is having a strong capacity to fight against oomycetes and the mechanism it
carries against the oomycetes is almost similar to the strategies against hastily
dividing cancer cells (Young et al. 1992). Plants can be protected from the water
molds by the use of taxol and similar compounds obtained from Taxus sp.
3 Interaction Between Root Endophytes and Plants: Their Bioactive Products and. . .
51
Table 3.2 Abilities of root endophytic symbionts to increase host resistance to pathogens
Symbiont
Rhizobia
Host
Legumes
like soybean, lentil,
chickpea,
and lupine
Indian
mustard
Pathogen
Fusarium,
Sclerotinia,
Cylindrocladium,
and Rhizoctonia
Response of host plant
Pathogen controlled by
mycoparasitism of bacteria within fungal
pathogen
References
Das et al.
(2017)
Sclerotinia
sclerotiorum
Significant control of
white rot disease
AMF
(Funneliformis
mosseae)
Barley,
tomato
Blumeria graminis
(powdery mildew of
barley), Tomato leaf
curl virus
Systematic resistance
reduced level of disease
by almost 80%
AMF
(Rhizophagus
irregularis)
Piriformaspora
indica
Potato
Phytophthora
infestans (late blight
of potato)
Fusarium head blight
Reduction in symptoms
but not under high disease pressure
Significant reduction in
symptoms and also
reduced the production
of mycotoxins by
Fusarium
Disease controlled by
stimulation of
antioxidative system of
plants
Chandra
et al.
(2007)
Mustafa
et al.
(2017),
Maffei
et al.
(2014)
Alaux
et al.
(2018)
Rabiey
and Shaw
(2016)
Mesorhizobium
loti
Wheat
Piriformaspora
indica
Chick pea,
barley
Alternaria brassicae,
Botrytis cinerea (fungal disease of leaves
and powdery mildew)
Trichoderma
spp.
Tomato
Meloidogyne hapla
(root nematodes)
Controlled severity of
disease by inducing
systematic resistance
and production of
phytoalexins
Waller
et al.
(2005),
Narayan
et al.
(2017)
Harman
et al.
(2004,
2018)
Certain techniques of fermentation are required for the characterization of bioactive secondary products synthesized by the living organisms, thus endophytic
organisms are extracted from the pure culture for their application in this methodology. For this method, certain medium like M-I-D medium rather than a complex
medium is used (Strobel and Daisy 2003). In the process of purification, certain
defined medium is applied, which further removes certain other products. For the
duration of 2–3 weeks, the fermentation process is carried out in big containers.
Certain compounds like butanol, ethyl acetic acid or methyl chloride can be used
initially for the extraction. After extraction, flash chromatography, TLC, and HPLC
are performed. Each step is followed by certain bioassay, where bioactive compounds are taken. Therefore, in the end, results are obtained in the form of crystals,
which is the most required form and finally can be analyzed by using X-ray
52
D. Kapoor and N. Kapoor
crystallography. Eventually, the data obtained from spectroscopic analysis must be
in correlation with the X-ray crystallographic analysis (Jones et al. 2008).
Globally, different endophytic isolates of Pestalotiopsis microspora are collected
from several plants which are present at different sites. This fungal species is one of
the most commonly isolated endophytic fungi specifically from temperate and
tropical regions. Many times it has been observed and allocated for various species,
mainly depends upon the host plant from where it is extracted.
3.4.1
Ambuic Acid
An isolate of Pestalotiopsis microspora gives rise to the formation of extremely
active cyclohexenone, which acts as an endophytic organism of Fagraea bodenii
and exists in plateau of Papua New Guinea (Li et al. 2001). This antifungal
compound has slight antifungal activities. Through solid-state NMR techniques, it
was found out that this compound is a first natural compound with the appropriate
structure which allows the spatial arrangement of hydroxyl group on the seventh
carbon atom (Harper et al. 2003a, b). Ambuic acid was considered as the most
important compound having anti-quorum sensing activity specifically in the Gramnegative bacteria and it was estimated from the primary work done on its extraction
and resolving the structure (Nakayama et al. 2009). Production of ambuic acid
causes inhibition of the quormones of Staphylococcus aureus and Listeria innocua,
which are cyclic peptide. Ambuic acid acts as a principal product, which further
leads to the formation of anti-pathogenic drugs targeting quorum sensing-facilitating
virulence expression in Gram-positive bacteria.
3.4.2
Cryptocin
One of the Asiatic plants namely Tripterygium wilfordii is having resilient immunosuppressive activities. Nature of endophytes of any living organisms must be
studied completely whether all of them are likely to synthesize the same or different
bioactive compounds. Apart from this, an endophytic fungus was extracted from one
of the key species of Cryptosporiopsis. Its conidiospores were found to be segmented, which is different from other members of the group (Li et al. 2000). Certain
unique antifungal properties were contained in the compounds namely cryptocandin,
which is a lipopeptide and cryptocin, that is tetrameric acid, of this particular
organism. Spectroscopic techniques and X-ray crystallography are used to extract
out and characterize this acid (Strobel 2014a). This particular compound plays a
significant role against fungi, which is specifically plant pathogen in comparison to
those which cause disease in human beings. Pyricularia oryzae is considered as the
most susceptible fungus, which acts as a pathogen for causing rice blast disease all
over the world, hence affects productivity.
3 Interaction Between Root Endophytes and Plants: Their Bioactive Products and. . .
3.4.3
53
Colutellin A
In the tropical forest region of Costa Rica, an endophytic fungus namely
Colletotrichum dematium was extracted out (Strobel 2014b). While dealing with
the different species of tropical regions, only this endophyte was isolated under
infrequent situations from that particular plant species outlandishly. Against Botrytis
cinerea and Sclerotinia sclerotiorum, respectively, this fungus leads to the synthesis
of a unique peptide antimycotic namely Colutellin A, which has least inhibitory
concentrations of 3.6 μg/mL at 48 h.
3.4.4
Pesatcin
Pesatcin is a unique benzofuran, which was obtained from a culture of an endophytic
fungus namely Pestalotiopsis microspora, extracted from Taxus wallichiana native
to the Himalayan foothills. It was very interesting fact that these endophytes also
lead to the formation of taxol along with the two unique antioxidants. 1,3-dihydroisobenzofuran a new compound is synthesized by the endophyte which is having
strong antioxidant potential superior to the trolox, a derivative of tocopherol. Mild
antifungal properties are also possessed by this unique compound (Harper et al.
2003a, b). With the help of methylene blue, extraction of culture fluid is done, which
is subsequently followed by the silica gel chromatography and hence leads to the
isolation of pestacin. Its structure and composition were estimated by X-ray diffraction method and 13C and 1H NMR spectroscopic techniques. According to the X-ray
analysis, it was found that pestacin exits in nature as a racemic mixture. These
analyses also include certain other features of the compound like its antioxidant
potential and post-biosynthetic racemization. Another compound namely
isopestacin has been synthesized by this specific endophyte and besides it contains
identical bioactivities as in pestacin (Strobel et al. 2002).
3.4.5
Torreyanic Acid
Certain specific endophytes existence is estimated in Florida Torreya plant as it is
considered as a rare and threatened species. From Torreya taxifolia plant, torreyanic
acid was extracted out, which is native to Northern Florida. Torreyanic acid is a
dimeric quinone acquired from the endophyte namely Pestalotiopsis microspora
(Lee et al. 1996). This specific compound exhibits the feature of cytotoxicity against
various human cancer cell lines.
54
3.5
D. Kapoor and N. Kapoor
Influence of Endophytes on Genetic and Phenotypic
Expression of Plants
In plants, endophytes play a significant role in stimulating induced systemic resistance against the pathogens (Kloepper and Ryu 2006). Host gene expression is
regulated by the endophytes which are foliar in their existence and further influence
the physiology and defensive responses of plants (Salam et al. 2017). Against the
attack of pathogens, certain plant growth regulators like salicylic acid and jasmonic
acid play significant roles to combat it (Khare et al. 2016). Resistance against
pathogens including insects are improved by gibberellin-synthesizing endophytes
by following jasmonic acid and salicylic acid pathway of ameliorating their toxic
effects (Waqas et al. 2015). According to Kavroulakis et al. (2007), induced systemic resistance is elicited by Fusarium solani against Septoria lycopersici, acts as a
pathogen for tomato by inducing the expression of pathogenesis-related genes in the
root tissues. Inoculation of Theobroma cacao was done by foliar endophytic fungi,
Colletotrichum tropicale, which further led to decrease in the infection caused by
Phytophthora sp (Mejia et al. 2008). In several plants like Arabidopsis, Theobroma,
etc. resistance against diseases is provided by inoculating Candida tropicalis, which
results in elicitating different constituents of the ethylene defense strategy and also
activates certain defensive genes (Mejía et al. 2014).
Certain volatile organic compounds are possessed by some endophytic bacteria,
which are also having antibacterial, antifungal properties and apart from this, they
can also combat the attack of other plant pathogens like nematodes. It was reported
that the endophyte present in black pepper namely Pseudomonas putida BP25
contributed in combating the diseases with the help of volatile organic compounds,
caused by various pathogens like Pythium myriotylum, Gibberella moniliformis,
Phytophthora capsici, Colletotrichum gloeosporioides, Radopholus similis
(a parasitic nematode), etc. (Sheoran et al. 2015). Macrophomina phaseolina, a
plant pathogen caused charcoal rot is controlled by siderophore contributing to
rhizobium formation in various crops (Arora et al. 2001). Pseudomonas fluorescens,
endophytic fungi were isolated from the olive roots, which is having antagonistic
property against different plant pathogens (Mercado-Blanco et al. 2004). Certain
endophytes have also been reported by Etminani and Harighi in 2018, which were
found to be having antagonistic properties against Bacillus, Pseudomonas, Serratia,
and Stenotrophomonas from Pistacia atlantica which further showed the regulation
of Pseudomonas syringae and Pseudomonas tolaasii.
Stimulation and expression of stress genes, production of secondary metabolites,
quenching of free radicals like hydroxyl radical, singlet oxygen, etc. are certain
defensive strategies activated by the endophytes (Lata et al. 2018). Osmotic stress
and other abiotic stresses are ameliorated by a plant growth regulator namely
abscisic acid, which further regulate the closing of stomata and plant growth and
survival (Waqas et al. 2012). Synthesis and signaling pathway of ABA get restrained
in the presence of significantly important microorganisms in the plant endosphere,
hence helpful in improving the development and productivity of plants in salt stress.
3 Interaction Between Root Endophytes and Plants: Their Bioactive Products and. . .
55
Lately, ABA signaling is modulated by salt-tolerant Dietzia natronolimnaea, which
is blamable for salt resistance in Triticum plants, was authenticated by the
upregulation of ABA responsive gene namely TaABARE and TaOPR1 genes
(Ilangumaran and Smith 2017). For the mutual symbiotic association of roots of
Arabidopsis and beneficial fungus Piriformospora indica, ABA is required (PeskanBerghofer et al. 2015). For the amelioration of abiotic stress from Oryza sativus
plant, upregulation of certain genes like dehydrin, aquaporin, and malonialdehyde
have been observed by inoculating endophytic Trichoderma harzianum (Pandey
et al. 2016).
According to a report, it was analyzed that the endophyte namely Enterobacter
sp. SA187 can form colonies on the surface as well as the inner tissues of roots and
shoots of Arabidopsis and caused salinity resistance by stimulating the ethylene
signaling strategies with the use of synthesized 2-keto-4-methylthiobutyric acid.
This strategy can be used to improve the salinity stress resistance and hence growth
and productivity by using the endophyte Enterobacter sp. Another endophyte
namely Curvularia protuberata, a fungal species leads to improving the survival
of grass Dichanthelium lanuginosum even at stressed conditions like increased soil
temperatures, specifically in the grass exist in Yellowstone National Park (Márquez
et al. 2007).
3.6
3.6.1
Significant Role of Root Endophytes
Endophytes Are Saprobic Decomposers
Certain reports suggested the role of endophytes as saprobes and their interactions
with each other (Tan et al. 2003). The thing is incidental; though, some of the
saprobes are obtained from the endophytes (James et al. 2002). There are various
reports which suggest that endophytes are helpful in regulating the components of
the host, endophytes residing inside host plants, and host specificity of saprobes
(Frank et al. 2017). There are various endophytes of wood and leaf tissues which are
specific to the host family, colony formation, and the matters within leaf and wood
(Lo Piccolo et al. 2010). Certain endophytes are pathogens whereas some possess
mutualism (White et al. 2014).
3.6.2
Endophytes as Producer of Antibiotics
Attack of various plant pathogens like fungus, bacteria, nematodes, etc. is overcome
by endophytes. With the hardwood species of Europe, a fungus species called
Pezicula cinnamomea is associated and Cryptosporiopsis quercina is considered
as its imperfect stage. It was extract out as an endophyte from a medicinal plant
Tripterygium wilfordii, which is native to Eurasia (Castanheira et al. 2017).
56
D. Kapoor and N. Kapoor
Outstanding antifungal property was reported in C. quercina against Candida
albicans and Trichophyton sp., human pathogens, when experiment was performed
on petri plates. From C. Quercina, antifungal compound cryptocandin was extracted
out and characterized, which acts as a distinctive type of peptide (Castanheira et al.
2017).
The compound cryptocandin and the related compounds are also used against
different fungi, which leads to skin and nail diseases. Another compound namely,
Cryptocin, which is an exclusive tetramic acid, also synthesized by C. Quercina
(Taghavi et al. 2009). This rare compound has persuasive action against Pyricularia
oryzae along with a number of phytopathogenic fungi. P. viridiflava is one of the
subordinate of an assembly of plant-related fluorescent bacteria. In the leaves of
grass species, it is positioned on and inside the tissues (Perrine-Walker et al. 2007).
Pseudomycins is alternative group of antifungal compounds which is synthesized
by a plants related pseudomonad (Paungfoo-Lonhienne et al. 2010). From the
Artemisia annua, another endophyte Colletotrichum sp. is isolated, which leads to
the formation of biologically active metabolites that possess diverse antimicrobial
activity. Artemisia annua is an old Chinese herb that is very familiar for the
production of an anti-malarial drug called artemisinin which is having potential to
reside over various geographically regions. In A. annua, Colletotrichum sp. is
present, which not only contributes in the synthesis of metabolites protecting against
human pathogen but also from bacteria, fungi etc., which cause diseases in human
beings and some of the fungus species in plants also (Baker and Orlandi 1993).
3.6.3
Antiviral Compounds
Antibiotic compounds obtained from endophytic fungi can be used for the reduction
of survival rate of viruses. Solid-state fermentation of endophytes specifically fungus
Cytonaema sp. leads to the isolation of protease inhibitors namely Cytonic acids A
and B which are two novel human cytomegalovirus protease inhibitors. With the
help of NMR and mass spectrometry techniques, their structures isomers were
illuminated (Ali et al. 2014). Because of the absence of relevant screening system,
compound discoveries program may face certain confines.
3.6.4
Endophytic Fungal Products as Anticancer Agents
Certain anticancerous compounds can be isolated from the endophytes like Paclitaxel and its derivatives, which is considered as the foremost group. Paclitaxel is
usually observed in almost in all species of taxus globally and it is an active
diterpenoid. This compound contributes to excluding tubulin molecules by depolymerization at the time of cell division (Doty 2017). This anticancerous compound is
3 Interaction Between Root Endophytes and Plants: Their Bioactive Products and. . .
57
considered to make world’s first anticancerous medicine, which is used in the
treatment of human tissue-proliferating diseases.
3.6.5
Antidiabetic Agents from Endophytes
From the endophytes namely Pseudomassaria sp., a nonpeptidal fungal metabolite
[L-783] was extracted out, which is gathered from an African rainforest near
Kinshasa in the Democratic Republic of the Congo (Germaine et al. 2006). This is
used as a substitute for the insulin without causing any damaging effects on the
digestive tract. A drastic inhibition in the blood sugar level was observed in two mice
models, when they have been orally given L-783,281. Such significant findings may
cause new development in therapies for diabetes (Germaine et al. 2006).
3.7
Conclusion and Future Insights
Endophytes are a rich and consistent source from which certain novel compounds
are extracted out. They are very diverse in nature, can lead to the synthesis of various
pharmaceutically important compounds which are highly bioactive in nature which
further seek attention from the scientific societies in extraction and examination of
their biotechnological potential. Comparatively uncharted ecological source is
represented by them, and they possess strong secondary metabolism as they have
metabolic interactions with the hosts. Plants are associated with the endophytes
closely. Therefore, a consistency of the research is required to avoid the vanishing of
different plant species that would further leads to disappearance of endophytic
potential. Collection, arrangement, and proper utilization of endophytes all over
the globe the world may lead to development in agricultural practices, industries, and
medicines. Management of microbial communities is a great challenge, which
further needs colonization of significantly important endophytes.
Conflicts of Interest The authors declare that they have no conflicts of interest.
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Chapter 4
Unravelling the Role of Endophytes
in Micronutrient Uptake and Enhanced
Crop Productivity
Kanchan Vishwakarma, Nitin Kumar, Chitrakshi Shandilya, and
Ajit Varma
Abstract Endophytes belong to the domain of bacterial and fungal species that
inhabit the host plant and associates symbiotically with plant tissue. The most
common forms of endophytes are found to be associated with the species of
Enterobacter, Colletotrichum, Phyllosticta, etc. Some of the potent methods of
emerging sustainable agriculture to make sure enhancement in crop production
having a minimal disturbance caused to environment are exploration of beneficial
microbial interactions with plants. The endophytic interactions with plants are
advantageous to both the host plant as well as for endophytic microorganism. The
ecological and environmental conditions of the host plant significantly influence the
associated endophytic population. Endophytes functions as plant growth promoters;
enhance uptake of minerals and nutrients; restrict the survival of phytopathogens and
increase plant tolerance to environmental stress. The study of functions and hostendophytic relations is important to focus on endophytic significance. Over recent
years, substantial studies have been carried out based on their potential to produce
valuable substances responsible to enhance plant growth, biocontrol potential as well
as their sustenance in adverse environment. The present article focuses on understanding the taxonomy of endophytes and their functions in nutrient uptake and
sustainable crop production.
Keywords Endophytes · Micronutrients · Crop productivity · Biocontrol ·
Biofertilization
K. Vishwakarma (*) · C. Shandilya · A. Varma
Amity Institute of Microbial Technology, Amity University Uttar Pradesh, Noida, India
e-mail: kvishwakarma@amity.edu
N. Kumar
Department of Biotechnology, Periyar Maniammai Institute of Science and Technology,
Thanjavur, Tamil Nadu, India
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_4
63
64
4.1
K. Vishwakarma et al.
Introduction
Endophyte is the mixture of two words taken together, i.e., endon meaning “within
the plant” and phyton meaning ‘plant’. Therefore, this term is a topological expression that includes all organisms collectively which are capable of colonization of
living internal tissues of plant hosts without giving any symptom along the uneven
period of their life cycle (Stone et al. 2000). This term has broad-spectrum literal
meaning with respect to its specificity to host plants and inhabitants (Kobayashi and
Palumbo 2000; Stone et al. 2000; Marler et al. 1999; Feller 1995). The hostendophyte interactions may have originated from the initial appearance of higher
plants on earth. This is supported by the manifestation of plant-related microbes that
were found to present on fossilized stem and leaf tissues (Strobel 2003; Andrzej
2002).
Increase in the plant growth occurs with the help of acquisition of enhanced
nutrients and/or stimulating the hormones (Dobbelaere et al. 2003). Plant growthpromoting rhizobacteria, i.e., PGPR releases the substances that enhance plant
growth and also important in macro- and micronutrient cycling. Micronutrients
important for plants, animals, humans include iron (Fe), zinc (Zn), and copper
(Cu) that helps in increased micronutrient density in staple crops such as wheat.
(Dobbelaere et al. 2003).
4.2
Major Groups of Endophytic Microbes
There are various distinguishable groups of endophytic microorganisms on the basis
of their source of plant organ, which is presented in Table 4.1.
Table 4.1 Different types of endophytes with respect to their host plant
S. No.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Types of endophytes
Endophytic Clavicipitaceae
Endophytic fungi of dicots
Systemic fungal endophytes
Fungal endophytes of lichens
Endophytic fungi of bryophytes and ferns
Endophytic fungi of tree bark;
Fungal endophytes of xylem
Fungal endophytes of root
Fungal endophytes of galls and cysts
Endophytic prokaryotes of plants (involves bacterial endophytes and actinomycetes)
4 Unravelling the Role of Endophytes in Micronutrient Uptake and Enhanced Crop. . .
4.2.1
65
Fungal Endophytes
The fungal symbionts pour extreme impact on ecological significance, fitness, and
evolution of plants (Brundrett 2006), forming communal plants (Clay and Holah
1999) and indicating profound impact on the biodiversity of integrated organisms
which may include insects and bacteria (Omacini et al. 2001). Fungal endophytes
inhabit completely within the plants and spread within roots, stems/leaves and
emerge to form spores at plant tissue senescence and are not similar to the mycorrhizal fungi that establish their colony in plant roots and spread to rhizosphere
(Sherwood and Carroll 1974; Stone et al. 2004). In effect, there are two broad
categories of endophytic fungi reported earlier which reflects the differences in
evolution studies, taxonomic studies, host plants and ecological properties:
clavicipitaceous endophytes (C-endophytes), infecting some grasses; and
non-clavicipitaceous endophytes (NC-endophytes), retrieved from asymptomatic
tissues of non-vascular plant hosts.
4.2.2
Class 1 Clavicipitaceous Endophytes
Majority of Clavicipitaceous endophytes impart resistance to insect attack on hosts
(Clay 1990; Patterson et al. 1991; Riedell et al. 1991). Kimmons et al. (1990) carried
out a study that enlists anti-nematodal activity of Class 1 endophytes. Since there are
multiple studies that do not support the defensive roles of C-endophytes to hosts,
some researchers put forward the questions on classifying them as defensemutualists (Faeth 2002). Many Class 1 endophytes release some substances that
inhibit the in vitro spread of other fungi. In a research conducted by Yue et al. (2000)
various indole derivatives, i.e., sesquiterpene and diacetamide were obtained from
Epichloë festucae. It is still undiscovered whether the disease-resistant mechanism is
credited to antifungal substances released by endophytes, substances secreted by
plants with respect to endophytes, tropical competition between fungi or any
physico-exclusion mechanism.
In a study carried out by Malinowski and Belesky (2000), extensive root systems
were developed after the infection of N. coenophialum. This further enabled the
acquiring capacity of plants to better capture water content in the form of moisture
and nutrients thereby resulting in resistance of abiotic stress such as drought
resistance and faster recovery from water-stress.
Non-clavicipitaceous Endophytes They are majorly found to be present in asymptomatic leaf tissues. Majority of research was focused on isolating and analyzing the
bioactive substances, using NC-endophytes as biocontrol agents, and executing
phylogenetic identification (Schulz 2006; Arnold et al. 2007; Kithsiri Wijeratne
et al. 2008). Despite the lack of functional understanding, some of the
NC-endophytes have been shown to display mutualistic behavior toward plants,
taking part in tolerating biotic and abiotic stress thereby giving fitness benefits,
66
K. Vishwakarma et al.
enhanced growth, productivity, and yield (Redman et al. 2002; Mucciarelli et al.
2003; Waller et al. 2005; Rodriguez et al. 2008).
4.2.3
Class 2 Endophytes
Class 2 endophytes involves variety of species, which are members of Dikarya
(Ascomycota or Basidiomycota). Majority of them are considered under Ascomycota
while only few come under Basidiomycota. It is observed that almost all Class
2 endophytes increase host root and/or shoot biomass. The reason may be dedicated
to the fact that plant hormones are induced by the host or fungi that biologically
synthesize plant hormones (Tudzynski and Sharon 2002). They are also thought to
give shelter to host plants to some extent against fungal attack by secreting secondary metabolites or inducing systemic resistance (Narisawa et al. 2002; Vu et al. 2006;
Campanile et al. 2007).
4.2.4
Class 3 Endophytes
Class 3 endophytes are differentiated according to their occurrence
1.
2.
3.
4.
Primarily or exclusively in the tissues above ground.
Transmissions made horizontally.
Occurrence of highly localized infections.
Potential to confer benefits or costs on hosts that are not necessarily habitatspecific.
5. Extremely increased in planta biodiversity.
The environmental roles of Class 3 endophytes are not much explored because of
their much-noted diverse species range within individual host or its tissues
(Rodriguez et al. 2009).
4.2.5
Class 4 Endophytes
Class 4 endophytes are also known as dark septate endophytes (DSE). These are
differently labeled on the basis of the functional group which characterizes dark
melanized septa and restrict to plant roots. They are shown to have very little host
specificity. Interactive relation of DSE with 600 plants has been reported, which
includes non-mycorrhizal plants and the plants which belong to Antarctic, Arctic,
and temperate zones and also African coastal plains and low-lands, and certain
tropical ecological systems (Jumpponen and Trappe 1998; Jumpponen 2001).
4 Unravelling the Role of Endophytes in Micronutrient Uptake and Enhanced Crop. . .
4.2.6
67
Bacterial Endophytes
Endophytic bacteria are observed to colonize internal host tissues. Their colonizing
number may be high, but it does not cause any damage to host and elicit strong
defense responses. These are not like endosymbionts that reside in living plant cells
and covered by a membranous chamber (Hurek and Hurek 2011). Endophytic
bacteria that are present inside the plants have been noticed for more than
120 years. The occurrence of bacterial endophytes within plants is thought to be
variable and sometimes transient but they are also seemed to elicit physiological
changes helping in modulating growth and productivity of plants (Zhang et al. 2006;
Hardoim et al. 2008; Santoyo et al. 2016).
In the endosphere, a strategic step has been taken by modulating plant physiology
by changing levels of plant ethylene. This is because any changes in the levels of
ethylene signal will impart major changes in bacterial diversity (Rouws et al. 2010).
Therefore, how bacteria respond to plant ethylene concentration signals is key to
their ecological success or competence as endophytes.
The phrase of “competent endophytes” is brought into context in order to dictate
path in characterizing those bacteria which have the required generic machinery to
colonize in the endosphere and persist in it. Whereas the “opportunistic endophytes,”
unlike the competent ones, have the competency to colonize the rhizosphere that
might turn into endosphere colonizer by entering the root tissues; they also lack
genes that play a key role in defining their ecological role inside the host plant.
4.2.7
The Ecology of Competent Endophytes
The stochastic events govern the diversity and composition of bacterial community
in endosphere, and these events are further affected by deterministic procedure of
colonization (Taghavi et al. 2009). The colonization of endophytes and their community structures are driven by several parameters that include genotype of plants,
several stages of growth and state of physiology, plant tissue type, soil environment
conditions, and agricultural practices (Chi et al. 2005; Ikeda et al. 2010). In addition
to characteristics that confer competence in the rhizosphere, numbers of other
characteristics are proposed to make competent endophytes successful in the plant
endosphere.
The comparison of observed endophytic bacterial communities and soil bacterial
communities revealed that bacterial endophytes were relatively simple than soilinhabiting communities, giving up to hundreds of bacterial types that differentiate
each other. Hence, it is observed that plants are capable of performing the function of
true “filters” for microorganisms of the rhizosphere, selecting those that are successful, competent endophytes (Shah 2009).
68
4.2.8
K. Vishwakarma et al.
Host Range
The plants that grow mainly in temperate, tropical and boreal forests are known to
carry the various classes of endophytes. Their host range includes herbaceous plants
with their habitats in extreme arctic, alpine, and extremely dry environment (Mushin
et al. 1989); moist temperate zones and tropical forests. Moss, ferns, angiosperms,
and gymnosperms along with palms, trees having broad leaves, estuary plantations,
and deciduous and evergreen forest trees are the carriers of endophytic fungi
(Ligrone et al. 1993; Schmid and Oberwinkler 1993). Although, from past studies,
it is evident that there are only 100,000 fungal species (approximately) described
together with endophytic fungus and around 900,000 fungi are still unknown
(Hawksworth and Rossman 1997). Since the number of new endophyte species
might exist in plants, they are considered to have the significant contribution to
microbial biodiversity (Clay 1992; Kandel et al. 2017).
In a study, 21 cacti species that occur in various zones within Arizona were found
to have the presence of 900 endophytic isolates that belonged to 22 fungal species.
Cylindropuntia fulgida possessed the highest endophyte species diversity, while
C. ramosissima harbored the most endophyte isolates (Suryanarayanan et al.
2005). It has been broadly accepted that there are very few plants that are free
from endophytes, and these include particularly herbs and shrubs (Gennaro et al.
2003). It is also reported that most frequent endophytes comprise bacteria and fungi
(Petrini 1991). Generally, fungal endophytes exhibit specificity to single host with
respect to plant. However, environment conditions do affect their specificity toward
host (Susan 2004). Only exception is Epichloë typhina with wide host range (Caruso
et al. 2000).
4.2.9
Physiological Role
It is known that endophytes perform potential and advantageous role in physiology
of plant hosts. It is also accepted that plants containing endophytes are healthier than
those deprived of endophytes (Waller et al. 2005). The reason may be attributed
partially to the fact that endophytes produce various phytohormones, for e.g., indole
acetic acid, cytokines, and plant growth-promoting compounds such as vitamins;
and partially owe to the property of endophytes to increase the absorption of
nutrients like N (Reis et al. 2000; Lyons 1990), P (Guo et al. 2000; Malinowski
et al. 1999) by host and regulating C: N ratio (Raps and Vidal 1998). For instance,
Kaldorf et al. (2005) inoculated Populus Esch5 roots with Piriformospora indica
and observed an increase in biomass of root with significant numbers of secondorder roots.
The plants infected with endophytes offer a range of advantages, i.e., increased
resistance to pests (Siegel and Schardl 1991; Breen 1994), acclimatization in drought
areas (Cheplick et al. 2000; Eerens et al. 1998), enhanced competition (Hill et al.
4 Unravelling the Role of Endophytes in Micronutrient Uptake and Enhanced Crop. . .
69
1991), improved tolerance to stress parameters like heavy metals (Monnet et al.
2001), acidity (Lewis 2004), high pH levels (Waller et al. 2005) and microbe-level
infections (Bacilio-Jimenez et al. 2001).
4.2.10 Ecological Role
Endophytic microbes have key ecological roles in systems which involves shaping
of plant host communities and governing their associations (Ganley et al. 2004).
Endophytic fungi colonized in few plants carry out novel ecological operations such
as studying thermal tolerance of plants that grow in geothermal soils (Redman et al.
2002). The ability of endophytes to impact communal biodiversity and interactions
between microbes has been considered to be useful determinants of biodiversity of
plants (Clay and Holah 1999). Toxic alkaloids are produced by the endophytic
microbial communities predominant in grass and herbs in order to prevent or hinder
herbivores (Wilkinson et al. 2000). They also play defensive roles to diminish or
prevent the damage caused by pathogens in woody plants (Miller et al. 2002; Arnold
et al. 2003). Complete ecological properties of endophytes residing in woody plants
are still not clearly understood but it can be associated with their species biodiversity
(Ganley et al. 2004). Clay and Holah (1999) carried out a study in eastern USA
observed that as the interaction between tall fescue and Neotyphodium
coenophialum was enhanced, the plant biodiversity was declined.
4.3
Micronutrients and their Role in Plant Growth
Elements necessary for plant growth and required in trace amount are called
micronutrients. In comparison to macro-minerals, their allowed dose is not more
than 100 milligrams/day. The following minerals are considered as micronutrients:
iron (Fe), cobalt (Co), chromium (Cr), copper (Cu), iodine (I), manganese (Mn),
selenium (Se), zinc (Zn), and molybdenum (Mo) (Fig. 4.1). Organic compounds like
vitamins and phytochemicals can also be included as micronutrient as they are also
required by organism in trace amount. Excellent source of micronutrients to growing
plants includes recycled organic matters consist of grass and tree leaves (White and
Brown 2010).
4.3.1
Boron (B)
In soil, both inorganic and organic forms of boron are found and are available to
plants in one of the two formats, i.e., boron composed minerals dissolved in soil and
soil organic matter. Around 10–300 mg/kg of boron is generally found in the upper
70
K. Vishwakarma et al.
Fig. 4.1 Relative
concentration of
micronutrients required for
plant growth
surface of soils of which only a small quantity of boron is available to plants (Howe
1998).
Boron has several functions in plant cell physiology. Primarily, it is involved in
structuring the cell and maintaining properties of plasma membrane (Brown et al.
2002; Wimmer et al. 2009; Atique-ur-Rehman et al. 2018). Boron deficiency causes
a reduction in plant growth by blocking the cell wall elongation at growth stage and
cause an increase in cellulose, uronic acid and hemicellulose and decrease in pectin
(Zehirov and Georgiev 2003). Symptoms for deficiency may include low flowering
rate, thickening, curling of leaves, and spots on fruits and leaves (Reid et al. 2004).
4.3.2
Zinc (Zn)
Zinc plays a bigger role in the regulation of early growth of plants by involving in the
enzymatic system and is considered important for root, seed, and fruit maturation,
stress alleviation, expression of plant growth regulators, and photosynthesis. Further
zinc help in many plant developmental processes by acting together with other
minerals like potassium, nitrogen, and phosphorus. Zinc is also required in the
synthesis of proteins and regulation of growth stages. Delay in maturity is shown
in zinc-deficient plants.
4.3.3
Manganese (Mn)
The application of manganese can be in the form of foliar or soil inclusions to
eradicate manganese deficiency. It is majorly involved in plants for being used as a
cofactor in the functioning of enzymes. It helps directly in photosynthesis mainly in
chlorophyll synthesis and activating many metabolic functions. It accelerates
4 Unravelling the Role of Endophytes in Micronutrient Uptake and Enhanced Crop. . .
71
maturation and germination of plants by increasing the availability of calcium and
phosphate (Millaleo et al. 2010).
4.3.4
Iron (Fe)
Generally, plants show deficiency of iron in soil due to the condition which makes
iron unavailable to the plant. The reason for the unavailability of iron to the plants is
basic pH of soil, oxygen scarce soil, high soil moisture, high temperatures and high
soil manganese, zinc, copper, and phosphorus levels. Unavailable iron is made
available by plants by secreting acidic compounds or phytosiderophores in
rhizospheric soil so that it complexes with iron at rhizosphere and make iron in
available form for the plant itself (Vishwakarma et al. 2018). Iron helps in synthesis
of chlorophyll and enzymatic metabolic pathways associated with the transfer of
energy, fixation and reduction of nitrogen, and formation of lignin in plants.
4.3.5
Copper (Cu)
Copper is necessary for carbon and nitrogen metabolism. The deficiency of copper
causes stunted growth of plants. It is also required in the process of lignin synthesis,
as it is needed for preventing wilt and strengthening cell wall. Deficiency symptoms
are pale greenish leaves, short growth, and yellow leaves. Generally young plants
show these symptoms.
4.3.6
Molybdenum (Mo)
Molybdenum is required in synthesis and activity of nitrate reductase and it is
important for symbiotic fixation of nitrogen by rhizobium species. It is also having
direct role in converting inorganic to organic phosphorus. pH of soil is the major
factor for deficiency of molybdenum, i.e., pH below 6.0 shows the major cause of
available molybdenum in soil. Molybdate (MnO42 ) is the form in which molybdenum is found in soil.
4.3.7
Chlorine (Cl)
Chlorine comes in essential nutrient elements and it exists in the soil in the form of
chloride anion (Cl ) (White and Broadley 2001). Chloride content in fertilizers can
affect the quality of crops. For example, tomato, potato, and tobacco need potassium
72
K. Vishwakarma et al.
nitrate and potassium sulfate fertilizers rather than potash. Common root rot, leaf,
and head fungal infections shown by small grains can be cured by the use of
chloride.
4.4
Mechanism of Micronutrient Uptake by Endophytes
Rhizospheric microbes provide plant nutrition by two universal mechanism. They
either transform the unavailable forms of nutrients to available form, for e.g.,
nitrogen fixation or phosphate solubilization or by increasing the transport of mineral
and uptake of nutrients by the plant (Hayat et al. 2010; Ahemad and Kibret 2014). It
has been reported that both the bacterial as well as fungal endophytes have the
potential to colonize their host plants for their plant-inhabiting strategies, promotion
of plant growth and protection at the time of biotic and abiotic stress and uptake of
soil nutrients and minerals (Johnston-Monje and Raizada 2011). As marine plants
absorb vitamins and minerals from water by the process of diffusion, the primitive
land plants (e.g., Aglaophyton major) had to develop the potential to absorb mineral
nutrients present in the soil. The actual root system is not found in the premature
bryophyte-like plants (e.g., mosses) therefore it may be possible that earlier mycorrhizal hyphae served this function in early land plant evolution (Brundrett 2002).
Endophytes are microbes that colonize the endosphere, a zone within the plants
establish them within the cells (Izumi et al. 2008; Izumi 2011). The most eminent
kind of symbiosis observed in plants has been observed by mycorrhizae. Some
properties indicate their availability to plants, and these are chemical properties,
mobility of macronutrients. Specific parameters for example soil microbes and their
consequences on the production of root exudates can substantially have an impact on
nutrient availability in soil (Vishwakarma et al. 2017a, 2017b). Extensive research
proved that mycorrhizal fungi are capable of increasing the uptake of various
micronutrients in association with their host plant which correlates the mobilization
of minerals and nutrients by the fungi. This becomes possible as a result of (1) the
secretion of various enzymes by fungi, (2) the association of the fungi with other soil
microorganisms, (3) the impact of fungi upon plant rhizosphere, and (4) the effect of
fungi on physiological and morphological (root growth) parameters of host plant that
influence the root exudation (Fig. 4.2). The extraradical mycelium extended from the
plant root to the encompassing soil is able to enlarge the surface area of plant roots
and enhance the nutrient uptake (Landeweert et al. 2001; Kariman et al. 2018).
The ancient fungal partner mainly belongs to the phylum of Glomeromycota as
the 90% of the land plants forming partnership with arbuscular mycorrhizal fungi
(AMF) belongs to Glomus species. More than 200 species of this AMF have
functions like transmission through soil spores, obligate biotrophy, and inability to
grow independently without a compatible host root. The AMF are capable of
elevating the supply of nutrients such as N, P, Cu, Zn, Ni, S, Fe, Ca, K, B, and
Mn to plant (Clark and Zeto 2000), mainly because the faster hyphae growth
explored soil volume more proficiently than roots. The area for nutrient absorption
73
Fig. 4.2 Various mechanisms of increasing root growth by endophytic bacterium
4 Unravelling the Role of Endophytes in Micronutrient Uptake and Enhanced Crop. . .
74
K. Vishwakarma et al.
increased by 100 times relative to root length by the association found in AM fungi
(Hetrick 1991). This mechanism proved to be advantageous for scavenging immobile nutrients like phosphorous and zinc, which are regarded as the primary macroand micronutrients made available to plants by the use of AM fungi. The difference
in the plant species is according to their dependence on AMF for gaining support to
acquire nutrients as well as relate to the level at which their growth responds to
colonization of AMF in soils (Lambers et al. 2008; Begum et al. 2019). Increased
concentration of P and Zn in soil makes AM fungi non-essential and even harmful to
the growth of plants as they become parasitic by imposing their drains on carbon
sources without causing any benefit to the plant host (Ryan and Graham 2002). The
role of AM fungi to enhance the nutrient use efficiency (NUE) in crop production
relies on certain factors. The role of AM seems to be not effective in nutrient-rich
soil, this may predict that AM are more effective in soils having less nutritional
content (Wright et al. 2005). More than 50 million years ago, three types of
mycorrhizal associations evolved besides AM, eventually enhancing host nutrient
interacting region which increases absorption of nutrients and makes available
various organic sources of N and P which can be absorbed by the fungi. Several
trees and shrubs having ectomycorrhizal symbiosis (e.g., pine trees), associates with
thousands of different species of zygomycetes, septate basidiomycetes and ascomycetes. Orchids are the second one associates with the basidiomycete mycorrhiza
additionally inhabitant the fungus as carbon supply. The third one is the ericoid
plants (e.g., tea) associates with the ascomycete mycorrhiza. These are the three
types of mycorrhizae that have the potential to utilize organic compounds by
producing extracellular enzymes such as phosphatases and carboxylases to catalyze
the mineralization of N and P from dead plant materials and various soil microorganism (Lambers et al. 2008). In addition, carboxylases also interact with aluminum
(Al) found in the soil and result in some Ca-rich acidic complexes; hence, in this
manner, Ca is released and taken up by the mycorrhiza (Lambers et al. 2008).
It was reported earlier that dark septate endophytes (DSEs) were observed in
more than 600 diverse plant species and establish them worldwide, frequently
associated with mycorrhizal fungi, even though various mycorrhizal plant roots
also exist who associates with DSEs. They may additionally enhance supply of P
to the host and leads to replacement of AMs and ectomycorrhizal fungi at regions
that deal with harsh environmental conditions (Mandyam and Jumpponen 2005).
Even though some DSEs have the capability to secrete plant hormones that show
plant growth, some probability arises that their newly recognized mycorrhizal
inhabit in combination with their saprophytic abilities that signify the key machinery
of DSE to increase nutrient uptake. Despite the fact that all plant life harboring DSEs
are not determined to have improved NUE, a number of studies are there that show
DSEs have the potential to enhance nutrient level in plants. It was reported previously that DSEs isolated from the roots of Carex species consequently increased
biomass and P concentration when re-inoculated in DSE-free plant of the same
Carex species (Haselwandter and Read 1982). The study of Pinus contorta inoculation in combination with Phialocephala fortinii (DSE) showed the increment of
leaf P and increase nutrient uptake from soil and increase biomass of plant
4 Unravelling the Role of Endophytes in Micronutrient Uptake and Enhanced Crop. . .
75
(Jumpponen et al. 1998). Nearly 90% of soil phosphorus is immobilized in natural
and organic sources, and DSEs may be capable of mineralizing P for their uptake and
utilization by plants, but their ability to try this has no longer but been explicitly
proven. On the other hand, DSEs have been proven to secrete a quantity of enzymes
such as cellulases, pectinases, lipases, laccases, pectinases, and polyphenol oxidases
capable of degrading organic materials and make nutrient available to plant host
(Mandyam and Jumpponen 2005).
4.5
Role of Endophytes in Plant Growth Promotion
A number of studies have been conducted on the plant growth-promoting potentials
of diverse endophytic microbes. The growth promotion mechanism adapted by them
involves both the direct method by enhancing the cycling of nutrients such as
nitrogen, phosphate, producing valuable metabolites as well as indirectly as biocontrol strains by inhibiting the growth plant pathogens (Garbaye 1994; Cardoso et al.
2011). The attributes of root endophytes as plant inoculants for promoting the
growth of plant has been significantly studied (Thakore 2006), and the present
research is moving toward the beneficial application to envisage the advantages of
endophytes in promoting plant growth and development at field conditions.
The mechanism adapted by endophytes to promote plant growth is very similar to
rhizobacteria which include the supply of nutrients by producing siderophore (Costa
and Loper 1994), solubilizing the insoluble phosphate (Verma et al. 2001; Wakelin
et al. 2004) and the production of growth regulators such as IAA (Lee et al. 2004) as
well as by providing essential mineral and vitamins to plants (Pirttilä et al. 2004).
The indirect method of promoting plant growth by endophytic bacterial was also
reported. The potential of bacterial endophytes to colonize plant niche in the same
manner as plant pathogens make them an appropriate biocontrol agent (Berg et al.
2005). The diverse role of endophytes on plant growth was well studied which
includes modification of stomata, regulating osmotic stress, altering root morphology, improving mineral uptake, and regulating nitrogen immobilization and mineralization (He et al. 2017; Chandrasekaran et al. 2019). The most promising field
which utilizes the potential of these growth-promoting endophytes comprises
rhizoremediation as well as revegetation.
The plant-inhabiting life strategies of endophytes follow three main categorizations, i.e., obligate, facultative, and passive endophytes. Obligate endophytes are
host-dependent endophytes that are not able to survive outside the plant and the
possible way of its transmittance is by means of seed (Hardoim et al. 2008; Gaiero
et al. 2013). Facultative endophytes are free-living endophytes that reside dominantly in soil and colonize plant roots as a result of infection. Passive endophytes are
the third class of endophytes that colonize plant roots because of any coincidental
damage to the root hairs (Hardoim et al. 2008, Gaiero et al. 2013).
The majority of the endophytes responsible for plant growth promotion belong to
the category of facultative endophytes due to their potential of colonizing plant roots.
76
K. Vishwakarma et al.
Fig. 4.3 Mechanistic illustration of biostimulation, biofertilization, and biocontrol
The most ordinary process adapted by root endophytes to colonize the epidermis and
infiltrate includes the sites of emerging lateral roots, beneath the zone of root hair,
and root cracks (Dong et al. 2003; Compant et al. 2005; Zakria et al. 2007) as well as
follows intercellular and intracellular establishment (Hurek et al. 1994; Zakria et al.
2007). Some endophytes can pass to different regions of the plant after preliminary
colonization via penetrating into the vascular tissues and spreading systemically
(Compant et al. 2005; Zakria et al. 2007; Johnston-Monje and Raizada 2011). Three
different as well as interconnected mode of action was involved for facilitating plant
growth promotion by bacterial endophytes comprises phytostimulation,
biofertilization, and biocontrol (Bloemberg and Lugtenberg 2001) (Fig. 4.3).
4.5.1
Phytostimulation
Phytostimulation involves direct method of plant growth enhancement via secreting
growth-regulating plant hormones (Bloemberg and Lugtenberg 2001). Probably the
most incredibly studied instance of phytostimulation includes the reduction of
ethylene levels (a plant hormone) by the enzyme 1-aminocyclopropane-1carboxylate (ACC) deaminase. A number of endophytes that produce ACC deaminase were observed to display plant growth and development, together with
Arthrobacter spp. and Bacillus spp. in Capsicum annuum (pepper) (Sziderics et al.
4 Unravelling the Role of Endophytes in Micronutrient Uptake and Enhanced Crop. . .
77
2007), in addition to Pseudomonas putida and Rhodococcus spp. in Pisum sativum
(peas) (Belimov et al. 2001). The mechanism of enhancing plant growth is not well
identified although ACC deaminase activity might also lower the abiotic stress by
means of balancing levels of plant ethylene production, because high ethylene
concentration inhibits replication, cell cycle as well as plant growth (Burg 1973).
Several reports are available that show the production of other phytohormones such
as abscisic acid, jasmonates, and indole-3-acetic acid by endophytes which enhance
plant growth (Patten and Glick 2002; Forchetti et al. 2007a, 2007b).
4.5.2
Biofertilization
Biofertilization is the process of enhancing the growth of plants and yield by
stimulating nutrient supply to plant required for its growth (Bashan 1998). Phosphate
solubilization, i.e., solubilization of insoluble phosphate and fixation of atmospheric
nitrogen, i.e., conversion of atm. Nitrogen to ammonia are the most common
mechanism of plant growth promotion which can categorize as a process of
biofertilization (Bargaz et al. 2018; Gouda et al. 2018). A few plant growthpromoting bacterial endophytes (PGPBEs) have been studied largely because of
their ability of fixing nitrogen inclusive of Azospirillum spp. (Hill and Crossman
1983), Pantoea agglomerans (Verma et al. 2001), and Azoarcus spp. (Hurek et al.
2002). To overcome the phosphorus stress in plants, endophytes start secreting
organic acids which solubilize the phosphate and enhances phosphorus availability
to the plant. These low molecular weight organic acids chelate the metal associated
with phosphorus and aid the plants to utilize the phosphorus (Kpomblekou-A and
Tabatabai 2003). A study of Forchetti et al. (2007a, 2007b) on the isolation and
characterization of phosphate solubilizing endophytes of sunflower (Helianthus
annuus) shows that Achromobacter xiloxidans and Bacillus pumilus were having
the phosphate solubilizing potential. Yazdani and Bahmanyar (2009) confirmed that
the addition of PGPBEs in fertilizer for corn (Zea mays) treatments decreased the
need for phosphorus and enhanced crop yield.
4.5.3
Biocontrol
The potential plant growth-promoting endophytic bacteria can inhibit the growth of
phytopathogenic organisms and indirectly increase the plant growth; this is referred
as biocontrol activity. Biocontrol mechanism includes the production of antibiotics,
antifungal compounds, siderophore production, and production of hydrogen cyanide. Siderophores, for instance, pyochelin and salicylic acid are iron-chelating
compounds therefore contribute to disease suppression indirectly by blocking phytopathogens for trace metals (Duffy and Defago 1999). Endophytes follow the
similar growth-promoting mechanism as explained for plant growth-promoting
78
K. Vishwakarma et al.
rhizobacteria which have been studied previously by several authors such as
Kloepper et al. (1999), Gray and Smith (2005) and Compant et al. (2010). Some
examples of antimicrobial compounds produced by endophytic bacteria are
2, 4-diacetylphloroglucinol (DAPG) that inhibits the growth of phytopathogens. It
was previously reported by Ramesh et al. (2008) that the chances of causing wilt by
Ralstonia solanacearum infection are reduced to 70% in eggplant. It was also
reported previously that endophytic microorganism reduced the chance of infection
or damage caused by nematodes (Hallmann et al. 1998), insects (Azevedo et al.
2000) or bacterial, viral and fungal species (Kerry 2000; Sturz et al. 2000; Ping and
Boland 2004; Berg and Hallmann 2006). It was also described in certain studies that
endophytes also promote plant growth in a stressed environment (Bent and Chanway
1998) and additionally support the acceleration of seedling emergence (Chanway
1997). A mechanism referred to as induced systemic resistance (ISR) is adapted by
several endophytes and is analogous on the basis of phenotype to systemic-acquired
resistance (SAR) (Harman and Uphoff 2019). SAR gets activated when plants
effectively initiate their defense mechanism against any primary infection caused
by a pathogen (van Loon et al. 1998). ISR proved to be successful for various
pathogenic organisms but differs from SAR in which the associated bacteria do not
produce symptoms which is visible on the host plant (van Loon et al. 1998).
Kloepper and Ryu (2006) studied broadly the role of bacterial endophytes in ISR.
Numerous studies are available on exploring the ability of PGPBEs as a potential
plant growth promoter and used as bioinoculant for agriculturally important crops
(Kloepper and Schroth 1978; Kuklinsky-Sobral et al. 2004).
4.6
Conclusion and Future Prospects
Ecologically, the association of host plants, pathogens, and herbivores with each
other is mediated by endophytes. The compositional and distributional species
of endophytes and their associations s within and among hosts, the reciprocal of
effective ecology of endophytic colonization on fitness of host and composition of
communal plants, ecological effects of endophyte colonization on host fitness and
the composition of plant communities, are some of general problems of interest to
endophytologists and plant ecologist. The upgradation of colonization of bacteria
stimulated by explicit carbonaceous exudates by plant roots and the limit of specific
microorganisms for modulation of metabolism of plants are key issues for future
investigation, as these can be major studies for understanding relationships of
potentially mutualistic plant–endophytes. Specific endophytes have significance in
the enhancement of growth and development of plants. If the transmission of such
endophytes is not on vertical base, (for example, by means of the seed), at that point
the development of productive physiological systems that empower their selectivity
from oil could have been the key fitness improving attributes that have upgraded the
transformative accomplishment of these plant species.
4 Unravelling the Role of Endophytes in Micronutrient Uptake and Enhanced Crop. . .
79
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Chapter 5
Dual and Tripartite Symbiosis of Invasive
Woody Plants
Robin Wilgan
Abstract The invasion of alien tree species is one of the most crucial threats for
biological conservation and the maintenance of biodiversity. Invasion of alien tree
species can change local, continental and global biodiversity, nutrient cycles and
ecosystem services and transform native habitats into novel ecosystems determined
by invaders. The overwhelming majority of tree species are associated with mutualistic symbionts: arbuscular mycorrhizal fungi, ectomycorrhizal fungi, and/or
N-fixing bacteria. These mutualistic symbionts play a key role in the proper development and functioning of trees, being able to profoundly influence the invasiveness
of alien tree species, and even make this invasion possible. That kind of key
interaction among invasive tree species and its mutualistic symbionts, what is
commonly named as “co-invasion,” is well known for alien ectomycorrhizal tree
species and its fungal symbionts, e.g., Pinaceae species and suilloid fungi in the
Southern Hemisphere.
This chapter is dedicated to the dual mycorrhizal and tripartite symbiotic relationships of alien and in particular invasive tree species. The ability to establish dual
mycorrhizal symbiosis with both ectomycorrhizal (ECM) and arbuscular mycorrhizal (AM) fungi and tripartite symbiosis between N-fixing bacteria, mycorrhizal fungi
(ECM and/or AM) and actinorhizal or legume trees, seems to support the global
expansion of alien tree species. The main assets of dual and tripartite symbiosis for
alien and invasive tree species are in seedling development, nutrient acquisition in
different and unfavorable habitat conditions, so tent to naturalization outside its
native range and relatively high tolerance to shift in climate conditions, such as soil
temperature and large fluctuations of the water table.
The global climate changes may significantly switch the global distribution of
trees, both native and invasive species, being favorable to tree species of multiple
symbiotic associations. Moreover, the climate changes can increase the distribution
of native tree species, thus release the ecological niches for other species, including
invasive and potentially invasive tree species. Understanding the multipartite
R. Wilgan (*)
Institute of Dendrology, Polish Academy of Sciences, Kórnik, Poland
e-mail: rwilgan@man.poznan.pl
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_5
87
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R. Wilgan
relationships between arbuscular fungi, ectomycorrhizal fungi, N-fixing bacteria,
and invasive tree species can allow us to better predict and counteract the future
invasions of alien tree species, what is necessary to maintain biodiversity and
preserve native ecosystems.
Keywords Forest ecosystem · Alien tree species · Biological invasions · Arbuscular
mycorrhiza · Ectomycorrhiza · Nitrogen-fixing bacteria
5.1
Introduction
The introduction of alien, i.e., non-native plant species has become a global phenomenon over the last centuries. It was resulting in the cultivation of alien tree
species as an economically important branch of forestry in many countries, but at the
same time, the widespread introduction of alien tree species brought the threat of its
invasion worldwide. Trees, as ecosystem engineers playing the fundamental role in
the functioning of the most terrestrial ecosystems and biomes, are having a severe
impact on the biosphere (Barrios et al. 2017; Henry et al. 2017). Thus the expansion
of invasive tree species (invaders) is important issue of the maintenance of biodiversity. The invasion of alien trees can drive the local, continental, and global-scale
changes in biodiversity, nutrient cycles, and ecosystem services (Dickie et al. 2011,
2017; Nuñez et al. 2017). By modifying the biotic and abiotic conditions in local
habitats, invasion of alien trees leading to habitat fragmentation and decline in the
ecological niches for native, and in particular rare and endangered species and makes
a “novel ecosystems”, determined by invaders (Nuñez et al. 2017; Haddad et al.
2015; Morse et al. 2014). The abovementioned features make the tree invasions one
of the greatest threats to global biodiversity.
The first studies of invasive plant species have been focused on the relationship
between invaders and pathogenic organisms and indicates that the invasive species
success is caused by the lack of their natural enemies, parasites, and pathogens in the
habitat they are introduced to (enemy release hypothesis; Keane and Crawley 2002;
Reinhart et al. 2003, Reinhart and Callaway 2004; Callaway et al. 2011). The role of
symbiotic organisms, e.g., mycorrhizal fungi and N-fixing bacteria in the process of
plant invasion has been studied since a few decades (Richardson et al. 2000;
Traveset and Richardson 2014), revealed the mutualistic symbionts to be a driver
of alien woody species invasion (Nuñez and Dickie 2014; Nuñez et al. 2017;
Policelli et al. 2019). It follows, that plant-symbiont interaction may play a key
role in the global management of invasive tree species (Dickie et al. 2017).
5 Dual and Tripartite Symbiosis of Invasive Woody Plants
5.1.1
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Mutualistic Symbiotic Relationship of Tree Species
The mycorrhizal symbiosis is thought to be established by almost all plant species,
including numerous tree species (Brundrett, Tedersoo 2018). The most common
type is arbuscular mycorrhizae, the relationship between Glomeromycota fungi and
about 80% of known plant species. The ectomycorrhizal symbiosis is established by
Ascomycota and Basidiomycota fungi (e.g., edible species of Tuber, Boletus,
Cantharellus), and, in most cases, tree species such as pines, oaks, beech or
eucalyptus. Dual mycorrhizal symbiosis (dual symbiosis) is considered as the
plant species ability to establish both ectomycorrhizal and arbuscular mycorrhizal
relationships (Fig. 5.1), what yet have been described as a characteristic feature of
Myrtaceae (Eucalyptus) and Salicaceae (Populus, Salix). Tripartite symbiosis is an
association between three types of organisms: plants, mycorrhizal fungi (arbuscular
and/or ectomycorrhizal), and N-fixing bacteria (Ryc. 1). Rhizobial bacteria are
associated with legume species (Fabaceae) and Frankia with actinorhizal plants
such as Alnus or Casuarina.
This symbiotic association with mycorrhizal fungi and N-fixing bacteria benefits
the plants. It is well recognized, that mycorrhizal fungus is responsible for the
efficiency of nutrient acquisition by plant roots and helping plants to tolerate the
environmental stress, i.e., drought, pathogens, high concentration of heavy metals in
soil (Smith and Read 2008). N-fixing bacteria, by transforming atmospheric nitrogen
into fixed nitrogen being usable by plants, are being provided with nitrogen availability, allows the legume and actinorhizal plants to inhabit extremely poor habitats
(Benson et al. 2004; Rascio and La Rocca 2008).
Fig. 5.1 The scheme of the multipartite symbiotic association of tree species: dual mycorrhizal
symbiosis involved both ectomycorrhizal (ECM) and arbuscular mycorrhizal (AM) fungi and
tripartite symbiosis between N-fixing bacteria, mycorrhizal fungi (ECM and/or AM) and
actinorhizal or legume trees
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5.2
R. Wilgan
Role of Mutualistic Associations in the Invasion of Alien
Tree Species
The mutualistic symbiotic relationships may profoundly influence the invasiveness
of alien woody species (Nunez and Dickie 2014). The lack of appropriate symbionts
can be a dispersal barrier for alien species outside its native range, like the presence
of appropriate symbionts can enable and support the naturalization and invasion of
alien trees (Nuñez et al. 2009; Pringle et al. 2009; Dickie et al. 2010). The “coinvasion,” i.e., simultaneously invasion of both alien tree species and its mutualistic
symbionts, has been well-known for ectomycorrhizal fungi and trees in the southern
hemisphere, e.g., Pinus (Nuñez et al. 2017; Policelli et al. 2019) and Salix (Bogar
et al. 2015) and Eucalyptus in tropics and subtropical zone (Sulzbacher et al. 2018;
Ducousso et al. 2012). These genera belong to the families with numerous invasive
species, i.e., Pinaceae (33), Salicaceae (23) and Myrtaceae (35 invasive species;
Rejmánek and Richardson 2013).
In contrast, the representatives of the Fagales order which involves a huge and
widely distributed families of mostly ectomycorrhizal forest tree species, are seldom
observed to be invaders. A few species of Fagaceae (5) and Betulaceae (3) have yet
been considered as invasive, in most cases in a narrow range of expansion (one
geographical region as listed in Fig. 5.2; Rejmánek and Richardson 2013). The only
exceptions are Alnus and Casuarina species, which are only widespread invaders of
Fagales (3–5 of 7 regions according to Fig. 5.2), and at the same time, the symbiotic
partners for n-fixing bacteria and both arbuscular and ectomycorrhizal fungi. That
Fig. 5.2 An approximate percentage of invasive tree species known to establish dual mycorrhizal
symbiosis (Teste et al. 2019; confirmed taxa) and/or tripartite symbiosis (Benson et al. 2004; Rascio
and La Rocca 2008) among all invasive tree species (invaders in any of seven world regions; left
bar), multi-continental (invasive in three of seven world regions; middle bar) and worldwide
invaders (invasive in five of seven world regions, i.e., North America, Europe, Australasia, Asia
(rest), Southern Africa, Africa (rest) with the Middle East or South America; right bar) according to
Rejmánek and Richardson (2013)
5 Dual and Tripartite Symbiosis of Invasive Woody Plants
91
relationship is rare among Fagales trees, being limited to Alnus and Casuarinaceae
species (Benson et al. 2004).
In general, dual and/or tripartite symbiotic relationships seem to be particularly
associated with widespread invasive tree species. Even up to 70% of tree species
worldwide (genera, e.g., Acacia, Robinia, Casuarina, Salix, Populus, Eucalyptus,
Fig. 5.2) are known to establish dual and/or tripartite symbiosis (Teste et al. 2019
(confirmed taxa only), Benson et al. 2004; Rascio and La Rocca 2008). It is around
50% among medium distributed invaders and 30% of the general number of invasive
tree species (Fig. 5.2; according to Rejmánek and Richardson 2013). These data
indicate that the multipartite symbiotic relations may be a determining factor for the
global-scale distribution of invasive tree species.
5.3
Benefits of the Dual and Tripartite Mutualistic
Relationship
The ability to establish symbiosis with arbuscular fungi may be one of the factors
supporting the invasion of ectomycorrhizal tree species. Ectomycorrhizal trees that
can form arbuscular symbiosis as seedlings and/or in unfavorable habitat conditions
(e.g., Salix, Populus, Eucalyptus) are highly competitive (Brundrett and Tedersoo
2018) and more likely to become invasive. The specificity of arbuscular mycorrhizal
fungi is relatively low, which is probably related to the higher naturalization success
of dual and arbuscular mycorrhizal plants (Moyano et al. 2020). That appropriate
arbuscular fungi are available for alien tree species (Moora et al. 2011; Majewska
et al. 2015), which leads alien plants to better acclimatization outside its native
range.
The legume trees are growing efficiently in symbiosis with native nitrogen-fixing
bacteria and arbuscular mycorrhizal symbionts (examined for Acacia and Robinia,
Rodríguez-Echeverría et al. 2009; Callaway et al. 2011). However, they are still
being invasive worldwide, establishing familiar associations (sensu Dickie et al.
2017), i.e., efficient and effective relationships with local, indigenous symbionts.
This ability to accept symbiotic partners from the indigenous pule of symbionts is a
crucial factor of the successful acclimatization of alien tree species outside of their
native range (Pringle et al. 2009; Nuñez and Dickie 2014, Dickie et al. 2017), and
subsequent its naturalization (Moyano et al. 2020) and ability to being invasive.
5.3.1
Facultative Mutualistic Relationship
Facultative dual mycorrhizal symbiosis is considered to be the plant's ability to
establish the different types of mycorrhizal associations in response to environmental conditions or plant age. For example, the predominance of ectomycorrhizal and
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R. Wilgan
presence of arbuscular structures have been examined for Quercus rubra seedlings
inside its native range (Dickie et al. 2001) and Carya seedling outside its native
range in Europe (Rudawska et al. 2018), where Q. rubra (Fagaceae, Fagales) is
invasive species but Carya species (Juglandaceae, Fagales) are naturalized and still
grown on the old European plantations abandoned for a half of a century (Paź et al.
2018; Wilgan et al. 2020). The general proportion of plant species that have
naturalized outside their native range is significantly higher for plants entering the
mycorrhizal symbiosis facultatively (76%) than obligatorily (52%). The obligative
dual mycorrhizal plants species are in the larger part naturalized (69%) than obligate
arbuscular (54%), obligate ectomycorrhizal (45%), and non-mycorrhizal (34%)
plant species (Moyano et al. 2020).
Eucalyptus is one of the best-known genera of invaders among dual mycorrhizal
tree species. The co-invasion of Eucalyptus and its ectomycorrhizal symbionts have
been described, e.g., from Iberian Peninsula (Díez 2005), Brasil (Sulzbacher et al.
2018), China (Dell et al. 2002), Africa and Madagascar (Ducousso et al. 2012). It is
considered, even more than 25% of alien ectomycorrhizal fungal species have been
introduced with Myrtaceae (mostly Eucalyptus; Vellinga et al. 2009). However, the
seedlings of Eucalyptus are rapidly colonized by arbuscular mycorrhizal fungi, being
replaced by ectomycorrhizal fungi along with tree age (dos Santos et al. 2001;
Adjoud-Sadadou and Halli-Hargas 2017), what is thought to be a major contributory
factor of Eucalyptus success outside its native range (aforementioned studies; Teste
et al. 2019).
The arbuscular and ectomycorrhizal fungi differ in nutrient acquisition strategy:
ECM fungi are having the greater capability to release N and P from soil organic
matter, but AM can transfer N after its mineralization by other organisms, e.g.,
N-fixing bacteria (Teste et al. 2019 and reference therein) and increase the efficiency
of nitrogen uptake for plants establish tripartite symbiosis (Takács et al. 2018).
Dual mycorrhizal symbiosis (in most cases facultative dual mycorrhizal symbiosis) can be established by a significantly wider group of tree species, that it yet has
been considered (Teste et al. 2019). However, it is poorly evidenced, usually once
for single or a few species in genera, what raises the question, is it a typical feature of
tree genera, accidental colonization or a relic of the evolutionary past.
Interestingly, the dual mycorrhizal symbiosis has been widely confirmed for
15 genera of Fabaceae tree species, e.g., 24 Acacia species (common tree invaders).
The interactions of arbuscular and ectomycorrhizal fungi into the tripartite symbiotic
relationship of invasive tree species may be an asset for worldwide invasive tree
species of legume trees. Teste et al. (2019) revealed mostly neutral or positive effects
of arbuscular and ectomycorrhizal fungal inoculations on the dual mycorrhizal plant
species, including dual into tripartite symbiosis, e.g., Acacia, Afzelia (legume tree
species) and Alnus (actinorhizal tree species).
Well-known dual mycorrhizal tree genera (Populus, Salix, Alnus, Eucalyptus)
and tree genera of dual mycorrhizae into tripartite symbiotic relationships (numerous
Fabaceae genera, e.g., Acacia) are predominant in ecosystems characterized by
large fluctuations in the water level, i.e., periodic flooding and/or drought. Higher
tree root colonization by arbuscular than ectomycorrhizal fungi have been observed
5 Dual and Tripartite Symbiosis of Invasive Woody Plants
93
in the soil of extreme water level, i.e., very dry and flooded. This shift may be
affected by poor oxygen availability in soil, which is unfavorable for
ectomycorrhizal fungi (Teste et al. 2019 and reference therein). The contribution
of dead ectomycorrhizal roots (i.e., the residue of former ectomycorrhizal colonization) increases along with moisture gradient (Aučina et al. 2019) to nearly 100% in
flooded hollows on pine-dominated peatland (personal observation; background
described in Aučina et al. 2019). Dual mycorrhizal tree species seem to have the
resilience to flooding or drought, by its ability to establish arbuscular or
ectomycorrhizal symbiosis depends on the extreme (very dry, flooded) or temperate
water level.
5.4
Distribution of Invasive Tree Species in the Time
of Global Climate Changes
The distribution of tree species depends on the climate (Taccoen et al. 2019), thus the
invasion of alien tree species can be significantly influenced by the changes in
climatic conditions. The native and non-native, including invasive tree species
moving into high elevation and high latitude ecosystems as a result of the climate
changes (Pauchard et al. 2016; Dyderski et al. 2017). Ectomycorrhizal symbiosis is
characteristic of temperature and boreal forests, while arbuscular mycorrhizal tree
species are widespread in forests of the tropic and subtropic zone. Dual mycorrhizal
tree species are prevalent in different climatic zones being native species (e.g., Alnus,
Salix) and non-native (e.g., Eucalyptus in India, China, Brazil, northern Africa, south
Europe). The prediction effect of climate changes on the distribution of tree species
showing a high decrease of ectomycorrhizal tree species distribution in the southern
range, what have not been observed for native arbuscular mycorrhizal trees
(Fraxinus excelsior) and invasive tree species holding tripartite symbiotic relationship (Robinia pseudoacacia; Dyderski et al. 2017).
Teste et al. (2019) suggest, that the distribution of ectomycorrhizal tree species
(mostly boreal and temperature climate) and arbuscular tree species (mostly tropics
and subtropical zone; Steidinger et al. 2019) is affected by the temperature of the soil
because arbuscular fungi are reduced in low soil temperatures, while ECM fungi are
not (for Alnus species Kilpelainen et al. 2017). If it could partly explain the
predominance of ECM tree species in cold climates, the same time it indicates, the
expansion of alien tree species holding dual and tripartite symbiotic relationships
increase in temperature and boreal zones along with a decrease of native
ectomycorrhizal trees caused by climate warming.
94
5.5
R. Wilgan
Conclusion
The invasion of alien species is one of the crucial issues of biological conservation
and the maintenance of biodiversity. In front of upcoming climate changes, the
world picture of tree invasions can be significantly different, that is considered to be
now. The dual and tripartite symbiotic associations seem to support the invasion of
alien tree species, especially the most widespread invasive trees, which nearly all are
known to establish dual and/or tripartite symbiosis.
The main assets of dual and tripartite symbiosis for alien and invasive tree
species:
1. Support the development of seedlings
2. Better and usually successful naturalization outside its native range
3. Higher possibility of nutrient acquisition in different and unfavorable habitat
conditions
4. Higher tolerance to shift in climate conditions (temperature of the soil, precipitation including flooding and drought)
Further research of the multipartite interactions among arbuscular fungi,
ectomycorrhizal fungi, and N-fixing bacteria and alien tree species are needed for
a better understanding of the relationship between invasive tree species and its
symbionts. It will allow us to better predict and counteract the invasions of alien
tree species invasions, what is necessary to maintain biodiversity, and preserve
native ecosystems in front of upcoming climate changes.
Acknowledgment Preparation of this chapter was supported by the Institute of Dendrology,
Polish Academy of Sciences.
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Chapter 6
Eco-friendly Association of Plants
and Actinomycetes
Saraswathy Nagendran, Surendra S. Agrawal, and
Aryaman Girish Patwardhan
Abstract Actinomycetes are filamentous Gram-positive spore-forming largest
dominant microbial population present in the soil. They are free-living rhizosphere
colonizing bacteria and producers of bioactive metabolites which helps in improving
the fertility of the soil, promote plant growth and development, provide biocontrol
action against phytopathogens, and have the ability to withstand various environmental stress. Entophytic Actinobacteria are characterized as those that are contained
within the internal structure of plants, making no obvious changes to their hosts.
Entophytic actinobacteria consitute a huge part of the rhizosphere. The symbiotic
association of Actinomycetes as endophytes have gained more importance because
they are considered to be reservoir for potential novel bioactive compounds which
finds in important applications in pharmaceutical and agricultural sectors. A notable
significant feature of actinobacteria is its ability not to contaminate the environment,
take active participation in pesticide degradation, phosphate solubilization,
siderophores production, and nitrogen fixation. Microbial resource possesses a
wide variety of plant growth potential thereby benefiting green and sustainable
agriculture.
Keywords Actinomycetes · Actinobacteria · Rhizosphere · Endophytes · Bioactive
metabolites · Streptomycetes · Frankia
6.1
Introduction
Actinobacteria are widely found in nature and positionedin the biggest scientific
categorization inside the Bacterial domain (Kuffner et al. 2010). Actinomycetes is an
important group of spore-forming rhizosphere colonizing microorganism which
shows its presence in the soil, have an eco-friendly association with the
S. Nagendran (*) · S. S. Agrawal · A. G. Patwardhan
Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, SVKM’s
NMIMS, Mumbai, India
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_6
99
100
S. Nagendran et al.
plants and acts as inhibitors against different kinds of pathogenic bacteria and fungi.
These inhibitions are by producing bioactive metabolites or compounds which are
harmful and toxic to disease causing pathogens, without having any harmful effects
on the environment [2-3]. (Zucchi et al. 2008; Costa et al. 2013). Actinomycetes, as a
biocontrol agent, play an important role in affording protection to the plants against
diseases. It also aids in production plant growth promoters, participation in crop
production, maintaining the integrity and protection of the ecosystem. It also plays
an significant role in nitrogen fixation as it has its own commitment in cycling of
nutrient, decomposition of organic matter and degradation of compounds that pollute
the environment (Bhatti et al. 2017; Doumbou et al. 2001). In soil microflora
association, a significant amount is engrossed by Actinomycetes. Among Actinomycetes, Streptomyces are found lavishly in the soil as an essential root colonizer
when compared to genera like Nocardia, Micromonospora and Streptosporangium
which are commonly present in lesser amount in comparison to Actinomycetes
(Pemila Edith Chitraselvi 2018; Kamal et al. 2014). Streptomycetes as a dominant
genus in the midst of Actinomycetes, has gained more importance toward their
participation in production of bioactive compounds, antibiotics, and extracellular
enzymes which encompasses the biocontrol and plant growth-promoting activities
(Kamal et al. 2014; Olanrewaju and Babalola 2019a). Endophytic Actinobacteria are
characterized by those that are contained within the internal structure of plants
and making no obvious changes to their hosts. These Actinobacteria assume explicit
jobs, for example, ensuring the host plants against insects and diseases. Endophytic
Actinobacteria establish a huge part of the rhizosphere, that are found inside plants
in which the broadly examined species are from the variety Frankia (Anandan et al.
2016). Actinorhizal symbiosis is the mutually benefiting relationship between
actinobacterial species of genus Frankia and plants which can form association
with these by means of nitrogen-fixing nodules present in their roots (Anandan
et al. 2016; Wall 2000). The word “actinorhiza” is derived from both the filamentous
bacteria Frankia which is an actinomycete and the root location of the nitrogenfixing nodules (Wall 2000).
6.2
Actinomyces as Soil Dwellers
Actinomycetes derived from the Greek word “actis” meaning ray and “mykes”
meaning fungi due to the fact that they possess the features of both fungi and
bacteria (Das et al. 2008). Actinomycetes are aerobic, spore-producing bacteria
filamentous Gram-positive microorganisms (Flores-Gallegos and Nava-Reyna
2019), with high G+C content Genome (Singh and Dubey 2018; Vivas et al.
2003), described by a highly intricate life cycle assuming a position within the
phylum Actinobacteria, in the domain of bacteria which are included in one of the
biggest ordered units among the 18 significant heredities. (Flores-Gallegos and
Nava-Reyna 2019). Actinomycetes are one of the most widely observed group of
soil microorganisms and surely understood for their antimicrobials generation to
6 Eco-friendly Association of Plants and Actinomycetes
101
control the microorganisms. They are well researched for their job in natural control
of plant pathogens, cooperation with plants, and their growth (Bhatti et al. 2017).
Actinomycetes show their presence in various habitat in natural environments on the
earth covering wide range of terrestrial and aquatic ecosystems. Strive to establish
themselves under wide variety of environmental circumstances, as aerobic, anaerobic as well at temperatures ranging 5–7 C and 45–70 C (Bhatti et al. 2017). They
are widely reported to show their establishment in soil, silt present in the water
reservoirs, air as well in plant remains. As a primary inhabitant Actinomycetes are
found sufficiently in soil compared to other microflora, their presence in the soil in
the form of hyphal thread-like filament identical to fungi gives a peculiar “earthy”
smell indicating the newly formed fresh and healthy soil (Bhatti et al. 2017; George
et al. 2012; Srinivasan et al. 1991; Kuster 1968; Rowbotham and Cross 1977).
Actinomycetes classification includes ten genera as follows; Actinomyces,
Nocardia, Streptomyces, Thermoactinomyces, Waksmania, Thermopolyspora,
Micromonospora, Thermomonospora, Actinoplanes, and Streptosporangium
(Babalola et al. 2009). Among these genus, Streptomyces is considered as a significant member of Soil bacteria and gained importance due to the presence of its rich
source of bioactive chemicals, antibiotics, and extracellular enzymes as well as its
importance in agriculture (Olanrewaju and Babalola 2019b).
6.2.1
Rhizospheric Actinobacteria
Rhizosphere is the zone surrounding the plant root and it is an important component
of the environment where the interaction between microbes and plants occur. The
large area of the soil microbiome in the plant root system is occupied by rhizospheric
Actinobacteria. Different Genus of the Actinomycetes present in the rhizosphere
zone are reported as follows Streptomyces, Sanguibacter, Phylum Actinobacteria
Rhodococcus, Pseudonocardia, Propionibacterium, Nocardia, Mycobacterium,
Micrococcus,
Microbacterium,
Frankia,
Corynebacterium,
Clavibacter,
Cellulomonas, Bifidobacterium, Arthrobacter, Actinomyces, and Acidimicrobium
(Franco-Correa and Chavarro-Anzola 2016; Yadav and Yadav 2019). As a major
resident of rhizosphere microflora, Rhizospheric Actinobacteria has received more
attention in recent years due to its active participation as plant growth promoters,
biocontrol agents against phytopathogens, efficient decomposers, nitrogen fixers as
well as able to withstand against various environmental stress thereby promoting
plant growth and productivity as well as in improving the fertility of the soil (FrancoCorrea et al. 2010; Alexander and Zuberer 1991; Jeon et al. 2003; Yadav et al. 2018).
102
6.2.2
S. Nagendran et al.
Endophytic Actinobacteria
Endophytes are a microbial community present inside the plant tissue without
causing any adverse effects on the host plant. The presence of Endophytic microbes
comprising of bacteria, fungi, and Actinomycetes are pervasive in most of the plant
species grown in agricultural fields (De Bary 1866; Schulz and Boyle 2006). These
endophytes proved their significant role in progressing plant growth, reduction of
diseases caused by pathogens, ability to withstand unfavorable environmental stress
conditions (Hasegawa et al. 2006). Endophytic Actinobacteria establish a huge part
of the rhizosphere, which are likewise found inside plants in which the broadly
examined species are from the variety Frankia (Wall 2000).
Recent years have seen the development of many genomes from Frankia
sp. strains and the improvement of strategies for controlling plant gene expression.
Understanding the bacterial interaction obligate utilization of a possibility of strategies that uncover the proteomes and transcriptomes from both refined and symbiotic Frankia. The image (Fig. 6.1) starting to rise gives some point of view on the
heterogeneity of Frankial populaces in the two conditions (Schwencke and Carú
2001).
Since the acknowledgment of the name Frankia in the Approved Lists of bacterial
names (1980), barely any revisions have been specified to the description of genus.
Progressive releases of Bergey’s Manual of Systematics of Archaea and Bacteria
have comprehensively clashing suprageneric attention to the genus with no advances
for subgeneric arrangement (Benson et al. 2011).
Endophytic Actinobacteria can be classified into two different subgroups: (Singh
and Dubey 2018; Rosenblueth and Martínez-Romero 2006)
Fig. 6.1 Frankia; a whole Alder root nodule gall
6 Eco-friendly Association of Plants and Actinomycetes
103
Fig. 6.2 Frankia–plant association (Sellstedt and Richau 2013)
1. Obligate—where survival of the bacteria is entirely dependent on plant metabolism and where plant-to-plant transmission occurs through various vectors or
vertical transmission (Hardoim et al. 2008)
2. Facultative—where during their life cycle, in particular stages, they are not
dependent on the host plant for survival and form an indirect association via
surrounding soil and the environment (Abreu-Tarazi et al. 2010)
In the light of phylogenomic investigations, Frankia ought to be viewed as a single
individual from the family Frankiaceae inside the monophyletic order, Frankiales. A
polyphasic methodology of joining genome to genome information and omniLog®
phenoarrays, together with classical methods, has permitted the assignment and an
altered depiction of a type strain of the type species Frankia alni. This has led to the
identification of 10 novel species comprising of both symbiotic and non-harmonious
taxa inside the variety (Benson et al. 2011).
Frankia strains are classified into four host-specificity groups based on their
ability to nodulate actinorhizal plants and into three major phylogenetic groups
(Wall 2000; Baker 1987). The actinorhizal plant species can be broadly classified
into four subclasses, eight dicotyledonous families (Betulaceae, Casuarinaceae,
Coriariaceae, Datiscaceae, Elaeagnaceae, Myricaceae, Rhamnaceae, and
Rosaceae) (Rosenblueth and Martínez-Romero 2006), and 25 genera containing
more than 220 species. (Wall 2000; Schwencke and Carú 2001) A marked genetic
variation in Frankia populations has been observed using molecular tools (Gtari et al.
2019a).
Frankia strains have been isolated into four hereditarily different groups dependent on 16S rRNA, nifH, and gln gene sequences (Sellstedt and Richau 2013).
The actinorhizal plants (Fig. 6.2) that are invaded by Gram-positive Frankia
species are individuals from three phylogenetically related gatherings: Fagales
(Betulaceae, Casuarinaceae, Myricaceae), Cucurbitales (Datiscaceae, Coriariaceae),
and Rosales (Rosaceae, Elaeagnaceae, Rhamnaceae) (Sellstedt and Richau 2013;
Berry et al. 2011).
The taxonomical and biological places of Actinomycetes that produce antibiotics
are all around perceived for their metabolic adaptability, generally accompanied by
the creation of essential and optional metabolites of financial importance. Different
methodologies including classical, chemo-taxonomical, numerical taxonomy, in
addition to atomic level have been routinely utilized for the recognizable proof of
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Actinomycetes. The metabolic point of view of Actinomycetes gives a fascinating
territory to inquire about and offer the probability of commercialization of the
metabolites created in the process (Ventura et al. 2007).
The plant microbiomes (epiphytic, endophytic, and rhizospheric) are usually secluded and which have the capacity to advance plant development are alluded
as plant growth-promoting (PGP) microorganisms. The plant microbiomes having
been sifted through from various sources are placed with every one of the three
domains archaea, bacteria, and fungi. Among these three the microbes under the
bacterial domain are all around described and reported as from differing abiotic
stresses, for example, basic soil, soil with high sodium chloride content, acidic soil
low temperature soil, high temperature soil, and drought (Yadav and Yadav 2019).
Research that utilize exponentially developing cultures have yielded data on a
complex proteolytic framework, including proteasomes, endo-and extracellular proteinases, and aminopeptidases, and furthermore on esterases, dehydrogenases, and
extracellular DNAses (Abdel-Lateif et al. 2012).
Early investigations on the subatomic science of Frankia were hampered by
troubles with developing enough cell material for DNA extraction and furthermore
by the low effectiveness of old-style lysis strategies of Frankia cells. The nif encode
the nitrogenase enzyme: nifH codes for the polypeptides of the Fe protein and nifDK
codes for the alpha and beta subunits of the MoFe protein. A few hereditary and
physical examinations have now evidently stated that in rapidly developing Rhizobium species the qualities associated with symbiotic relationship, especially the nif,
are situated on enormous plasmids. Hypothetically DNA can be presented in beneficiary cells by four techniques: Transduction, Conjugation, Transformation, and
Protoplast combination (Cervantes and Rodriguez-Barrueco 1992).
The symbiosis receptor kinase SYMRK plays a role in a signaling pathway that
has been demonstrated for leguminous plants that are necessary to trigger the
development of nodules and the acceptance of bacteria or fungi into their root
cells as endosymbionts (Sellstedt and Richau 2013) as explained in Fig. 6.3.
Flavonoids are a gathering of secondary metabolites obtained from the
phenylpropanoid pathway. They are omnipresent in the plant realm and have
numerous assorted capacities including key functions at various degrees of root
endosymbiosis. While there is a great deal of data on the job of specific flavonoids in
the Rhizobium-legume beneficial interaction. Whereas the defined function during
the foundation of arbuscular mycorrhiza and actinorhizal symbioses stays vague
(Singh and Dubey 2018).
Arbuscular mycorrhiza embodies an exceptionally basic kind of root endosymbiosis, creating the groundwork of symbiosis made out of land plants and which fall
under phylum Glomeromycota (Sellstedt and Richau 2013).
Frankial populaces in root knobs appear to keep up a somewhat strong metabolism that incorporates fixation of nitrogen, generous biosynthesis and pathways that
create energy, alongside an altered ammonium assimilation. Until this point in time,
specific bacterial qualities have not been embroiled in root knob development yet a
6 Eco-friendly Association of Plants and Actinomycetes
105
Fig. 6.3 Actinorhizal symbiosis signaling (Ostrowski and Jakubowska 2008)
few theories are rising as to how the plant and microorganism figure out how to exist
together. Specifically, frankiae appear to show a nonpathogenic existence to the
plant that may have the impact of limiting some plant protection reactions
(Schwencke and Carú 2001).
Types of Compatible Frankia Interactions
1. Root Hair Infection (Wall 2000)
Gymnostoma papuanum was infected by infiltration of Frankia into twisted
root hairs and trailed by improvement of a prenodule region including one to
many nodule lobe primordia in the root cortex. Frankia hyphae developed in a
straightforward manner through cell walls from cell to cell, colonizing cells of the
prenodule preceding invasion of nodule lobe cells (Racette and Torrey 1989).
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2. Intercellular Penetration (Wall 2000)
Shepherdia argentea roots were invaded by Frankia by means of intercellular
entrance into the root epidermis and cortex with direct invasion of cells of the
nodule lobe primordia. No prenodule locale was found to be present.
So far, the method of invasion seems, by all accounts, to be unique for every one
of the plant families (Liu and Reid 1992). The type of interaction is governed by the
host plant species. Mature nodules consist of multiple lobes with modified lateral
roots containing infected cells in the expanded cortex. The absence of root cap and a
superficial periderm is observed (Schwencke and Carú 2001).
6.3
Plant Growth-Promoting Rhizobacteria (PGPR)
These are considered as having overridig importance in agriculture due to constructive good impact on well being of plant growth and development by inhibiting the
disease-causing pathogens as well as improving their nutrient requirement and
consumption. Plant growth-promoting rhizobacteria (PGPR) are a gathering of
microorganisms that colonize the rhizosphere and underlying foundations for various
plant species (Santamarina Siurana et al. 2004). Actinobacteria are considered as
plant growth-promoting rhizobacteria because of the ecological role played in the
crop growth and productivity as well the maintaining the fertility of the soil by
nutrient cycling (Franco-Correa and Chavarro-Anzola 2016; Franco-Correa et al.
2010; Jiang et al. 2006; Pathom-Aree et al. 2006).
6.3.1
Machinery Involved in Plant-Growth Promoting
Rhizobacteria
The Plant growth-promoting rhizobacteria are influenced by several mechanisms as
shown in Fig. 6.4.
Fig. 6.4 Machinery underlying PGPR
6 Eco-friendly Association of Plants and Actinomycetes
6.3.1.1
107
Production of Plant Growth Regulators (PGRs)
Plant organ arrangement and their ensuing advancement are interceded by inner
variables of essential significance. PGRs (“Plant Growth Regulators”) are also
widely recognized as plant hormones. At low concentration, PGRs are small molecules that influence plant development (Franco-Correa et al. 2010; Neilands 1995).
The capability of the rhizospheric bacteria depends upon the ability to encourage the
development of the root system with reference to plant growth regulators (Fett et al.
1987; Torres-Rubio et al. 2000).
Indole acetic acid (IAA) is a plant growth hormone and an active structure of
auxins. It assumes a significant role in plant development through its life cycle
(Vessey 2003; Ostrowski and Jakubowska 2008; Pawlowski and Demchenko 2012).
IAA induces the development of the radicular framework (Liu and Reid 1992; Sylvia
et al. 2005; López Nicolás et al. 2004), because of the improvement of lateral roots
and divisions of the growing tip of the plant that results in roots lengthening
(Rosenblueth and Martínez-Romero 2006; Abreu-Tarazi et al. 2010). The generation
of IAA has been broadly contemplated in Actinobacteria (Hardoim et al. 2008).
Streptomyces genus and Frankia species have been extensively investigated for their
IAA producing capabilities (Péret et al. 2008; Sousa et al. 2008; Flores et al. 2003).
6.3.1.2
Production of Siderophores
Microorganisms have been constrained by natural limitations and biologic goals to
deliver explicit particles that can contend positively with hydroxyl ions for ferric
condition/ form of iron. Siderophores are mixes delivered by different microorganisms in soil. These living beings depend on the process of chelation to help their
natural action. Siderophores are fluorescent pigments found outside the cell that bind
to iron (III) in high affinity and they are soluble in water having a low molecular
weight (500–1000 Da) (Prasad et al. 2011).
These compounds go about as chelate agents that act specific for ferric ion,
leaving accessible the ionic structure (Fe+2), which is effectively consumed by
microorganisms (Vessey 2003; Pawlowski and Demchenko 2012).
Most of the nitrogen-fixing microorganisms produce siderophores to acquire iron.
This is essential for the proper functioning of the enzyme nitrogenase. The compound is made out of a few protein units; a sum of 36 iron atoms are necessary for
working appropriately (Froussart et al. 2016).
6.3.1.3
Non-Symbiotic Nitrogen Fixation
The Actinobacteria are heterotrophic living beings that require carbon as a source to
derive the energy required for fixing N2. Accordingly, every one of the various
microbes contrasts in such a way that carbon is processed with characteristic
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capacity of nitrogen fixation, demonstrating various rates of acetylene reduction
assay (ARA). This test depends on identifying in an indirect fashion, the presence of
nitrogenase protein, which is responsible for the reduction of nitrogen (N2) to
ammonium ion. ARA evaluates the enzymatic reduction of acetylene to ethylene
(NH4+) (Aranibar et al. 2003). Some non-leguminous, nitrogen-fixing plants convey
dual symbiotic infection including both mycorrhizal parasites and actinorhizal
actinomycetes. Examples include Arthrobacter, Agromyces, Corynebacterium,
Mycobacterium, Micromonospora, Propionibacteria, and Streptomyces (Sellstedt
and Richau 2013).
6.3.1.4
Chitinase Production
The chitin is a homopolymer containing N-acetyl-D-Glucosamine residues with α-1,
4 bonds. It is extensively found in the environment as a basic component of fungi
(22–44%), insects and crustaceans (25–58%), and protozoa (Azcón-Aguilar and
Barea 1997; Hoster et al. 2005; Nehl and Knox 2006; Bhattacharyya 2012). The
chitin is hydrolyzed by a complex chitinase that contains three proteins which
are exochitinase, endochitinase, and N-acetyl-D-Glucosamine (Franco-Correa and
Chavarro-Anzola 2016).
Actinobacteria are considered as the predominant creatures associated with the
deterioration of chitin in soil (Ames 1989) and furthermore encouraging antagonistic
agents for biocontrol because of the hydrolysis response over the mycelium fungi
(Gomes et al. 2001). The species that come under Streptomyces class are considered
as the primary chitinolytic microbe in soil, because of its ability to breakdown this
polymer (Metcalfe et al. 2003).
6.3.1.5
Antagonistic Activity Against Phytopathogenic Fungi
(Franco-Correa and Chavarro-Anzola 2016)
In phytopathology, antagonism refers to the action of any organism that suppresses
or interfere with the normal growth and activity of a plant pathogen, such as the main
parts of bacteria or fungi.
Different Actinobacteria may behave as hostile microorganisms toward
F. oxysporum by antimicrobial synthesis (antibiosis). These synthesized chemicals
diffuse through the medium hindering the development of phytopathogenic growth.
Molano et al. (2000) decided in vitro inhibition of Fusarium oxysporum development by actinomycin, an anti-infection created by Nocardia sp., strain confined
from rhizosphere soil sample lichen (Mosquera, Colombia). The generation of such
optional metabolites was lethal to the phytopathogenic organism.
6 Eco-friendly Association of Plants and Actinomycetes
6.3.1.6
109
Mycorrhiza (MA) Helper Bacteria (Franco-Correa
and Chavarro-Anzola 2016)
By and large, the capacity of specific microorganisms to impact the arrangement and
working of the symbiotic MA through different types of activities, for example,
initiation of parasitic propagules in infective pre-symbiotic stages (Azcón-Aguilar
and Barea 1997; Giovannetti et al. 2002), and encourage the development of sources
of input points into the root and thereby augument the development rate.
(Giovannetti et al. 2002; Carpenter-Boggs et al. 1995). The portrayal of the variety
Frankia, the microsymbiont in the actinorhizal advantageous interaction, is moving
toward maturity however the ideal use of techniques previously created requires the
thought of the genuine commitment of mycorrhiza related with the actinorhizal
symbioses. Mycorrhizal species that are found in most actinorhizal plant genera so
far inspected in both ectomycorrhizal and vesicular-arbuscular mycorrhizal symbioses. The nearness of a mycorrhizal beneficial interaction in a plant species relies
generally upon many soil attributes. In this way, contamination of Myrica gale by
both ecto- and vesicular-arbuscular mycorrhiza is limited to well-drained soils, being
missing in wet soils (Gtari et al. 2019b).
6.4
Ecological Contributions of Actinomycetes
For quite a long time plants have been broadly utilized as the reservoir of bioactive
chemicals for their therapeutic effect. In the present day scenario, plant-related
microorganisms have been observed to create substances of high therapeutic potential (Vivas et al. 2003; Hoster et al. 2005). The microorganisms living inside plant
tissues, generally in symbiosis, may incorporate different communities, for instance,
fungi and Actinobacteria (Singh and Dubey 2018; Pimentel et al. 2011).
Customarily, serious research has been conducted on free dwelling soil
actinobacteria. Generally, advantageous Actinobacteria dwelling as endophytes
inside the plant tissues have produced enormous enthusiasm as a potential wellspring of novel mixes, which may find applications in medication, horticulture, and
environment as given in Fig. 6.5 (Kuffner et al. 2010).
Fig. 6.5 Ecological importance of Actinomycetes
110
6.4.1
S. Nagendran et al.
Handling of Abiotic Stresses
There are advancements in research on transgenic actinorhizal plants on actinorhizalexplicit qualities and proteins (actinorhizins) associated with symbiotic communications, infectivity, and host particularity. Actinorhizal plants are quickly developing
species, ready to grow in N-poor soils, and for specific species, in unforgiving
natural pressure conditions (Vivas et al. 2003).
6.4.1.1
Salinity
Antioxidative defence system and entire cell proteome description is dependent on
spectrophotometric investigation. SDS-PAGE and 2-dimensional gel electrophoresis have been investigated among salt-tolerant and salt-sensitive Frankia strains.
Proteomic premise giving basis for salinity tolerance in the recently identified
Frankia strains from Hippophae salicifolia. Salt-tolerant strain HsIi10 shows greater
quantity of superoxide dismutase, catalase, and ascorbate peroxidase when compared with salt-sensitive strain HsIi8. Differential 2-DGE profile has uncovered
differential profiles for salt-tolerant and salt-sensitive strains. Proteomic affirmation
of salt resilience in the strains with inbuilt proficiency of flourishing in nitrogeninsufficient regions is a distinctive benefit for these organisms. This would be
similarly valuable for the development of soil nitrogen status. Proficient protein
modulation in HsIi10 recommends further investigation for its latent capacity to be
used as biofertilizer in saline soils, strains (Srivastava et al. 2017).
6.4.1.2
pH
Neutrophiles and acidophiles are two characteristically different groups observed in
Streptomyces species of two pine forest soils and it is recognized that the acidophilic
group is more widely found and of significance to the soil more than what was
known previously (Williams et al. 1971).
6.4.2
Auxiliary Metabolite Generation
Actinobacteria assume an important role in reusing unmanageable biomaterials by
deteriorating complex blends of polymers in dead plants, animals and contagious
materials. They are considered as the biotechnologically significant microbes that are
exploited for its auxiliary metabolite generation. Around, 10,000 bioactive metabolites are created by Actinobacteria, which is 45% of all bioactive microbial metabolites found (Anandan et al. 2016).
6 Eco-friendly Association of Plants and Actinomycetes
6.4.3
111
Fertility
Actinorhizal plants play a very important role in improving fertility of agroforestry
ecosystems, as sources of biomass for generating energy or for carbon storage, for
remediation of soils in harsh environmental stress conditions, reclamation of land,
amenity planning for preventing erosions and as coastal windbreaks (Pemila Edith
Chitraselvi 2018; Kamal et al. 2014). The actinorhizal advantageous interaction is a
significant supporter of the worldwide nitrogen spending plan, assuming a prevailing
job in environmental progressions following unsettling influences. The components
included are still inadequately known; however aided in understanding of kinase that
transmit the symbiotic signal and thereby transmission of the Rhizobium Nod signal
in legumes. On the microbial side complementation with Frankia DNA of Rhizobium nod mutants prevented identification of genes thought to be symbiotic
(Simonet et al. 2018).
Of late land degradation has expanded extensively because of climatic reasons
and human mediation bringing about a decrease in fertility, biodiversity, and profitability. Since tropical nations are portrayed by increasing population density, the
populace reliance on biological systems is bringing about environment degradation.
Consequently, the restoration of these lands is essential. To beat the issue of the
absence of fertility in soil, rapidly developing nitrogen-fixing trees, for example,
actinorhizal plants in blend with biofertilizers are utilized (Diagne et al. 2013; Mahdi
et al. 2010; Sayed 2011). PGPRs have high economical benefit and ecological
significance as biofertilizers. Example: Azospirillum (Fuentes-Ramirez and
Caballero-Mellado 2005).
6.4.4
Phytoremediation
Phytoremediation of Cd mediated in sunflower plant by S.tendae and mobilization of
Zinc(Zn) and Cadmium(Cd) by secondary metal-binding metabolites of Salix caprea
clearly define the scope of endophytic Actinobacteria in heavy metal removal from
the soil and increased mobilization in soils contaminated by heavy metals (Das et al.
2008; Dimkpa et al. 2009; Baoune et al. 2018). Endophytic Streptomyces sp. have
been accounted for improving phytoremediation efficiency of oil-polluted soil by
their petroleum degradation property (Karthikeyan et al. 2018).
6.4.5
Miscellaneous Contributions
Auxin, siderophore production, solubilization of phosphates, and complementing
VA Mycorrhizal fungi are also some of the major functions of actinomycetes and
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S. Nagendran et al.
their isolates. They are also potential biocontrol agents and all of these properties
play an essential role in improving agricultural productivity and quality and they
should therefore be taken into consideration as an alternative tool for promotion of
eco-friendly as well as sustainable farming practices unlike harmful chemicals which
are detrimental to the environment (Pemila 2018; Sharma et al. 2014).
6.5
Conclusion
Plant and actinomycetes and their eco-friendly association form an integral and
essential component of the ecosystem as a whole. Actinomycetes are classified
using classical and taxonomic classification and can be obligative or facultative.
The symbiotic relationship is supported due to various complex signaling pathways
between the actinorhizal plant and the particular strain of actinomycetes, with
different species, each acting in a unique and characteristic way. The interaction
between the plant and actinomycetes occurs either by infection into root hair or
penetration between the cells of host plant. Actinomycetes, once they form the
symbiotic relationship are a major influencer of the plant growth and regulates
various synthesis pathways and systems such as auxin and chitinase production
and nitrogen fixation. With the increasing concern for the environment, actinomycetes has been considered as possible alternative for a number of objectives such as
Biofertilizers or in land reclamation or in phytoremediation. Therefore, the existence
of Actinomycetes and their eco-friendly association with plants forms the essential
basis and foundation for future research including their benefit to the ecosystem.
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Chapter 7
The Arbuscular Mycorrhizal Symbiosis
of Trees: Structure, Function,
and Regulating Factors
Leszek Karliński
Abstract The majority of terrestrial plants establish symbiotic relationships with
fungi, which mostly co-create various types of mycorrhizae, differing in phylogenetic history, anatomy, and functional features. Arbuscular mycorrhiza is evolutionary the oldest and most common form of the symbiosis of plants and fungi belonging
to phylum Glomeromycota. Arbuscular mycorrhiza plays a significant role in mineral nutrition of plants, protection against stress factors and pathogens, and supports
the soil structure and fertility. Arbuscular fungi dominate in relatively nutrient-rich
and phosphorus-limited soils. The highest diversity of arbuscular fungi was observed
in the tropics, where fungal communities revealed significant spatial heterogeneity
and non-random associations with the different hosts. Arid areas and wetlands are
also often inhabited by shrubs or trees associated with arbuscular mycorrhizal fungi,
presenting high tolerance to environmental conditions. A few tree species form
tripartite symbiotic associations with arbuscular and ectomycorrhizal fungi, whose
contribution in the microbiome of trees is mainly shaped by soil conditions and tree
genotype.
This review presents a brief summary of the main types and evolutional history of
mycorrhizal associations with trees with special emphasis on arbuscular fungi, their
characteristic structures, taxonomical classification, distribution, and factors
impacting them.
Keywords Symbiosis · Microorganisms · Host genotype · Glomeromycota
L. Karliński (*)
Institute of Dendrology, Polish Academy of Sciences, Kórnik, Poland
e-mail: leszekk@man.poznan.pl
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_7
117
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7.1
L. Karliński
Types of Mycorrhiza
The majority of terrestrial plants establish symbiotic relationships with fungi, which
mostly co-create four main types of mycorrhizae (Smith and Read 2008), differing in
phylogenetic history, and anatomical and functional features (Brundrett and
Tedersoo 2018). Arbuscular mycorrhiza is the most common type of plant–fungi
symbiosis, occurring in more than 71–90% of colonized vascular plant species. The
presence of structures of arbuscular mycorrhizae has been demonstrated in angiosperms, gymnosperms, and pteridophytes having roots as well as gametophytes of
some mosses, lycopods, Psilotalus, which do not have true roots (Smith and Read
2008; Błaszkowski 2012). Next to arbuscular mycorrhiza is Orchid mycorrhiza,
which is found in 10% of plant species (Orchidaceae) and characterized by specific
for Orchidaceae, hyphal peletons within root cells, colonization from the root surface
mycelia or neighboring cells. Two percentage of plant species (2 Gymnosperm and
28 Angiospermae lineages) establish symbiotic relationships with ectomycorrhizal
fungi, characterized by external differentiated hyphal mantle, current Hartig network
and lack of intracellular colonization of the plant host. An even smaller group of
species are plants belonging to Ericaceae and Diapensiaceae and forming together
with fungi ericoid mycorrhizas (1.5%) characterized by structures such as hyphal
coils in cells, cells colonized separately from the root surface, presence of mantle
(Brundrett and Tedersoo 2018). Sometimes two other types of mycorrhizae are also
distinguished, i.e., mycoheterotrophic monotropoid mycorrhiza, specific for the
Monotropa genus and arbutoid mycorrhiza found in genera Arctostaphylos and
Arbutus (Garg and Chandel 2010). However, both types of mycorrhiza are included
in ectomycorrhizas (Smith and Read 2008).
7.2
Evolution of Mycorrhizal Symbiosis
Arbuscular mycorrhiza, apart from the fact that it is most common in the world of
plants, is also evolutionarily the oldest form of the symbiosis of fungi with plants,
probably accompanying them from the moment the plants enter the land. Arbuscular
mycorrhiza could appear in plant tissue structures over 450 million years ago in
Ordovician (Brundrett 2002). However, problems with the interpretation of these
findings mean that the first well-preserved fossils of mycorrhizal rhizomes are
structures similar to modern counterparts from early Devon, whose age is estimated
at 407 million years old. The Devonian land plants revealed the presence of
mycorrhiza-like intracellular structures similar to arbuscules of symbiotic
Glomeromycotina and hyphae reminiscent Mucoromycotina, having the same
ancestor somewhere between 358 and 508 million years ago (Martin et al. 2017).
However, the first AM found fungus-like spores are 50 Million years older and come
from Ordovician (Brundrett and Tedersoo 2018). In contrast, molecular data of plant
symbiosis genes indicated that the relationships between fungi and plants are much
7 The Arbuscular Mycorrhizal Symbiosis of Trees: Structure, Function, and. . .
119
older than the presence of plants on land and already occurred in their ancestors in
the aquatic environment (Martin et al. 2017). Even if arbuscular fungi did not
directly participate in leaving the aquatic environment by plants, they significantly
supported the plants to colonize the terrestrial environment in a relatively short
period (Chen et al. 2018; Delaux 2017). Along with the changes in environmental
conditions on Earth and the ongoing process of plant species radiation, the development or decline of symbiotic associations with fungi has also taken place. The
presence of arbuscular mycorrhizal structures has been found, among others, in late
Carboniferous (over 300 Million years) in woody plants Lepidodendron and early
relatives of conifers—Cordaites (Martin et al. 2017). Then the symbiotic relationships of plants with arbuscular fungi develop in gymnosperms cycads and conifers,
evolve, and constitute the main form of mycorrhizal symbiosis from Triassic to
Cretaceous. Next, in the Early Cretaceous (145 Million years) the arbuscular mycorrhiza also occurs in basal angiosperm trees, and monocots, and eudicots. During the
Cretaceous, most of the non-tree plant species established symbiotic associations
with arbuscular fungi and this relationship continues to this day (Martin et al. 2017).
Earlier, in the Jurassic, for the first time, ectomycorrhiza had evolved from
arbuscular mycorrhiza, establishing relationships with the early Pinaceae. The next
evolutionary wave takes place in Crataceus with the origins of the Orchidaceae,
Ericaceae, and multiple families with ECM, parasitic plants, carnivorous plants, and
nitrogen-fixing symbioses (Brundrett 2002; Brundrett and Tedersoo 2018; Martin
et al. 2017). Over the last 65 Million years (Paleogene), another wave of radiation
has been underway mainly associated with ectomycorrhizal and non-mycorrhizal
species, leading to complete transformations of mycorrhizal types from arbuscular
mycorrhiza to ectomycorrhiza, but also to the joint coexistence of both mycorrhizal
types on the roots of the same tree species (e.g., the genus Salix, Casuarinaceae,
Alnus, Eucalyptus), whose contribution is conditioned by many environmental and
tree-host-genetic factors (Brundrett and Tedersoo 2018).
7.3
Classification of Arbuscular Fungi
Arbuscular fungi as one of the most abundant groups of soil organisms, establish
symbiotic relationships with more than 200 thousand of plants (Lee et al. 2013).
Despite their common prevalence, arbuscular fungi are known to show low species
diversity. About 240 species of arbuscular fungi are described mostly based on their
morphology and grouped in the phylum Glomeromycota (Błaszkowski 2012; Lee
et al. 2013). According to classification presented by Błaszkowski (2012), phylum
Glomeromycota consists of four orders: Archaeosporales, Diversisporales,
Glomerales, and Paraglomerales, comprising nine families and thirteen genera,
belonging to the class Glomeromycetes of the phylum Glomeromycota. The only
Geosiphon pyriformis of the family Geosiphonaceae (Archaeosporales) forms
endocytosymbiosis with cyanobacteria (Nostoc sp.) but does not form arbuscular
mycorrhizae (Błaszkowski 2012). Members of Glomeromycota phylum turned out
120
L. Karliński
to be closer to fungi belonging to Ascomycota and Basidiomycota than to those from
Zygomycota as previously thought (Błaszkowski 2012).
The factors limiting the estimation of species diversity of arbuscular fungi and
their taxonomical position were the difficulty in extracting their spores from the soil
and maintaining the fungi in cultures (Błaszkowski 2012). However, molecular
methods used in recent years, have suggested that diversity of arbuscular fungi
may be underestimated (Husband et al. 2002; Wubet et al. 2003; Baltruschat et al.
2019). Also, it has been shown that individual species and single spores present high
genetic diversity, resulting in differentiation of important functions such as colonization rates, growth of extraradical hyphae, or phosphorus uptake by arbuscular
fungi (Lee et al. 2013). The highest diversity of arbuscular fungi was observed in the
tropics, where fungal communities revealed significant spatial heterogeneity and
non-random associations with the different hosts. Fungal communities presented
also a high variation of species composition in time (Husband et al. 2002).
7.4
Structure of Arbuscular Mycorrhizae
The term arbuscular mycorrhiza has evolved over many years with the development
of science. Originally, arbuscular mycorrhiza was called endomycorrhiza. However,
after finding its distinctness in terms of functionality and evolution in comparison to
the other types of endomycorrhiza, the name vesicular-arbuscular mycorrhiza
(VAM) was adopted and used for several years. Because only 80% of VAM forms
vesicles, this name was finally abandoned and the new name arbuscular mycorrhiza
(AM) has been started to use without indicating the existing structural traits.
The arbuscular mycorrhiza is formed of its characteristic extra- and intraradical
structures. The extraradical structures are spores, extraradical hyphae penetrating the
soil and connecting spores with roots, and auxiliary cells (formed instead of the
vesicles—only by species belonging to Gigasporaceae and Pacisporaceae)
(Błaszkowski 2012). Extraradical hyphae penetrate to the roots through the appressorium. Colonization of plant roots by arbuscular fungi is linked with the presence of
intraradical hyphae located between the cells of the plant root (intercellular) or
penetrating inside the cells (intracellular). Other structures whose arbuscular fungi
owe their name are arbuscules—intracellular tree-like transformations of mycelium,
responsible for the exchange of nutrients between the host and the fungus. Some
arbuscular fungi also create intraradical oval structures—vesicles. Vesicles
containing abundant lipids and numerous nuclei are thought to be important storage
organs and as propagules play a role in recolonization of roots in the soil. Fungal
species in the genera Gigaspora and Scutellospora produce auxiliary vesicles (sometimes called auxiliary bodies or cells) in the extraradical mycelium (Peterson et al.
2004). Among arbuscular fungi, there are two main anatomical types—Arum-type
and Paris-type, taking their names from the names of plants in which they were first
observed by Gallaud at the beginning of the twentieth century (Gallaud 1904, 1905).
In the case of the Arum-type, hyphae penetrate the epidermis generally forms a coil
7 The Arbuscular Mycorrhizal Symbiosis of Trees: Structure, Function, and. . .
121
either in the epidermal cell or first cortical cell layer before it enters the intercellular
spaces of the cortex. Hyphae also branch many times. Inki Arum-type, roots can be
rapidly colonized along the root axis due to the free movement of hyphae in the
intercellular spaces (Peterson et al. 2004). Arbuscules arise depending on the species
of fungus inside cortical cells. Vesicles may also be present. In the case of woody
species, the presence of Arum-type was found, among others, in the roots of Sorbus
torminalis (Rosaceae) (Bzdyk et al. 2016).
Paris-type is characterized by the presence of intracellular coiled hyphae spreading from cell to cell. Plant species containing this type of mycorrhiza show a lack of
conspicuous intercellular spaces and only intracellular hyphae is present. Coils
develop from that intracellular hyphae and next, the coils give rise to fine lateral
branches. These coils are often accompanied by arbuscules or arbusculate coils. The
hyphal coils and the fine branches have a perifungal membrane and an interfacial
matrix of host-derived cell wall components (Peterson et al. 2004). The Paris-type is
the most common type of arbuscular mycorrhiza in the plant world (Smith and Read
2008). It occurs in several herbaceous plants present in forests as well as in woody
plants such as Acer saccharum, Ginkgo biloba, or Taxus baccata (Smith and Read
2008). In addition to both types, there are also many intermediate types containing
characteristic traits of both Arum- and Paris-type. One should also take into account
the seasonal variability of mycorrhizal structures, their durability, and the impact of
soil conditions. An example would be arbuscules, whose life span may be limited to
1–15 days (Harley and Harley 1987). For example, in the case of horse chestnut
depending on the period of observation of colonization of roots by arbuscular fungi,
both arbuscules and coils or only coils were observed (Karliński et al. 2014;
Tyburska-Woś et al. 2019). Also, the common presence of vesicles in fine roots of
Aesculus hippocastanum in late autumn is characteristic for the end of the growing
season (Peterson et al. 2004; Karliński et al. 2014). Hence, to fully assess the
structure of fungal symbionts in tree root tissues it is important to repeat observations
at different time points.
7.5
Global Distribution of Arbuscular Mycorrhizal Trees
Arbuscular mycorrhiza is the most common form of symbiosis in the plant community and thus also among woody species (Table 7.1). The main factors determining
global distribution and diversity of trees colonized by arbuscular fungi seem to be the
soil nutrients abundance, climatic conditions, and the evolutionary heritage of
arbuscular symbiosis (Chen et al. 2018; Tedersoo and Bahram 2019; Soudzilovskaia
et al. 2019). The long history of the evolution of arbuscular symbiosis and their
prevalence in different (often extreme) habitats underline high adaptation and good
metabolic fit of arbuscular fungi and trees (Chen et al. 2018). Also, the low host
specificity supports the potential success of arbuscular fungi in the establishment of
symbiotic relations with different tree partners (Smith and Read 2008). On the other
hand, arbuscular fungi are known for their intraspecific diversity of spores (Lee et al.
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L. Karliński
Table 7.1 Selected shrubs and trees, and their mycorrhizal symbionts
Type of
mycorrhiza
AM
AM and ECM
Shrubs and trees
Acer sp.; Aesculus hippocastanum L.; Araucaria sp.; Arecaceae; Artemisia sp.;
Asimina Adans.; Buxus sp.; Camellia sinensis (L.) Kuntze; Carica papaya L.;
Carya ovata (Mill.) K. Koch; Cedrus Trew; Citrus sp.; Cocos nucifera; Coffea
sp.; Cornus florida L.; Cupressus sp.; Elaeagnus angustifolia L.; Erythrina
caffra Thunb.; Euonymus; Euphorbia pulcherrima Willd. ex Klotzsch; Fagus
sp.; Ficus sp.; Fraxinus sp.; Ginkgo biloba L.; Hevea brasiliensis; Juglans
nigra L.; Juniperus sp.; Ligustrum sp.; Liquidambar styraciflua L.; Magnolia
L.; Malus sp.; Mangifera indica L.; Musa sp.; Myrica L.; Olea europaea L.;
Oxydendrum arboreum; Parthenium argentatum; Persea americana; Pistacia
vera L.; Pittosporum tobira (Thunb.) W.T.Aiton; Platanus L.; Podocarpus
Persoon; Prosopis L.; Prunus sp.; Rhaphiolepis Lindl.; Rhus L.; Robinia
pseudoacacia; Robus sp.; Rosa sp.; Sequoia sempervirens (lamb. Ex D. Don)
Endl.; Sequoiadendron giganteum; Shibataea kumasasa; Sorbus torminalis
(L.) Crantz; Taxus baccata L.; Theobroma cacao; Triadica sebifera
Acacia sp.; Alnus sp.; Carya laciniosa (F.Michx.) G.Don; Carya cordiformis
(Wangenh.) K.Koch; Casuarina sp.; Eucalyptus sp.; Myrtaceae; Populus sp.;
Quercus rubra L.; Salix sp.
2013), which may selectively favor different plants (Chen et al. 2018). A more
diverse arbuscular fungal community can maintain plant diversity (van der Heijden
et al. 1998; Husband et al. 2002; Lovelock et al. 2003).
Arbuscular fungi dominate in relatively nutrient-rich and phosphorus-limited
soils of tropics (Tedersoo and Bahram 2019; Soudzilovskaia et al. 2019), in contrast
to temperate and northern nitrogen-limited habitats dominated by ectomycorrhizal
forests. The picture of temperate areas seems to be the most complex because of
human-driven land transformations (Soudzilovskaia et al. 2019). It can be assumed
that due to the rapid global climate changes the contribution of trees establishing
symbiotic relationships with arbuscular fungi will increase in this area. Arid areas
and wetlands are also often inhabited by arbuscular mycorrhizal shrubs or trees
because of high tolerance of arbuscular fungi to unfavorable conditions.
7.6
Factors Impacting Trees and Their Fungal Symbionts
Soil conditions, their diversity, and frequent changes (much higher than in the
aquatic environment) have been the main factor that arbuscular fungi have faced
since the beginning of their colonization of the lands. The impact of environmental
factors on the communities of fungi associated with roots of plants is well reflected
by the multi-seasonal woody plants. In this respect particularly interesting is the
small group of trees that establish symbiotic associations with arbuscular as well as
ectomycorrhizal fungi. The presence of both groups of fungal symbionts on the roots
of the one host tree allows to analyzing not only the mutual relationship of trees and
7 The Arbuscular Mycorrhizal Symbiosis of Trees: Structure, Function, and. . .
123
fungi but also to compare the reactions between these two different fungal worlds
and host trees.
Environmental conditions determine the global distribution of mycorrhizal fungi
from the tropical forests being the habitat of mainly arbuscular mycorrhiza and rich
plant communities, to the less differentiated northern forests, dominated by
ectomycorrhiza and much slower nutrient cycling than in the tropics. Soil conditions
are thought to be an important factor in mycorrhizal colonization (Gonçalves and
Martins-Loução 1996; Smith and Read 2008; Karliński et al. 2010, 2013, 2020).
Arbuscular fungi are characterized by a greater amplitude of tolerance to adverse soil
conditions than ectomycorrhizal fungi (Smith and Read 2008). In case of poplars as
well as the majority of other members of Salicaceae, they primarily establish
symbiotic associations with arbuscular fungi. Later, arbuscular fungi are partly
replaced by ectomycorrhizas (Dominik 1958; Smith and Read 2008). Arbuscular
fungi giving way to ectomycorrhizal fungi, tend to partially move into deeper soil
layers with limited oxygen and nutrients concentration (Neville et al. 2002; Karliński
et al. 2010). Also, the floods are important disturbances impacting dual mycorrhizal
colonization symbiosis of trees, e.g., Populus, Salix, Alnus, or some species of
Quercus (Watson et al. 1990; Teste et al. 2019). Wet and poorly aerated conditions
favor arbuscular fungi and negatively impact ectomycorrhizas of Alnus
(Truszkowska 1953), Populus, Salix (Lodge 1989) or Quercus rubra (Watson
et al. 1990). On the other hand, also drought stress negatively impacts the
ectomycorrhizas in contrast to dominating arbuscular fungi (Gehring et al. 2006;
Quereyeta et al. 2009; Kilpeläinen et al. 2017). Also, forest fires to a greater extent
reduce ectomycorrhizal inoculum than arbuscular fungi, which are able more
quickly to support the regeneration of disturbed areas (Lapeyrie and Chilvers
1985; Horton et al. 1998; Teste et al. 2019). The neighborhood of other trees may
also affect the root colonization of young seedlings. The proximity of
ectomycorrhizal trees supports the higher ectomycorrhizal colonization of seedlings
roots and vice versa, the trees colonized by arbuscular fungi may increase arbuscular
mycorrhizal colonization of seedlings (Dickie et al. 2001).
A significant impact of soil temperature on the range of root colonization by both
fungal groups was also found. In high soil temperatures, ectomycorrhizal colonization was found to be decreased (McGee 1988). On the other hand, the low temperature was the limiting factor for arbuscular fungi, but ectomycorrhizal fungi were not
affected (Kilpeläinen et al. 2016). Different factors have been suggested to influence
the arbuscular and ectomycorrhizal colonization such as litter accumulation negatively impacted arbuscular fungi (Conn and Dighton 2000; Piotrowski et al. 2008) or
N and P fertilization (Baum and Makeschin 2000).
The negative impact of heavy metal pollutants on the biomass of arbuscular fungi
and other groups of fungi has been reported by several authors (e.g., Frostegård et al.
1993; Hagerberg et al. 2011; Pennanen et al. 1996). The analyses of microbial
communities in the rhizosphere of mature poplars growing in the vicinity of the
copper smelter revealed that the negative effect of heavy metal pollution was more
pronounced for the biomass of fungal part of soil microbiome than for bacterial
biomass (Karliński et al. 2020). This result is in line with the other observations
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L. Karliński
obtained near copper or zinc smelters, which showed a decrease in phospholipid
fatty acids (PLFAs) as indicators of the fungal biomass (e.g., Pennanen et al. 1996).
Lower tolerance of fungi than bacteria to heavy metal pollution (Cu, Zn) was also
confirmed in laboratory experiments (Rajapaksha et al. 2004).
The negative impact of heavy metal pollution was observed especially in the case
of the biomass of arbuscular fungi in the rhizosphere of poplars (Karliński et al.
2020). To a lesser extent, soil pollution decreased the biomass of ectomycorrhizal
fungi. On the other hand, the colonization of poplar roots by fungal endophytes was
significantly higher at the polluted site than at the unpolluted sites (Karliński et al.
2010). This group of fungi seems to be more tolerant of high concentrations of heavy
metals and to compete with arbuscular fungi in the root colonization of poplars. The
increase of dark septate endophytes in the roots at polluted sites was also observed
by Routsalainen et al. (2007) and Jumpponen and Trappe (1998).
Plants, as primary suppliers of carbon exudates and other plant-derived materials,
are important players in shaping the microbiome of the soil environment (e.g.,
Bulgarelli et al. 2015; Lottmann et al. 2010; Schweitzer et al. 2008). The role of
plant genotypes in shaping fungal communities associated with plants has mainly
focused on short-lived crop plants, e.g., barley, tomato, cucumber, sweet pepper, and
chickpea (Bulgarelli et al. 2015; Ellouze et al. 2013; Ravnskov and Larsen 2016). To
a lesser extent, the impact of the genotype has been analyzed in case of trees,
especially those growing in different environmental conditions and impacted by
the long-lasting natural or anthropogenic factors (Karliński et al. 2020). The observations of different poplar clones and hybrids revealed their different response to
environmental conditions, which was particularly evident in the polluted area.
Poplars clearly reflected different rooting strategies expressed in differentiated fine
roots production and their different distribution in the soil profile (Karliński et al.
2010), perhaps due to the increased environmental pressure caused by heavy metal
contamination of soil. At the poplar rhizosphere, the biomass of arbuscular fungi and
other soil microorganisms was mainly determined by site conditions and to a lesser
extent by genotype. But the contribution of microorganisms in poplars microbiome
was significantly impacted by tree genotype (Karliński et al. 2020). The poplar
genotype determined the contribution of the biomass ratio of arbuscular fungi and
other soil fungi despite their different character of interactions with host trees. Also,
the biomass ratio of fungi (including arbuscular and ectomycorrhizal symbionts of
poplars) and bacteria (F:B ratio) in poplar rhizosphere appeared to be a good
indicator of the poplar genotype (Karliński et al. 2020). However the F:B ratio
was also shown as a suitable indicator of changes in the microbial community
depending on the soil moisture gradient and changes in soil management or soil
pollution (Bailey et al. 2002; Zhang et al. 2016). Generally, fungi (including
arbuscular fungi) seem to be more related to poplar genotype, while bacteria to a
greater extent depend on the site (Karliński et al. 2020). On the other hand, the soil
microbiomes of the poplars showed relatively stable proportions of groups of
microorganisms different sites, which confirm high adaptability of poplars to different soil conditions and their ability to shape communities of microorganisms associated with them (Karliński et al. 2020).
7 The Arbuscular Mycorrhizal Symbiosis of Trees: Structure, Function, and. . .
125
Both arbuscular and ectomycorrhizal fungal species may belong to commonly
occurring generalists/cosmopolitans and to the specialists, whose occurrence is
limited by several environmental, geographical, or physiological factors. Up to
now, more studies describing fungal communities have been concentrated on
ectomycorrhizal fungi associated with woody species in natural conditions, nurseries, or at disturbed areas (e.g., Karliński et al. 2013; Leski et al. 2019; Rudawska
et al. 2018, 2019). Communities of arbuscular fungi, despite their widespread
occurrence, were thought to be less diverse (Smith and Read 2008). However, the
use of new molecular techniques causes this image to change in recent years
(e.g. Öpik et al. 2008, Helgason and Fitter 2009; Baltruschat et al. 2019). However,
as in case of ectomycorrhizal fungi, also in the case of arbuscular fungi, the main
factors shaping their communities are soil conditions. The tree genotype only plays a
limited role here (Karliński et al. 2013). The main role in the colonization of young
plants by mycorrhizal fungi plays the local network of mycorrhizal fungi, shaped by
geographical and ecological factors. Most of the fungi as a generalist establish
symbiotic relationships with many plant species. Thus, poplars growing in different
conditions show a common set of root bacteria as well as endophytic and
ectomycorrhizal fungi associated with them, and no single taxon or consortium of
microbes is indicative of a particular Populus genotype (Karliński et al. 2013; Bonito
et al. 2019). This means that possible differences between communities of mycorrhizal fungi associated to poplar genotypes are marked rather locally, in specific soil
conditions and general trends can be expected at the level of groups of organisms
rather than individual species
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Chapter 8
Effectiveness of Arbuscular Mycorrhizas
in Improving Carob Culture
in the Mediterranean Regions
Abdellatif Essahibi, Laila Benhiba, Cherki Ghoulam, and Ahmed Qaddoury
Abstract Carob (Ceratonia siliqua L.) tree is considered among the most important
forest-fruit species native to the Mediterranean region. It has various uses and great
valorization potential, all parts of this plant could be exploited as a source of income,
as human food or livestock fodder as well as source of raw materials for pharmaceutical, cosmetic, or food industries. Moreover, due to its particular agroecological
features, carob tree offers the advantage of growing in poor and unfertile soils in the
Mediterranean and Mediterranean-like regions of the world. Thus, carob trees are
suitable for the rehabilitation of marginal and sub-marginal areas, helping to compensate for the expanding land desertification in these regions where it can play the
role of pioneer and productive species. Carob has been intermittently explored over
the last 20 years as a potential tree crop industry in low rainfall areas. The importance
of developing the industrial agroforestry potential of the carob tree is hurdled by the
lack of options for agroforestry, especially in Mediterranean regions with low
rainfall (below 500 mm), and by the need to develop suitable practices for the
sustainable management of natural resources. Viable commercial carob cultivation
will require mastering efficient farming practices with detailed attention to water
requirements and soil fertility. It would improve agricultural productivity in low
rainfall areas, help manage water and land degradation, diversify farmers’ incomes,
and contribute to the development of export industries contributing to balance the
economy of the country. This chapter will provide current knowledge regarding the
use of mycorrhizal symbiosis for the improvement of carob culture and productivity
in the context of Mediterranean ecosystems. An overview on the multipurpose
potential of the carob tree and how spreading its cultivation will benefit people
and the environment in marginal areas is highlighted.
Keywords Carob tree · Multiplication · Mycorrhiza · Biofertilizer · Sustainability ·
Abiotic stresses
A. Essahibi · L. Benhiba · C. Ghoulam · A. Qaddoury (*)
Plant Biotechnology and Bioengineering Laboratory, Department of Biology, Faculty of
Sciences & Technology, University Cadi Ayyad, Marrakech, Morocco
e-mail: a.qaddoury@uca.ma
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_8
129
130
8.1
A. Essahibi et al.
Introduction
The carob tree (Ceratonia siliqua L.) is a flowering woody species belonging to the
legume family, Fabaceae, native to the Mediterranean region. This evergreen forestfruit tree may reach, under favorable conditions, 20 m in height, up to 3.5 m in trunk
girth, and up to 200 years of longevity (Ait Chitt et al. 2007). Carob trees have been
widely exploited for food and forage since antiquity. It is an important component of
the arboreal flora of the Mediterranean region which is widely cultivated for industrial, ecological, ornamental, environmental, agricultural, and land restoration purposes (Batlle and Tous 1997). It has various uses and great valorization potential:
human food, livestock fodder as well as ornamental, carpentry, beekeeping, and
traditional medicines purposes (Morton 1987; Rejeb et al. 1991; Ait Chitt et al.
2007). The nutritional value of carob fruits (seeds and pulp) is indisputable; it is in
high demand for pharmaceutical, chemical, food, and cosmetic industries (Rejeb
et al. 1991; Makris and Kefalas 2004; Konate 2007; Custodio et al. 2011a, b). On the
other hand, carob tree develops strong and deep roots, which probably account for its
ability to grow under harsh conditions including drought, salinity, and nutrient-poor
soils (Vertovec et al. 2001; Sakcali and Ozturk 2004; Correia et al. 2010; Ozturk
et al. 2010). In addition, due to its ability to preserve and enrich the soil, carob
cultivation facilitates the establishment of other plant species, being particularly
useful for the rehabilitation of difficult areas where it can simultaneously play the
role of pioneer and productive species. In fact, this tree has been used to restore
numerous marginal and semiarid and arid lands around the Mediterranean basin
(Ozturk et al. 2010; Ouahmane et al. 2012; Bakry et al. 2013). Nowadays, it has
caused to lost due to problems as urban sprawl, illegal cut of trees and overgrazing,
and global climate changes, leading to damage of natural flora and ecosystems. In
addition, carob cultivation in the Mediterranean region is limited to marginal and/or
semiarid areas characterized by a harsh climate and poor soil conditions that
negatively affected plants establishment, thus limiting the success of reforestation
programs. Moreover, the large scale cultivation of carob is hampered by the traditional methods of its propagation that fail to meet the growing demand for high value
plants. The detrimental effects of these constraints exacerbated by the global climate
changes caused not only reduction in the carob production, a considerable source of
incomes for millions of humans in the marginal and semiarid areas, but also an
imbalance of the natural ecosystems causing a serious threat to plant resources and
sustainable development in these difficult environments.
While carob is reported to be tolerant of water shortage and saline soil, a
commercial carob cultivation will require reliable yields that can only be achieved
with rainfall of 500 mm or greater. In areas of low or unpredictable rainfall, irrigation
will be necessary to achieve required yields. In addition, correct agroforestry practices including irrigation and fertilizer applications will also be required to achieve
optimum yields. One of the main challenges for the sustainable development of
carob cultivation is the combination of the proper agroforestry practices based on
plant technology innovations and the management of the agroecosystem services
8 Effectiveness of Arbuscular Mycorrhizas in Improving Carob Culture in the. . .
131
that has been at the forefront of generating and promoting sustainable agricultural
production. In this context, arbuscular mycorrhizal fungi (AMF), often referred to as
“ecosystem engineers,” “biocontrol agents,” “biofertilizers,” or “bioenhancers,” are
the most important providers of these ecological services. Arbuscular mycorrhizal
fungi can form symbiotic associations (mycorrhiza), to the mutual benefit of the host
plant and fungus, with roots of 80% of terrestrial plant species including trees,
shrubs, herbs, and crop plants (Harley and Smith 1983; Smith and Read 2008).
Mycorrhizal plants show not only high nutrient and water acquisition efficiency, but
also less susceptibility to disease and high tolerance to adverse conditions (Evelin
et al. 2012; Beltrano et al. 2013; Ruiz-Lozano et al. 2016; Wu 2017). The benefits
from AMF are thought to be highest when the colonization occurs as early as
possible during the vegetative growth of the plant (Scagel 2001; Essahibi et al.
2017). Carob tree is among the vascular plants that can establish mycorrhizal
associations and strongly depends on its functioning to grow under severe environmental constraints (Manaut et al. 2015; Essahibi et al. 2018, 2019). This chapter will
provide an overview regarding the multipurpose potential of the carob tree and how
spreading its cultivation will benefit people and environment in marginal areas. It
discusses also the current knowledge concerning the use of mycorrhizal symbiosis
for the improvement of carob culture and productivity in the context of Mediterranean ecosystems. The most effective applications of AMF for improving carob
performance in terms of multiplication, growth, nutrition, and protection against
adverse effects of environmental stresses are highlighted.
8.2
Carob Origins and Cultivation
Carob tree has been grown since antiquity in most countries of the Mediterranean
basin. The origin of carob is not clear, it was considered by De Candolle (1883) and
Vavilov (1951) as native to the eastern Mediterranean region, mainly Turkey and
Syria. However, according to Schweinfurth (1894) carob is native to the southern
Arabia (Yemen). Recently, Zohary (1973) considered carob as originating from
Indo-Malesian flora. The only known carob-related species is Ceratonia
oreothauma, which is considered to be native to the southeast Arabia (Oman) and
the African horn (north of Somalia) (Hillcoat et al. 1980). The origin of carob
populations throughout the Mediterranean basin has been associated with an historical process of dissemination by humans since its domestication in the Middle-East
around 4000 BC. Its value was recognized by the ancient Greeks, who brought it to
Greece and Italy and by Arabs who disseminated it along the North African coast
and into Spain and Portugal. Lately, it has also been successfully introduced in
Australia, South Africa, the USA, Chile, Argentina, Philippines, and Iran (Fig. 8.1)
(Morton 1987; Batlle and Tous 1997; Sbay and Abourouh 2006). This indicates that
the general environmental conditions (e.g., rainfall, soils, temperature, etc.) of these
regions do not impose significant limitations on carob growth. However, commercial
cultivation will require detailed attention to carob needs. For example, while the
132
A. Essahibi et al.
Fig. 8.1 World carob distribution (Batlle and Tous 1997)
literature often reports that carobs are drought tolerant carob trees have little yield
under low rainfall conditions but increase with increasing rainfall; 500 mm/year
considered necessary for reliable yields.
8.3
Economic Potential of Carob Tree
Carob is predominantly grown in the Mediterranean and Mediterranean-like climate
regions of the world. The World production of carob is estimated at about 420,000 t/
year, and the main producers of pulp and seeds, respectively, are Spain (36%, 28%),
Morocco (24%, 38%), Italy (10%, 8%), Portugal (10%, 8%), Greece (8%, 6%),
Turkey (4%, 6%), and Cyprus (3%, 2%) (Bouhadi et al. 2017). The economic
importance of carob tree comes from the multiple uses of its products in pharmaceutical, chemical, food, and cosmetic industries (Makris and Kefalas 2004;
Custodio et al. 2011a, b; El Kahkahi et al. 2016). Indeed, Carob tree has various
uses and great valorization potential; all parts of the tree are valued as food or as a
source of income. Carob fruits or pods consist of a pulp wrapping seeds, presenting
respectively 90 and 10% of the total fruit weight. The pulp is currently used in the
food industry for the production of juices, biscuits, and cocoa, and as a substituent
for the manufacture of chocolate, since it contains no alkaloids, caffeine, or theobromine (Craig and Nguyen 1984; Sbay and Abourouh 2006). It is also used for the
production of alcohol (ethanol) and citric acid (Rejeb et al. 1991). Moreover, the
gum extracted from the endosperm is used as stabilizer, fixer, and gelling agent in
many industrial sectors including food (cheese, mayonnaise), cosmetics (creams,
toothpaste), and pharmaceuticals (medicines, syrups) (Batlle and Tous 1997). Carob
pod is rich in carbohydrates, sucrose (437.3 mg/g dry weight), glucose (395.3 mg/g
dry weight) and fructose (42.3 mg/g dry weight), and proteins (5–8 g protein per
100 g dry weight). Carob pod contains also vitamins A and B, and minerals (K, P,
8 Effectiveness of Arbuscular Mycorrhizas in Improving Carob Culture in the. . .
133
Ca, and Mg, Fe, Mn, Zn, and Cu), and is fat-free (Khatib and Vaya 2010). Carob
flowers are very useful for beekeepers; leaves serve for animal feed, while the wood
of carob tree is valued in carpentry and charcoal manufacturing (Rejeb et al. 1991;
Gharnit et al. 2006). Carob processing could contribute to regional employment and
enhance the viability of rural communities, and would help increase the potential of
export industries to compete in the global carob bean gum market valued at $100
million/year (Hogan 1995), contributing to balance the economy of the country.
Viable commercial carob cultivation will require mastering efficient farming practices with meticulous attention to water requirements and soils fertility. It would
improve agricultural productivity in low rainfall areas, help manage water and land
degradation, and diversify and increase farmers’ incomes. Moreover, economic
returns need maintaining access to higher value markets. The economic returns
will fall in direct relationship with the decline in tree yields because of the major
fall in profitability due to the low rainfall dryland scenario.
8.4
Agroecological Potential of Carob Tree
The Mediterranean basin is a global hotspot of biological diversity. However, the
various environmental biotic and abiotic constraints prevailing in this region negatively influence plant growth and development. Drought it is a normal recurrent
feature of the climate that occurs in both developing and developed countries,
especially in arid and semiarid regions. This multidimensional stress is the consequence of several factors including low rainfall, salinity, high temperatures, and high
intensity of light. Carob is reported to be highly tolerant of water scarcity and saline
soil. The optimum annual rainfall for the carob tree is approximately five hundred
millimeters. It is generally assumed that poor quality pod production is predictable
under conditions of less than 300 mm per annum; however, carob trees are able to
survive in areas with a rainfall of 250 mm/year (Esbenshade and Wilson 1986).
Carobs have rarely been intensively cropped and there is little information available
on optimum water requirements. According to Curtis and Race (1998), an average
yield of 6-7 t/ha was obtained by mature trees receiving 300 L of water per tree per
year in addition to the natural rainfall (500 mm/year) in the Catalina region of Spain.
For low rainfall areas, irrigation (or access to groundwater) will be a critical factor in
obtaining consistent yields and establishing desirable growth characteristics during
the early years of development (Esbenshade 1994). Thus, the carob’s ability to
survive under marginal conditions should not be the basis for commercial production; long-term monitoring will be required to determine optimum irrigation practices. In any case, due to its particular agroecological features, this robust and rustic
tree has a high capacity to withstand the harsh climatic and edaphic conditions of the
Mediterranean regions (Correia et al. 2001) and is considered suitable for the
rehabilitation of marginal and sub-marginal lands. According to Nunes et al.
(1989), Ceratonia leaves can, to some extent, maintain turgor under situation of
soil drought, using different strategies according to the season. The most common
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A. Essahibi et al.
strategy adopted by carob to withstand drought is through water stress avoidance
which is mostly related to its ability to maintain cell water homeostasis mainly by
prohibiting water loss and increasing the water inlet to the cells, which eventually
leads to normal cell functions (Correia et al. 2001; Vertovec et al. 2001; Essahibi
et al. 2018). A variety of other particular morphological and physiological characters
appeared to be involved in carob tolerance to drought: (1) the efficient regulation of
the transpiration rate and gas exchanges depending on the general plant water status,
(2) the occurrence (only on the epidermis of the lower (abaxial) leaf surface), the size
(very small), and the frequency (about 217 stomata mm 2) of stomata (Nunes and
Linskens (1980), (3) the importance of wax deposition on leaf cuticle, which
contributes in improving stomatal resistance (El Kahkahi et al. 2016), and (4) the
extensive root system with quite long tap root which penetrates deeply into the sol
(down to twenty meters) maintaining a continuous flow of water to the leaves even
during an extended period of the topsoil drying (Rhizopoulou and Davies 1991), and
the high efficiency of its xylem in term of water transport (Kikuta et al. 1997). In
addition, carob tree appears to be very resistant to cavitation, tolerating leaf water
potential values below the turgor loss point, with minor losses in hydraulic conductivity. It can, therefore, quickly restore the continuity of the water film within the
xylem after re-watering (Salleo and Gullo 1989).
On the other hand, salinization affects about 16 million hectare of soils in the
Mediterranean basin, more than 20% of arable lands in arid and semiarid areas are
affected by increased salinization, of which 350,000 ha in Morocco (Hamdy and
Lacirignola 1999). The presence of salt in the soil results in reduction of the water
availability. Thus crops reaction to salinization is very similar to their reaction to
drought stress; despite the presence of water in the soil, the plants react as if the soil
was dry or almost dry. In fact, in addition to striving to alleviate the toxic effect due
to Na+ and Cl ions, plants seek to reduce water loss and maintain normal vital
functions (Zraibi et al. 2012). Carob tree is characterized by its high tolerance to
salinity; it appears to maintain almost all its physiological processes and grow
successfully under NaCl concentrations up to 3% (Winer 1980; Batlle and Tous
1997; Correia et al. 2010); it may have considerable merit as a cash crop for saline
lands. According to El Kahkahi et al. (2015), Carob conserves relatively stable
contents of proline and soluble sugar under salt stress.
Carob tree is indifferent to the nature of the substrate, having the ability to adapt
to several types of soils and wide range of pH (from 6.2 to 8.6) (Eshghi et al. 2018).
Esbenshade and Wilson (1986) reported that carob prefer a calcareous, well-drained,
low-clay soil, but can grow on almost any type of soil which is well drained and
aerated, including sands, clay loams, limestone, and alkaline or moderately acid
soils. However, carob cannot tolerate acidic soil or overly wet conditions
(Esbenshade and Wilson 1986; Sbay and Abourouh 2006).
Thus carob, as pioneer and productive forest-fruit tree has great potential for
reestablishing vegetation in degraded areas around the Mediterranean basin and in
other Mediterranean-like dry areas of the world and has considerable merit for
improving productivity in lands which would be marginal for other crops.
8 Effectiveness of Arbuscular Mycorrhizas in Improving Carob Culture in the. . .
8.5
135
Importance of Mycorrhiza in Improving Carob
Cultivation
Along with root nodules, mycorrhiza is considered to be the most important symbiosis that helps feed the world’s population, conserve ecosystems, and preserve
biodiversity. Arbuscular mycorrhiza is a highly evolved mutualistic symbiotic
relationship that forms between soil-borne fungi and plant roots. The fungus colonizes the root system of a host plant, providing increased water and nutrient
absorption capabilities and increased protection against biotic and abiotic stresses,
while the plant provides the mycorrhizal fungus with carbohydrates formed from
photosynthesis. Nowadays, the fundamental role of arbuscular mycorrhizal fungi
(AMF) at the interface soil–roots is well documented. The potential of arbuscular
mycorrhiza to enhance the multitrophic and protective interactions that affect plants
productivity, competitiveness, and survival both in natural ecosystems and in managed fields has been widely demonstrated (Baslam et al. 2014; Fouad et al. 2014;
Benhiba et al. 2015; Essahibi et al. 2018, 2019). Arbuscular mycorrhizal fungi
(AMF) can form symbiotic associations with roots of over 80% of terrestrial plant
species including most agricultural and horticultural crop species (Harley and Smith
1983; Smith and Read 2008). Fossil evidence (Remy et al. 1994) and DNA sequence
analyzes (Simon et al. 1993) suggest that arbuscular mycorrhizal symbiosis appeared
400–460 million years ago when the first terrestrial plants started colonizing dry
land, suggesting that AMF helped plants to colonize and adapt to diverse terrestrial
habitats (Morton 1990). Arbuscular mycorrhizal fungi are multifunctional and
provide a variety of ecosystem benefits. The extraradical mycelium of the fungus
grows into the surrounding soil (reaching up to 10–14 m cm 1), developing an
extensive mycelium network exploring the environment for water and mineral
nutrients (Smith and Read 2008), thus increasing the root absorbing surface area
100 or even 1000 fold (Barea et al. 2011). Moreover, due to their narrow diameter,
the thin hyphae are able to extend into the microsites of rocks and soil pores that are
inaccessible to roots or even root hairs (Barea et al. 2011). Nowadays, it is well
established that AM symbiosis provides complementary characteristics that increase
host plant’s tolerance to water-related stresses including drought and salinity (Evelin
et al. 2012; Ruiz-Lozano et al. 2016; Wu 2017). It is also well known that AMF play
an important role in phosphate (P) acquisition mainly in soils with low availability
and/or mobility of P (Bagayoko et al. 2000; Beltrano et al. 2013). In addition,
benefits from AMF are not limited only to the host plant, but concern the entire
ecosystem; strong potential of AMF for ecosystems engineering leading to sustainable improvement in soil quality and plant growth in semiarid and/or degraded areas
have been demonstrated (Rillig 2004; Manaut et al. 2015; Chitarra et al. 2016; Tyagi
et al. 2017; Chen et al. 2018).
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8.5.1
A. Essahibi et al.
Occurrence of Mycorrhizal Symbiosis in Carob Tree
The majority of wild plant species in natural ecosystems forms mycorrhizal symbiosis. Difficult habitats with extreme environmental conditions are generally lacking
in water and nutrient and most plants growing there are endowed with adaptive
strategies allowing acquisition and conservation of water and nutrient; mycorrhizal
symbiosis being one of them. Regarding the adverse conditions prevailing in the area
of carob distribution, it can be assumed that carob has evolved highly mycotrophic as
a strategy to overcome the environmental constraints. Currently, the mycotrophic
status of carob tree is well established (Ouahmane et al. 2012; El Asri et al. 2014;
Essahibi 2018). In fact, Carob tree always forms mycorrhizal associations regardless
of its age, the area of growth or the type of soil (Mohammad et al. 2003; El Asri et al.
2014; Essahibi 2018). Moreover, the roots of carob tree have been shown receptive
not only to the arbuscular mycorrhizal fungi but also to other endophytes (El
Asri et al. 2014; Mohammad et al. 2003). In fact, roots of adult trees of Ceratonia
siliqua sampled from different Mediterranean regions showed very high mycorrhizal
frequency (> 90%) confirming its high mycotrophy. Moreover, carob roots naturally
colonized by AMF showed the presence of all arbuscular mycorrhizal structures
(vesicles, arbuscules, and hyphae), proving the presence of functioning mycorrhiza
(Mohammad et al. 2003; El Asri et al. 2014; Essahibi 2018). The high mycorrhizal
status of the adult carob tree is related to the abundance and diversity of the
community of mycorrhizal fungi in the rhizospheric soil of the carob groves. In
fact, high spore density (2100 spores/100 g of soil) was reported in Ourika valley,
regions of Marrakech, Morocco (Ouahmane et al. 2012), while less than 160 spores/
100 g of soil) were reported in Zaio and Afourar, in the center of Morocco (El Asri
et al. 2014). The identification of the native AMF species based on spore morphology showed between 12 and 18 AMF morphotypes belonging to six genera: Glomus,
Acaulospora, Scutellospora, Gigaspora, Entrophospora, and Pacispora. The Glomus genera is the most abundant (Ouahmane et al. 2012; El Asri et al. 2014; Essahibi
2018), confirming its occurrence in a wide range of environmental and soil conditions, particularly in arid and semiarid areas (Stutz et al. 2000). The high mycotrophy
of the carob tree along with the abundance and diversity of AMF community in the
rhizospheric soil of the carob groves prove the strong mycorrhizal-dependency of
this tree. Thus, the integration of AMF in the commercial propagation of carob
(nurseries activities), the economic carob cultivation, and the restoration and/or
creation of new carob plantations would have considerable merit.
8.5.2
Importance of AMF in Improving Carob Propagation
In general, Carob plants can be propagated by seeds germination or asexual propagation techniques such as grafting and cutting. Carob seeds are difficult to germinate and require scarification; its coat is extremely hard and does not absorb water. In
8 Effectiveness of Arbuscular Mycorrhizas in Improving Carob Culture in the. . .
137
addition, seedlings show high heterozygoty and 50% of them are potentially nonproductive males. Moreover, seedlings are not true to type and normally result in
fruits of varying size and quality. Propagation by cuttings is problematic because
carob has been described as one of the most difficult to root species (Hartmann et al.
1997); this method is not commercially used Batlle and Tous (1997). Grafting is,
generally, used to maximize the number of female trees, by grafting explants from a
female or hermaphrodite tree onto a male one (Curtis and Race 1998). Carob grafting
takes more than two years to obtain grafted nursery material Batlle and Tous (1997).
The first attempts of in vitro propagation of carob tree were performed by MartinsLouçao and Rodriguez-Barrueco (1981). Then, many efforts have been made to
elaborate efficient protocol for carob micropropagation using explants from different
origin, diversified culture media, and hormonal combinations (Romano et al. 2002;
Radi et al. 2013; Aguinaz et al. 2017). More recently, the plant production potential
of the cuttings method has been substantially improved through controlling cuttings
water status (using fog system), increasing rooting ability (using auxin treatment and
arbuscular mycorrhizal fungi), and determining the best sampling period (El-Soda
et al. 2016; Essahibi et al. 2017; Essahibi 2018).
Currently, it is well known that the integration of AMF into the plants’ propagation cycle is one of the main practical and effective applications of these microorganisms (Scagel 2001, 2004; Davies 2008; Fouad 2015). The utilization of AMF in
the different phases of carob propagation by cutting strongly improved the potential
of this method. Indeed, the rooting capacity of carob cuttings was strongly enhanced
by the inoculation of the rooting substrate with a mixture of AMF strains
(Funneliformis mosseae, Rhizophagus fasciculatus, and Rhizophagus intraradices)
(Essahibi et al. 2017). In fact, rooting performance in terms of rooting percentage
and number of roots per cutting were higher in the presence of AMF compared to the
control. Several studies have reported the positive effect of AMF on the rooting
ability of cuttings in other plants species (Scagel 2004; Davies 2008; Fouad 2015).
Scagel (2004) explained the positive effects of AMF on adventitious roots formation
before colonization by the existence of a pre-colonization signal between cuttings
and the propagules of AMF. This signal, relatively similar to that existing in the
presence of host plant roots is triggered in the basal ends of the cuttings by the
liberation of CO2 or other metabolites able to activate AMF propagules (Gadkar
et al. 2001; Tamasloukht et al. 2003). AMF exudates released in response to these
metabolites may induce changes in cuttings’ metabolism, thereby enhancing adventitious roots initiation (Larose et al. 2002); after the colonization of the first roots
AMF induces the formation of new roots (Larose et al. 2002). Inoculation with AMF
has also positive effects on the acclimatization of carob rooted-cuttings. According
to Essahibi et al. (2017), AMF increased rooted-cuttings survival to the transplantation and hardening shocks. Moreover, mycorrhizal rooted-cuttings showed higher
performance in terms of growth, physiology and biomass production and high
tolerance to water and nutrient deficiency during the post-acclimation development
compared to non-mycorrhizal plantlets (Essahibi et al. 2017; Essahibi 2018). The
positive effects of AMF on cuttings performance have been reported in other plants
species (Binet et al. 2007; Fouad 2015). According to Ouahmane et al. (2012),
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mycorrhizal seedlings of carob exhibited high growth and biomass production and
high open field establishment capacity, being more suitable for the restoration of
degraded areas.
8.5.3
Importance of AMF in Enhancing Carob Tolerance
to Drought
In the Mediterranean basin, especially the southern regions, drought is one of the
most serious abiotic stress limiting plants growth and development. In fact, due to
the global climate changes effects, which are more exacerbated in these areas,
drought is becoming longer, more extreme, and more frequent negatively impacting
all plants functions. Due to its particular agroecological features, carob trees can
stand severe conditions (drought, salinity, and nutrient-poor and degraded soils),
being suitable for the rehabilitation of marginal and sub-marginal areas of the
Mediterranean basin (Correia et al. 2001). Carob can survive dry climates without
irrigation and it is well adapted to dry environments with annual average rainfall
between 250 and 500 mm year 1. Although carob tree can withstand long periods of
drought, large amount of water is required for vigorous growth and high yields; in
these regions, the groundwater is the main source to meet carob water needs.
Therefore, limited precipitation along with the overexploitation of groundwater
can significantly threatened the cultivation and the long-term productivity of carob
trees. Bibliographic data agree on the importance of the mycorrhizal inoculation in
strengthening carob tolerance to drought and other abiotic constraints (El Asri et al.
2014; Essahibi 2018; Essahibi et al. 2018, 2019; Boutasknit et al. 2020). In fact,
mycorrhizal carob plants subjected to severe water deficit have maintained almost
normal growth and biomass production (Essahibi et al. 2018; Boutasknit et al. 2020).
The performance of mycorrhizal plants under water stress implies several tolerance
strategies including (1) improved water balance and nutrient status, (2) maintained
stomatal conductance at high levels (3) preserved cellular turgor by increasing cell
wall rigidity and osmolytes accumulation, and (4) enhanced defense systems
involved in oxidative stress alleviation, including enzymatic and nonenzymatic
compounds (Essahibi et al. 2018). Indeed, due to their highly developed
extrametrical hyphae network, AMF showed high efficiency in taking and
transporting water improving thereby the host plant water status even in soils with
low water availability. According to Smith et al. (2010), the length of the AMF
hyphae associated with roots is estimated at more than 100 m per gram of soil. In
addition, the average diameter of these hyphae varies between 2 and 20 μm which is
one or two degrees lesser than the diameter of the absorbing hairs (Ruiz-Lozano et al.
2012). This very large and very fine extraradical hyphae network can get water from
pores inaccessible to the non-mycorrhizal roots, allowing better water supply to the
host plant. Thus mycorrhizal plants could maintain stomatal conductance at higher
level compared to non-mycorrhizal plants (Chitarra et al. 2016). According to these
8 Effectiveness of Arbuscular Mycorrhizas in Improving Carob Culture in the. . .
139
authors, the high stomatal conductance in mycorrhizal plants is associated with low
ABA concentration in the xylem sap. Indeed, under the same conditions of water
stress, the roots of mycorrhizal plants perceive less the effect of water stress and
therefore produce less ABA than non-mycorrhizal plants (Chitarra et al. 2016). The
high stomata conductance and increased water and mineral nutrient acquisition in
mycorrhizal plants allow maintaining high photosynthetic activity. The
photosynthesized carbon products are then used both for growth and osmotic
adjustment as water potential of the soil becomes low. In addition, AMF provide
host plant with complementary characteristics to avoid the oxidative stress induced
by water stress through their action on the antioxidant system involved in the
elimination of reactive oxygen species (ROS). AMF increase both the activity of
antioxidant enzymes, superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, and catalase (Baslam et al. 2014; Fouad et al. 2014; Benhiba et al. 2015), as
well as the content of abscisic acid, glutathione, carotenoids, and anthocyanins
(Baslam and Goicoechea 2012). At the molecular level, AMF regulate the expression of p5cs genes encoding a rate-limiting enzyme in the biosynthesis of proline
(Porcel and Ruiz-Lozano 2004), genes encoding aquaporins (Porcel et al. 2006), and
nced genes encoding a key enzyme in the biosynthesis of ABA (Aroca et al. 2008),
allowing mycorrhizal plants to maintain a better water status (Porcel et al. 2004).
8.5.4
Importance of AMF in Improving Carob Tolerance to P
Deficiency
In addition to drought, another serious limitation of plants development and crop
production in the Mediterranean regions is the deficiency of available phosphorus
(P). In fact, the high affinity of P for soil particles and the facility of its precipitation
by free Al3+ and Fe3+ in acidic soils and by Ca2+ in alkaline soil (Havlin et al. 2005)
make it inaccessible to plants. The importance of AMF in improving P acquisition by
the host plant, particularly in soils with low P availability and/or mobility has been
demonstrated by several investigations (Cardoso et al. 2006). This is due to the
narrow extra-radical mycelial network, which penetrates wider and deeper into the
soil (Turk et al. 2006), increasing thereby P absorption of the host plant. In addition,
AMF have the ability to mobilize other forms of P unavailable to plants through the
solubilization of inorganic phosphorus (Pi) (Duponnois et al. 2005; Gholamhoseini
et al. 2013). In fact, AMF can interact with phosphate-solubilizing bacteria to
improve P availability and can produce with the associated roots organic acids and
hydrolytic enzymes involved in the decomposition of organic matter and dissolution
of Pi (Gholamhoseini et al. 2013; Goussous and Mohammad 2009). It is also well
established that mycorrhizal symbiosis induces the expression of Pi transporters in
plants (Xie et al. 2013; Walder et al. 2015). In tomato, three Pi transporter (PT) genes
(LePT3, LePT4, and LePT5) are upregulated in colonized roots (Nagy et al. 2005).
MtPT4 genes are regulated in root cells of Medicago truncatula to absorb P supplied
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A. Essahibi et al.
by mycorrhizae (Javot et al. 2007). According to Essahibi et al. (2019), under P
deficiency, arbuscular mycorrhizal symbiosis significantly improved growth and
biomass production of carob plants. Moreover, mineral nutrient acquisition, photosynthetic activity, stomatal conductance, total chlorophyll content, and soluble sugar
accumulation were also strongly improved in mycorrhizal plants in comparison with
non-mycorrhizal ones. In addition, under P deficiency, mycorrhizal plants showed
strongly increased acid phosphatase activity in the roots and the rhizospheric soil
than Non-mycorrhizal plants. Furthermore, mycorrhizal plants maintained high
membrane integrity (over 80%) and low hydrogen peroxide (H2O2) and
malondialdehyde (MDA) contents, associated with increased activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (G-POD),
and catalase (CAT) compared to non-mycorrhizal plants.
8.5.5
Importance of AMF in Improving Field Establishment
of Carob Tree
Ecosystem restoration using pioneer plants is considered to be necessary and useful
and has achieved great progress in recent years. Because of its hardiness and
adaptation to environmental constraints, carob tree is included in the list of priority
as forest resources for conservation in many countries in the Mediterranean basin. It
is often used for afforestation and reforestation of areas affected by erosion and
desertification (Rejeb et al. 1991). However, successful restoration is very difficult,
because of the severity of abiotic stress, affecting negatively plants installation and
making them growing hardly. The potentialities of ecological engineering strategy
based on the use of AMF for improving afforestation programs with carob trees in
degraded environments have been demonstrated (Manaut et al. 2015). These authors
revealed the high potential of this approach in improving sustainably the growth and
nutrient status of carob trees plantation. Indeed, in addition to benefit host plants’
growth via alleviating various environmental stresses, AMF improve aggregate
stability, through their fine hyphal mesh and glomalin production thereby enhancing
soil quality and stability (Rillig 2004; Chen et al. 2018).
8.6
Conclusion
Carob (Ceratonia siliqua L.) tree is one of the most important species existing in the
Mediterranean regions due to its exceptional socioeconomic and environmental
benefits. It is evident from the above discussion that arbuscular mycorrhizal fungi
are very effective for promoting carob culture in these regions. Indeed, this mycorrhizal technology has been proved useful for promoting the successful mass propagation of carob (a rooting-recalcitrant species) using herbaceous semi-herbaceous
8 Effectiveness of Arbuscular Mycorrhizas in Improving Carob Culture in the. . .
141
cuttings under mist system. Moreover, AM symbiosis has been shown to provide
complementary characteristics that increase carob plant’s tolerance to drought and P
deficiency, the most serious abiotic stresses limiting plants growth and development
in the Mediterranean regions. The positive effects of AMF on carob growth and
physiology were not only observed in greenhouse but also in open field conditions,
showing their high potential for reinforcing the reforestation programs of degraded
areas. While the mycorrhizal fungus is effective when used singly or in combination,
the specificity of strain in this regard is also important so that maximum benefits can
be obtained from AM symbiosis.
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Chapter 9
Leaf Endophytes and Their Bioactive
Compounds
Parikshana Mathur, Payal Mehtani, and Charu Sharma
Abstract Endophytes can reside in every part of a plant and show mutualistic
relationship but leaf tissue is considered as an ideal habitat for such microorganisms.
Endophytic microflora is a great source of secondary metabolites that can have
various biological activities like antibacterial, antifungal, anticancerous, etc. This
chapter focuses on such endophytic microbes that inhabit leaf tissues and produce
numerous bioactive compounds. Further various classes of secondary metabolites
obtained from leaf endophytes are discussed along with some applications of the
bioactive compounds.
Keywords Anticancer · Antimicrobial · Endophytic fungi · Mutualism · Plant ·
Secondary Metabolites
9.1
Introduction
Endophytes are microorganisms that colonize internal parts of plant at least once in
their lifetime and cause asymptomatic and imperceptible infections in the plant.
Endophytes have proved to play an important role in the adaptation and evolution of
plants to their environment. With due course of time plants have developed coping
mechanisms to deal with external stress but still most plants fail to survive in hostile
environment. However, due to the presence of microorganisms in association with
plants, they have developed various adaptations to deal with stressful environmental
conditions. In order to help plants in surviving in an unfriendly environment,
endophytes are hold responsible in host secondary metabolite synthesis either
partially or completely (Chutulo and Chalannavar 2018). Every plant is found to
cohabit endogenous microbes like bacteria, fungi, algae, actinomycetes, etc. that are
P. Mathur · P. Mehtani (*) · C. Sharma
Department of Biotechnology, IIS (Deemed to be University), Gurukul Marg, SFS, Mansarovar,
Jaipur, Rajasthan, India
e-mail: payal.mehtani@iisuniv.ac.in
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_9
147
148
P. Mathur et al.
HOST PLANT
ENDOPHYTIC FUNGI
ENDOPHYTIC BACTERIA
BIOACTIVE
COMPOUNDS
Fig. 9.1 Association between host plant and endophytic microflora
located in all plant parts including stem, leaves, root, flowers, etc. It has also been
said that the phenotypic expression of plants is also a result of their interactions with
the microorganisms inhabiting them. In vitro and in vivo studies have confirmed that
removal of endophytes from the plant has led to loss of their adaptative and resistant
properties toward an unfavorable environment where they survive with presence of
the endophyte (Li et al. 2019). Variation is found in the population of endophyte in
plants depending on host species, developmental stage of the host, and the environmental conditions (Gouda et al. 2016).
The bioactive compounds synthesized by endophytes as a part of their defense
and survival mechanisms have shown potential applications in medicine, pharmaceutical industries, agriculture, etc. (Fig. 9.1). With the extraction of Taxol from
endophytic fungi Taxomyces andreanae residing in the plant Taxus brevifolia and its
application as an anticancer compound, the interest of researchers shifted toward
finding novel bioactive products from endophytic flora and exploring their applications (Stierle et al. 1993; Strobel et al. 2004). Research in the last few decades has led
to the exploration of many valuable applications of these bioactive compounds as
anticancer agents, insecticides, and antimicrobial agents (Zhang et al. 2006). This
chapter primarily focuses on the endophytes dwelling in the leaves of the plants and
the bioactive compounds they synthesize. We have also discussed some applications
of the bioactive products produced by the leaf endophytes.
9 Leaf Endophytes and Their Bioactive Compounds
9.2
149
Leaf Endophytes
Taylor et al. (1999), have discovered endophytic fungi from fossils of stems and
leaves. Leaf tissue is reported to be ideal for exploring endophytic microflora and
their secondary metabolites as it has shown the highest species richness and endophyte colonization. Colonization of endophytic microbes is majorly found in xylem
tissues, leaf veins, mesophyll cells trichomes, cut sections of leaf pieces, and
substomatal areas (Fig. 9.2). In different studies on leaf endophytes of Azadirachta
indica basal leaflets showed higher colonizing frequency than apical or middle
leaflets with significant increase during the rainy season (Verma et al. 2011). Similar
results were obtained by Chareprasert et al. in 2006 on endophytes of Tectona
grandis L and Samanea saman Merr. with more colonization frequency in mature
leaves compared to young leaves. Eighty-five endophytic fungal isolates were
isolated from the leaves of Azadirachta indica (Tenguria and Khan 2011). Higher
isolation frequency from petiole than blade segments from leaves of Neolitsea
sericea were reported in a study conducted by Hata and Sone in 2008 though
blade segments showed more variation in endophyte pattern.
The presence of Burkholderia sp. strain PsJN has been reported in xylem and
substomatal chambers of inoculated leaves of grapevine plants by FISH and other
microscopy techniques. Klebsiella variicola was found in the mesophyll cells of
sugarcane leaves; Herbaspirillum sp. in young leaves and shoots of wild rice;
Herbaspirillum seropedicae Z67 in leaf vein, mesophyll cells, and substomatal
cavities of rice leaves and Serratia marcescens in the leaf sheaths and leaf aerenchyma of rice plants (Kandel et al. 2017). Lepanthes orchids of tropical region have
Fig. 9.2 Diagrammatic
representation of parts of
leaf which may have
colonization of endophytes
150
P. Mathur et al.
reported the presence of Xylaria and Rhizoctonia fungi in the leaves of the plant
more than the roots (Bayman et al. 1997).
9.3
Endophytic Secondary Metabolites and Their Biological
Activities
Secondary metabolites are compounds biosynthetically derived from primary metabolites and are classified on the basis of their biosynthetic origin (Fig. 9.3). The
following section throws light on some of the bioactive compounds obtained from
endophytes isolated from leaves and their applications.
9.3.1
Phenolic Compounds
Phenolic compounds comprise the largest class of secondary metabolites. The term
phenolic compounds include a wide range of plant substances which are watersoluble and have a common aromatic ring having hydroxyl substituents. Almost all
plant phenolics are produced by phenylpropanoid metabolism from products of the
shikimic acid pathway. This class includes important subclasses like lignans and
flavonoids. Flavonoids further include subclasses like anthocyanin pigments, tannins, isoflavonoids, flavanones, flavones, leucoanthocyanidins, catechins, chalcones,
and aurones. Tannins are feeding deterrents and wood protectants. Isoflavonoids are
used as signaling molecules and also used in defense mechanism of plants (Croteau
et al. 2000) (Fig. 9.4).
Endophytic fungi Pestalotiopsis mangiferae residing in the leaves of Mangifera
indica has been reported to synthesize a phenolic compound 4-(2,4,7-trioxa-bicyclo
[4.1.0]heptan-3-yl) phenol which has shown significant antibacterial and antifungal
activities against Candida albicans, Pseudomonas aeruginosa, Escherichia coli,
Klebsiella pneumoniae, Micrococcus luteus, and Bacillus subtilis. The compound
is reported to form pores in cell wall leading to destruction of the bacterial cell
(Subban et al. 2013).
Phomopsis sp. BCC 1323 isolated from the leaves of Tectona grandis produces
Phomoxanthones A and B shows in vitro antitubercular activity against Mycobacterium tuberculosis strain. Another strain of the same fungi isolated from Laurus
azorica leaves produces cycloepoxylactone and cycloepoxytriol B has also shown
activity against Bacillus megaterium, Microbotryum violaceum, and Chlorella fusca
(Deshmukh et al. 2015). Table 9.1 includes some of the other phenolic compounds
isolated from leaf endophytes and their properties.
151
Fig. 9.3 Major classes of secondary metabolites
9 Leaf Endophytes and Their Bioactive Compounds
152
Fig. 9.4 Structures of some phenolic compounds obtained from leaf endophytes
P. Mathur et al.
9 Leaf Endophytes and Their Bioactive Compounds
153
Table 9.1 Some phenolic compounds produced by leaf endophytes
Endophyte
Trichoderma
harzianum
Unknown fungi
Fungi PM0651480
Colletotrichum
gloeosporioides
9.3.2
Source plant
Ficus elastica
Compound
Isocoumarin
Property
Antimicrobial
Quercus
coccifera
Mimusops
elengi
Phlogacanthus
thyrsiflorus
Hinnuliquinone
Antiviral
Ergoflavin
Anticancerous
Phenol,2,4-bis
(1,1-dimethylethyl)
–
References
Ding et al.
(2019)
Geetanjali
(2017)
Deshmukh
et al. (2009)
Devi and
Singh (2013)
Terpenoids and Steroids
Terpenoids and steroids are major class of substance biosynthetically derived from
isopentenyl diphosphate synthesized by way of the acetate/mevalonate pathway or
the glyceraldehyde 3-phosphate/pyruvate pathway. Terpenoids are commercially
important in the fragrance industry and food industry as flavoring agents (Ohloff
1990). Terpenoids are classified by the number of five-carbon units(isoprene) they
contain. Diterpenes commonly include phytol, gibberellin hormones, and resins.
Triterpenoids include phytoalexins and toxins whereas sesquiterpenes majorly
include essential oils (Croteau et al. 2000). Diterpenoids, triterpenoids, and sesquiterpenes produced by endophytic fungi are mainly responsible for their antimicrobial
activity (Geetanjali 2017). More than ten varieties of terpenes synthesized by
endophytic fungi Alternaria alternata isolated from the leaves Azadirachta indica
are reported to exhibit antimicrobial activity against Bacillus subtilis, Listeria
monocytogenes, Staphylococcus aureus, Salmonella typhimurium, and Escherichia
coli. The same compounds also showed strong antioxidant activity (Chatterjee et al.
2019) (Fig. 9.5). Antimicrobial activity is reported against Enterococcus faecalis by
compound Pestalotiopens A produced by endophytic fungi Pestalotiopsis sp. isolated
from the leaves of Rhizophora mucronate (Deshmukh et al. 2015) (Fig. 9.6).
9.3.3
Alkaloids
Alkaloids is one of the most important fungal metabolites from pharmaceutical and
industrial point of view. They are a class of nitrogenous organic compounds
biosynthesized from amino acids. They are known for their diverse biological
properties, such as antifungal, anticancer and antiviral activities and are an important
source for drugs (Wang et al. 2011). Production of spiroquinazoline alkaloids
alanditrypinone, alantryphenone, alantrypinene, and alantryleunone by endophytic
fungi Eupenicillium sp. isolated from leaves of Murraya paniculata is reported by
Barros and Rodrigues-Filho in 2005 (Fig. 9.7). Fungal endophyte Neotyphodium
sp. are known for the production of ergot alkaloids (Panaccione et al. 2006). Ergot
154
P. Mathur et al.
Fig. 9.5 Structures of terpenes obtained from leaf endophytes of Azadirachta indica (Chatterjee
et al. 2019)
Fig. 9.6 Structure of Pestalotiopens A
Fig. 9.7 Structures of some spiroquinazoline alkaloids obtained from endophytic fungi
Eupenicillium sp. isolated from leaves of Murraya paniculate. (a) Alanditrypinone (R¼
3-indolyl); Alantryphenone (R¼ Ph); Alantryleunone (R¼ CHMe2), (b) Alantrypinene
9 Leaf Endophytes and Their Bioactive Compounds
155
alkaloid synthesis is also reported by an endophytic fungus of tall fescue
Acremonium coenophialum (Bacon 1988).
9.3.4
Lipids
Lipids consist of group of natural compounds like waxes, essential oils, fixed oils,
sterols, fat-soluble vitamins (such as vitamins A, D, E, and K), phospholipids and
others. Waxes are long aliphatic chains having one or functional groups and are
esters of fatty acids. Very popular Jojoba wax is reported to have wound healing and
antiaging properties. They are also used in the cosmetic industry. Essential oils and
fatty acids are derived from propionyl CoA, acetyl CoA, and methyl malonyl CoA
precursors. Fixed oils are high molecular aliphatic long-chain fatty acids, e.g., oleic
acids. They have antioxidant and anti-inflammatory properties. Essential oils are low
molecular weight volatile compounds like linalool, menthol, menthone, camphor,
etc. They possess sedative, analgesic, antiseptic, anesthetic, and spasmolytic activities (Hussein and El-Anssary 2018). Linoleic acid and cyclodecasiloxane produced
by endophytic fungi Alternaria sp. isolated from Pelargonium sidoides showed
antibacterial activity against Bacillus cereus, Enterococcus faecium, and
E. gallinarum (Manganyi et al. 2019) (Fig. 9.8). 1-Hexadecene, 1-Hexadecanol,
Hexadecanoic acid, octadecanoic acid methyl ester, and 1-Nonadecene synthesized
by endophytic fungi Colletotrichum gloeosporioides residing in leaves of
Phlogacanthus thyrsiflorus is reported by Devi and Singh (2013).
Apart from the abovementioned reports, there are many other studies and applications of leaf endophytes. However, these studies were conducted on crude extracts
and not on purified compounds. Majority of these applications include antimicrobial
activity of the crude extract as summarized in Table 9.2. One hundred and sixty-one
fungal endophytes were isolated from leaves of Calotropis procera with 35.1%
colonization rate. All these isolates were tested for antifungal activity against
Fig. 9.8 Structures of (a) Linoleic acid and (b) Cyclodecasiloxane obtained from leaf endophytes
of Pelargonium sidoides
156
P. Mathur et al.
Table 9.2 Antimicrobial activity of certain leaf endophytes
Endophyte
Penicillium sp
Trichoderma longibrachiatum and
Syncephalastrum racemosum
Unidentified fungal isolate
Cochliobolus intermedius
Pestalotiopsis neglecta
Cladosporium sp. and Curvularia sp.
Pestalotiopsis sp.
Alternaria alternata
Chaetomium globosum and
Myrothecium verrucaria
Nigrospora sphaerica and
Pestalotiopsis maculans
Plant source
Azadirachta
indica
Markhamia
tomentosa
Mangifera
indica
Sapindus
saponaria
Cupressus
torulosa
Cupressus
torulosa
Pinus
caneriensis
Coffea
arabica
Calotropis
procera
Indigofera
suffruticosa
Property
Antibacterial and
antifungal
Antifungal and
antiproliferative
Antibacterial
Antibacterial
Antibacterial
Antibacterial and
antifungal
Antibacterial and
antifungal
Antibacterial and
antifungal
Antifungal
Antibacterial
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(2017)
Ibrahim et al.
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Alternaria alternata, Fusarium oxysporum, Botrytis cinerea, and Pythium ultimum.
Out of all the isolates, four strains of Chaetomium globosum and three strains of
Myrothecium verrucaria showed maximum efficacy against the selected pathogenic
fungi (Gherbawy and Gashgari 2014). Leaves of Indigofera suffruticosa are
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Pestalotiopsis maculans presented antimicrobial activity against Staphylococcus
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from Coffea arabica. Their crude extracts showed antimicrobial activity against
Staphylococcus aureus, Escherichia coli, and Candida albicans. The isolate
Alternaria alternata showed maximum efficacy along with the production of phenol.
The extract also displayed antitumor activity against HeLa cell lines (Fernandes et al.
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fungi Pestalotiopsis neglecta isolated from leaves of Cupressus torulosa (Sharma
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In a study on endophytes of mango leaves, two bacterial endophytes and five
fungal endophytes were isolated. These isolated bacterial strains showed the production of IAA and has potential to be used as biofertilizers (Dasari et al. 2015).
Secondary metabolites extracted from Geotrichum sp. AL4 isolated from the leaf of
Azadirachta indica is reported to produce novel bioactive compounds having nematicidal activities (Li et al. 2007). Lipophilic antimicrobial peptides from an endophytic bacterial strain EML-CAP3 isolated from Capsicum annuum is reported to
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Conclusion
Endophytes are still considered a lesser exploited source of secondary metabolite
extraction. Leaf endophytes are easier to isolate and produce all types of secondary
metabolites. As per the abovementioned reports, there are several classes of secondary metabolites and all of them have various biological activities. Therefore, the
endophytic microflora of leaves can be explored further to obtain novel bioactive
compounds. These can also be studied for gene expression of specific genes that
contribute to such type of plant–microbe interaction.
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Chapter 10
Role of Endophytic Fungus Piriformospora
indica in Nutrient Acquisition and Plant
Health
Neha Sharma and Ajit Varma
Abstract Piriformospora indica is a unique endophytic fungus belongs to
sebacinales order of Basidiomycetes. This endophytic fungus colonized roots of
many plant species including model plants like Arabidopsis thaliana, Hordeum
vulgare (barley), and various monocots and dicots. P. indica has a beneficial impact
on plant growth promotion. Interestingly, symbiotic relationship of P. indica does
not depend on host specificity and thus it can form symbiotic associations with wide
variety of terrestrial plants. Colonization of P. indica improves crop productivity and
enhances the tolerance of host plants against different biotic (root pathogens) and
abiotic (drought, salinity, cold, high temperature) stresses. The positive interactions
of P. indica with different model plants are used to explore the molecular mechanism
of plant–microbe interactions. Furthermore, P. indica also has a significant role in
nutrient uptake and transport. In this chapter, we discussed the possible beneficial
role of P. indica in difference aspects of plant growth.
Keywords Piriformospora indica · Root endophyte · Crop health · Biotic and
abiotic stress · Phytohormones · Plant growth promotion
10.1
Introduction
Piriformospora indica, the entophytic fungus that colonized roots of monocots as
well as dicots plants (Verma et al. 1998). This symbiotic fungus has growthpromoting activity in wide variety of plants (Verma et al. 1998). This entophytic
fungus can also function as a supporting factor in the enhancement of the host
tolerance in plants against abiotic and biotic stresses (Waller et al. 2005). In barley,
P. indica inoculated plants become more resistance to stress and various root
pathogens. Plant with P. indica inoculation also induces disease resistance in plants
N. Sharma (*) · A. Varma
Amity Institute of Microbial Technology, Amity University, Noida, Uttar Pradesh, India
e-mail: nsharma27@amity.edu
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_10
161
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N. Sharma and A. Varma
(Serfling et al. 2007; Waller et al. 2005; Waller et al. 2008). Different from other
mycorrhizal fungi P. indica is easy growing on different substrates.
P. indica, belongs to the Basidiomycota division behaves similar in many ways
like Arbuscular Mycorrhizal Fungi (AMF) that also belong to Sebacinaceae family
(Qiang et al. 2012). Similar to AMF, P. Indica also promotes plants growth,
increases the resistance in colonized plants against fungal pathogens, and to various
abiotic stress and overall beneficial for plant growth (Harman 2011). It can modify
secondary metabolites of many economical relevant plants and also can promote
seed production in many plants. Similar to AMF, P. indica did not invade two strains
of Brassica and myc mutants of glycine max. However, different from AMF,
P. indica can cultivate axenically (Varma et al. 2012a) and it also has the potential
to colonize the model plant Arabidopsis thaliana.
Plant colonized with P. indica exhibits enhancement of plant growth. This
entophytic fungus increases nutrient uptake, makes plant susceptible to survive
under salt stress, water stress and temperature stress and also provides resistance
to heavy metals, toxic elements, pathogens. Many researchers have reported that
P. indica treatment can improve plant health and biomass production of diverse host
(Varma et al. 1999, 2001; Achatz et al. 2010; Sun et al. 2010). More than hundred
important medicinal, agricultural, horticultural plants are reported to interact with
P. indica (Sahay and Varma 1999, 2000; Varma et al. 2012b; Sun et al. 2010).
Genome of P. indica has been sequenced and publically available since 2011
(Zuccaro et al. 2011). It possesses 6 chromosomes with genome size of 15.4 to
24 Mb. Genome of P. indica contains 50.7% of GC content and 11,768 putative
protein coding genes which are responsible for mutualistic interactions, early
biotrophic and late necrotrophic phases of the fungus. Moreover, P. indica can be
transformed stably by random genomic integration of foreign DNA (Zuccaro et al.
2009). P. indica acquires a relatively small genome as compared to other members of
the Basidiomycota that makes it good model system to study symbiotic relationship
in plants (Zuccaro et al. 2009).
10.2
Role of P. indica in Plant Growth Promotion
Symbiotic fungus P. indica interacts beneficially with large number of crops to
promote growth by enhancing root and shoot biomass, secondary root formation,
increase in vegetative growth and early and increase flower and seed production
(Oelmüller et al. 2009; Qiang et al. 2012; Varma et al. 2012c, 2014; Johnson et al.
2014; Shrivastava et al. 2018). Once endosymbiosis is initiated in the roots, fungus
gets access to various plant nutrients, which ultimately promotes colonization and
proliferation of symbiotic fungus and thus significantly increases plant growth
(Oelmüller et al. 2009; Johnson et al. 2014; Su et al. 2017).
P. indica interaction promotes growth in almost all economically important crops
like wheat (Triticum aestivum), maize (Zea mays), rice (Oryza sativa), sugarcane
(Saccharum officinarum), tomato (Solanum lycopersicum), potato (Solanum
10
Role of Endophytic Fungus Piriformospora indica in Nutrient Acquisition. . .
163
Fig. 10.1 (a) Functional annotation of P. indica. (b) Ultra structure of P. indica spores (Shrivastava
et al. 2019)
tuberosum), and gram (Cajanus cajan). Also in model plant Arabidopsis thaliana, in
medicinal plants Abrus precatorius, Bacopa monnieri, Chlorophytum tuberosum,
Curcuma longa, Stevia rebaudiana growth enhancement have been observed
(Franken 2012; Varma et al. 2012c, 2013). P. indica also efficiently colonized
roots of winter wheat varieties and further result in enhanced root and shoot biomass
specifically when wheat crops were grown under nutrient limiting conditions
(Serfling et al. 2007). Interaction of P. indica with barley also results in more
grain yield (Waller et al. 2005). Similarly in maize colonization with P. indica
exhibits enhanced biomass production, root length and root number (Kumar et al.
2009, 2012). P. indica also promotes growth of tropical leguminous plants like
chickpea, mung bean, peas, and soybean (Varma et al. 2012c). P. indica is considered as potential candidate to enhance the biomass production along with various
value additions in the form of active ingredients. It significantly increases the
vegetative growth of plant and boost immunity to sustain in diverse environment
as compared to control. The important pharmaceutically important metabolites are
also found increased many folds in fungal treated A. vera host. Various functional
properties imparted from P. indica during interaction with host is depicted in
Fig. 10.1.
10.3
Role of P. indica in Nutrient Transport
Symbiotic plant–microbe interaction is mainly helpful in assimilation of nitrogen,
phosphorus, and other vital micronutrient that help in plant growth (Varma et al.
2013). Symbiotic fungus P. indica also promotes plant growth by transportation and
absorption of nutrients from soil. This fungus is able to extract and transport
macronutrients like nitrogen, phosphorus, potassium, sulfur and magnesium and
micronutrients like iron, zinc, manganese, and copper (Shahollari et al. 2007).
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N. Sharma and A. Varma
Phosphorus is essential nutrient for plant growth and is taken either directly by its
own transporters or indirectly through mycorrhizal associations. P. indica is an
excellent phosphate mobilizer and also a good phosphatase enzyme producer. It
has been shown that P. indica mediated growth enhancement in Arabidopsis is
associated with an uptake of radiolabeled phosphorus from growth medium
(Shahollari et al. 2007). Phosphorus uptake and transport is also stimulated by
P. indica colonized roots of maize (Yadav et al. 2010). P. Indica colonized mung
beans plants also exhibit significantly high levels of nitrogen, phosphorus, and
potassium (Kumar et al. 2012).
It has been reported that root colonization with P. indica increases nitrogen
uptake. Colonization of roots with P. indica in Arabidopsis is accompanied by
requisition of nitrogen from environment (Peskan-Berghofer et al. 2004). Similarly,
P. indica colonized tomato plants also exhibit better nitrogen acquisition (Cruz et al.
2013). Moreover, P. indica stimulated various plastid localized genes involved in
sulfur metabolism in A. thaliana (Oelmüller et al. 2009). P. indica can efficiently
mobilize micronutrient from soil and increase their availability to plants through its
colonization with roots (Achatz et al. 2010). Growth enhancement in mung bean
through P. indica colonization is correlated with the significant uptake of nitrogen,
phosphorus, and potassium from the soil (Kumar et al. 2012). Overall, P. indica
plays a significant role in delivering various nutrients for plants growth and
development.
10.4
Role of P. indica in Phytohormones Regulation
Phytohormones like auxin, cytokinin, gibberellin, and ethylene play significant role
in P. indica induced growth enhancement in Arabidopsis and Hordeum vulgare
(Sirrenberg et al. 2007; Schäfer et al. 2009). It has been found that P. indica
inoculated plants exhibit significant promotion in growth that is associated with
the higher levels of auxin and gibberellin in colonized roots. Furthermore, cytokinins
also play an important role in growth promotion in P. indica colonized Arabidopsis
(Vadassery et al. 2008).
Interestingly, it has been found that in that many genes involved in auxin
signaling and metabolism are upregulated in the P. indica colonized roots of Chinese
cabbage (Lee et al. 2011; Johnson et al. 2013). Significant role of gibberellins has
been reported in P. indica colonized roots of barley plants (Schäfer et al. 2009).
Although, ethylene biosynthesis and signaling that are necessary for plant growth are
inhibited by P. indica (Schäfer et al. 2009; Khatabi et al. 2012). Abscisic acid and
brassinosteroids are also synthesized or modulated by P. indica (Schäfer et al. 2009).
Thus, P. indica colonized plants have better compatibility for endosymbiosis
between this endophyte and host plant. Apart from that P. indica promotes robust
root architecture by producing IAA.
10
Role of Endophytic Fungus Piriformospora indica in Nutrient Acquisition. . .
10.5
165
P. indica as a Biocontrol Agent in Disease Resistance
Symbiotic fungus like AMF is reported to induce systemic resistance in hosts (Pham
et al. 2004). Similarly, P. indica is also beneficial in protecting host from pathogenic
fungus (Waller et al. 2005). P. indica provides bioprotection against root parasite
Fusarium verticillioides in maize plants (Kumar et al. 2009). This fungus inhibits the
colonization by F. verticillioides. Antioxidant enzyme Catalase activity was also
found to be significantly high in F. verticillioides-P. indica colonized roots. This
minimized the probabilities of oxidative burst and ultimately F. verticillioides could
be protected from the oxidative defense system during colonization.
P. indica can act as a bioprotector and biofertilizer in barley plants (Waller et al.
2005). Similarly, in wheat, P. indica is beneficial as a biocontrol agent against
pathogenic fungus Pseudocercosporella herpotrichoides. Overall, P. indica has
the potential as a beneficial biocontrol against major cereal pathogens those are
harmful for economical important crops. P. indica also biosynthesizes important
secondary metabolites like hydroxamic acids which can function as natural pesticides (Varma et al. 2001).
10.6
Role of P. indica in Stress Response
P. indica can help in stress tolerance like salt and nutritional stress in plants. Tomato
plants co-cultivated with P. indica promotes stress resistance through antioxidant
metabolism activation. Furthermore, P. indica inoculated fruit plants can sustain
lycopene content better independent of growth conditions. P. indica also found
beneficial in survival of plantlet, phosphorus content, and nutrient acquisition in
Chlorophytum sp. (Gosal et al. 2010). Similar results were found in sugarcane
plantlets in which P. indica inoculation enhanced the survival rate up to 12% after
transfer to soil. There was a significant effect on cane yield, cane height, and tillering
in P. indica inoculated sugarcane plants. Also in ratoon crop Fe and Cu uptake were
promoted in P indica colonized plants. Moreover, the cell wall extract of P. indica
was found significant in increasing cadmium tolerance in rice plants (Varma et al.
2012c). Thus P. indica can make plants resistance to various stresses.
10.7
Interaction of P. indica with Model Plants Arabidopsis
thaliana and Nicotiana attenuate
Different from AMF, P. indica also interacts with the non-mycorrhizal host like
Arabidopsis thaliana and promotes plant growth in this model plant (PeskanBerghofer et al. 2004). This growth enhancement effect was explained during the
entire life cycle of plant. First, A. thaliana seedlings were co-inoculated with the
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P. indica under in vitro conditions, there was enhancement found in root and shoot
biomass production of seedlings. When these seedlings were transferred to soil
leaves were larger in size. Later on, the A. thaliana plants grew faster with more
number of leaves and early flowering occurs. Also seed yield per plant was higher in
P. indica inoculated plants (Peskan-Berghofer et al. 2004; Johnson et al. 2011).
Similar observations were also seen in another model plant Nicotiana attenuata.
Cocultivation of N. attenuata results in increased seed germination and enhanced
stalk elongation and plant growth (Barazani et al. 2005). Seedlings with P. indica
colonization exhibit significant increase in root and shoot biomass in soil experiments (Johnson et al. 2011).
The ability of this entophytic fungus to maintain endosymbiosis with wellestablished model plants like A. thaliana, N. attenuata, N. tabacum, H. vulgare
makes these plants a powerful model system to study plant–microbe interactions.
10.8
Effect of P. indica on Transgenic Plants
P. indica inoculation found to be significant in wide variety of plants. P. indica
found to be associated with an increase in fresh and dry matter of shoot in rice plants.
P. indica has a stimulatory effect on eco-physiological parameters like carboxylation
efficiency, photosynthetic rate, stomatal conductance, intrinsic water use ability.
However, research done on transgenic rice, overexpressing the vacuolar H+-PPase
shows that P. indica inoculation was more pronounced in wild type plants in
compare to transgenic rice plants (Bertolazi et al. 2019). In wild type rice plants,
P. indica inoculation was promoting stimulation of Hc pumps while in transgenic
plants ATPase were found to be inhibited. Interestingly, nutrient uptake in roots and
shoots of wild type and transgenic plants were increased. But nutrient uptake was
less in transgenic rice plants compare to transgenic plants (Bertolazi et al. 2019).
This shows that in transgenic rice plants P. indica colonization is inefficient.
Reasons possible for this insufficient symbiosis need to study more.
10.9
Conclusion and Discussion
In brief, P. indica can be utilized as a plant promoter, biofertilizer, bioprotector,
bioregulator, and biotization agent. P. indica can significantly affect plants growth
and total biomass. Easy cultivation of P. indica with variety of synthetic media
makes it an easy to use as biofertilizer. The root endophyte P. indica colonization has
potential to enhance plant growth, better nutrient uptake, allows plants to survive
under various stress like salt stress, temperature stress, and protection from pathogens. P. indica is also helpful in stimulating growth and seed production. P. indica
very well interact with model plants like Arabidopsis thaliana and Nicotiana
10
Role of Endophytic Fungus Piriformospora indica in Nutrient Acquisition. . .
167
attenuata and makes it an excellent model system to study plant–microbe interactions. P. indica also plays an important role in phytohormones regulation.
More knowledge of P. indica properties will create new horizons for various
biotechnological application of this multifunctional fungus especially for agriculture
to enhance the crop resistance against different abiotic stresses like temperature and
drought. Deeper understanding of molecular mechanism and biomolecules involved
in the symbiotic plant–fungus association will give provide significant input for
future biotechnological applications of P. indica. Overall, P. indica is an excellent
eco-friendly biofertilizer that can improve crop health, soil health, and can help in
fighting hunger problems across the globe.
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Chapter 11
The Role of Symbiotic Fungi in Nutri-Farms
Saumya Singh and Ajit Varma
Abstract Small holder farms comprise about 78% of the entire country’s agricultural system. These farms are very important for Indian economy, as it produces
around 41% of the country’s total food grain production. The Indian government and
different agricultural institutes have started taking initiatives to convert these farms
into nutrition farms, by growing biofortified varieties. Nutri-farms are supposed to
be an important link in order to bridge the wide gap between agriculture and
nutrition. However, the use of biofortified varieties can be further assisted with
various other biofortification techniques, such as use of arbuscular mycorrhizal
fungi (AMF) to enhance agricultural output on a broader spectrum. AMF form
symbiotic association with the majority of the cultivated crops and play an important
role in improving plant nutrition, abiotic and biotic stress tolerance and soil fertility.
Incorporation of AMF as bio-fertilizers can help farmers to support a cost-effective
and sustainable agriculture.
Keywords Arbuscular mycorrhizal fungi · Nutri-farms · Biotic and abiotic stresses ·
Bio-fertilizer · Sustainable agriculture
11.1
Introduction
Traditionally, economists believed that health of people in the developing countries
was directly related to increased calorie intake. By increased calorie, they meant
increased energy to work. In order to attain this objective, the main concern was to
increase agricultural productivity. Higher energy intake was thought to enable
labours work more efficiently, which would ultimately increase their wages and
higher wages would empower them to buy enough food. This was the economic
cycle, thought to be governing the society (Meenakshi 2016). This was the main
S. Singh (*) · A. Varma
Amity Institute of Microbial Technology, Amity University, Noida, Uttar Pradesh, India
e-mail: ajitvarma@amity.edu
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_11
171
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underlying idea of the “Green Revolution” in the late 1960s. Dr. M. S. Swaminathan
started Green Revolution in India, which helped farmers to increase their agricultural
output, mainly cereals such as wheat, rice and maize, thereby, freeing India from the
threat of prolonged hunger. Very less importance was given to the nutritional impact
of agricultural output on human health.
It is only in recent times, that agricultural researchers have started paying greater
attention to the impact of agricultural performance on nutritional outcomes. It has
been found that vitamins and micronutrients in non-staple foods and animal products
were more strongly correlated to human health benefits. Whereas in developing
countries, the poorer and rural communities are majorly dependant on staple foods.
In order to overcome malnutrition, frequently called as ‘hidden hunger’, major
challenge is improving the nutritional quality of these staple food crops.
There can be several way-outs to fight this hidden hunger. First, several fortified
products, supplemented with essential minerals and vitamins are available off-theshelf, worldwide. A very common example in the category is, common salt enriched
with Fe and I, developed by the National Institute of Nutrition, Hyderabad, India
(Sesikaran and Ranganathan 2009). Second, dietary patterns can change in favour of
increased consumption of red meat, fruits and vegetables (Prasad et al. 2014). The
major drawback for the two approaches is, that these are restricted to urban and semiurban masses in the developing countries. It is in this context, that the biofortification
of staple crops has the potential to avoid calorie-micronutrient trade-off in the
underprivileged section of the society. Considering the adversity of the problem,
different research universities and government programmes have come up with
genetic biofortification. Genetic biofortification involves both traditional breeding
and biotechnological tools. Traditional breeding is labour and time intensive. However, the biofortified cultivars thus produced must be essentially high yielding, to be
accepted by farmers, particularly in developing countries. Further, the genetically
modified crops developed through genetic engineering face problems in the acceptance in several countries. On the contrary, the use of ferti-fortification, that is use of
chemical fertilizers to increase yield as well as micronutrients in grains have gained
popularity among agronomists. However, only 3–5% of the applied fertilizer can be
utilized by plants (Singh et al. 2017). Therefore, repetitive use of chemical fertilizers
leads to environmental pollution and disturbance in the native ecological niche.
Another less intervened approach to increase micronutrients and vitamins content in
grains could be using symbiotic fungi or other biological agents (Rana et al. 2012).
There are two important aspects of agriculture in India, i.e. family farming and
smallholder farming. FAO 2014 has declared that family farming plays an important
role in world agriculture (Haque 2016). It exists in both developed as well as
developing countries. Though, the relative size of family farms is small in developing countries in the Asia-Pacific region. On the other hand, smallholder farms, in
India particularly, are less than 2.0 ha (Singh et al. 2002). They are alone responsible
for country’s 41% of the food grain production. In both farming styles, farmers have
adapted the local techniques to support their agriculture. Major advantages of these
farming systems are agroforestry, crop rotation and intercropping (Oruru and Njeru
2016). These practices help to improve soil texture, health and microflora. Therefore,
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Fig. 11.1 A diagrammatic representation of symbiotic fungal functions to improve plant health,
nutrition and productivity (Begum et al. 2019)
there has been surging interest to incorporate sustainable agriculture to these farms
and convert these farms to nutritional farms or Nutri-farms.
Nutri-farms of fruits and vegetables were supposed to provide the household with
direct access to important micronutrients that may otherwise not be accessible within
their economic reach. Therefore, Nutri-farms were thought to be one of the alternatives to enhance access and generate conditions for better consumption of nutritive
food. Influenced by the idea, in the budget 2013–2014, United Progressive Alliance
(UPA) government, elicited a proposal to establish nutrition farms, also known as
Nutri-farms in India (Singla and Grover 2017). This initiative was supported by
Harvest Plus, an international organization that works on Nutri-farms. Biofortified
staple crops, such as maize (rich in lysine and tryptophan), wheat and rice (rich in
zinc) and pearl millet (rich in iron) were grown (Ravi and Usha 2017). Nutri-farms
were thought to improve nutritional availability for poorer people especially women
and children. Therefore, Nutri-farms create a cost-effective way to challenge the
escalating problem of malnutrition in India.
Nutri-farms are supposed to involve low input cropping systems. Therefore, the
use of bio-fertilizers can contribute to control plant pathogens and crop productivity
for a wider range of crops. Symbiotic mycorrhizal fungi form an association with
about 80% of the terrestrial plants, thus can prove to be useful in improving
nutritional values of crops and protection from different abiotic and biotic stresses
(Fig. 11.1).
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S. Singh and A. Varma
AMF as Bio-Fertilizer
It is supposed that in order to support the world population, agricultural production
must be doubled by the year 2050 (Igiehon and Babalola 2017). In order to increase
crop productivity, agriculture widely depends on chemical fertilizers and pesticides.
Continuous use of chemical-based inorganic fertilizers, herbicides and pesticides
deteriorate food and soil quality and air and water systems (Begum et al. 2019). In
addition to ecological and environmental problems, it causes harmful effects on
human and plant health. Therefore, using mixture of naturally occurring substances
or microorganisms as bio-fertilizers can prove to be a sustainable source to improve
soil quality. Further, different studies have shown that AMF can possibly lower the
use of chemical fertilizers by about 50% (Begum et al. 2019). However, this
percentage would depend on host and fungal species and environmental conditions
prevailing.
11.3
AMF and Mineral Nutrition
Rigorous studies have shown that AMF symbiosis can enhance the accumulation of
macro and micronutrients in plants. These in turn increase photosynthate production
in host plants which in turn increases biomass production. Sometimes nutrients are
insufficiently phytoavailable in soil. This may be due to either low absolute nutrient
amounts or formation of metal complexes which makes them less mobile. Solubility
of these metal complexes increases with increasing soil pH and this is a major
problem as 30% of the agricultural soil worldwide is alkaline (Lehmann and Rillig
2015). AMF develop symbiosis with host plants to obtain carbon resource from them
and in return provide them with essential nutrients such as N, P, K, Zn, Fe, Cu, Mg,
Mn, Ca and S.
P is an important mineral that limits plant growth. AMF have been found to
beneficially affect plants, in the conditions where soil P levels are low. This may be
associated with extra radicle hyphae (ERH) formed by AMF. ERH helps plants to
explore greater soil volume, particularly beyond the depletion zone, as compared to
root hairs of non-AMF plants. This has been experimentally proved by many
researchers. Rhodes and Gerdemann (1978) and Marschner and Dell (1994)
performed pot experiments with onions and clover, respectively, and observed that
AMF colonized roots were able to explore greater distance in soil, under P deficiency. Since pot conditions are different from field conditions, Mai et al. (2019)
planned experiments with AMF colonized cotton under field conditions. They also
found that AMF colonization in roots increased their P acquisition range by 15 folds,
under P deficiency.
Furthermore, not all P present in the soil is available for plants. In particular,
20–80% of the total P in soil consists of organic phosphates and insoluble inorganic
salts (Sato et al. 2019). Thus, the hydrolysis of these phosphates into plant-available
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form is an important aspect. It has been reported that some plants secrete alkaline
phosphatases in their root exudates, to hydrolyse unavailable P and increase plantavailable inorganic P (Pi) pool. Zhang et al. (2011) found that Glomus mosseae
increased maize growth even in the presence of organic fertilizers. Increased ability
of plants to absorb P, infected by AMF, is predicted to be caused by the production
of certain organic acids and phosphatase enzymes (Aswitha et al. 2019). Similar
study was carried out by Pel et al. (2018) to establish if extraradical hyphae of
different Glomus strains were capable of releasing Pi from resilient apatite matrix.
It has been found that mobility of inorganic P is much lower than inorganic
N. Therefore, the role of AMF to increase acquisition of N is less significant than for
the acquisition of P. However, there may be situations when available nitrogen in
soil limits plant growth. For instance, AMF can be useful to increase N uptake in
semiarid and arid conditions, when the mobility of nitrate is reduced substantially or
under water stress conditions (Miransari 2011; Shrivastava et al. 2018).
Moreover, apart from the direct use of nitrogen by plants, AMF have been
observed to enhance the activities of certain enzymes like, nitrate reductase, glutamine synthetase and glutamine synthase (Miransari 2011). These enzymes help
plants to sustain through drought conditions. AM fungi release certain hydrolytic
enzymes such as pectinases, xyloglucanase, in the mycorrhizosphere, to organic N
patches present in soil. In addition to this, AMF sometimes affect the activities of
other soil microbes, which may in turn enhance mineralization of organic matter
in soil.
Moreover, AMF balance the dynamic ratio of Ca+2 and Na+, important to
improve plants’ overall performance (Begum et al. 2019). Tran et al. (2019) found
that inoculation of AM fungi Rhizopus irregularis with durum wheat, improved the
plant’s availability of zinc and iron. Farzaneh et al. (2011) found that AMF inoculation had positive impact on acquisition of P, K, Fe, Mn and Cu in chickpea.
However, they observed that amount of N decreased under the effect of AMF. This
was supposed to be due to the dilution effect due to increased biomass. Liu et al.
(2000) also found that colonization of Glomus intraradices had a positive influence
on uptake of nutrients such as Zn, Cu, Mn and Fe in Zea mays. Subramanian et al.
(2013) have observed that AMF colonization help plants to increase Fe and Zn
uptake in both calcareous and non-calcareous soils. In order to hydrolyse the tightly
bound nutrients, AMF acidifies surrounding soil.
11.4
AMF and Anti-Nutrients
Phytates (inositol hexakisphosphate) is the most abundant source of organic P in
soil. This P cannot be utilized by plants directly. In order to hydrolyse this phytate
organisms use various phosphatases, mainly phytase and phosphomonoesterase
e.g. acid phosphatases. Early researchers believed that fungal hyphae only produced
acid phosphatases in order to hydrolyse lower order inositol phosphates. However,
later it was found that fungal hyphae possess phytase activity along with acid
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phosphatases. Though, the phytase activity was less than 5% of the total acid
phosphatase activity (Wang et al. 2017). Subramanian et al. (2013) demonstrated
that use of AMF not only increased the accumulation of zinc and iron in maize grains
but also helped in circumventing the impact of anti-nutritional compounds like
phytate. Zhang et al. (2018) carried out tripartite experiments using AMF strain
Rhizopus irregularis, a phosphate solubilizing bacteria Rahnella aquatilis and two
plants root organ cultures of carrot and whole plant of Medicago truncatula. They
demonstrated that few rhizospheric bacteria are associated with AMF. These
rhizobacteria provide P to AMF in return to C. These sugars not only work as C
source but they also play an important role as signal molecules for rhizobacteria.
These signal molecules stimulate expression of phosphatase genes in bacterium.
11.5
AMF and Abiotic Stresses
11.5.1 Heavy Metals
Researchers have been suggesting, that strigolactones are the molecules responsible
for the germination of fungal spores and formation of extra radical hyphae. Further,
it was found that optimal or increased concentration of various nutrients in rhizosphere, restrain the secretion of strigolactones from the plant roots (Konieczny and
Kowalska 2017). These in turn reduce the mycorrhizal colonization and formation of
hyphae. Thus, enabling AM fungi to act as a buffer and inhibit accumulation of any
nutrient beyond a required amount. This suggests that AMF increases micronutrient
content under nutrient deficiency but prevents over accumulation of minerals when
their concentration in soil is close to being toxic.
Joner et al. (2000) demonstrated through experiments, that fungal hyphae are
extremely capable of binding heavy metals. Kaldorf et al. (1999) performed experiments with different strains of Glomus and maize. They found that AM fungi
colonized plants when grown in heavy metal contaminated soils, increased the
concentration of essential minerals such as K, P and Mg but acquisition of heavy
metals (HM) such as Ni, Fe, Zn or Cu were decreased in aerial parts of plants, as
compared to non-AMF colonized controls. Microbeam analysis to study element
localization have shown that heavy metals which entered roots, were deposited in the
root parenchyma cells. As a result of this study, it was difficult to establish, whether
the HM were deposited in plant roots or fungal cells as, most of the fungal structures
such as intraradical hyphae, arbuscules and vesicles are present in root parenchyma
cells. In later studies, electron dispersive X-ray spectrometry confirmed that the HM
were deposited in electron-dense granules present in fungal cell cytoplasm or their
vesicles (Hildebrandt et al. 2007). AM fungal hyphae were found to produce some
insoluble glycoproteins called as glomalin, which was capable of binding HM and
toxic elements.
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11.5.2 Drought
Drought stress adversely affects plant growth by affecting its enzyme activity, ion
uptake and nutrient absorption (Begum et al. 2019). Various studies have established
strong evidence that AMF helps to lessen drought stress in different crops such as
wheat, barley, maize, soybean, strawberry and onion (Begum et al. 2019). Wu and
Zou (2017) have outlined potential mechanisms regarding AMF induced drought
tolerance in plants under different headings such as morphology, physiology, biochemistry and molecular.
AMF colonization reduced epicuticular wax and lower cuticle weight in rose
leaves during drought stress. This led to the abscission of leaves (Henderson and
Davies 1990). Further, increased deposition of starch in palisade mesophyll tissues
enables mycorrhized plants to recover more quickly from wilting after drought
recovery (Henderson and Davies 1990). Recently, Liu et al. (2016) found that
mycorrhization influence root morphology of host plants by increasing its total
root length, projected area, surface area, root diameter and volume under both
well-watered and drought circumstances (Fig. 11.2). These changes in the root
morphology may be due to regulation of endogenous polyamine metabolism and
phytohormone equilibrium such as root putrescine synthetases and ornithine decarboxylase, IAA (Wu and Zou 2017).
Further, mycorrhizal hyphae have been well known to increase transport and
uptake of micronutrients from soil. However, researches have now shown that fungal
hyphae mediate to transport water to host plants, along with different nutrients.
Fungal hyphae are thinner than root hairs and hence can penetrate soil pores,
Fig. 11.2 Difference in root morphology of plants under drought stress (a) without AMF, (b) with
AMF (Wu and Zou 2017)
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inaccessible to root hairs. Furthermore, due to less or no septa in hyphae, it transports
water to plant cells from distant places.
Liu et al. (2016) found that mycorrhization increased levels of following phytohormones in trifoliate orange seedlings: IAA, ABA, methyl jasmonate (MeJA) and
zeatin riboside (ZR) under well-watered conditions and IAA, ABA, MeJA, ZR and
brassinosteroids under drought conditions. Variations in these phytohormones could
be responsible for drought tolerance in plants. Further, mycorrhization causes
increased osmotic adjustments in plants. This leads to increased accumulation of
soluble starch, glucose, sucrose and K+ and Ca+2 in leaves and roots (Wu and Zou
2017). These osmolytes tend to protect and stabilise macromolecules and cellular
structures from osmotic damage and maintain a water potential gradient for water
absorption from soil to roots.
AMF not only imparts resistance against drought to plants but also helps plants to
be drought tolerant. AMF colonization enhances antioxidant enzymatic and
non-enzymatic activities in plants, thereby protecting plant organs against oxidative
damage of reactive oxygen species, thus enhancing drought resistance (Wu and Zou
2017).
Nichols (2008) demonstrated an indirect influence of AMF to prevent plants
against drought stress. He found that AMF release Glomalin-related soil protein
(GRSP) which helps to maintain a good structures soil in comparison to
non-mycorrhizal soils. GRSP forms a hydrophobic layer on fungal hyphae which
helps to lessen water loss in soil aggregates.
11.5.3 Salinity
Increased concentration of Na+ and Cl in plants adversely affects its normal growth
by damaging its foliage, causing nutrient deficiencies, inhibiting carbonic anhydrase
and nitrate reductase activities, damage to cellular organelles, decreased photosynthesis by damaging PS II reaction centre (Borde et al. 2017). [Na+] > 40 mM within
the root zone creates an extremely hyperosmotic condition for the plants which leads
to damaging effect on plants. This leads to an ionic imbalance within plant system,
by increasing Na+ and Cl concentration and decreasing K+, Ca+2, NO3 and
inorganic phosphorous. Under such stress conditions AMF colonization help plants
to maintain the ionic balance by reducing Na+ uptake and increasing K+ uptake
(Porras-Soriano et al. 2009). K+ helps to preserve pH balance within plant cells and
increase osmotic potential within cellular vacuoles. Porras-Soriano et al. (2009) have
shown that AMF colonized olive plants have the highest K+ in shoots. Thus, K+ as an
osmotic solute was able to maintain high tissue water level even under adverse
conditions of osmotic deficiency.
In addition to osmotic imbalance, salinity stress leads to increased production of
reactive oxygen species (ROS) like superoxide radicals (O2 ), hydroxyl radical
(OH ) and singlet oxygen (O1 ) within plant cells. High levels of these species
negatively affect plant cellular metabolism through oxidative damage to lipids,
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proteins and nucleic acids (He et al. 2007). In response to these destructive elements,
plants produce ROS-scavenging enzymes such as superoxide dismutase (SOD),
peroxidase (POD), APX and catalase (CAT) in order to neutralize ROS, produced
as a result of stress. SOD converts superoxide to oxygen and H2O2, and CAT
converts this H2O2 to oxygen and water. He et al. (2007), reported that AMF
colonization in tomato plants helps to enhance host’s antioxidant defense
mechanism.
11.5.4 Temperature
Temperature stress (high or low) inhibits plants growth and biomass production. Zhu
et al. (2011) reported that mycorrhized maize had higher water conservation potential and relative water content. However, it was observed that low or high temperature stress reduces plant’s ability to take up water. AMF colonization improves this
water status by enhancing water uptake through extraradical hyphae and improving
hydraulic conductivity of roots (Zhu et al. 2017).
High temperatures also affect plant’s photosynthetic ability. Mathur et al. (2018)
investigated effect of AMF colonization in maize under high temperature stress.
They reported that mycorrhization increased number of active PS II reaction centres,
improved quantum efficiency of PS II, linear e transport, excitation energy trapping, thus enhancing the overall photosynthesis rate even at 44 C. AMF colonization increased total chlorophyll content in leaves.
Furthermore, temperature stress affects integrity of plant plasma membrane by
altering its composition and structure (Zhu et al. 2017). Such damages increase
membrane permeability for electrolytes. However, Evelin et al. (2009) have reported
that mycorrhization helps plants to maintain ionic balance by improving stability of
plasma membrane. In addition to these effects, temperature stress also leads to
production of ROS. Mycorrhized plants’ response to these ROS have already been
explained previously.
11.5.5 Biotic Stress
AMF symbiosis have been widely accepted to reduce the damage caused by soilborne pathogens such as Fusarium, Rhizoctonia, Erwinia carotovora, Phytophthora,
Pythium (Whipps 2004), certain root parasitic plants such as Striga and Orobanche
(Bouwmeester et al. 2003) and to some extent above ground diseases (Whipps
2004).
Several mechanisms have been proposed that may be responsible for the
enhanced resistance offered to mycorrhized plants. It may be possible that AMF
offers a strong competition for the photosynthates to the soil-borne pathogens
(Cordier et al. 1998). In addition to this, AMF colonization results in modification
180
S. Singh and A. Varma
of root architecture, morphology and composition of root exudates. These changes
may be responsible for alterations in the dynamics of infection. Moreover, AMF
colonization reduces production of strigolactones from plant roots. This reduced
production of strigolactones can be positively correlated to the reduced susceptibility
of hosts to parasitic plants (Pozo et al. 2010).
11.6
AMF and Secondary Metabolites
These endosymbionts only improve plants’ nutritional status and their tolerance for
different abiotic and biotic stresses, as described above, but also enhance quality of
crops. For example, Bajaj et al. 2014 and Su et al. 2017 observed that colonization of
Curcuma longa and Brassica napus with Piriformospora indica increased productivity along with volatile oil and curcumin content. Curcumin has several medicinal
and ROS scavenging properties. Dry rhizomes have importance as flavouring and
colouring agent in Asian diets. Similarly, Liu et al. (2019) reported that symbiotic
association of Funneliformis mosseae with Astragalus membranaceus promotes its
medicinal properties by increasing active ingredients such as astragaloside IV,
calycosin-7-glucoside, astragalus polysaccharide and Se. Similarly, CRIŞAN et al.
(2018) described several AMF and host combinations, that have been used to
demonstrate usefulness of symbiotic fungi to increase active secondary metabolites
in hosts.
11.7
Conclusion and Future Prospects
Over the years several researchers have published work related to beneficial work of
AMF to increase plant productivity and tolerance for different abiotic and biotic
stress. Therefore, this chapter aims to combine the existing information regarding
AMF and their potential use in Nutri-farms. By combining the concepts of Nutrifarms and use of AMF could possibly provide nutritional security among
populations of developing nations. Green Revolution particularly focused to
increase crop yield by employing improved quality seeds to farmers, increased use
of chemical fertilizers and pesticides and improved irrigation facilities. But even
after Green Revolution, there is a missing link between increased productivity and
nutritional security. Therefore, there is a need of Nutritional Revolution, specially in
developing countries.
In order to meet this need, converting small holder farms to nutritional farms can
prove to be useful. In order to achieve this, the Indian Government has already taken
initiatives to promote more establishment of Nutri-farms. However, only biofortified
varieties are being employed in these farms, as of now. The major drawback of this
could be, wide acceptability of the limited biofortified varieties thus available. Thus,
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measures could be taken to increase the effectiveness of this concept, by involving
other methods of biofortification like the use of AMF.
However, there are some points of concern that should be taken care of, in order
to make this technique more acceptable. Most of the smallholding farmers of a
particular geographic area, use old landraces having similar properties and genetic
composition. Therefore, these genotypes could be tested for their response efficiency
to AMF colonization. Moreover, AMF diversity patterns need to be mapped and
their efficiency determined in order to encourage different agroecosystems. Farmers
should be educated for the beneficial and correct use of AMF so that they are more
likely to adopt this sustainable technique. Apart from training the farmers, it is
important to mass cultivate AMF for its cost-effective availability to farmers.
Cheap fungal inocula need to be produced through different techniques such as
mass in vitro production, on-farm multiplication and use of nursery inoculated crops.
Large scale production of AMF is a cost-intensive technique, which is borne by
small farmers and nursery owners. An alternative for this technique could be on-farm
method to develop mycorrhizal inoculum of either indigenous or introduced AMF
strains. Thus, all these methods would collectively help to increase number of Nutrifarms in developing countries, thereby bridging the gap between agriculture and
nutrition.
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Part II
Bacterial Symbiosis
Chapter 12
Understanding the Evolution of Plant
Growth-Promoting Rhizobacteria
Pratyusha Sambangi, Vadlamudi Srinivas, and
Subramaniam Gopalakrishnan
Abstract Soil is an integral part of the complicated natural environment which is
very much alive with complex ecosystem of microbes. Among them, the symbiotic
association of rhizobacteria with plants especially on agriculturally important crops
is very much advantageous in improving the soil and plant health. These plant
growth-promoting rhizobacteria (PGPR) have evolved over the years and involved
in many plant functions such as growth promotion, root development, colonization,
production of metabolites and in eliciting plant defence mechanism against abiotic
and biotic agents. The PGPR’s ability to fix the atmospheric nitrogen, solubilize
phosphate, potassium and zinc, produce siderophore along with wide variety of
phytohormones and secondary metabolites such as antibiotics have attributed to their
significance as biocontrol agents. These functions lead to their application as
biofertilizers, biopesticides, bioprotectants and phytostimulators. The employment
of these PGPR is very much important in agricultural fields as they reduce the burden
of chemical fertilizers and pesticides to the farmers and in turn promises an increased
crop yield. This chapter discusses the symbiotic association of PGPR with plants in
detail including their direct and indirect mechanisms and basis of their induced
systemic defence mechanism. It also highlights the use of bioinoculants and nanoformulations of PGPR as an effective tool towards enhanced agricultural production
and to combat the plant diseases in an eco-friendly manner.
Keywords Rhizobacteria · PGPR · Symbiosis · Biocontrol · Antifungal ·
Agriculture
P. Sambangi · V. Srinivas · S. Gopalakrishnan (*)
International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru,
Telangana, India
e-mail: s.pratyusha@cgiar.org; s.vadlamudi@cgiar.org; s.gopalakrishnan@cgiar.org
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_12
187
188
12.1
P. Sambangi et al.
Introduction
The plant root system interacts with a large mixture of microorganisms and these
interactions define the extent of association between the plant and microbe. This
relationship between the soil bacteria i.e. rhizobacteria and plant is very precise and
often influences a lot of factors such as plant growth, soil health, microbiome and the
environment (Muller et al. 2016). Rhizobacteria such as rhizobia, root nodule
bacteria of the many leguminous plants, undergo symbiotic association and facilitate
in biological nitrogen fixation (Peix et al. 2015). In the arid and semi-arid regions,
this rhizobia-legume symbiosis is extensively investigated and many studies have
reported for their significant source of Nitrogen (N) input in the agricultural fields
(del Pozo et al. 2000; Buhian and Bensmihen 2018). The bioavailability of nutrients
and minerals at a given soil location is highly dependent on the type of residing
rhizobacteria. These nutrient transformations occur depending on the variety of
plant–microbial symbiosis. The symbiotic association between the plant and
microbe is the key driving factor for the plant growth and even affects the local
soil ecosystem (van der Heijden et al. 2008; Verbon and Liberman 2016).
With greater demand for sustainable agriculture, the application of PGPR to crops
is beneficial and essential. These root-associated bacteria are diverse in nature and
colonize a wide variety of agricultural crops. The genera of rhizobacteria that
exhibits the plant growth promotion include Azospirillum, Azotobacter, Bacillus,
Burkholderia, Enterobacter, Erwinia, Flavobacterium, Mycobacterium,
Mesorhizobium, Pseudomonas, Rhizobium and Streptomyces. These ecological
engineers have an association with many agriculturally important crops namely
barley, corn, canola, chickpea, groundnut, oats, maize, wheat, rice, lentils, peas,
rye and radicchio (Podile and Kishore 2006). These PGPR directly synthesize
compounds and provide them to the plant to assist in their well-being. Sometimes
they indirectly also facilitates the plant root system to absorb certain nutrients from
the soil environment. In this manner, either directly or indirectly the rhizobacteria
symbiotically benefit the plant and also fight-off against disease-causing pathogens
(Maksimov et al. 2011).
During recent years, several research studies have reported the significance of
rhizosphere microbes in playing an important role in the plant growth promotion, in
formation of important microbial consortia and disease resistance in host plants
(Bhattacharyya and Jha 2012; Alekhya and Gopalakrishnan 2017; Vijayabharathi
et al. 2018; Anusha et al. 2019; Gopalakrishnan and Vadlamudi 2019; Kim et al.
2019). Also, they emerged as a potential alternative for chemical fertilizers and have
shown promising crop yield outputs in agricultural fields (Laslo and Mara 2019).
These PGPR, with high abundance and low cost could be further exploited for their
applicative advantages in sustainable agriculture over conventional practices.
PGPR are mainly known to associate with the agriculturally important cereals and
leguminous crops. These cereals and legumes are the vital food source for humans
and they were also widely used for the livestock (FAO 2018). Hence, the PGPR role
as cereals/leguminous plant growth and yield promoters is very much in need of the
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Understanding the Evolution of Plant Growth-Promoting Rhizobacteria
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hour with ever-increasing global population. In the present chapter, the mechanisms
and applications of the rhizobacteria were comprehensively analysed to further
exploit them and to understand the pre- and post-colonization strategies with changing times. The underlying cellular and molecular mechanisms of rhizosphere
microbiota were also studied for their efficacy as bioinoculants.
12.2
Biology of PGPR
Rhizobacteria are a soil bacterium that infects the host plant root system and help
plants in many ways. They form a symbiotic association with the host plants and
facilitate its growth and development by the exchange of nutrients and metabolites.
The main mechanisms that aid in these host-microbe interactions are symbiosis,
nitrogen fixation and growth promotion.
12.2.1 Symbiosis
The symbiosis between the rhizobia and legumes triggers the nodulation process and
they are fully functional in 3–4 week old plants. The specificity of these nodules
usually depends on the type of microbe associated to it. The plant flavonoids are the
important metabolites that are utilized by these soil rhizobia to recognize the host
system and initiate the symbiotic nodule association. Especially, the aglycones play
a key role in the activation of rhizobial Nod genes namely NodA, NodB and NodC
(Perret et al. 2000). Apart from the flavonoids, the levels of calcium also alter the
plant roots hair structure to develop the nodules (Ehrhardt et al. 1996; Downie and
Walker 1999). It is reported that NIN, a transposon, is involved in the nodule
formation and it is the first cloned gene to successfully develop nodules in the host
plant (Schauser et al. 1999). During the formation of these nodules an infection
thread is formed between the symbiotic bacteria cell surface and host plant wall.
From many studies, it is evident that the root lectins facilitated this attachment of
rhizobial infection thread and the nodulation process in the plants (Kijne et al. 1997;
Van Rhijn et al. 2001). Galibert et al. (2001) reported higher content of G + C in the
nodulating and nitrogen fixation genes.
Rhizobia present in soil usually reside in large colonies and it is very much
essential to communicate among them. It is evident from various research studies
that rhizobacteria have a complex quorum sensing system to form a symbiotic
relationship with the host plant. Some of the rhizobia namely, Rhizobium fredii,
Rhizobium leguminosarum and Sinorhizobium meliloti are known to have a wellestablished quorum sensing signalling for nodulation and nitrogen fixation. Mainly
this quorum sensing is mediated by the production of N-acyl homoserine lactones
(AHLs) by rhizobial strains, which involves the chemical crosstalk (González and
Marketon 2003). The purpose of understanding this diverse legume-rhizobial
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chemical and molecular symbiosis is important because of their ability to fix
atmospheric nitrogen and pathogen suppression. By recognizing the evolution of
host-rhizobial symbioses in agriculturally important crops, we may have better
applicative value towards sustainable agriculture.
The biological nitrogen fixation is a major contribution of soil rhizobia to the
plant kingdom. Nitrogen is one of the major atmospheric gases and very much
essential for the plant growth and photosynthesis (Wagner 2012). But the available
form of nitrogen (NH3) is only made available by these rhizobacteria through the
process of biological nitrogen fixation in the root nodules of the plant. Rhizobia are
the best-known group of symbiotic soil bacteria that fix nitrogen in relation with a
wide variety of agriculturally important crops (Peoples et al. 1995; Dawson 2008;
Lindstrom and Mousavi 2010). Applications of chemical fertilizers have significantly reduced in agriculturally important crops due to rhizobia being an efficient
source of nitrogen and nutrients (David and Ian 2000). The process of biological
nitrogen fixation is regulated by a group of bacterial nif (nifH, nifD and nifK) genes.
The structure and function of this nif gene are similar in many diazotrophs such as
Azotobacter vinelandii, Bradyrhizobium japonicum, Herbaspirillum seropedicae
and Pseudomonas stutzeri (Fischer 1994). Due to this property of biological nitrogen
fixation, many rhizobacterial strains were inoculated in legume plants and a significant increase in nodulation and nitrogen fixation was observed. The size, weight and
number of nodules and fixed nitrogen were found to significantly enhance in the
PGPR inoculated plants compared to un-inoculated plants (Islam et al. 2013; Kuan
et al. 2016; Gopalakrishnan et al. 2017, 2018). Hence, these bacteria are regarded as
renewable source of nitrogen in the fields and environment that majorly contribute to
the conservation of the soil health.
12.2.2 Growth Promotion
Many research studies have reported that the treatment of PGPR has enhanced the
plant growth and nutrition status. This proves the fact that these PGPR have the
ability to increase the soil fertility and microbial diversity through the production of
various root exudates namely extracellular metabolites, hormones, signal compounds and antibiotics (Van Loon 2007; Wani and Gopalakrishnan 2019). PGPR
have the capacity to synthesize the phytohormones that directly aid in the plant
development. Many reports have identified the synthesis of indole acetic acid (IAA)
by PGPR which is mainly responsible for the maturation of plant root system (Patten
and Glick 2002; Remans et al. 2008). Cytokinins were also observed and regulated
the cell division and root–shoot development of the host plants when inoculated with
PGPR (Hussain and Hasnain 2009). In addition, ethylene, gibberellic acid and
abscisic acid were also emitted by PGPR that aids in the plant development (Dodd
et al. 2010).
The importance of these soil bacteria is mainly attributed to their ability to
produce siderophores, which greatly assists in the iron uptake of the host plants. In
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191
the presence of metal competition these siderophores aid in the solubilization and
diffusion of iron into the plant cell walls (Crowley 2006). This siderophore production also assists in the PGPR colonization by evading other microbial and fungal
pathogens. In iron-deficient agricultural fields, the siderophore expressing PGPR are
promising alternative for the bioavailability of iron to soil and plants (Sayyed et al.
2013). Fertilizer applications will secure enough phosphorous (P) in the agricultural
fields but, these PGPR are the main solubilizers that provide soluble form of
phosphate to the crops. PGPR such as Bacillus, Pseudomonas, Rhizobium and
Streptomyces species are known to enhance the phosphate uptake in the inoculated
plants (Ramaekers et al. 2010). Apart from the plant growth and development, these
PGPR also influence the yield of the crops, by increasing the mineral density of the
seeds (Sathya et al. 2016). Enhanced content of Fe, Zn, Mg, Mn, Ca and Cu were
reported in the seeds of PGPR inoculated crops such as wheat, rice chickpea and
pigeon pea (Rana et al. 2012; Sharma et al. 2013; Gopalakrishnan et al. 2016a). This
ability of biofortification by the PGPR in the agricultural inoculated crops will
provide greener choices for better nutrition intake in humans.
In evidence, various greenhouse and field research studies conducted at the
International Crops Research Institute for the Semi-Arid Tropics (ICRISAT),
based at Patancheru, Hyderabad, India, with different strains of PGPR have shown
multiple growth potentials by enhancing the plant growth-promoting and biocontrol
traits in the PGPR inoculated host crops (Table 12.1). This emphasizes the significance of PGPR utilization in the future for sustainable agricultural practices in
legume crops.
12.3
Role of PGPR as Biocontrol Agents
It is reported that PGPR have antagonistic activities against wide array of bacterial,
viral and fungal pathogens. These soil rhizobacteria, within its habitation, exhibit a
variety of defence mechanisms to control and fight against the invaders. They trigger
the host plant induced systemic resistance i.e. alters the plant cell wall, pathways and
metabolites in response to the pathogen infection. Hence, the utilization of PGPR for
the management of soil-borne pathogens is highly beneficial in the agriculturally
important crops, as it reduces the use of chemical fungicides and eco-friendly in
nature (Gopalakrishnan and Vadlamudi 2019). The anti-oxidant enzymes namely,
peroxidase, phenylalanine ammonia-lyase, superoxide dismutase and polyphenol
oxidase are elicited in the infected host plants by the PGPR to trigger the defence
pathways (Gopalakrishnan et al. 2019). This in turn initiates the production of plant
defence metabolites such as phenolic compounds, phytoalexins, lytic enzymes and
antibiotics (Conrath et al. 2001; Walters et al. 2005). The antibiotics produced by
these soil bacteria, especially by Bacillus spp. were known to suppress many
pathogenic bacteria (Maksimov et al. 2011). Metabolic compounds such as lipids
produced by Bacillus and Pseudomonas are effective biocontrol agents against many
bacteria, fungi and protozoans (Raaijmakers et al. 2010). PGPR especially, the
PGP properties
Biocontrol properties
NCBI no.
JQ247013
IAA
Sid.
+
HCN
Cel.
+
Lip.
+
Pro.
+
Chi.
+
β
KM250376
+
+
+
+
+
+
+
+
KM250377
+
+
+
+
+
+
+
+
KM250378
+
+
+
+
+
+
+
+
KM250375
+
+
+
+
+
+
+
+
JQ247010
+
+
+
+
+
+
+
MF359733
+
+
+
MF359733
+
+
+
+
+
+
+
MF359737
+
+
+
+
+
+
+
MF370070
+
+
+
+
+
+
+
MF370069
+
+
+
+
+
+
+
JQ247009
+
+
+
+
+
+
1,3
P
Sol
+
Crops
evaluated
(greenhouse/
field)
Chickpea;
Rice
References
Gopalakrishnan et al. (2012),
Sreevidya and Gopalakrishnan
(2015), Anusha et al. (2019)
+
+
+
P. Sambangi et al.
PGPR
Acinetobacter
tandoii (SRI-305)
Bacillus
sp. (VBI-4)
Bacillus
sp. (VBI-19)
Bacillus
sp. (VBI-23)
Bacillus
sp. (SBI-23)
Bacillus altitudinis
(SRI-178)
Bacillus
xiamenensis
(BS-10)
Bacillus safensis
(BS-15)
Bacillus subtilis
(BS-17)
Bacillus altitudinis
(BS-19)
Bacillus altitudinis
(BS-20)
Brevibacterium
antiquum
(SRI-158)
192
Table 12.1 In vitro evaluation of PGPR strains for PGP and biocontrol traits
+
+
+
+
+
+
KX611375
+
+
KY800376
+
JQ247011
+
+
+
+
JQ247012
+
+
+
+
JQ247008
+
+
+
JQ247014
+
+
MF373465
+
KX583493
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
KX583495
+
+
+
KX583492
+
+
+
Chickpea
Gopalakrishnan et al. (2017)
Rice
Gopalakrishnan et al. (2012)
Rice;
chickpea
Gopalakrishnan et al. (2017, 2018)
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Understanding the Evolution of Plant Growth-Promoting Rhizobacteria
KX583496
12
Chryseobacterium
indologenes
(ICKM-4)
Chryseobacterium
sp. (ICKM-17)
Chryseobacterium
indologenes
(ICS-31)
Enterobacter
ludwigii (SRI-211)
E. ludwigii
(SRI-229)
Pseudomonas
plecoglossicida
(SRI-156)
Pseudomonas
monteilii (SRI-360)
Paraburkholderia
kururiensis
(IC-76A)
Pantoea dispersa
(ICKM-1)
Pseudomonas
geniculata (ICKM7)
Pseudomonas
geniculata (ICKM12)
(continued)
193
194
Table 12.1 (continued)
PGP properties
Biocontrol properties
PGPR
P. geniculata
(ICKM-14)
NCBI no.
KX611373
IAA
+
Sid.
+
HCN
+
Cel.
+
Lip.
+
Pro.
+
Chi.
+
Pseudomonas
geniculata
(ICS-30)
Rhizobium pusense
(IC-59)
Stenotrophomonas
maltophilia
(IC-2002)
Stenotrophomonas
pavanii (ICKM-9)
Stenotrophomonas
maltophilia
(ICKM-15)
Stenotrophomonas
acidaminiphila
(ICS-32)
KX611376
+
+
+
+
+
+
+
MF372582
+
+
+
+
+
+
+
MF372584
+
+
+
+
+
+
KX583494
+
+
+
+
+
+
+
KX611374
+
+
+
+
+
+
+
KX611377
+
+
+
+
+
+
+
β
+
1,3
P
Sol
Crops
evaluated
(greenhouse/
field)
References
+
+
+
+
+
P. Sambangi et al.
IAA Indole Acetic Acid (μg/ml); β, 1–3 β-1,3-glucanase (mg/ml); Sid siderophore; HCN hydrocyanic acid; Cel cellulase; Lip lipase; Pro protease; Chi chitinase.
For HCN production, the following rating scale was used: 0 ¼ no colour change; 1 ¼ light reddish brown; 2 ¼ medium reddish brown; and 3 ¼ dark reddish
brown
12
Understanding the Evolution of Plant Growth-Promoting Rhizobacteria
195
Streptomyces spp. produce various hydrolytic enzymes and acids that show antifungal ability against different agriculturally important fungal pathogens (Alekhya and
Gopalakrishnan 2017; Vijayabharathi et al. 2018; Kim et al. 2019; Gopalakrishnan
et al. 2019). This PGPR-plant interaction also enhances the jasmonic acid, salicylic
acid and ethylene production, which in turn activates the induced and systemic
acquired resistance to subdue the disease (Vleesschauwer and Höfte 2009). As the
agriculturally important crops namely chickpea, pigeon pea, groundnut and soybean
are more prone to the soil-borne pathogens, it is essential to utilize these
rhizobacteria for their broad spectrum of biocontrol and plant growth-promoting
activities.
12.4
Application of PGPR in Agriculture
As these soil rhizobacteria are advantageous in many ways and confer multiple
benefits to the agriculture, application of these beneficial microbes led to their
exploitation as biofertilizers and biopesticides. Nowadays many PGPR based
bioproducts with high competence are prevailing in the agri-market.
Actinorhizobium spp., Azotobacter spp., Azospirillum spp. and Rhizobium spp.
based biofertilizers are the promising nitrogen suppliers in the agriculture fields
(Marketsandmarkets 2014). The Bacillus spp. and Pseudomonas spp. were also
widely used as biopesticides to increase the plant growth and suppress the pathogen
(Sallam et al. 2013). Due to their unique specificity and less toxicity, PGPR are
further formulated with inoculants to enhance their shelf life. Inoculants such as peat,
compost, talc, alginate and chitosan are widely used to entrap these beneficial
microbes (Vijayabharathi et al. 2016). PGPR bioinoculants, especially Rhizobium
spp. could increase the bioavailability and shelf life of the bacteria in the field
conditions and also protects against the adverse climate conditions and native
microbial flora (Gopalakrishnan et al. 2016b).
In recent years, with the development of nanotechnology, techniques like micro
and nano-encapsulation are also being utilized for the efficient delivery of these
rhizobacteria. Nanoparticles such as silver, silica and chitosan, which are known to
enhance the plant growth are used to encapsulate these rhizobacteria and their
metabolites. This will aid in the improved efficacy and better management of plant
growth and yield (Nayana et al. 2020). Nowadays, nanofibers are also being used to
immobilize these microbial cells for targeted delivery (John et al. 2011). In order to
maintain the viability of these beneficial PGPR, the inoculum will be coated over the
seeds using spun nanofibers (De Gregorio et al. 2017). Apart from these, new
applicative approaches must be identified in the future to explore these PGPR for
more innovative bioproducts.
196
12.5
P. Sambangi et al.
Commercialization
Various research studies have confirmed the potential of PGPR such as Azospirillum,
Bacillus, Pseudomonas, Serratia and Streptomyces as growth-promoting and biocontrol agents in many horticultural and agricultural crops (Reddy 2014; Wani and
Gopalakrishnan 2019). These strategies have led to their successful commercialization as bioproducts in the agri-market. From strain discovery, lab to field, formulations and mass production many steps are to be undertaken to successfully
commercialize a PGPR product. Procedures such as documentation, regulations
and registrations of the PGPR bioproduct are the main challenges during commercialization. Over the years, the manufacturing industry of these rhizobial inoculants
has increased steadily in many countries. They are successfully applied either as
single inoculants or co-inoculants to various crops namely legumes, maize, wheat,
rice and sugarcane (Santos et al. 2019). But, factors such as long term efficacy,
viability and expenditure act as the limiting factors in their commercialization
(Hungria et al. 2005). Apart from that, the global market of PGPR products is also
influenced by the different patenting policies and legislations in each continent of
EU, US and Asia (Backer et al. 2018). But, measures are being undertaken to
establish new legislations, alternative technologies, educating farmers and to provide
financial support for successful product commercialization, as these PGPR reduce
the cost of synthetic agrochemicals for the farmers and lead towards low-cost
agricultural practices.
12.6
Conclusion
It is a well-established fact that these soil rhizobacteria are evolving day-by-day in
their plant–microbe association. Hence, for sustainable agricultural practices, the
researchers and entrepreneurs are attracted to these symbiotic microbes for their
plant growth and development, biofertilization, rhizo-remediation, biofortification
and disease resistance properties. The bioproducts of these PGPR are successfully
enhancing the agricultural yields but are often discouraged by their inefficacy and
less viability over a long time. Environmental factors also affect the growth and
proliferation of these rhizobacteria in the field conditions. Hence, these limitations
should be addressed by multidisciplinary research team for better PGPR formulations and crop protection.
Acknowledgement We thank Mr. PVS Prasad for his significant contribution in collecting the
literatures.
Conflict of Interest None.
12
Understanding the Evolution of Plant Growth-Promoting Rhizobacteria
197
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Chapter 13
Rhizobia–Legume Symbiosis During
Environmental Stress
Sriram Shankar, Ekramul Haque, Tanveer Ahmed, George Seghal Kiran,
Saqib Hassan, and Joseph Selvin
Abstract The rhizobia are microorganisms present in the soil that interact with
leguminous plants. Legumes form a unique symbiotic relationship with bacteria
known as rhizobia, which they allow to infect their roots. This in turn leads to root
nodule formation where bacteria are accommodated for carrying out the process of
nitrogen fixation. This symbiotic nitrogen fixation allows legumes to thrive in
habitats with limited nitrogen availability. In the present time various environmental
stresses such as desiccation, alkalinity, acidity, toxic doses of fertilizer, global rise in
temperature, salinity, etc. not only suppress the growth and symbiotic characteristics
of rhizobia but also change the effect the nodulation processes. Extreme environmental conditions have been found to adversely affect rhizobia–legume interactions
wherein rhizobial partner utilizes endogenous or exogenous osmolytes and secretes
specific proteins to alleviate the problem of aridity, salinity and toxicity. In this
chapter, we discuss about the diversity of rhizobia in soil, environmental stresses
affecting the rhizobia–legume symbiosis and their regulation.
Keywords Rhizobia · Nodulation · Salinity · Legume · Symbiosis
S. Shankar · E. Haque · S. Hassan · J. Selvin (*)
Department of Microbiology, School of Life Sciences, Pondicherry University, Puducherry,
India
T. Ahmed
Department of Biotechnology, School of Life Sciences, Pondicherry University, Puducherry,
India
G. S. Kiran
Department of Food Science and Technology, School of Life Sciences, Pondicherry University,
Puducherry, India
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_13
201
202
13.1
S. Shankar et al.
Introduction
The chief concern for the global agricultural market lies in offering sustainable food
supplies to approximately 8 billion people, wherein 40% of the global agricultural
lands are affected by the environmental stress related to drought, salinity and soil
toxicity. The cultivation of short duration crops, use of excessive fertilizers and
the extensive usage of herbicides and pesticides which played an important part in
the green revolution are the primary reasons for the degradation of soil quality. The
adverse soil conditions negatively affect the rhizobia–legume symbiosis. The agricultural significance of rhizobia–legume symbiotic interactions cannot be taken
lightly. This symbiotic relationship fixes 45 106 metric tons of N2 per year and
gives almost half the amount of nitrogen utilized in agriculture (Brockwell et al.
1995). For maintaining a sustainable supply of food, the amount of N2 required to
meet the global requirements is expected to double by 2030 (Tilman 1999). Therefore, the rhizobia–legume symbiosis will have positive impact on both environment
and agriculture. Moreover, generation of reactive oxygen species (ROS) such as
superoxide ion, hydrogen peroxide and hydroxyl radical can negatively affect the
survival of the cells of rhizobia. Drought area is mostly characterized by relatively
low humidity, high temperature, high evaporation and scanty rainfall, all of which
lead to soil salinity. It also results in the accumulation of salts and solutes,
hyperosmotic stress, deterioration of metabolism due to reduction in the water
activity and generation of ROS. In the symbiotic relationship, the legumes maintain
the production of protein-rich seeds while enhancing the efficiency of various cereals
and different crops on the basis of crop rotation (Graham and Vance 2003). These
exclusive characteristic features make legume plant farming very important for
sustainable agriculture which improves the quality of soil. However, the symbiotic
relationship is often adversely affected by many natural conditions, like drought and
soil salinity, soil pH, temperature, etc. (Zahran 1999). It is recorded that about 60%
production of legume in the developing countries happens in extreme drought
conditions (Graham and Vance 2003).
13.2
Diversity of Rhizobia in Soil
Rhizobia refers to polyphyletic group of genera that produces root nodules and fix
atmospheric nitrogen in a symbiotic relationship with legumes. They belong to the
largest and most remarkable metabolically distinct order Rhizobiales of the phylum
Alphaproteobacteria. The Genera includes Ochrobactrum, Bradyrhizobium,
Azorhizobium, Methylobacterium, Mesorhizobium, Devosia, Sinorhizobium,
Phyllobacterium and Rhizobium. Generally, rhizobia are isolated by smashing
nodules, spreading their contents on solid media rich in nutrients and examining
the colonies for the observance of abundant amount of exopolysaccharide slime.
There is a noticeable specificity among the species of rhizobia and legume. A
13
Rhizobia–Legume Symbiosis During Environmental Stress
203
specific rhizobial species can infect only a particular species of a legume. A group of
similar legumes which can be infected by a specific rhizobial species is known as
cross-inoculation group. N2 fixing nodules get established on roots only when
inoculated with an appropriate rhizobial strain.
Learning about rhizobial diversity is an indispensable biological resource and
endeavours to discover bacterial strains with fascinating features to boost agricultural productivity (Dai et al. 2012). Moreover, rhizobial residents in surface soil are
diverse because of environmental factors. Also, the distribution and genotype of
legume plants influence the existence and prevalence of rhizobia in soil. For
instance, Mimosa affinis, Phaseolus vulgaris vary in its nodulation selectivity for
legumes. While, Phaseolus vulgaris is quite nonselective for its nodulating companion, it gets nodulated by different rhizobia that include Rhizobium giardinii,
R. gallicum, R.etli, R. tropici Bradyrhizobium spp. and R. leguminosarum
bv. phaseoli. In contrast, M. affinis is quite selective and becomes nodulated by
R. etli only (Wang et al. 1999). Bradyrhizobium strains nodulate Genistoid host plant
(brooms) of Morocco and Spain which is a part of four different evolutionary
ancestries that contain B. canariense, B. japonicum and two anonymous genospecies
(Vineusa et al. 2005). Further, the diversity of rhizobial players in soil get affected by
abiotic factors which include pH, soil type, rainfall and temperature, and the type of
soil can further disturb the constitution of rhizobial community. This can be typically
demonstrated by a host plant where a legume growing in distinct terrestrial sites gets
nodulated with diverse rhizobial genera. For instance, Glycine max (soybean) is
frequently nodulated by B. japonicum worldwide; Nevertheless, at Xinjiang region
in China, Sinorhizobium fredii and Mesorhizobium tianshanense have been
retrieved from this legume. Furthermore, Rhizobium leguminosarum bv. trifolii
and bv. viciae were noted in bean nodules inhabiting Leon, France, while, additionally S. fredii, R. etli, and R. gallicum also have been documented in bean plants
thriving in Andalusia region (Velázquez et al. 2001). Conventionally
Mesorhizobium mediterranean and M. ciceri were documented from nodules of
Cicer arietinum, on the other hand, Ensifer meliloti (previously Sinorhizobium
meliloti) has been documented from nodules of this host plant growing in waterdeficient conditions in Tunisia (Romdhane et al. 2009). Likewise, E. meliloti was
also reported in C. arietinum host plant inhabiting Terai and Almora area of
Uttarakhand Himalayas, India (Rajwar et al. 2013). The belief that diversity of
rhizobia is determined by the type of soil is ascertained by recognition of distinct
rhizobia from the host Caragana plant inhabiting multiple ecological regions in
China contrasting in type of soil. Mesorhizobium genospecies I, II, IV, VI and VII
were identified from host Caragana inhabiting sandy soils of Mongolia.
R. yanglingense, M. tianshanense, M. temperatum, M. septentrionale, Rhizobium
sp. IV and M. genospecies III were identified in saline/alkaline soils and Rhizobium
sp. IV, M. genospecies VII and V and M. plurifarium growing in nutrient-rich forest
soils of Northwestern Yunnan locality (Lu et al. 2009). Furthermore, by studying
rhizobial diversity stress-tolerant natural isolates can be identified very easily
(Zahran 2001). Moreover, when non-hostile environments are tested, inhabitants
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S. Shankar et al.
regularly hold strains tolerant to non-acting stresses, possibly as flexibility to
acclimatize to novel challenges (Giller et al. 1997).
13.3
Interaction Between Legume and Rhizobia
One of the significant mutualistic interactions of great importance to humans is that
of legume nitrogen-fixing bacteria, a plant bacterial symbiosis. Angiosperms that
bear their seeds in pods which include plants of agricultural significance such as
peas, clover, soybeans, alfalfa and beans are termed as legumes. Food and agricultural sectors greatly depend on legumes and the ability of the legumes to grow
without the addition of nitrogen encourages agriculturalists economically as there is
no need to spend huge amount of money towards fertilizers and also minimizes the
land pollution which is caused due to the addition of chemical fertilizers. The
symbiosis partners are called symbionts and nitrogen-fixing symbionts are called
rhizobia. Formation of root nodules takes place when rhizobia infect the legume
roots where the bacteria help in nitrogen fixation. The steps in formation of root
nodules are as follows:
1. Both plants and the bacterium recognize the correct partner followed by the
adherence of bacteria to the root hairs.
2. Bacterium secrets signalling molecules known as Nod factors.
3. Invasion into the root hairs by the bacterium.
4. Formation of infection thread and migration to the main root.
5. Formation of bacteroids (modified bacterial cells) within plant cells followed by
the cell division to form a mature root nodule where the nitrogen fixation takes
place under anaerobic conditions involving the scavenging of oxygen molecules
by leghaemoglobin.
13.4
Role of Exopolysaccharides in Legume–Rhizobia
Interaction
The development of an efficient symbiosis does not rely only on the genes that are
required for symbiosis but also on genes required for the synthesis of different kinds
of cell surface polysaccharides. The rhizobia cell surface comprises of many different polysaccharides, which include), cyclic β-(1,2) glucans, lipopolysaccharide
(LPS), outer membrane-confined capsule polysaccharide (CPS exopolysaccharides
(EPS), gel-forming polysaccharide (GPS), neutral polysaccharide (NP or
glucomannan) and K-antigen polysaccharide (KPS) (Skorupska et al. 2006;
Janczarek and Skorupska 2011; Laus et al. 2006). LPS is present in the outer
membrane and is composed of lipid A, O-antigen polysaccharide and a core oligosaccharide, for symbiosis to occur lipopolysaccharide is very important (Fraysse
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Rhizobia–Legume Symbiosis During Environmental Stress
205
et al. 2003). Neutral CPS is present as an insoluble polysaccharide matrix and is
synthesized in the idiophase stage of Rhizobium trifolii and R.leguminosarum
growth (Zevenhuizen 1986). Whereas, periplasmic space contains cyclic neutral
β-(1, 2) glucans and play a prominent role in plant infection and hypo-osmotic
adaptation (Breedveld and Miller 1994). Rhizobial KPS structurally is similar to
K-antigens of E. coli (Becker et al. 2005). Whereas, glucomannan, that is entirely
localized towards a particular pole of the bacterial cell, provides high-affinity
adherence to lectin and nodulation of rhizobia (Laus et al. 2006; Williams et al.
2008). Rhizobia synthesizes heteropolymeric exopolysaccharides that are chemically different, species specific and made of linear or branched monomers comprising monosaccharides, like D-galactose, D-glucose, L-rhamnose, D-mannose acid
and D-galacturonic acid typically substituted with non-carbohydrate residues
(e.g. pyruvyl, acetyl, 3-hydroxybutanoyl groups and succinyl,) and D-glucuronic
(Cremers et al. 1991; Laus et al. 2005; O’Neill et al. 1991). Moreover, during stress,
EPS supports the survival of microorganisms by excluding toxic compounds such as
chloride ions and by forming a barrier to oxygen that encourages the higher
persistence of rhizobia (Lloret et al. 1998).
The secreted polysaccharides are given extraordinary importance due to their
functions (biofilm formation, safeguarding environmental stresses, adherence to
abiotic surfaces and roots and nutrient acquirement) which confers an adaptive
environmental advantage to the bacteria (Downie 2010; Skorupska et al. 2006).
Although the exact role played by EPS in symbiosis has been studied in detail, the
mechanism of action is still under investigation. Very few studies have been reported
about their mechanism in symbiosis. A possible mechanism is that EPS plays a
significant role in the advanced stages of infection thread initiation and in bacterial
release during symbiosis with Lotus japonicas (Kelly et al. 2013). The role played by
EPS in initiating the infection process has been intensively explored using Rhizobium leguminosarum and S meliloti symbioses. Mutants incapable of producing EPS
give rise to root hair curlings however, they lack the capacity to form infection thread
and nitrogen-fixing nodules (Cheng and Walker 1998; Leigh et al. 1985).
13.5
Role of ROS in Nodulation
The plant–microbe interaction consists of both the symbiotic relationship and plantphytopathogen association. The plant’s response to both the interactions are quite
similar in the initial stages. Microbes adhere to a compatible host plant tissue and
initiate the invasion process wherein the plant protects by the generation of reactive
oxygen species (ROS). ROS include singlet oxygen (1O2), superoxide radical
(O2 ), (H2O2), hydroxyl radical (•OH) and hydrogen peroxide. ROS are highly
known to have a negative impact on biomolecules like protein and nucleic acids by
oxidizing them. The plant’s defence mechanism reacts strongly to the high built up
of ROS that includes the restricting the spread of microorganisms through the
formation of strong cell wall barriers and induction of hypersensitive response
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S. Shankar et al.
which activates the secondary cell signalling pathways that is responsible for
additional defensive mechanisms. For instance, hydrogen peroxide is reported to
be the most important signal molecule for the induction of plant defensive responses
to both biotic and abiotic stresses (Bartoli et al. 2013; Djébali et al. 2011). Even in
the legume–rhizobia symbiosis the conventional plant defence response is carried
out. When rhizobia infects the legume host, the generation of hydrogen peroxide and
superoxide radical has been reported at the site of infection (Santos et al. 2001). This
resembles the hypersensitive response of plants in incompatible plant–pathogen
interactions. Even though this resembles the regular hypersensitive responses
exhibited by the plant, legume–rhizobia the host plant implements a distinct defence
mechanism (Mithöfer 2002). The nodule, an organ formed to confine the invading
bacteria has been widely reported as a defence mechanism in the legume–rhizobia
interaction. This is well understood when there is a defect in the surface polysaccharides of rhizobia it in turn affects nodulation leading to susceptibility towards
pathogenic responses (Oldroyd and Downie 2008). So a controlled ROS level must
be maintained by both the invading bacteria as well as by the legume host to weaken
the oxidative stress response, thus, the legume host and the invading rhizobia
inherently possess a collection of antioxidant molecules in the form of enzymes
that include superoxide dismutase, catalase and different peroxidases (Becana et al.
2000; Matamoros et al. 1999).
A few studies have reported that Sinorhizobium meliloti contains an array of
antioxidant enzymes which includes three catalases and two superoxide dismutases
(Minchin et al. 2008; Becana et al. 2010; Jamet et al. 2007). So the absence of any of
the three catalases or two superoxide dismutases in the mutants leads to amplified
sensitivity of S.meliloti to ROS. However, the prevention of nodule formation was
not observed (Jamet et al. 2007). Also, nod factors (NFs, signalling molecule)
released by rhizobia inhibits the efflux of ROS from the roots of the legume host
(Shaw and Long 2003). Nod factors play a significant role in suppressing the
generation of ROS by the plant which is highly essential for the compatible association among the host plant and rhizobia (Chang et al. 2009). Thus, for a successful
nodulation, the host defence system against the oxidative stress exhibited by ROS is
highly essential, as catalase deficit due to RNA interference in Medicago truncatula
roots, has been found to reduce nodulation, particularly during osmotic stress
situations. Even though the suppressing ROS is essential for nodule formation and
the compatible symbiosis, ROS like hydrogen peroxide is vital for the
transpeptidation and strengthening of plant cell wall formation. Previously, Chang
et al. (2009) has reported that the root hair curling and infection thread formation is
prevented when ROS production is inhibited. Finally, the difference in the ROS
accumulation differentiated at distinct time period during the symbiosis describes the
indispensable role of ROS at multiple steps in the nodulation.
13
Rhizobia–Legume Symbiosis During Environmental Stress
13.6
207
Environmental Stresses and Their Regulation
13.6.1 Temperature Stress
The temperatures close to the soil surface may be too high in arid and semiarid areas.
Egyptian sandy soils can reach 59 C when the atmospheric temperature is 39 C.
The temperature of soil declines quickly through penetration, reaching a modest temperature of 35 C, at 0.15 m. It looks as if rhizobial strains are unaffected by high
temperatures in soil than in laboratory conditions. The temperature plays a vital role
in the transfer of environmental signals among rhizobia and their host plant, consequently decreasing nodule formation. Low temperature prevents inter-organismal
signalling among the symbiotic associates. It has been revealed that low temperature
obstructs the synthesis and secretion of ligands (signal molecules) required in
initiating the nodulation process (Abd-Alla 2001).
The ideal temperature for rhizobial growth is 24–30 C (Zhang et al. 1995). Most
of the research on rhizobial temperature stress tolerance concentrates on common
bean and soybean microsymbionts. Soybean isolates develop feebly at 40 C and
none of the isolates was found to be capable to grow at 45 C (Chen et al. 2002).
Temperature stress can be of two types, cold and heat shock. The heat shock
response is analogous to the acid stress response considering the synthesized proteins. The heat shock proteins (HSPs) assist in tolerating heat by providing protection against heat to the bacteria thus maintaining internal homeostasis (Yura 2000).
Cold shock is just the reverse of heat shock. Instead of proteins denaturing and
misfolding, cells experiencing cold shock have to struggle with disorientation of
membrane integrity, cytosol fluidity and with the maintenance of secondary structures of DNA/RNA (Phadtare et al. 2000). The impact of temperature stress affecting
nodulation and nitrogen fixation has long been recognized, since the first studies on
this topic can be traced back to 1960s. Even prior to the nodule formation, the
temperature of root zone affects the rhizobia prevalence in soil, along with the
mutual transfer of molecular signals among the symbiotic associates (Sadowsky
2005). High temperature has been found to have an inhibitory influence on adherence of rhizobia to root hairs, root hair development and infection thread initiation
(Hungria and Vargas 2000). Nodule functioning is also affected by high temperatures. Specifically, affecting the rate of reactions involved in the production of
leghemoglobin or in nitrogenase activity. Low temperatures also influence nodulation, as cold temperatures interrupt nodulation initiation or even totally inhibit the
process and appear to affect nodule occupancy (Graham 1992). Also, prior studies
with B. japonicum highlight the fact that Nod factor synthesis markedly drops at
17 C or 15 C, in spite of the fact that its biological action remains unchanged
(Duzan et al. 2006). Soybean and bean have related threshold, whereas lentil is extra
tolerant and nodulation is considerably delayed only at subordinate temperatures
(10 C) (Junior et al. 2005)
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S. Shankar et al.
13.6.2 Salt Stress
Salinity is among the chief ecological threats towards agriculture and affects about
7% of the global total land space (Türkan and Demiral 2009). Biomass and defence
mechanisms are restricted due to salt stress (Zheng et al. 2009), and it also obstructs
the mechanism of symbiotic biological nitrogen fixation of the legume plants. The
negative impact of salt on biological nitrogen fixation in legumes has been widely
described, for instance, the effects include reduced photosynthate substrate supply to
the nodule, minimum supply of respiratory requirements to the bacteroids, and
variations in the oxygen diffusion barrier (Soussi et al. 1998).
The salt stress has been found to affect the activity of nodules in a few ways.
The higher concentration of solutes can hinder nodulation and affect nitrogenase
activity. There is a threshold level of few ions in the soil and if it increases
beyond that level, it acts as a factor that can contribute to salt stress. Previous
studies using soybean demonstrated that the contact between Bradyrhizobium
japonicum and root hairs in increasing salt concentrations (0% to 1.8%) affected
the properties of the nodule. Inhibition was apparent in the flexibility of the hairs
at a concentration of 1%, a phenomenon noticeably emphasized at increasing
concentrations. Growth was gradual at 0.2% salinity and the nodulation was
affected at 1.2% salinity.
A few studies using Vigna unguiculata (cowpea) and Vicia faba (pea) demonstrated that the administration of various concentrations of salt at various stages of
development of root hairs decreases the colonization of roots, coiled hairs, organisation of the hypodermic hair and hair cells, the quantity and weight of nodules and
activity of nitrogenase (Zahran and Sprent 1986; Georgiev and Atkins 1993).
Another study documented the impact of various NaCl concentrations on growth
and accumulation of polyhydroxybutyrate (PHB) in four distinct Sinorhizobium
strains (Arora et al. 2006). Oxygen flux into the nodule gets affected due to the
salt stress that alters the oxygen diffusion barrier (Serraj and Drevon 1998). The drop
in N2 fixation is due to the minimum supply of carbon to bacteroids, principally
because of malate limitation and salinity-induced inhibition of nodule through the
inhibition of the enzyme sucrose synthase (Salah et al. 2009). The accumulation of
compatible solutes like sucrose, D-pinitol and proline, in the nodules of some
legumes such as cowpea and alfalfa has been described and they employ an
osmoregulatory activity during salt stress (Irigoyen et al. 1992).
In the legume- rhizobia symbiosis, the bacterial partner can tolerate salt stress
efficiently than their host which displays disparity in the tolerance level (Zurayk
et al. 1998). 100 mM NaCl can inhibit the growth of several rhizobia (Yelton et al.
1983), but the previous studies have described that several species can withstand salt
concentration ranging from 300 to 700 mM (Mohammad et al. 1991). Another study
reported that R. leguminosarum can survive even upto 350 mM NaCl concentration
(Breedveld et al. 1991). The rapidly growing strains of chickpea and soybean
rhizobia can also survive upto a concentration of 340 mM of NaCl related to slowgrowing bacterial strains (Elsheikh and Wood 1995). Mashhady et al. (1998) have
13
Rhizobia–Legume Symbiosis During Environmental Stress
209
documented that S. meliloti developed productive mutualism with M. sativa in
salinity conditions, i.e. 100 mM NaCl (Mashhady et al. 1998). Among the tree
species, Prosopis articulata, P. pallida and P. tamarugo can grow and fix nitrogen
in less than 300 mM NaCl concentration (Felker et al. 1981). Australian Salttolerant Acacia (A. stenophylla and A. auriculiformis) can grow in the range from
1.7 to 1.8 M NaCl (Aswathappa et al. 1987).
In Phaseolus vulgaris (common bean), a negative impact of NaCl on the expression of nod genes and nodulation factors by Rhizobium etli, Rhizobium tropici was
observed (Dardanelli et al. 2008). The preincubation of B. japonicum using the
signal molecule genistein, in saline conditions, was designated as a method to
improve the stressful impact of salt on soybean–B. japonicum symbiosis (Miransari
and Smith 2009). High salt (NaCl) concentrations modify the metabolism of rhizobia
and also influence nitrogen fixation (Dowling and Broughton 1986). Nevertheless,
rhizobia strains, that are salt-tolerant and effective nitrogen fixers, have been isolated
(Rai 1983). The hunt for strains of rhizobia that can tolerate salinity can perhaps be
an alternative for successful symbiosis in conditions wherein crop growth is not
altered (Douka et al. 1984; Rosas et al. 1996).
13.6.3 Drought Stress
Crop production and symbiotic N2 fixation are affected due to drought. The biological N2 fixation is also extremely sensitive to water deficit. When exposed to soil
water deficit, several rhizobia exhibit a drop in nitrogen fixation (Zahran 1999;
Pimratch et al. 2008). Regulation of water loss is the primary mechanism by
which plants escape drought stress and is controlled either by morphological
changes or by physiological adaptations. Structural changes include stiffening of
waxy cuticle and expansion of root systems. Moreover, responsive stomata, leaf
rolling and plant hormone secretions are the physiological adaptations that plants
carry out to combat drought (Turner et al. 2001). Therefore, plants with water
potential lower than that of soil persist (Tiaz and Zeiger 2002). Both root development and root hairs have been reported to be greatly affected by drought stress that
results in the inhibition of nodule formation, it has also been observed that watering
the soil helped in the renewal of growth. Reduction in the soil water potential
has been observed to affect the formation of infection threads and has been reported
to cause complete inhibition of nodulation (Worrall and Roughley 1976). After the
start of infection, a restricted supply of water may slow the nodulation and quicken
its senescence. There is also a reduction in nitrogenase activity complemented by the
reduction in the respiration of common bean and nodules of soybean (Weisz et al.
1985; Ramos et al. 2003) A curb in the metabolism of bacteroids and oxidative
damage of cellular constituents are causative factors for the inhibition of nitrogenase
enzyme activity in nodules of alfalfa (Naya et al. 2007). Furthermore, the transfer of
fixed nitrogen out of the nodule is lessened, because of an inadequate supply of
photosynthates in leaves and stems that are under stress (Huang et al. 1975). A
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S. Shankar et al.
subordinate rate of water flow out of the nodule throughout drought stress can
control the export of N2 fixation metabolites and restrict nitrogenase activity through
a feedback mechanism (Serraj and Drevon 1998). Oxidative stress in nodules is
stimulated due to drought. This results in an overall reduction in the antioxidant
performances that are associated with nodule senescence (Porcel et al. 2003). It has
been reported that water stress prevalent during vegetative growth is unfavourable
for nodule formation and biological nitrogen fixation than the water stress that
occurs during the reproductive phase (Pena-Cabriales and Castellanos 1993). The
exact factors regulating desiccation are unknown. A few mentioned mechanisms are
the capacity to limit cellular metabolism, the better catalase activity and also the
existence of particular plasmids for drought tolerance. A substantial known evidence
has demonstrated the changes in desiccation tolerance of distinct rhizobial species
(Al-Rashidi et al. 1982; Vriezen et al. 2007). Free-living rhizobia have the potential
to survive during lower water potential or drought stress (Fuhrmann et al. 1986).
Although, population densities tend to decrease during the maximum desiccated
conditions and increase as the conditions improve. The survival and activity of
microbes rely on their distribution within microhabitats and variations in soil
moisture (Orchard and Cook 1983).
A few studies have presented that a mutualistic association can happen while
working with legumes and rhizobia strains selected for desiccation tolerance (Soria
et al. 1996), while, most legumes are sensitive to surplus water (flooding). The
capability of aerobes to use nitrogenous oxides, as terminal electron acceptors,
allows them to persist and grow during stages of anoxia. This may be beneficial to
the survival of rhizobial species in soils (Zablotowicz et al. 1978). Jenkins et al.
(1987) found a rhizobial species that nodulate arid legumes (Jenkins et al. 1987).
Another study demonstrated that osmotolerant rhizobial species can migrate even
during scarce moisture conditions (Wadisirisuk et al. 1989). Athar and Johnson (1996) identified that osmotolerant strains of S. meliloti performed well than
that of the non- tolerant alfalfa rhizobial species and developed effective symbiotic
association during drought stress (Athar and Johnson 1996).
13.6.4 Agrochemical Based Stress
The expanding global population causes insufficiency in natural resources, especially in the requirement of food. Therefore, to improve crop protection and increase
production, numerous agrochemicals are being utilized. Agrochemicals, such as
pesticides are highly essential in crop protection against several pests. The extreme
utilization of pesticides over many years has triggered environmental complications
such as aggregation of agrochemicals in groundwater and soil. Moreover, merely
15% reach the target when agrochemicals are applied to a plant. Moreover, factors
like leaching, soil sorption, volatilization, surface runoff and their acquisition by
plants determine their destiny in the soil. Agrochemicals in soil influence both micro
and macroorganisms comprising bacteria, fungi, actinomycetes, earth-worms,
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Rhizobia–Legume Symbiosis During Environmental Stress
211
Table 13.1 Frequently used agrochemicals and their impact on rhizobia
S. no.
1.
2.
3.
4.
5.
6.
7.
8.
Pesticides
Thiamethoxan
(I)
Glyphosate
(H)
Metribuzin (H)
Mancozeb (F)
Captan (F)
Thiaram (F)
2,4-D(H)
Lindane (I)
Atrazine (H)
Hexaconazole
(F)
Effects
Reduction in the synthesis of Auxin (IAA) and siderophore
Reduction in the growth of rhizobia by altering membrane composition
Inhibit the growth of Sinorhizobium, Rhizobium, Bradyrhizobium,
Mesorhizobium
Reduction in rhizobium and Mesorhizobium population
Alteration of membrane fluidity
Inhibit the growth of Rhizobium japonicum
Reduction in nodule numbers
Inhibition of dehydrogenase system in Rhizobium
I insecticides; F fungicides; H herbicides; IAA indole acetic acid
crustaceans, nematodes, arthropods and legumes. Soil microbes have a crucial role
in the detritus organic matter degradation along with nitrification, nitrogen fixation
and discharge of numerous nutrients from the soil (Pandey and Singh 2004; Seghers
et al. 2003). Therefore, the applicability of agrochemicals poses a main risk to
beneficial soil microorganisms and affects the sustainability of agronomy. Likewise,
they unite with rhizobia and decrease the number of existing root sites that are
required for initiating infection (Anderson et al. 2004). Additionally, agrochemicals
block the transfer of signals among rhizobia and legumes and hinder the communication between them (Fox et al. 2007).
Pesticides are agrochemicals that safeguard agricultural plants from insects,
weeds or pathogens. Although pesticides are utilized for protecting crops, they
eventually reach soil system and affect microbes and plants. Pesticides can have
an impact on the diversity of rhizobia in soil and rhizobia–legume symbiosis
(Ahemad and Khan 2013). Pesticides affect rhizobia–legume symbiosis in various
means. Their impacts are summarized in Table 13.1. Firstly, they result in decrease
of root biomass as there is reduction in the sites available for initiating infection
which ultimately affects the transport of sugars to the prevailing nodules with
subsequent reduction in the capability of rhizobia to initiate infection. Accordingly,
the activity of meristem falls which is crucial for nodule development (Anderson
et al. 2004). Secondly, they inhibit nodulation by blocking the signalling molecules
(Flavanoids and nod factors). Finally, the competence of pesticides and rhizobia for
common adhering site in the roots.
For instance, glyphosate [N-(phosphonomethyl) glycine] is a nonspecific herbicide that competes for adherence sites on plant roots and gets transported via
phloem. Glyphosate blocks synthesis of aromatic amino acids and other phenolic
compounds like flavones (Ishikura et al. 1986) cinnamic acid (Cañal et al. 1987) by
targeting shikimate acid pathway that ultimately reduces plant growth. The activity
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S. Shankar et al.
of this herbicide reduces starch synthesis (Geiger and Bestman 1990) causes inequity
in phytohormones (Lee 1984), decreases synthesis of chlorophyll (Kitchen et al.
1981) and its precursor δ-aminolevulinic acid (ALA), and affects the performance of
nitrogenase enzyme complex (Mallik and Tesfai 1984). The quantity of total nitrogen fixed by leguminous plants is indirectly decreased and photosynthesis is
prevented by the action of herbicide. Fluchloralin and Metribuzin prevent the
movement of electrons from compound Q to plasto-quinone in the electron transport
chain of photosystem II, inhibiting reduction of NADP+ which is required in Calvin
cycle (Fedtke 1982). Although, in the presence of herbicide the activity of nitrogenase remains unaffected.
A huge number of studies are carried out using soybeans which is the first legume
cultivated on a large-scale globally; and in various instances the results related to
agrochemicals were contrary. For instance, trifluralin, a herbicide has been reported
to have an inhibitory effect on nodulation in soybean (Parker and Dowler 1976).
Furthermore, it has been found that trifluralin influences the number and weight of
nodules formed and activity of nitrogenase enzyme (Bollich et al. 1985), whereas
Eldin et al., previously proposed that greater dosage of trifluralin than generally
administered promoted soybean nodulation (Eldin et al. 1981). Likewise, a study
suggested that pentachlorobenzene (PCNB), a fungicide decreases nodulation in
soybean (Curley and Burton 1975). Whereas another study indicated that it is
harmless even when ten times higher dosages were administered than normal
(Mallik and Tesfai 1984). Laboratory scale studies also exhibited the negative
impact of pesticides and fungicides on the persistence of microorganisms.
13.6.5 pH Stress
Rhizobia are generally known to grow in optimum pH between 6 and 7 (Hungria and
Vargas 2000). They greatly vary in tolerating acidic conditions. Although, a few
mutants of Rhizobium leguminosorum can grow at a pH near to 4.5 (Chen et al.
1993). Sinorhizobium meliloti has been found to grow only when pH is near to 5.5
(Foster 2000). S. fredii has a higher range (4 to 9.5) of pH for survival, whereas
Bradyrhizobium japonicum lack the extreme extend of pH for their survival
(Fujihara and Yoneyama 1993). The efficacy of symbiosis between legume–rhizobia
is significantly determined by the pH of the soil (Glenn and Dilworth 1994). The
limiting factor for a successful symbiotic association is the pH of the soil as rhizobial
strains can grow only in a neutral or acidic soil. Rhizobium Alkaline or acidic soil has
a negative impact on the survival or multiplication of rhizobia and can affect both
symbiotic companions (Zahran 1991). Most of the agricultural lands are alkaline
having a pH ranging from 7.0 to 8.5. Thus, stress conferred by the alkalinity can
interfere with Rhizobium growth and successive formation of nitrogen-fixing symbiosis with legumes. Hence, it is logical to select rhizobia isolates that can withstand
the alkaline pH which results in the capability of the rhizobia to nodulate (Farissi
et al. 2014). Well characterized rhizobial defence mechanism has been recorded
13
Rhizobia–Legume Symbiosis During Environmental Stress
213
at acidic pH and can also be applicable to basic pH (Fujihara and Yoneyama 1993).
A common mechanism employed by the bacterium such pH stress is by raising the
internal pH of the cell with the help of ABC transport system and other transport
mechanisms either expelling H+ ions out of the cell or by influx of basic ions into the
cell (Priefer et al. 2001). Alternative mechanism against this acid shock is by
synthesizing acid shock proteins (ASPs) by the bacterium. However, they do not
modify the internal pH of the cell but provide acid protection (Foster 1993). Kurchak
et al. (2001) reported almost 20 genes that play a key role in protecting Rhizobium
leguminosarum from acid stress and are termed as act genes (Kurchak et al. 2001). A
form of cellular signalling must be carried out by the bacteria to sense the acid stress.
The key players involved in this type of signalling consist of a sensor and a regulator
and a typical acid stress sensing signal coded by actR gene (Regulator) and actS
(Sensor) in S.meliloti (Tiwari et al. 1996).
13.6.6 Waterlogging Stress
A few research studies have highlighted the effect of waterlogging on nodulation
capacity. Legumes like alfafa, soybean and pea display reduction in nodule weight
when they are treated under hypoxic conditions (Minchin and Pate 1975; ArreseIgor et al. 1993; Sung 1993).When 0.1% of oxygen concentration was maintained
Medicago truncatula exhibited 45% reduction in nodulation capacity and was not
having any impact when treated under 4.5% of oxygen concentration (Pucciariello
et al. 2019). Effective nodulation was demonstrated after few weeks by 21 species of
legumes that included waterlogging sensitive species like Medicago sativa and
Melilotus albus (Nichols et al. 2008). It is uncertain whether the type, nature of
nodule could demonstrate distinct mechanisms of combating waterlogging stress as
Medicago spp., Melilotus spp. and Pisum spp. possessing indeterminate nodules
show persistent meristem and continuous growth characteristics compared with
Glycine spp., Lotus spp. and Vigna spp exhibiting non-persistent and limited growth
characteristics. A few phenotypical and physiological adaptations can be seen in
nodulation process of flood-tolerating legumes. Nodules formed on Melilotus
siculus, in the course of waterlogging stress has been observed on adventitious
roots (Rhizopoulou and Psaras 2003).
13.7
Conclusion
The effect of environmental factors on legume–rhizobia interactions is of great
importance as they control and regulate the symbiotic process. Survival and proliferation of rhizobia in rhizosphere and surface soil are greatly affected by abiotic
factors as they can restrict the infection thread formation, metabolism of nodule and
alteration of legume growth. The rhizobial population is distinctive in their
214
S. Shankar et al.
efficiency to tolerate major environmental stresses like pH, temperature, salinity,
drought and agrochemicals, thus selecting resistant strains is considerably preffered
option. Many strains of rhizobia that can tolerate various environmental stresses
have been isolated for different crops and wild legumes and this seems to be a
favourable strategy that may promote the efficient interaction among rhizobia and
legume resulting in increased agricultural productivity. Moreover, innovative and
efficient mixtures of formulation using tolerant strains and factors (Flavanoids, Nod
factors) involved in the nodulation process appears to be a promising technological
tool for the betterment of agronomy. These products can be used to extend the
cultivation of native or naturalized legumes and increase the efficiency and potential
of rhizobia–legume interaction which can increase agricultural productivity.
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Chapter 14
Archaeal Symbiosis for Plant Health
and Soil Fertility
Ranjith Sellappan, Senthamilselvi Dhandapani, Anandakumar Selvaraj,
and Kalaiselvi Thangavel
Abstract In plant microbes play an indispensable role for their growth and immunity. Microbes are capable of colonizing the plant rhizosphere, phyllosphere and
internal part of the plant (endophytes). Plant growth-promoting microorganisms
(PGPM) like bacteria, fungi and archaea improve the plant growth through nutritional (nutrient solubilisation, phytohormones production) and non-nutritional
(induce defence) mode. Archaea are an important group of microbes which have
distinct character and disseminated not only in an extreme environment, they have a
broad range of habitat. Plant growth-promoting archaea (PGPA) play a pivotal role
in biogeochemical cycle and making accessible of important nutrients like C, N, S
and P to the plants through fixation, solubilisation. It also helps in plant growth by
phytohormones and siderophore production. Archaea elicit a defence response of the
plant against both biotic and abiotic stress through inducing systemic resistance
(ISR) of the plants. Soil the substrate for plants, contains huge microbial (bacteria,
fungi, archaea) population which improves the soil health through nutrient cycle. So
archaea are an important group of microorganism having special attention in agricultural production. However, archaea occupy 20% of world biomass and play
important role in plant growth similar to bacteria and fungi it not well studied.
Understanding archaeal plant interaction is necessary for improving the plants grown
in different environmental conditions. Only a few studies focused on archaeal plant
interaction. This chapter briefs the immense role of archaea in plant development
and soil fertility improvement.
Keywords Archaea · Plant growth-promoting archaea · Soil health · Induced
systemic resistance
R. Sellappan (*) · S. Dhandapani · A. Selvaraj · K. Thangavel
Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore,
Tamil Nadu, India
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_14
221
222
14.1
R. Sellappan et al.
Introduction
Archaea are single-celled prokaryotic organisms that have a distinct character from
bacteria. Archaea are extremophiles because of their inhabitants in varied stress
environmental conditions like high temperature, pH, salinity, anoxic condition and
terrestrial soil (Flemming and Wuertz 2019). Most culturable and well investigated
archaeal species are phylum Euryarchaeota and Crenarchaeota and the other phylum
Nanoarchaeota, Thaumarchaeota, Aigarchaeota and Korarchaeota have also been
reported (Huber et al. 2002; Baker et al. 2006). For a long time archaea consider as
the organisms which grow only in the extreme environment, but at the end of the
twentieth century, it was found that the extremophiles (archaea) also present in nonextreme conditions (Reitschuler et al. 2016) using metagenomics approaches. They
are abundantly present in marine, freshwater, normal terrestrial soil.
Soil the hotspot of the microorganisms, their population varies based on the soil
conditions. Plant roots have an intimate association with numerous soil microorganisms including bacteria, archaea and fungi. These microbes modulate plant health in
both beneficial and detrimental manner. Among these microbe plant growthpromoting microorganisms play a pivotal role in plant health through nutritional
and non-nutritional mode. Microbes not only present in the root, it also presents as
epiphytic and endophytic in the plant. Archaea are the third-largest domain after
bacteria and fungi in soil (Hassani et al. 2018). Plant growth-promoting archaea play
the major role in biogeochemical cycling and protection against pathogen in plants.
The role of plant growth-promoting bacteria and fungi for plant development was
studied, but the role of archaea in plant growth promotion was not fully studied.
Archaea represent an important component in plant microbiome, but their impact on
the host is still unclear. This chapter discusses the important role of soil archaea in
soil fertility improvement and plant growth and development.
14.2
Distribution of Archaea in Soil
Archaea exist ubiquitously and as a foremost portion of global biomes like marine,
sediments, freshwater, soil and hydrothermal vents (Lee et al. 2019) may contribute
up to 20% of earth’s biomass (Yadav et al. 2017). After the recognition of the
archaeal domain (Woese et al. 1991) understanding of archaeal diversity, biology
and ecology were not fully understood. Euryarchaeota and Crenarchaeota the two
major cultivated archaea defined by Carl woese. Of these, Crenarchaeotaes are
majorly distributed and known to dwell environments like hot springs, geysers and
ocean vents which are inhospitable to many other organisms (Yadav et al. 2017).
Commonly Crenarchaeota phylum is abundant in cultivated soil (Bates et al. 2011),
but the structure of the soil community and functional diversity are remain poorly
understood.
14
Archaeal Symbiosis for Plant Health and Soil Fertility
14.3
223
Plant Archaeal Microbiome
Distributions and ecological assortment of soil archaeal communities in agricultural
ecosystem are essential for increasing the crop productivity and soil fertility.
Archaeal microbiome varies depending on different soil conditions and plant genotype (Müller et al. 2015). Based on the 16s rRNA Crenarchaeota and Euryarchaeota
retrieved from the soil (Borneman and Triplett 1997). In a later analysis of seven
diverse soil, Crenarchaeota was found abundant (0.5–3%) in bulk sandy soil samples, but Festuca ovina grown in same soil only 0.16% archaea were retrieved (Buée
et al. 2009). Lee et al. (2019) reported the archaea in different rhizocompartment of
tomato plant root and the dominant archaeal phylum was Thaumarchaeota followed
by Euryarchaeota. These phyla also dominated in Jatropha curcas (Dubey et al.
2016) rhizosphere. Archaeal population is more in mycorrhizosphere using glycogen
as food sources (Taffner et al. 2018). Phyllosphere microbes present in surface of
plant the leaves; plays an important role in plant growth and health. Occurrences of
the phyllospheric archaeal community were low compare to rhizosphere (Taffner
et al. 2019; Bringel and Couée 2015). In rice plant, methanotrophs are abundant in
phyllosphere region (Knief et al. 2012). Archaea associated with arugula
phyllosphere are Thaumarchaeota and Euryarchaeota groups. Endophytes reside
inside the plant which improves the plant growth and health. Plant endophytic
archaeal taxa belong to phyla Crenarchaeota, Thaumarchaeota and Euryarchaeota
(Hassani et al. 2018) but their function in plants remain to be clarified. Wassermann
et al. (2019) isolated the endophytic archaea in alpine plant seeds. It is dominated by
Thaumarchaeota and Crenarchaeota, but Euryarchaeota is less represented. Colonization levels of archaea in the rhizosphere and endosphere are high compared to
phyllosphere (Taffner et al. 2019).
14.4
Role of Archaea in Biogeochemical Cycling
Microorganisms play the important role in biogeochemical cycle, which improves
the availability of like N, P, S and K to plant for growth and increase soil fertility.
Microbial inoculum improves the plant growth in nutrient-poor soil and it also
improves the soil nutrient status. An archaea domain serves as enhancing agricultural
production because of their different habitats and plays the major role in nutrient
cycling.
14.4.1 Carbon Cycling
Carbon is the main building block of the plant metabolism. Catabolic degradation of
organic substrates by chemoorganotrophic microbes results in the production of CO2
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as the main end product and absence of terminal electron acceptor results partially
oxidized compounds. Numerous archaea are growing organotrophically in both
aerobic and anaerobic conditions. Archaea play the major role in carbon mineralization process major groups are Euryarchaeota and Crenarchaeota.
CH4
Methanotrophs
Methanogen
CO2
Carbon mineralization
Organic carbon
After the mineralization process, CO2 converts into methane by methanogenic
archaea. Methanogenic archaea (Methanobacteria spp., Methanococci spp., etc.) in
the carbon cycle contribute immensely to the decomposition of organic matter by
removing hydrogen. Euryarchaeota colonizes the rice roots, which utilize the carbon
of plant and produce methane (Buée et al. 2009). Methylotrophic archaea
(Methanosarcinales spp., Methanoplasmatales spp., etc.) convert this methane into
CO2 by anaerobic oxidation. Methanogens and anaerobic methane oxidation are two
processes of global importance that are performed exclusively by archaea.
14.4.2 Nitrogen Cycle
Most of the organisms uptake the nitrogen in the form of NH3 (inorganic form) or
nitrate (NO3) /organic forms. The atmospheric nitrogen enters into the biogeochemical cycle by a group of bacteria/archaea as ammonia (NH3) followed by nitrification
and denitrification process occurs. Hitherto we recognized only lithotrophic group of
bacteria involved in nitrogen fixation. But recent discoveries of methanogens are
recognized as the broadened nitrogen fixer in archaea. Diazotrophic methanogens
belong to three major taxonomic classes, i.e. Methanobacteria, Methanococci and
Methanomicrobia. These methanogenic archaea are ubiquitous in aerated soils and
become active under anoxic conditions (Angel et al. 2012). Ammonium oxidizing
archaea (AOA) belong to the Thaumarchaeota (Santoro and Casciotti 2011) which
oxidize the ammonia and convert it into nitrite then nitrate by bacteria. Only few
cultivable archaea are recognized as the denitrifiers, for example, Pyrobaculum
aerophilum.
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225
-
Nitrogen (N2)
N fixation
Nitrate (NO3 )
Nitrite (NO2 )
Ammonia (NH3)
Nitrification
AOA (Thaumarchaeota)
Nitrification
14.4.3 Sulphur Cycle
Sulphur reduction (Euryarchaeota,
Crenarchaeota, Thermocladium)
In plant sulphur presents as amino acids like cysteine, methionine and some substance like thiamine, biotin. It also indirectly influences the plant chlorophyll and
nodulation in legumes. Bacteria, archaea, fungi involve in sulphur cycling. Elemental sulphur may respire with H2 or organic compounds as electron donors. These
metabolisms are performed by Crenarchaeota and Thermoplasmata groups (Dubey
et al. 2016).
Hydrogen sulphide (H2S), Elemental sulphur (So)
Sulphur oxidation
(Euryarchaea)
Sulphur reduction (Thaumaplasmatales
and Crenarchaea)
Sulphate (SO 2-)
Dimethyl sulphide (DMS) is one of the sulphur-based compound on sulphur
cycle which is used for cloud formation have an advantage in reducing the global
warming.
14.4.4 Phosphorous Cycle
Phosphorous (P) is one of the essential elements after nitrogen for plants. On the
average content of P in soil is 0.05%, but only 0.1% of these available for plants.
This problem is mitigated by phosphorus solubilizing microorganisms. They are
enormous soil bacteria, fungi are identified as phosphate solubilizer but in the case of
archaea only the meagre report available (Alori et al. 2017; Yadav et al. 2017).
Yadav et al. (2015) identified the P solubilizing halophilic archaea from plant
Abutilon, Cenchrus, Dichanthium, Sporobolous and Suaeda nudiflora. Among the
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isolates Natrinema sp. and Halococcus hamelinensis solubilized phosphorus 134.61
and 112.56 mg/L. They produce organic acid to solubilize the phosphorus (gluconic,
citric, succinic, oxalic, lactic). These archaea groups may play the role in P
solubilisation for crops growing in saline environment.
14.5
Archaea in Plant Health Improvement
Plant growth-promoting rhizobacteria (PGPR) improves the plant growth through
nutritional and non-nutritional mode. PGPR improves plant growth by nutrient
availability and elicits defence of plants against the pathogen and herbivores were
well studied. Similar to PGPR, archaea also improve the plant growth and immunity.
Hence it is referred to as a plant growth-promoting archaea (PGPA). Several studies
reported that archaea improve the plant growth through siderophore production
(Dignam et al. 2018), phosphorus solubilization (Yadav et al. 2015), nitrogenfixing methanogens (Leigh 2000) and auxin production (Taffner et al. 2018). But
only a few studies have focused on the archaeal role in disease resistance. PGPA
induce the systemic resistance (ISR) against the plant pathogenic bacteria via
salicylic acid- independent signalling pathway. Song et al. (2019) reported
Nitrosocosmicus oleophilus MY3 determinants stimulate ISR against both
necrotrophic and biotrophic pathogen by colonizing on the root of Arabidopsis
thaliana. Simultaneously, it also releases volatile compounds that elicit the defence.
Archaea also provide resistance to abiotic stress to plant through osmoprotectants
(Smith-Moore and Grunden 2018), antioxidant enzymes (Grunden et al. 2005).
14.6
Conclusion
Archaea contribute nutrient cycle and offered various biotic and abiotic stress
tolerances to plant. Because of its uniqueness, archaea adopt several ecosystems
having special attention in agriculture, industry and medicinal application. Archaea
are still an understudied area of plant microbiome and their role in plant growth and
immunity. Understanding the plant archaeal interaction paves the way for new
insight in plant grown in different environment conditions and their health
improvement.
Acknowledgements Thank to Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore.
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227
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Chapter 15
Microbial Symbionts of Aquatic Plants
Tejaswini Dash, Klaus-J. Appenroth, and K. Sowjanya Sree
Abstract Aquatic plants have been used over time in several ways which include
use as food and feed supplement, for bioenergy production, for phytoremediation
and wastewater treatment, to name a few. Like in the terrestrial ecosystem, interactions between microorganisms and plants are diverse in an aquatic ecosystem. In the
present chapter, one of the positive interactions, symbiosis between the microorganisms and aquatic plants has been discussed focusing on how this interaction can
prove to be beneficial to aquatic plants. Some of the features like biofiltration,
nitrogen fixation, phytohormone production, and bioenergy production from aquatic
plants and the role of microbial symbionts in these processes have been discussed.
One of the re-emerging model plants, duckweeds have been considered as an
example in order to describe the abovesaid. Lemnaceae (duckweeds) constituting
the fastest-growing flowering plants have gained attention in terms of their practical
applications because of their huge potential for biomass production. As a future
prospect, sustainable ways of use of aquatic plants and their biomass production
should be a prime focus in aquatic research.
Keywords Aquatic plants · Bioenergy production · Biofiltration · Duckweeds ·
Lemnaceae · Microbial symbionts
T. Dash · K. S. Sree (*)
Department of Environmental Science, Central University of Kerala, Periye, India
e-mail: ksowsree9@cukerala.ac.in
K.-J. Appenroth
Matthias Schleiden Institute – Plant Physiology, Friedrich Schiller University of Jena, Jena,
Germany
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_15
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15.1
T. Dash et al.
Introduction
Symbiosis is a positive ecological interaction between biotic components of an
ecosystem, in which all the interacting partners benefit from this relationship. Such
relationships can exist in both terrestrial (Unnikumar et al. 2013) as well as aquatic
(Appenroth et al. 2016) ecosystems. In the present chapter, we will focus on this
symbiotic relationship between microorganisms and plants in a freshwater aquatic
ecosystem and how this interaction proves to be beneficial to the plant community.
The multitude of organisms living in either lotic or lentic ecosystems, like any other
ecosystem, need to adapt themselves to the existing environmental conditions. So
also the aquatic plants, which are the primary producers of an aquatic ecosystem.
These adaptations can either be through changes or modifications in morphology,
anatomy, physiology or can be through their positive and negative interactions with
the rest of the biota in their environment. The plants belonging to several different
groups of lower plants as well as angiosperms have adapted to these aquatic
environments. Some of the examples are water hyacinth (Eichhornia crassipes),
Indian lotus (Nelumbo nucifera), water spinach (Ipomoea aquatica), water mimosa
(Neptunia oleracea), mosquito fern (Azolla pinnata), water lettuce (Pistia
stratiotes), and duckweeds (Lemnaceae, e.g., Wolffia globosa). The aquatic plant
community is also diverse in its ability to inhabit different strata of the water body.
They can be free-floating, submerged, or rooted. Equally diverse are the microbes of
the aquatic ecosystem. They belong to several different groups of microorganisms
and can differ in their capacity to interact with the plant body, e.g., ectophytes and
endophytes. The positive interaction between these two groups of biotic community
has been tapped by several fields of applied biology.
15.2
Uses of Aquatic Plants
Several uses of aquatic plants and their economic benefits have been reviewed by
Wersal and Madsen (2012). They not only provide ecosystem supporting services
but also such of the benefits that are being utilized by human beings.
Aquatic plants can uptake nutrients from contaminated water and consequently
purify the water (Zimmels et al. 2004). This phenomenon has been put to practical
applications as treatment of wastewater by phytoremediation is an environment
friendly approach (Ziegler et al. 2016, 2017, 2019; Calado et al. 2019). Various
aquatic plants have been used for remediation of wastewater contaminated with
several industrial pollutants. Heavy metal removal by E. crassipes, Hydrilla,
Jussiaea repens, Lemna minor, Pistia stratiotes, and Trapa natans was studied
and L. minor was found efficient for remediating the particular wastewater (Mishra
et al. 2013). In other respect, halophytic plants like Salicornia europaea, Salsola
crassa, and Bienertia cycloptera have been investigated for salt removal capacity
(Farzi et al. 2017). Some other halophytic species like Atriplex barclayana have
15
Microbial Symbionts of Aquatic Plants
231
been found to act as biofilters to remove nutrients from wastewater (Brown et al.
1999).
Duckweeds are some of the well-studied aquatic plants. Duckweeds include the
smallest and fastest-growing free-floating flowering monocotyledonous plants (Sree
et al. 2015a, b; Ziegler et al. 2015) that have the capacity to grow at a rapid rate in
nutrient-rich waters. These plants belonging to the family Lemnaceae are classified
into five genera, which are Spirodela, Landoltia, Lemna, Wolffia, and Wolffiella, and
36 species (Bog et al. 2019). These plants are distributed throughout the world
except the polar regions and deserts. Different species of duckweeds have been used
for the treatment of municipal, industrial, domestic wastewater (Oron 1994), secondary effluents (Sutton and Ornes 1975), aquaculture wastewater (Porath and
Pollock 1982), and Swine wastewater (Mohedano et al. 2014). Lemna minor has
been studied for removal of heavy metals by Kara (2004), Bianconi et al. (2013),
Chaudhuri et al. (2014), and Sree et al. (2015c) and has been reviewed by Ziegler
et al. (2016, 2017, 2019).
Different duckweed species viz., Spirodela polyrhiza, Landoltia punctata, Lemna
minor, Lemna gibba, Wolffiella hyalina, and Wolffia microscopica were investigated
for their nutritional value and were found suitable for human consumption
(Appenroth et al. 2017, 2018). The duckweed plant extracts also did not show any
cytotoxicity and antiproliferative effect on human cell lines (Sree et al. 2019). Owing
to their nutritional quality, duckweeds can also be utilized as fish feed (Paul et al.
2013) and animal feed (Culley et al. 1981).
From ancient times, plants have been used as good source of medicines (Shakya
2016). Some of the aquatic plants used for medicine include Typha domingensis,
Ipomoea aquatica, and Marsilea minuta (Panda and Mishra 2011).
Antioxidants play an important role in defense mechanism in our body against
oxidative stress. Plants have the ability to synthesize antioxidants, which are capable
of reducing the level of Reactive Oxygen Species (Kasote et al. 2015). Gulcin et al.
(2010) studied the antioxidant, antibacterial, and anticandidal activities of the duckweed Lemna minor against 21 different bacteria and fungi and confirmed the
antibacterial, antifungal activities of Lemna minor, which is a source of antioxidants,
food, medicines, and pharmaceuticals. Zinc supplement to Spirodela polyrhiza
increased the antioxidant activity and ROS scavenging activity (Upadhyay and
Panda 2009). Further, Tipnee et al. (2017) found antioxidant, anti-inflammatory
properties in another duckweed, Wolffia globosa.
15.3
Microbial Symbionts
Positive interaction of microorganisms with plants not only helps to promote plant
growth but also plays a vital role in nutrient cycling (Ishizawa et al. 2020). There are
three regions in plants where microorganisms can associate and interact with them.
These are phyllosphere, rhizosphere, and endosphere (Compant et al. 2019).
Depending on the habit and habitat of the aquatic plant species, the exposure of
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these regions to different abiotic factors such as temperature, moisture, and radiation
will vary. The scale of these abiotic factors may affect the diversity of microorganisms associated with a specific sphere. These microorganisms use carbon, nitrogen,
carbohydrate, and ammonium as source of energy (Knief et al. 2012). The microbes
that are found in the endosphere region, e.g., inside the root tissue are considered as
endophytes. Some of these associated microorganisms are beneficial in nature such
as growth-promoting microorganisms (Appenroth et al. 2016; Ishizawa et al. 2020).
Some endophytic bacteria have the capacity to produce plant growth hormones such
as auxins and some plant growth-promoting rhizobacteria also defend the plants
from their pathogens (Sturz et al. 2000).
15.4
Benefits of Microbial Symbiosis in Aquatic Plants
In this mutually beneficial interaction, as implied by the term mutual, both the
aquatic plants as well as the associated microorganisms gain advantages from each
others’ company. In the present chapter, we will focus on the benefits gained by the
aquatic plants from this symbiotic relationship. In the following, we have mainly
taken the example of the symbiotic relationship between microorganisms and
members of the aquatic plant family Lemnaceae (duckweeds). Whereever appropriate we have also included details on the investigations on other aquatic plants.
There are several benefits of this microbial association to the aquatic plants, a few
are detailed below.
15.4.1 Biofiltration
Plant-associated microorganisms enhance the uptake capacity of plants and help in
removal of nutrients, heavy metals, xenobiotics, and so on from a water body.
Hence, this partnership can be made use of in wastewater remediation. Apart from
studies in duckweeds (see below), investigations have been carried out on several
other aquatic plants. Investigations showed that microbes like Nitrobacteria
irancium associated with E. crassipes help in removal of chromium and zinc
(Abou-Shanab et al. 2007).
As already mentioned in the previous section, duckweeds as biofilters are promising candidates for use in wastewater remediation especially because of their
extremely high growth rates (Sree et al. 2015a; Ziegler et al. 2015) and by their
capacity to interact with a huge diversity of microorganisms (Acosta et al. 2020;
Appenroth et al. 2016; Ishizawa et al. 2017a, 2019a). Deeper understanding of the
molecular mechanism of plant–microbe interaction is meanwhile facilitated by the
availability of genome sequences of several plant growth-promoting and inhibiting
bacteria (Sugawara et al. 2015; Ishizawa et al. 2017b, 2018). We present here the
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effects of duckweed-bacteria interactions on removal of nutrients, heavy metals, and
organic xenobiotics.
15.4.1.1
Removal of Nutrients from Eutrophic Wastewater
Excessive use of fertilizers in modern agriculture results finally in eutrophication of
surface water bodies, which is a worldwide problem. Because of the very high
growth rates of duckweed by vegetative propagation and by the additional effect
of plant growth-promoting bacteria, duckweed-bacteria systems show excellent
performance in the uptake of excess nutrients from wastewater. The advantage of
the system lies in the point that the huge amount of biomass generated this way can
be harvested from the surface of the water body and can be utilized for several
practical applications depending on the quality of the wastewater (Appenroth et al.
2015).
Bacteria associated with duckweeds play an important role in nutrient removal
from water bodies. Bacterial growth was found to be a key factor for the degradation
of organic matter (Körner and Vermat 1998). Nitrogen assimilation was especially
well investigated (review in Appenroth et al. 2016). Duckweed provides additional
adherence space for bacteria both via roots as well as the submerged plant body as
shown in the rootless duckweed species of the genus Wolffia. Moreover, duckweed
provides oxygen for the bacterial nitrogen assimilation (Appenroth et al. 2016).
Ying-ru et al. (2013) showed that a strain of Pseudomonas species found in the
rhizosphere of L. minor helps in removal of nitrogen together with promotion of
plant growth.
Root exudates from some of the duckweed species like S. polyrhiza and L. minor
showed the presence of Pseudomonas fluorescens (Lu et al. 2014). However,
Ardiansyah and Fotedar (2016) found an abundance of heterotrophic bacteria like
Gram-negative cocci and Gram-negative bacilli associated with L. minor. Also,
surface area of a plant influences the growth and structure of bacterial communities
(Leonard et al. 2000). More recently, Shen et al. (2019) showed that the association
of heterotrophic nitrifying bacteria like Acinetobacter sp. with L. gibba enhanced the
rate of removal of ammonium and total nitrogen from the water body.
In terms of the practical application of a specific duckweed-bacteria association
that would have been studied in detail in a laboratory, a certain problem exists
concerning the stability of the association under non-axenic conditions, i.e., when
other bacteria or microbes present in the environment can compete for a possible
association with the surface of duckweed. In this respect, very recently, Ishizawa
et al. (2020) suggested a two-step cultivation process with the plant growthpromoting bacterium, Acinetobacter calcoaceticus P23 comprising a “colonization
step” and a subsequent “mass cultivation step,” for increasing the nutrient removal
capacity, thereby enhancing the biomass production.
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15.4.1.2
T. Dash et al.
Removal of Heavy Metals
The interaction of bacteria and duckweed was investigated hoping that also the
uptake of heavy metals by duckweed could be stimulated by certain bacteria and that
this will enhance the phytoremediation potential of duckweeds. Related experiments
were carried out with cadmium (Cd) and chromate (Cr (VI)). It turned out that, in the
toxic concentration range, the uptake of these two heavy metals was not enhanced,
but was rather diminished. Several mechanisms might be responsible for these
effects but in summary, it can be concluded that the associated bacteria rather play
a role in phytoprotection than in enhanced phytoremediation potential of duckweed
(Appenroth et al. 2016). The growth promotion activity of Aquitalea magnusonii
strain H3 on L. minor increased remarkably when the plant was subjected to copper
(Cu) and zinc (Zn) stresses, which itself inhibited the duckweed growth by ca. 40%.
This plant growth-promoting bacterium also enhanced the capacity of the duckweed
to accumulate and tolerate the two heavy metals (Ishizawa et al. 2019b). In another
study, oxidation of As(III) by bacteria from phyllosphere of Wolffia australiana was
reported (Xie et al. 2014).
15.4.1.3
Removal of Organic Xenobiotic
Wastewater is often contaminated by dissolved organic material like herbicides,
pesticides, antibiotics, or other drugs because of their illegal delivery into the
environment. As early as 1985, Pignatello et al. (1985) showed that L. minor
contributed to removal of pentachlorophenol from wastewater. These authors also
showed that not only duckweed but also associated bacteria were responsible for the
degradation of this phenolic compound. A phenol degrading bacterium,
Acinetobacter calcoaceticus associated with Lemna aequinoctialis was isolated by
Yamanga et al. (2010). Muerdter and LeFeyre (2019) recently reported transformation of the insecticides Imidacloprid and Thiacloprid (Neonicotinoids) into their
metabolites by an unsterile culture of Lemna sp. After surface sterilization, the
metabolites could not be observed demonstrating the role of duckweed-microbe
interactions in this transformation reaction. The interaction of rhizospheric bacteria
with S. polyrhiza for degradation of organic compounds has been investigated by
several groups. This association has been found to be capable of degrading surfactants (Mori et al. 2005) and degrading a variety of aromatic compounds (Toyama
et al. 2006; Hoang et al. 2010). Strains of Pseudomonas and Cupriavidus were
isolated from the rhizosphere region of S. polyrhiza and were found to be able to
degrade 3-Nitrophenol which is also an aromatic compound (Kristanti et al. 2012).
Bacterium, Sphingobium fuliginis associated with S. polyrhiza was found capable of
degrading 4-tert-butylphenol and using it as a sole carbon and energy source (Ogata
et al. 2013).
In the last 20 years, investigations concerning the degradation, removal, and
uptake of a broad range of organic xenobiotics has increased to a large degree
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(Appenroth et al. 2016; Ziegler et al. 2016, 2017). In several cases, the effects of
interactions between duckweeds and different bacteria were demonstrated. More
investigations are required before large-scale biotechnological applications can be
expected. Ziegler et al. (2019) pointed to the opportunity to even identify the nature
of contamination by biotests with duckweed species.
15.4.2 Nitrogen Fixation
Besides assimilation of nitrate and ammonium, interaction of duckweed with
nitrogen-fixing bacteria has also been reported, although the mechanism of interaction is still unknown. Nitrogen-fixing heterotrophic bacteria have been found to be
associated with duckweed mat that can recycle nitrogen produced by the denitrifiers
to produce organic compounds (Zuberer 1982). Nitrogen-fixing free-living bacteria
have also been isolated from three aquatic plant species Azolla filiculoides, Lemna
gibba, and Ricciocarpus natans (Quisehuatl-Tepexicuapan et al. 2016).
15.4.3 Production of Phytohormones
The role of auxin in plant–microbe interaction has been a topic of discussion
(Spaepen and Vanderleyden 2011). Gilbert et al. (2018) isolated endophytic bacteria
from surface-sterilized duckweed species and found that they are capable of producing indole-3-acetic acid and other indole related compounds. However, the role
of these indole related molecules in the association of duckweeds and the bacteria
producing them needs to be more clearly established. It is interesting to note that the
same study had found a correlation between the duckweed species and the indole
related molecules produced by the associated bacteria. Extrapolating this finding to
other metabolites and functions, this might have an impact on the use of a particular
duckweed species for a particular practical application.
15.4.4 Bioenergy Production
Fuel produced from plant biomass is an alternative source of energy that is safe for
the environment. Water hyacinth as a candidate for bioethanol production has been
investigated by several researchers (Hronich et al. 2008; Wang and Calderon 2012).
Methane producing capacity of eight aquatic plants such as Salvinia molesta,
Hydrilla verticillata, Nymphaea stellate, Azolla pinnata, Ceratopteris sp.,
Scirpus sp., Cyperus sp., Utricularia reticulata was investigated by Abbasi et al.
(1990) and found that Salvinia molesta has the highest yield.
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The plant growth-promoting capacity of the microbes can play a role in enhancing the yield of the plants suitable for biofuel production. Mishima et al. (2008)
compared the effect of two microbes Saccharomyces cervisiae and Escherichia coli
on ethanol production from water hyacinth and water lettuce. It was found that
E. coli had more ethanol-producing capacity than S. cervisiae. Duckweeds have also
been investigated for use as potential feedstock for biofuels such as bioethanol (Chen
et al. 2012; Cui et al 2011; Xu et al. 2011; Soda et al. 2015) and biobutanol (Li et al.
2012) production. These studies might owe to the fact that duckweeds include some
of the fastest-growing plants which can result in huge amounts of biomass at a fast
rate. Xiao et al. (2013) found that a duckweed species, Landoltia punctata is a
suitable crop for bioenergy production as it was found to have a high-starch
accumulation capacity. Another study was conducted by Xiu et al. (2010) for
bio-oil production from duckweeds. Lemna minor was found to be a potential crop
plant for biogas production (Muradov et al. 2012). Further, ethanol and methane
production capacity were investigated in four duckweed species S. polyrhiza,
L. minor, L. gibba, and L. punctata after being grown in effluent from municipal
and swine wastewater. It was found that, out of the four, S. polyrhiza had highest
nitrogen removal capacity as well as highest ethanol and methane production
capacity (Toyama et al. 2018). Biomass of L. minor produced from swine lagoon
wastewater was found to be capable of ethanol production using yeast (Ge et al.
2012). Biomass of duckweed obtained from wastewater points toward the conditions
where duckweeds are not growing in isolation but are in association with other
beings of the aquatic ecosystem especially pointing toward a closer association
between duckweeds and microbes.
15.5
Conclusion
The symbiosis between microorganisms and aquatic plants has a significant role in
enhancing the different capacities of aquatic plants. When tapped in a sustainable
manner, the aquatic plant resources can prove to be beneficial with several alternatives for practical applications. The enormous growth of different aquatic plants in
the nutrient-rich wastewaters has been considered for a long time as a sign of bad
health of the ecosystem. However, the huge biomass thus produced can be harvested
and efficiently put to use for several applications like biogas, biofuel, or biofertilizer
production or bioplastic production based on the quality of wastewater on which the
plants had grown. Regular harvest of biomass will ensure the safe health of the
ecosystem and will also be a bioresource. In this sustainable use of aquatic plants,
microorganisms, especially symbionts of these aquatic plants play a crucial role in
supporting and enhancing several of the processes as described in this chapter.
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mats. Appl Environ Microbiol 43:823–828
Chapter 16
Rhizobium Presence and Functions
in Microbiomes of Non-leguminous Plants
Alexandra Díez-Méndez and Esther Menéndez
Abstract The genus Rhizobium is well known in the context of its interaction with
leguminous plants. The symbiosis Rhizobium-legume constitutes a significant source
of ammonia in the biosphere. Rhizobium species have been studied and applied as
biofertilizers for decades in legumes and nonlegumes, due to the potential as N-fixer
and plant growth promoter. Since its discovery, conventional culture-dependent
techniques were used to isolate Rhizobium members from their natural niche, the
nodule, and their identification was routinely performed via 16S rRNA gene and
different housekeeping genes. Biotechnological advances based on the use of omicsbased technologies showed that species belonging to the genus Rhizobium are
keystone taxa in several diverse environments, such as forests, agricultural land,
Arctic, and Antarctic ecosystems, contaminated soils and plant-associated
microbiota. In this chapter, we will summarize the advances in the study of the
Rhizobium genus, from culturomics strategies to modern omics methodologies,
mostly based on next-generation sequencing approaches. These cutting-edge molecular approaches are fundamental in the study of the behavior of Rhizobium species in
their interaction with Non-leguminous plants, supporting their potential as an ecological alternative to chemical fertilizers in the battle against Climatic Change.
Keywords Rhizobia · Plant growth promotion · Plant microbiome · Biofertilizers ·
Non-legumes
A. Díez-Méndez
Universidad Católica de Ávila, Ávila, Spain
e-mail: alexandra.diez@ucavila.es
E. Menéndez (*)
Mediterranean Institute for Agriculture, Environment and Development (MED), Institute for
Advanced Studies and Research (IIFA), Universidade de Évora, Évora, Portugal
e-mail: esthermenendez@uevora.pt
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_16
241
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16.1
A. Díez-Méndez and E. Menéndez
The Genus Rhizobium at a Glance
The genus Rhizobium as known nowadays was firstly described 131 years ago, in
1889 when B. Frank reclassified the Bacillus radicicola as named by Beijerinck in
1888 onto the name Rhizobium leguminosarum. The name of the genus comes from
“Gr. fem. n. rhiza, a root; Gr. masc. n. bios, life; N.L. neut. n. Rhizobium, that which
lives in a root.” The genus Rhizobium is the type genus of the family Rhizobiaceae,
which was described by Conn in 1938, and this family belongs to the order
Rhizobiales, named many years later, in 2006 by Kuykendall. The species Rhizobium leguminosarum is the type species of the genus Rhizobium, being USDA2370T
its type strain.
According to the List of Prokaryotic names with Standing in Nomenclature—
LPSN (Parte 2018; accessed for the last time March 22, 2020) at the time of writing,
there are 89 validly published species within the genus Rhizobium. In the last
10 years, there has been a boom in describing new species belonging to this genus
(Table 16.1) and interestingly, the reclassification of some of the species into newly
described genera, such as Neorhizobium (Mousavi et al. 2014) and Pararhizobium
(Mousavi et al. 2015), is becoming more frequent due to the use of the NGS tools
(Ormeño-Orrillo et al. 2015; Checcucci et al. 2019; González et al. 2019).
The members of the genus Rhizobium were traditionally isolated from legume
nodules; nevertheless, in the last years, some reports showed that other sources can
be considered for the isolation of Rhizobium. Table 16.1 shows in bold characters the
newly described species originally isolated from non-leguminous plants. Moreover,
the surface of minerals, contaminated soils, and water are also sources of new
species of this genus.
It is implicit that the definition of “rhizobium” is based on the ability to elicit the
formation of root nodules in leguminous plants and the latter action of fixing
nitrogen within these newly formed structures. This symbiotic relationship has
been extensively studied (Oldroyd et al. 2011; Wang et al. 2018; Taylor et al.
2020). However, Rhizobium has many features apart from the classical function in
Nitrogen Fixation, this group of bacteria interacts also with other plants than
legumes and has plant growth promotion and biocontrol functions. Member of this
genus is able to interact and produce benefits in a wide range of crops, such as the
most important grain food crops, including rice, maize, wheat, and barley; vegetable
crops, including lettuce, carrots, potatoes, and spinach; or bioenergy crops, such as
canola, sunflower, or switchgrass. The functions and properties that Rhizobium exert
in Non-leguminous plants, especially the ones related to plant growth promotion, are
extensively reviewed in the last years (Vargas et al. 2017; Nag et al. 2019;
Yoneyama et al. 2019; Velázquez et al. 2019; Mahmud et al. 2020). The involvement of Rhizobium in biocontrol actions against pathogens an alleviation of stresses
is a not well-studied characteristic; however, there are some studies showing the
potential of this feature; for example, Bellabarba et al. (2019) reviewed the importance of plant growth-promoting rhizobia in the alleviation of salt and osmotic
stresses as well as in contaminated soils. Volpiano et al. (2019) also reviewed the
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Rhizobium Presence and Functions in Microbiomes of Non-leguminous Plants
243
Table 16.1 New species of Rhizobium validly described in the last 10 years (2010–2020)
Name of the species
Rhizobium acidisoli
Rhizobium
aegyptiacum
Rhizobium aethiopicum
Rhizobium aggregatum
Rhizobium altiplani
Rhizobium alvei
Rhizobium anhuiense
Rhizobium aquaticum
Rhizobium arenae
Rhizobium azibense
Rhizobium azooxidifex
Rhizobium
bangladeshense
Rhizobium binae
Rhizobium calliandrae
Rhizobium capsici
Rhizobium cauense
Rhizobium chutanense
Rhizobium
ecuadorense
Rhizobium
endolithicum
Rhizobium
endophyticum
Rhizobium esperanzae
Rhizobium favelukesii
Rhizobium flavum
Rhizobium freirei
Rhizobium gei
Rhizobium grahamii
Rhizobium
halophytocola
Rhizobium helianthi
Rhizobium ipomoeae
Rhizobium jaguaris
Rhizobium laguerreae
Rhizobium lemnae
Rhizobium lentis
Rhizobium leucaenae
Rhizobium marinum
Isolated from
Phaseolus vulgaris
Trifolium alexandrinum
References
Roman-Ponce et al. (2016)
Shamseldin et al. (2016)
Phaseolus vulgaris
Hexachlorocyclohexane dump site
Mimosa pudica
Freshwater river
Vicia faba and Pisum sativum
Water of a crater lake
Sand
Phaseolus vulgaris
Arable soils
Lens culinaris
Aserse et al. (2017)
(Hirsch and Müller 1986)
Kaur et al. (2011)
Barauna et al. (2016)
Sheu et al. (2015)
Zhang et al. (2015a)
Mathe et al. (2018)
Zhang et al. (2017)
Mnasri et al. (2014)
Behrendt et al. (2016)
Rashid et al. (2015)
Lens culinaris
Calliandra grandiflora
Capsicum annuum var. Grossum
Kummerowia stipulacea
Phaseolus vulgaris
Phaseolus vulgaris
Rashid et al. (2015)
Rincon-Rosales et al. (2013)
Lin et al. (2015)
Liu et al. (2012)
Huo et al. (2019)
Ribeiro et al. (2015)
Beach sand
Parag et al. (2013)
Phaseolus vulgaris
López-López et al. (2010)
Phaseolus vulgaris
Medicago sativa
Soil
Phaseolus vulgaris
Geum aleppicum
Dalea leporina, Leucaena
leucocephala and Clitoria ternatea
Rosa rugosa
Cordeiro et al. (2017)
Torres Tejerizo et al. (2016)
Gu et al. (2014)
Dall’Agnol et al. (2013)
Shi et al. (2016)
Lopez-Lopez et al. (2012)
Helianthus annuum
Ipomoeba aquatica
Calliandra grandiflora
Vicia faba
Lemna aequinoctialis
Wei et al. (2015)
Sheu et al. (2016)
Rincon-Rosales et al. (2013)
Saidi et al. (2014)
Kittiwongwattana and
Thawai (2014)
Rashid et al. (2015)
Ribeiro et al. (2012)
Liu et al. (2015)
Lens culinaris
Leucaena leucocephala
Seawater
Bibi et al. (2012)
(continued)
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A. Díez-Méndez and E. Menéndez
Table 16.1 (continued)
Name of the species
Rhizobium mayense
Rhizobium
mesoamericanum
Rhizobium
metallidurans
Rhizobium
naphthalenivorans
Rhizobium
oryziradicisoryziradicis
Rhizobium oryziradicis
Rhizobium
paknamense
Rhizobium paranaense
Rhizobium
petrolearium
Rhizobium populi
Rhizobium puerariae
Rhizobium rhizoryzae
Rhizobium
rosettiformans
Rhizobium smilacinae
Rhizobium soli
Rhizobium sophorae
Rhizobium
sophoriradicis
Rhizobium
sphaerophysae
Rhizobium
straminoryzae
Rhizobium subbaraonis
Rhizobium
taibaishanense
Rhizobium tarimense
Rhizobium tubonense
Rhizobium
tumorigenes
Rhizobium vallis
Rhizobium vignae
Rhizobium viscosum
Isolated from
Calliandra grandiflora
Phaseolus vulgaris, siratro, cowpea,
and Mimosa pudica
Anthyllis vulneraria
References
Rincon-Rosales et al. (2013)
Lopez-Lopez et al. (2012)
Grison et al. (2015)
Sediment of a polychlorinated-dioxintransforming microcosm
Oryza sativa
Kaiya et al. (2012)
Arachis hypogaea
Lemna aequinoctialis
corrig. Khalid et al. (2015)
Kittiwongwattana and
Thawai (2013)
Dall’Agnol et al. (2014)
Zhang et al. (2012)
Phaseolus vulgaris
Oil-contaminated soil
Zhao et al. (2017)
Oryza sativa
Hexachlorocyclohexane dump site
Rozahon et al. (2014)
Boonsnongcheep et al.
(2016)
Zhang et al. (2014)
Kaur et al. (2011)
Smilacina japonica
Soil
Sophora flavescens
Sophora flavescens
Zhang et al. (2015a, b)
Yoon et al. (2010)
Jiao et al. (2015)
Jiao et al. (2015)
Sphaerophysa salsula
Xu et al. (2011)
Oryza sativa
Lin et al. (2014)
Beach sand
Kummerowia striata
Ramana et al. (2013)
Yao et al. (2012)
River soil
Oxytropis glabra
Cane gall tumors on thornless
blackberry
Phaseolus vulgaris, Mimosa pudica
and Indigofera spicata
Nodules of multiple legume species
Soil
Turdahon et al. (2013)
Zhang et al. (2011)
Kuzmanovic et al. (2018)
Populus euphratica
Pueraria candollei var. candollei
Wang et al. (2011)
Ren et al. (2011)
(Gasdorf et al. 1965) FloresFelix et al. (2017)
(continued)
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Rhizobium Presence and Functions in Microbiomes of Non-leguminous Plants
245
Table 16.1 (continued)
Name of the species
Rhizobium wenxiniae
Rhizobium
wuzhouense
Rhizobium yantingense
Rhizobium zeae
Isolated from
Zea mays
Oryza officinalis
References
Gao et al. (2017)
Yuan et al. (2018)
Surfaces of weathered rock (purple
siltstone)
Zea mays
Chen et al. (2015)
Celador-Lera et al. (2017)
Rhizobium species originally isolated from Non-leguminous plants are marked in bold
Fig. 16.1 Strategies based on current omics tools that are useful to study of the belowground
bacteria–plant–environment interactions
mechanisms and the efficacy of Rhizobium and related genera on biocontrol of
several pathogen-caused diseases affecting all kinds of plants.
Fortunately, nowadays, there is a plethora of multidisciplinary approaches that
can be applied to elucidate aspects that remain unclear (diCenzo et al. 2019), above
all the aspects and features attributed to this interesting genus.
16.2
The Study of Rhizobium in the Era of the Omics
Omics-based technology is a holistic approach to understand biological systems
using predictable tools to know their properties and functions. These omics strategies describe the cell, tissue, and organism features around the dogma central due to
the identification of genes (genomics), mRNA (transcriptomics), proteins (proteomics), and metabolites (metabolomics) (Arivaradarajan and Misra 2019). Due to the
immense potential of omics approaches and the current competitive prices, scientists
from many disciplines began to use them as their primary resource (López-Mondéjar
et al. 2017; Menéndez and Paço 2020). In the pursue for a sustainable agriculture,
these omics-based technologies have become essential tools to face agricultural
challenges (Fig. 16.1).
It is well known that Plant Probiotic Bacteria (PPBs) could be applied as
biofertilizers or biopesticides (Menendez and Garcia-Fraile 2017). There is a trend
to integrate them as an ecological innovation to face climate change and improving
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A. Díez-Méndez and E. Menéndez
crop productivity without compromising agriculture, which become more sustainable (Compant et al. 2019; Menéndez and Paço 2020). Thus, for the development of
eco-friendly agriculture based on the application of safety bacteria, such as those
belonging to the genus Rhizobium, omics—strategies will be essential tools to
improve our knowledge on many features and in the interaction among the inoculant
bacteria, the environment, and the host plant. Omics-based techniques should be
coordinated with the classical techniques developed to allow the cultivation of
microorganisms. This knowledge will help in the selection of a correct inoculum
or consortium for each crop.
16.2.1 Classical Techniques: Culturomics
The first culturomic techniques were the ones used by Louis Pasteur, which became
a pillar in the field of microbiology (Sarhan et al. 2019). The term culturomics was
introduced latter by the group of Didier Raoult and Jean-Christophe Lagier as a
strategy to bring more bacterial isolates into cultivation in the laboratories from
environmental microbiomes (Lagier et al. 2018). In combination with these culturing
approaches, PCR amplification of the ubiquitous 16S ribosomal RNA (rRNA) has
been used commonly to identify bacterial isolates (Turner et al. 2013). Classical
classification of Rhizobium species is based on PCR amplifications of 16S rRNA
gene and different housekeeping genes, such as recA and atpD (Gaunt et al. 2001),
amongst others. Nowadays and despite the use of the genome sequences in taxonomical classification, those identification techniques are essential to identify microorganisms, and various reports still endorse these methodologies for the
classification of the members of the genus Rhizobium (García-Fraile et al. 2007;
Kaur et al. 2011; López-López et al. 2010; Rincon-Rosales et al. 2013; Zhang et al.
2012). Moreover, other techniques became complementary to the identification of
species of Rhizobium, such as the MALDI-TOF mass spectrometry (MALDI-TOF
MS). For example, a recent study revealed the usefulness of a combination between
MALDI-TOF MS and classical PCR amplification for the taxonomic identification
of isolates of Rhizobium laguerreae from nodules of Phaseolus vulgaris and Lens
culinaris (Flores-Félix et al. 2019).
The obtention of pure cultures and their correct identification are two essential
concerns in the design of biofertilizers (Menéndez and Paço 2020). The members of
the genus Rhizobium are easy to culture and to identify as well as it is a proven Plant
Probiotic Bacteria, which is very interesting for the design of biofertilizers. The most
used culture media for the growth of Rhizobium is the Yeast Mannitol Agar (YMA;
Vincent 1970) and/or the Tryptone Yeast agar (TY; Beringer 1974).
Despite its importance, the “culturomics” has many limitations being the most
import one the still limited capacity for the cultivation of some bacterial taxa.
Nowadays, the culturome (strains that we are able to culture in laboratory conditions) does not represent the total microbiome (Martiny 2019; Steen et al. 2019).
Thus, current studies are focused on the improvement of a variety of factors involved
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Rhizobium Presence and Functions in Microbiomes of Non-leguminous Plants
247
in microbial growth such as culturing conditions (Light/dark, anaerobic/aerobic,
proper temperatures), times of incubation, growth media (providing environmental
and nutritional conditions), according to the bacteria nutritional requirements
(Sarhan et al. 2019; Compant et al. 2019). These studies propose to combine
plant-based culture media plus multiple growth factors such as vitamins, amino
acids, etc. to improve isolation techniques of plant probiotics, such as Rhizobium
species (Sarhan et al. 2019).
16.2.2 Genomics
The term genomics was introduced by Tom Roderick in 1986 when mapping the
human genes and referred to study of the structure, function, and interaction of the
genome, i.e. examine a complete organism set of DNAs, including both non-coding
and protein-coding genes (Womack 2019). However, the interest in these techniques
has increased in recent years since many disciplines might benefit from their use.
Since Plant Probiotics have demonstrated their role supporting benefits effects for
plant growth and health, there is an urgent need to bring microbial innovations into
agriculture practices (Compant et al. 2019). Thus, the selection of appropriate
microbiota inoculants is an urgent need to face current challenges in crop production.
In this sense, Biological Nitrogen Fixation (BNF) is an ecologically and agriculturally process performed by two prokaryotic groups. Among them, the fixation of
N2 gas into ammonia by root nodule bacteria (called rhizobia) is an efficient process
in terms of supplying nitrogen to the plant (diCenzo et al. 2019) For decades,
researchers have generated massive data to increase crop yield using rhizobial
species based on culture-dependent approaches. However, in the first decade of the
XXI century, genomic approaches become increasingly, and rhizobial genomes are
becoming to be sequenced, according to the proposed minimal standards of the
taxonomy of prokaryotes and for the description of new rhizobia and agrobacteria
(Chun et al. 2018; De Lajudie et al. 2019). These strategies have resulted in an
increased trend on genome sequencing projects by different methodologies to the
present. Genomes Online Database (GOLD) is a World Wide Web resource,
powered by DOE Joint Genome Institute, for comprehensive access to information
regarding genome and metagenome sequencing projects, and their associated metadata around the world (https://gold.jgi.doe.gov/), which showed the increased data
from genome sequencing projects over time (Fig. 16.2).
Public databases as NCBI (National Center for Biotechnology Information) and
GOLD show an increasing number of reports showing the genome sequences of
strains belonging to the genus Rhizobium. At the time of writing, there are
691 (NCBI) and 521 (JGI GOLD) sequenced genomes from different species in
both databases. All these data are improved due to technological advances, which led
to the development of the second-generation sequencing (SGS) technologies
improving limitation from Sanger sequencing, such as read lengths, assembly, and
determination of complex genomics regions, methylation detection, and isoform
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Fig. 16.2 Genome Sequencing Project from the last decades (modified from GOLD Database)
detection. Nowadays, this methodology has evolved using the next-generation
platforms such as Illumina, Roche 454, Ion Torrent, and PacBio (Rhoads and Au
2015; Smits 2019).
Recent studies based on genomics research have demonstrated that the genus
Rhizobium forms part of the core microbiome of many plants (Bulgarelli et al. 2015;
Yeoh et al. 2017; Oberholster et al. 2018; Pérez-Jaramillo et al. 2019). Besides, NGS
platforms such as Illumina and PacBio are nowadays indispensable in the classification via genomes of the members of the genus Rhizobium (Ormeño-Orrillo et al.
2015; González et al. 2019). Some studies already report the use of PacBio (longer
reads than Illumina add more possibilities of complete the genome) to generate
genomes of novel species, such as Rhizobium jaguaris CCGE525T isolated from
Calliandra grandiflora nodules (Servín-Garcidueñas et al. 2019) or completing
genomes sequence such as Rhizobium sp. strain 11515TR isolated from tomato
rhizosphere cultivated in the Philippines (Montecillo et al. 2018) and novel
non-nodulating Rhizobium species isolated from Agave americana (Ruíz-Valdiviezo
et al. 2017), amongst others.
16.2.3 Proteomics
Proteins are essential molecules that have direct involvement in cellular function.
The term “proteome” was introduced by Marc Wilkins in 1994 (Wasinger et al.
1995), defining it as the study of the entire range of proteins in a single cell. The
“proteomics” is focused on the identification and characterization of proteins to
understand physiological events (Zhao and Lin 2014).
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Rhizobium Presence and Functions in Microbiomes of Non-leguminous Plants
249
Plant proteomics will be a crucial research tool in developing new technologies to
further improve agricultural production and its sustainability. Focusing on the
Rhizobium-legume symbiosis perspective, there are two types of approaches involving proteomics: (i) the study of the legume root proteome after the inoculation with
their respective microsymbiont and, (ii) the studies focused on the Rhizobium partner
(Larrainzar and Wienkoop 2017).
Regarding the first approach, proteomics methodologies are focused on the
identification of proteins in the early stages of symbiosis. Morris and Djordjevic
(2001) focused on roots proteome of clover cultivar Woogenellup, and the results
showed that ethylene levels are upregulated during the early stages of infection
(48 h) by the inoculation of the bacterial strain ANU843 identified as
R. leguminosarum bv trifolii, but that does not turn in the induction of common
pathogenesis mechanisms. However, inoculation with a rhizobial non-nodulating
bacterial as the strain ANU743 produced aberrant nodules. This phenotype may be
related to the induction of alpha-fucosidase by the bacterial strain on the roots of this
clover.
On the other hand, Irar et al. (2014) reported a proteomic approach relating to
nodule response to drought on Pisum sativum. Pea plants were inoculated with a
strain of R. leguminosarum and grown under “normal well-irrigated” conditions, and
the other was subjected to drought issues. The results showed a total of 18 proteins
expressed during drought stress: 11 are encoded by Rhizobium leguminosarum and
seven nodule proteins encoded by Pisum sativum. These proteins are related to
flavonoid metabolism, sulfur metabolism, and RNA-binding proteins. All these
data provide new targets to improve legumes tolerance to drought.
In the second approach, proteomics strategies have been used to identify specific
proteins during the symbiosis comparing free-living Rhizobium cells vs bacteroids,
their nitrogen fixation symbiotic forms. Despite the importance of these approaches,
authors have focused their study on model organisms, such as Sinorhizobium or
Bradyrhizobium species. Tullio et al. (2019) showed that this omic-based methodology is useful to identify proteins related to tolerance to environmental stress, such
as soil acidity, using the bacterial strain Rhizobium freirei PRF81. Thus, further
studies should be performed to understand the proteomic side of the genus
Rhizobium.
16.2.4 Metabolomics
Metabolome includes a wide range of molecules such as amino acids, peptides,
carbohydrates, nucleic acids, organic acids, polyphenols, alkaloids, minerals, etc.; all
chemical compounds in a cell, which are metabolized or synthesized (Pandey and
Dubey 2019).
In the last decades, the genus Rhizobium has been tested for growth promotion of
Non-leguminous plants, such as maize, rice, oat, and others cereals (Mia and
Shamsuddin 2010; Nag et al. 2019). Thus, in the biofertilizer-technology, Rhizobium
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species are becoming sustainable tools for agriculture of nonlegumes and legumes
by substituting agrochemicals gradually. Therefore, the identification of their
metabolome is needed because they are the key in the symbiotic relationship,
rhizosphere, soil niche, and communications with other organisms (Jacoby et al.
2018).
A recent study aimed to identify the exo-metabolomes produced by Rhizobium
etli CFN42T, Rhizobium leucaenae CFN299T, Rhizobium tropici CIAT899T, Rhizobium phaseoli Ch24-10 from free-living culture by nuclear magnetic resonance
(Montes-Grajales et al. 2019). The exometabolomic profile was carried out growing
cultures using minimal medium containing sucrose and glutamate. The results show
that there is a different pattern among the bacterial strains, being acetone and
C4-dicarboxylate the only compounds secreted by all of them. It has been observed
that this amino acid stimulates nitrogen fixation of bacteroids isolated from soybean
root nodules (Kouchi et al. 1991). Ornithine was only detected in the culture
supernatant of R. tropici CIAT 899T. It has been observed that this compound is
involved in symbiotic efficiency and resistance to stress conditions, such as acidity
(Rojas-Jiménez et al. 2005; Vences-Guzmán et al. 2011).
There are few studies about the Rhizobium metabolomes; therefore, we should
highlight the necessity of more works on this, aiming to achieve more knowledge on
the role of the metabolome of Rhizobium species in improving crop performance.
16.3
Rhizobium and co.: Interactions with Non-leguminous
Plants
It is well known that the Green Revolution improved crops yield saving people from
starvation. The objectives of this revolution were focused on applying a massive
amount of chemical fertilizers for growing a single crop. However, global crop
production cause not only adverse effects on the environment, such as climate
change, pollute fresh and marine water, loss of biodiversity, and alterations of
biogeochemical cycles, but also harmful impacts on human health (García-Fraile
et al. 2017). Although, agriculture is an important pillar that maintenances society
staple food, there are a billion people who still suffer inadequate food supplies and
have unhealthy diets (Tilman and Clark 2014). Thus, EAT-Lancet Commission
revealed that global food systems need a transformation to focus mainly on environmental sustainability of food production and the health consequences of final
consumption (Willett et al. 2019).
Plant probiotics are eco-friendly tools that help face agriculture challenge. However, before using a biofertilizer, it is necessary to know the effects of the microorganisms on the environment and human health (García-Fraile et al. 2012; Menéndez
and Paço 2020). For that reason, there is a plethora of studies of new ecological
alternatives based on Plant Probiotics. Amongst them, the genus Rhizobium represents a group of interest not only for their biosafety features after decades of legume
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Rhizobium Presence and Functions in Microbiomes of Non-leguminous Plants
251
inoculations (Bashan 1998; Bhattacharjee et al. 2008) but also because of their
potential to improve the performance of Non-leguminous crops (Celador-Lera
et al. 2017; Menendez and Garcia-Fraile 2017; Nag et al. 2019).
Different reports have been shown that these probiotic bacteria have diverse
direct and indirect plant growth promotion (PGP) mechanisms in vitro, such as
phosphate solubilization, siderophores, or the production of phytohormones as acid
indole acetic IAA (Table 16.2). These potential abilities result in the better development of cereal and horticulture crops (Table 16.2). For example, the improvement
of grain yield and plant growth in Oryza sativa produced by the strain E11 identified
as Rhizobium leguminosarum bv trifolii under gnotobiotic and field conditions
(Yanni et al. 2001). Few years later, a report by Hafeez et al. (2004) showed that
the same strain (E11) was able to improve dry root weight and root length of cotton
(Gossypium hirsutum) under growth room conditions. Moreover, the same strain has
been used with excellent results in rice crops grown under field conditions (Yanni
and Dazzo 2010; Jha et al. 2020). These pieces of evidence may be related to the
bacterial ability to synthesize phytohormones.
Other example is the enhance in production of a variety of crops belonging to the
genus Brassica by two Rhizobium species. First, seed inoculations of B. campestris
and B. napus with R. leguminosarum VF39SM improved early seedling root growth
under chamber conditions (Noel et al. 1996). Second, R. rubi increased chlorophyll
content, quality, and plant weight under field conditions (Yildirim et al. 2011). Both
strains showed PGP in vitro mechanisms associated with the production of phytohormones like IAA and/or cytokinin and resulted in beneficial to Brassica crops.
Root colonization is an essential step of a productive relationship between the
macro and microsymbiont (Compant et al. 2019). For this attachment, bacteria must
produce a variety of surface polysaccharides, being cellulose a synthesized widespread among the genus Rhizobium and the principal component of their biofilms
(Robledo et al. 2012). In this sense, some reports showed the behavior of
GFP-tagged strains on the surface of the roots by using fluorescence microscopy,
such as spinach (Jiménez-Gómez et al. 2018), strawberry (Flores-Félix et al. 2015),
tomato, and pepper (García-Fraile et al. 2012) and carrots and lettuce (Flores-Félix
et al. 2013). Besides, all these bacterial strains showed in vitro PGP, which result in
the improvement of plant growth increasing different parameters as nutritional
values, number of flowers, chlorophyll content, and so on (Table 16.1). Interestingly,
the work presented by Marks et al. (2015) suggest that the presence of Rhizobium
itself might be not necessary in some occasions. The addition of LCOs extracted
from R. tropici CIAT 899 to an Azospirillum-based inoculant, to the seed or by
spraying, increases several parameters in maize cultivated on Brazilian soils.
However, there is enough evidence to claim that the best performance Rhizobium
inoculants are those able to enter and colonize internal tissues and persist there. This
should be addressed with microscopy or other approaches (Romano et al. 2020).
Thus, plant growth promotion (PGP) Rhizobium can be applied in agriculture
because of their capability to confer beneficial effects on plant growth also, their
abilities to colonize the rhizosphere and the roots efficiently and their safety for
human and the environments.
Strain
Br5
In vitro PGP mechanisms
IAA
Nonlegume host
Gossypium hirsutum
Rhizobium sp.
nd
Eruca sativa
Rhizobium sp.
YAS34
Rhizobium sp.
PEPV13
R. leguminosarum
bv. phaseoli
LR30/MR2
R. leguminosarum
bv viciae
USDA2370T
IAA
Siderophore production
Exopolysaccharide
production
IAA
Siderophore production
Phosphate solubilization
IAA
Catalase
Exopolysaccharide under
simulated drought conditions
Phytohormones and
increased colonization
R. leguminosarum
bv. trifolii
R. leguminosarum
IAT168
Probably phytohormones
Triticum aestivum
VF39SM
IAA.
Cytokinin.
R. leguminosarum
bv phaseoli
R. leguminosarum
bv phaseoli
P31
Phosphate solubilization.
IAA
Phosphate solubilization.
IAA
Brassica campestris
cv. Tobin
Brassica napus
cv. Westar
Zea mays
R1
Parameters improved
Plant height
Boll weight and number of bolls per plant
Seed cotton yield
Root system
References
Qureshi et al. (2019)
Rubio-Canalejas et al.
(2016)
Alami et al. (2000)
Heliant
hus annum
Dianthus caryophyllus
Growth promotion
N uptake
Plant growth in the first
stages of development
Triticum aestivum
Plant growth
Biomass
Drought stress
Hussain et al. (2014)
Oryza sativa
SDW
Plant height
Shoot N
Grain yield
Shoot dry matter
Grain yield
Root length.
Seedling growth.
Chi et al. (2005)
Lactuca sativa
Plant height
Dry weight.
Increased lettuce P
concentration.
Menéndez et al. (2016)
Hilali et al. (2001)
Noel et al. (1996)
Chabot et al. (1996)
A. Díez-Méndez and E. Menéndez
Species
Rhizobium sp.
252
Table 16.2 Rhizobium species described as PGPB of non-leguminous plants
IAA.
Root colonization.
Solanum lycopersicum var.
cerasiformein and Capsicum
annuum
R. leguminosarum
bv trifolii
R. leguminosarum
bv trifolii
R. leguminosarum
sv. trifolii
R. leguminosarum
sv. phaseoli/viciae
R. leguminosarum
bv trifolii
R. leguminosarum
E11
Oryza sativa L.
Consortia
2/4 strains
TV-13
Phosphate solubilization.
IAA
Phytohormones
Oryza sativa L.
IAA
Lactuca sativa
R1-R5
IAA
Wheat greenhouse
E11
IAA
Gossypium hirsutum L
PEPV16
Siderophores.
IAA.
Phosphate solubilization.
Root colonization.
Cellulose.
Fragraria ananassa
Daucus carota
Lactuca sativa
R. laguerreae
PEPV40
Siderophores.
IAA.
Phosphate solubilization.
Root colonization.
Cellulose
Spinacia oleracea
García-Fraile et al.
(2012)
Yanni et al. (2001)
Yanni and Dazzo
(2010)
Schlindwein et al.
(2008)
Etesami et al. (2009)
Hafeez et al. (2004)
Flores-Félix et al.
(2015)
Flores-Félix et al.
(2013)
Jiménez-Gómez et al.
(2018)
(continued)
253
Dry weight.
Fresh weight.
Number of flowers.
Nutritional value (P, K,
and Mg).
Grain yield.
Plant growth.
Straw biomass.
Reduced N-fertilizers
Progressive damages in
seed vigor and growth
Growth
NPK uptake
Dry root weight.
Root length.
Number of stolons,
flowers, and fruits.
Nutritional value (Fe,
Zn, Mn y Mo).
More citric acid and
malic acid.
Dry weight.
Nutritional values
(N, P, Ca, K, S).
Number of leaves.
Leaf size.
Fresh weight.
Dry weight.
Chlorophyll content.
Nutritional value (N, P,
K, Mg, Ca)
Rhizobium Presence and Functions in Microbiomes of Non-leguminous Plants
PEPT01
16
R. leguminosarum
bv trifolii
254
Table 16.2 (continued)
Species
R. etli
Strain
CFN42
In vitro PGP mechanisms
Not data
Nonlegume host
Zea mays
R. rubi
Not data
Phosphate solubilization.
Brassica oleracea L., var. italica
Parameters improved
Root weight.
Shoot weight and
length.
Nutritional value Mn,
Fe, Cu, Ca.
Increase yield.
Plant weight,
Chlorophyll content.
References
Gutiérrez-Zamora and
Martı́nez-Romero
(2001)
Yildirim et al. (2011)
A. Díez-Méndez and E. Menéndez
16
Rhizobium Presence and Functions in Microbiomes of Non-leguminous Plants
16.4
255
Finding Rhizobium in the Microbiomes associated
with nonlegumes
The members of the order Rhizobiales and the family Rhizobiaceae are composed of
diazotrophic bacteria usually living in the soils associated with plant roots. There are
some exceptions, such as Pseudorhizobium pelagicum R1-200B4T, which was
isolated from the Mediterranean Sea (Kimes et al. 2015), Rhizobium daejeonense
L61T, which was isolated from a cyanide treatment bioreactor (Quan et al. 2005) or
Rhizobium alvei TNR-22T, which was isolated from a freshwater river (Sheu et al.
2015), amongst others. Recent studies based on computational inference
(omics-based) showed that the order Rhizobiales is a keystone taxon in several
environments, such as forests, agricultural land, Arctic, and Antarctic ecosystems,
contaminated soils and plant-associated microbiota (Banerjee et al. 2018; LeBlanc
and Crouch 2019). More specifically, some of these works identify the genus
Rhizobium as a keystone taxon in core microbiomes of several plant crops rhizospheres, such as tropical crops, sunflower, and sorghum (Bulgarelli et al. 2015; Yeoh
et al. 2017; Oberholster et al. 2018), as well as their well-known presence and
functions on legume nodule microbiome (Velázquez et al. 2017; Martínez-Hidalgo
and Hirsch 2017; Cardoso et al. 2018; Muresu et al. 2019; Velázquez et al. 2019;
Zheng et al. 2020). Other genera belonging to the order Rhizobiales and closely
related to the genus Rhizobium, such as Agrobacterium, Bradyrhizobium, and
Devosia, were found forming part of the maize rhizospheric core microbiome in
long- term assays (Walters et al. 2018). According to Walters et al. (2018), members
of the family Rhizobiaceae and some other Rhizobiales members appeared to form
part of the heritable fraction of maize rhizosphere microbiome.
Interestingly, over the last years, many reports and studies have been published
about the presence of Rhizobium and related genera in the rhizosphere, endosphere,
and phyllosphere of Non-leguminous crops. This is the result of the high interest in
the exploration of the crop microbiomes with the aim of finding native rhizobial and
non-rhizobial bacteria that might be endophytes to produce benefits in those crops
and also, to be friendly with the microbiomes (Menéndez and Paço 2020).
Rhizobium, which fixes Nitrogen within legume nodules, and other endophytic
diazotrophs were usually found in Non-leguminous crops (Yoneyama et al. 2017,
2019). Using nifH gene amplification and cloning (not HiSeq/MiSeq or
pyrosequencing, but somehow comparable) from various, some works reported the
existence of members of the genus Rhizobium in several plant structures and
compartments, such as Rhizobium sp. in roots and stems of maize plants grown in
fields (Roesch et al. 2008), R. etli in roots of one cultivar of sorghum grown with low
and high doses of nitrogen fertilizers (Rodrigues Coelho et al. 2008),
R. leguminosarum in sweet potato tubers (Terakado-Tonooka et al. 2008),
R. helanshanense in roots and shoots of switchgrass (Bahulikar et al. 2014) and
R. daejeonense in stems and roots of sugarcane cultivated in Japan and Brazil
(Thaweenut et al. 2011).
256
A. Díez-Méndez and E. Menéndez
Using NGS techniques, Lay et al. (2018) performed a comparative analysis
between rhizosphere and endosphere of canola, pea, and wheat grown in Canadian
prairies. R. leguminosarum was found in different relative abundancies in the
endospheres and rhizospheres of the three crops.; however, related members of the
family Rhizobiaceae, such as Agrobacterium sp. was only found associated with
endosphere of canola and wheat, but not pea (Lay et al. 2018).
Moreover, Essel et al. (2019), in a study focused on the selection of adequate
agronomic practices for agricultural soils, found that Rhizobium is only present in the
rhizospheric soils of wheat and pea cultivated in rotation and not in the bulk soils.
This finding represents that Rhizobium is more abundant in the soils closely attached
to the roots, exposing the specialized function of the genus Rhizobium in its
interaction with crops.
Jha et al. (2020) revealed Rhizobium as a dominant OTU amongst other
diazotrophs in rice fields. Other related OTUs, such as unclassified Rhizobiales
and unclassified Rhizobiaceae, as well as other OTUs of other rhizobia were also
found with less predominance. The addition of a strain of Rhizobium leguminosarum
as inoculant with or without a low dose of urea fertilizers reduced the OTU richness;
Rhizobium continued as relevant OTU but other OTUs belonging to AlphaProteobacteria are less abundant. Nevertheless, the beneficial effects produced by
inoculation and inoculation+lowNdose on the rice growth and yield upon inoculation is the proof of Rhizobium addition is beneficial for this crop, suggesting that the
communities are not negatively affected, at least regarding the plant.
Most of the studied crops are those cereals or similar crops with agro-economic
importance. Nevertheless, in the last years, some research has been performed in
microbiomes of vegetable plants, trees, and shrubs, all of them also with agroeconomic importance. Members of the genus Rhizobium and related genera are
also present in those microbiomes, due to their importance in plant growth promotion and biocontrol. For example, Rhizobium spp. were detected in bulk and
rhizospheric soils of cucumber plants (Jia et al. 2019). Using DGGE and not
amplicon sequencing or metagenomics, Marasco et al. (2013) found various Rhizobium species in grapevine roots, both in the rhizosphere and in the inner tissues.
Remarkably, members of the complex Allorhizobium–Rhizobium/
Pararhizobium–Rhizobium were found only in Xylella-infected and Xylellauninfected olive trees of the variety “Leccino” (tolerant to Xylella infection). They
were found by NGS in the phyllosphere and endosphere of leaves and branches
(Vergine et al. 2020). Rhizobium was found only in the resistant and not in the
susceptible cultivar, suggesting that this taxon might exert some roles related to this
cultivar resistance to pathogens.
16.5
Concluding Remarks
Rhizobium species are essential in the development of ecological agriculture not only
for its ability to fix nitrogen but also its qualities as plant probiotic bacteria for
different agronomic crops. Moreover, their easy growth under laboratory conditions
makes them an ecological alternative to chemicals fertilizers.
16
Rhizobium Presence and Functions in Microbiomes of Non-leguminous Plants
257
Molecular approaches based on “-omics” revealed their selves as essential tools
for the study of this particular genus. Overall, all these techniques showed that
members of the genus Rhizobium and related ones are always present in the tissues or
the rhizospheres of the Non-leguminous crops, suggesting their functions are essential for the plants and the environment.
To understand their role in those diverse ecosystems where it has been found,
further studies are necessary with a combination of methodologies, improving the
techniques of isolation as well as the molecular strategies. In this way, the design of
biofertilizers based on Rhizobium strains alone or in combination with other beneficial bacteria will be more effective from the economic point of view as well as
sustainable with our agricultural systems.
Acknowledgments EM acknowledges the FCT research contract from the Individual Call to
Scientific Employment Stimulus 2017 (CEECIND/00270/2017). This work is funded by National
Funds through FCT—Foundation for Science and Technology under the Project UIDB/05183/
2020.
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Part III
Insect–Fungus Mutualism
Chapter 17
Symbiotic Harmony Between Insects
and Fungi: A Mutualistic Approach
Saraswathy Nagendran, Surendra S. Agrawal, and Sheba Abraham
Abstract Microbes are known to interact with a variety of organisms belonging to
different classes, genera, or species through their own diverse and specific pathways
and mechanisms. Such an interaction, which exists between microbes and herbivores
like insects, has become a topic of great importance for researchers far and wide.
Since such interfaces occur in the form of mutual interactions, which in turn leads to
the participating organisms achieving rich and important advantages that are necessary for their survival and development. Much of the research on reciprocal interactions between insects and microbes have focused on bacterial associations with
insects, more or less ignoring the fact that interactions between insects and fungi are
equally important which usually follow the same mechanisms and pathways as
associations between insects and Bacteria.
This chapter deliberates the various aspects and properties of fungal interactions
with mushroom growers such as leaf-cutters (Attina ants), termites, and ambrosia
beetles. These interactions are based on a complex and interesting evolutionary line,
which finally introduces the concept of mutuality into this insect community. The
benefits of these interactions range from nutrition to the spore dispersal of fungi as
well as protection from predators and competitors. The interaction between yeast and
insects has also been discussed in ample detail, with our focus mainly on the areas in
which each participant in the interaction benefits.
Keywords Fungi · Insects · Beetles · Ants · Termites · Yeast · Mutualism
S. Nagendran (*) · S. S. Agrawal · S. Abraham
Shobhaben Pratapbhai Patel School of Pharmacy & Technology Management, SVKM’s
NMIMS, Mumbai, India
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_17
269
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Introduction
Nature has its own way of astonishing us at the intricate details found in each of its
naturally occurring phenomena, as well as the beauty of the complexity that revolves
around it. The harmony of the ecological interactions between different organisms of
different species and the balance that it offers for the environment as a whole is such
that, ecologists all over the world always leave with awe and wonder at the
perfection of nature’s creativity.
Ecological interactions, which indicate that certain organisms are primarily
dependent on one another for their survival, are classified according to whether
these interactions have neutral, advantageous, or harmful effects for one of the
interacting organisms. Symbiotic interactions contribute to a main category of
such interactions in which both interacting organisms are closely related. Depending
on whether the interaction partners produce a positive, negative, or neutral effect,
symbiotic relationships are divided into mutuality, parasitism, or commensalism
(Molles 2015; Smith et al. 2012).
Mutualism as a positive category of symbiotic relationships is examined in more
detail and investigated, which brings us closer to the complexity of mutual interactions between different species or organisms that cannot be taxonomically related,
but thrive together and contribute to mutual survival in a competent and challenging
environment. Although there are mutual relationships between almost all organisms
on earth, including humans, this chapter mainly focuses on the characteristics and
evolutionary history of such an interaction between different types of fungi and
insects.
So what exactly is understood when the term “mutualism” is being referred to?
Mutualism involves an interaction between two kinds of organisms, a host and a
symbiont, belonging to different class, species, or taxa. It is different from other
types of interactions as mentioned in Fig. 17.1, wherein both the organisms are
equally benefitted from the interaction, in contrast to commensalism or parasitism in
which either of the organisms gets no benefit, or they are caused harm by the other.
In short, mutualism is a reciprocatory positive interaction between a pair of organisms (Lu et al. 2016). For example, the interaction between ants and aphids in which
the ants rear aphids for their source of food which the aphids produce and in turn the
aphids are protected by the ants from potential predators. Another example of
mutualism, within the aquatic kingdom is that of clownfish and sea anemone. The
clownfish lives within the poisonous tentacles of the anemone, being immune to its
poison and act as a bait for luring other small sea animals close to the vicinity of the
anemone’s tentacles. The association of sharks with remora fish is another of the
many wonders of mutualistic interaction in the sea world. Last, but not the least, we
Fig. 17.1 Different Types
of Mutual Associations
Mutualism
Obligate
Facultative
Trophic
Defensive
Dispersive
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Symbiotic Harmony Between Insects and Fungi: A Mutualistic Approach
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humans ourselves survive on the basis of a mutualistic relationship with microbes
such as bacteria which thrive well in the intestinal microenvironment depending on
the host’s nutrition, and at the same time see to it that we remain protected against the
harmful effects of ingested pathogenic microbes (Roossinck 2008).
17.2
Fungi and the Ecosystem
It is a well-known fact that many species belonging to kingdom fungi live in
symbiotic associations with lower organisms such as cyanobacteria as well as higher
organisms belonging to kingdom plants or animals. While some species may have a
parasitic relationship with the associated organism wherein they derive food and
thereby have harmful effects on the host, it is found that some other species survive
through a mutually beneficial exchange of services between them and their associated partner. One such relationship that observed, is between fungi and plant roots.
Mycorrhiza is the term used to refer to such an alliance in which the fungus is
benefitted by the food accessed from the plant and in return the fungi mycelia help to
absorb water and aid nutrition thereby providing nourishment to the plant.
The mutual association between fungi and photosynthetic organisms, usually
cyanobacteria or green algae, is called lichen. The fungus grows around its host
and absorbs food prepared by photosynthesis in exchange for water and nutrients
(https://www.Ck12.Org/Biology/Fungi-Symbiosis/Lesson/Symbiotic-Relationships-Of-Fungi-Bio/).
When it concerns microbe-insect mutualism, much importance is directed
towards exploring associations within the bacterial society, more or less, limited
importance being given to the fungal associations that are equally common and
important. In contrast to bacterial mutualism, fungal associations with insects are
mostly facultative and horizontal, and it is reported that the microbial cells live
extracellularly in hemolymph, fat bodies, or other specialized structures of the
associated insects (Klepzig et al. 2009). A well-characterized and general example
of such a relationship is that between leaf-cutter ants and fungi, in which the ants
cultivate the fungi to gain access to their source of nourishment. A similar kind of
mutualistic interaction is seen between certain species of bark beetles and fungi too.
Other examples of insect–fungi mutualism which may not be too well known
include that of wood wasp Sirex noctilio and wood decay fungus Amylostereum
areolatum. The fungus contains wood degrading enzymes which enable the wasps to
degrade and colonize tree barks (Nielsen et al. 2009; Kukor and Martin 1983; Talbot
1977) Another example, out of the several species that can be quoted in this context,
is that of Epichloe species of fungi and Botanophila flies. The flies consume the
fungal spores and cause deposition of their fecal matter onto unfertilized stroma
rendering cross-pollination (Bultman et al. 1998; Bultman and Leuchtmann 2008).
Fungal mutualists are now also reported to play a pivotal role as producers of
chemical indicators for insect communication. The fungi Pichia pinus and
Hansenula capsulate produce are capable of converting cis and trans verbenol to
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verbenone which acts as an anti-aggregation pheromone for bark beetles (Hunt and
Borden 1990; Leufvén and Nehls 1986).
This chapter also focuses the attention on the evolutionary history as well as on
the features that include the mutual relationship between fungi and insects such as
leaf-cutting ants, ambrosia beetles, termites, and arthropods.
17.3
The Evolutionary Antiquity of Fungus-Farming
Insects
Extensive work and research in this area have proposed that the evolution of an
obligate mutualism proceeds primarily through five stages: (a) consistent and
extended contact (b) avoidance of lethal harm during contact (c) coadaptation
leading to reduced virulence and increased tolerance (d) further co-adaptation leading
to dependence or interdependence (e) still further coadaptation leading to permanence and stability in the association. These stages give an overview of the progress
of any agonistic or antagonistic interaction to stable mutualism (Taylor and FJR
1983).
Since time immemorial, even when the idea of cultivating plants for food to
sustain oneself dawned in man’s mind, the lineages of three insects namely, termites,
ants, and beetles, rose to ecological importance by evolving into fungus cultivating
farmers. Since they were completely dependent on the cultivated produce as the sole
source of their nutrition several tasks partitioned societies all playing their own
individual role in raising of food for their kind and thus in the process, became
important players of the ecosystem (Wilson 1971). These insects, mostly being pests
and detested by the human population, were put into the task of being exterminated
but only recently it had come to the knowledge of the evolutionary glory that runs
through their veins.
The transit of termite, ants, and beetles to the arena of fungi culture follows
different evolutionary pathways. In termites, fungi probably had been an important
source of nutrition derivation before they turned to cultivation. Many non-farming
termites are known to feed on fungus infested woods and the termite fungi culture is
now believed to be an expansion of such feeding habits of the ancestors (Batra 1979;
Rouland-Lefèvre et al. 2006). Studies reveal that out of the 2600 species of identified
termites, about 330 species survive in obligate association with a fungus basidiomycete genus, Termitomyces. Termitomyces are grown in subterranean combs
within the heart of termite mound nest (Batra 1979; Abe et al. 2000a). Consumption
of the fungal spores and deposition of fecal pellets consisting of the consumed fungal
spores and plant forage within the comb serves as the seed for the growth of new
fungi cultivar (Mueller and Gerardo 2002). Such a fungi culture practice also
serves as a boon for obtaining the genetic material of the fungus from termites,
sidestepping the tedious task of nest excavation. To reassemble the evolutionary
history, researchers first compared the DNA of Termitomyces with that of
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non-domesticated fungi, and secondly, comparison was drawn between the DNA of
termite farmers and non-farming relatives (Aanen et al. 2002). Studies conducted
revealed that the fungus cultivation began in termites with the Termitomyces species
of cultivars which eventually differentiated into several other cultivar species each
almost exclusively associated with a particular termite group. Moreover, each of
these cultivar groups has been found to be exchanged between the termite lineages
within these group of termites. This led to the adaptation of termite groups to
specific fungus cultivars at the same time fungus cultivars have evolved to adapt
and survive only in association with certain farming groups (Mueller and Gerardo
2002).
A striking evolutionary parallel can be drawn when comparing the emergence of
termite farmers with that of beetle and ant cultivators. In contrast to the ancestral
feeding habits of termites that evolved into termite fungi culture, an ancestral
vectoring system is responsible for the evolution of beetles into fungus cultivators.
Whereas in ants it is still not clear whether fungus cultivation emerged from
ancestral mycophagy or ancestral vectoring (Seifert et al. 1993; Mueller et al.
2001). Whatever may be the reason, it is now known that ants started growing
fungus (mostly basidiomycetes) in their backyard about 50–60 million years ago
(Mueller et al. 2001) and since then till date, roughly about 200 species of fungusgrowing ants have emerged (Schultz and Meier 1995). Ants raise their fungus
cultivation in subterranean chambers providing manure to their growth in the
form of vegetable debris, or in the case of leaf-cutter ants, leaf fragments from
live plants. The leaf-cutter ant’s sustenance is completely dependent on the fungi
they grow and hence, form an obligatory association with their fungal partners
(Mueller et al. 2001). As in the case of ants, certain species of beetles commonly
known as ambrosia beetles are found in obligatory association with fungi as they
grow them as their primary source of food and to derive important nutrition in order
to complete their life cycle (Farrell et al. 2001; Batra 1966). The fungus provides
nutrition to most of the beetle developmental stages while the insects carry the
fungal spores along with them, infecting new trees (Harrington 2005; Paine et al.
1997). The fungi also produce that degrade the indigestible wood into nutritious
matter for the insects (Valiev et al. 2009). In stark contrast to termite and ant fungi
culture which arose just once in each group and later led to diversification, studies
suggest multiple origin of fungus cultivating habit in ambrosia beetles which arose
at least seven times giving rise to sheer diversity of beetle species with respect to
their feeding habits (Farrell 1998). No records of an evolutionary reversal to a nonfungus-cultivating pattern of life in any of these nine known, independently evolved
farmer lineages have been found, suggesting a similar trend to that of humans where
transition to way of living depending on agriculture has contributed to a radical and
irreversible change that probably has bridled subsequent evolution (Diamond and
Renfrew 1997).
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Mutualism Between Leaf-Cutter Ants and Fungi
Leaf-cutter ants are considered major players of the ecosystem owing to the significant effects they elicit on local flora (Wirth et al. 2003), seedling recruitment (Costa
et al. 2008), distribution of soil nutrients (Sternberg et al. 2007) and human agriculture (Cherrett and Peregrine 1976). These species of ants have known to be in
association with fungi species since as long as 40–50 million years which it is
believed, originated on the Amazon basins (Schultz and Brady 2008). Fungusgrowing ants (Myrmicinae:Attini) cultivated fungus for their young ones using
organic debris such as dead insect parts and feces as manure for their garden
(Weber 1972; Mueller and Wcislo 1998; Mueller et al. 2005). A significant behavioral change in the ancestors Acromyrmex and Atta in replacing leaf fragments and
other plant parts as compost for their fungus gardens instead of the usual organic
debris contributed majorly to the deviation in the evolutionary pattern of the species
(Schultz and Brady 2008; Hölldobler and Wilson 2010). Such a change in the pattern
of cultivation contributed to an astounding increase in colony size, social structure,
and ecological footprint. Leaf-cutter ants have now emerged to be the most dominant
and diverse in Neotropical ecosystems, harvesting about 2–17% of the foliar biomass of annual leaf production of the forest and savanna woody plants (Wirth et al.
2003; Costa et al. 2008; Weber 1972; Hölldobler and Wilson 2010; Wheeler 1907).
Although the most common associate of attine ants remains to be basidiomycetes
fungus, a larger population of the species is involved in farming lepiotaceous fungus
of the genus Leucoagaricus while a smaller population are farmers of a distantly
related pterulaceous group of fungi (Herz et al. 2007; Chapela et al. 1994). The leafcutter ants are precise and definite about the plant species, the individual plant, and
the leaves within the plant that they cut. Factors that steer the assumption that leafcutter ants prefer relatively easy to cut, less defended leaves with high nutritional
values include selection of younger leaves than older ones, woody rather than
herbaceous and light demanding rather than shade-tolerant species (Villesen et al.
2004; Blanton and Ewel 1985; Coley and Barone 1996; Farji-Brener 2001). The
worker ants are involved in cutting the leaves, carrying the fragments to the nest,
cleaning, and processing them to form suitable for the fungus to grow and thrive in
underground chambers inside the nest (Schultz and Brady 2008). In return, the
fungus cultivar partially degrades the leaf material which serves as a source of
nutrition to the ant colonies and their developing larvae. Thus the mutualistic
interaction endures on the basis of services offered by ants ranging from weeding
and grooming to the disposal of various antimicrobial compounds while reaping the
benefits of a healthy fungus cultivar (Wirth et al. 2003; Schultz and Brady 2008;
Barke et al. 2010; Currie and Stuart 2001, Fernández-Marín et al. 2006, 2009).
Fungus gardens are principally composed of only one fungal mutualist (Aylward
et al. 2012) and factors that contribute to the low diversity of fungi in the fungus
gardens include cautious cleaning by ants to maintain the hygiene of the fungal
cultivars. Three main hygienic practices have been observed in fungus-growing ants
which include: (a) weeding removal of dead fungal debris (b) fungus grooming-
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removal of spores of foreign fungi (Wirth et al. 2003) (c) application of fecal droplets
to the fungal matrix. The fecal droplets of some species if attine ant is reported to
contain fungal chitinases and lignocellulases which contributes to both plant biomass degradation as well as eradication of fungal pathogens (Rønhede et al. 2004,
2008; Martin et al. 1973).
Apart from the predominant fungus mutualist, microbes such as Actinobacterium
of the genus Pseudonocardia, that produces antibiotic against fungus attacking
parasites (e.g., Escovopsis), have been found in association with fungus gardens
(Currie 2001; Currie et al. 1999a, b). Many of the bacterial species isolated from
these ecosystems have been established to play a major role in not just antibiotic
mediated extermination of pathogens but also in nutrient biosynthesis. One particular study carried out on Klebsiella and Pantoea species of nitrogen-fixing bacteria
isolated from leaf-cutter ant nests proves the significance of these bacteria in fixing
nitrogen and thus being important nutritional players in the ant–fungi ecosystem
(Pinto-Tomás et al. 2009; Hölldobler and Wilson 2009).
The central role of fungus gardens cultivated by ants is the conversion of plant
biomass into useful compounds important for the nutrition of the ants. The integrated
biomass of fungus gardens includes a rich source of cellulose, hemicellulose, lignin,
protein, simple sugars, and other compounds. In gardens of higher attine ants, these
compounds are converted to hyphal swellings known as gongylidia, rich in lipids,
carbohydrates, and other nutrients (Mueller et al. 2001; Martin et al. 1969).
Gongylidia serves as an important food source for the entire colony and is an
exclusive nutrient source for the developing larvae and brood (Hölldobler and
Wilson 2009; Weber 1966; Nygaard et al. 2011; Suen et al. 2011).
17.5
Adaptation of the Ant Genome
The sequenced genomes of the leaf-cutter ants Atta cephalotes and Achromyrmex
echinatior have opened up a sea of knowledge about the symbiotic association of
these species. Obligate dependence of ants on their fungi associate have led to
reductions at the genomic level (Suen et al. 2011; International Aphid Genomics
Consortium 2010). On examination, genomes of attine ants were found to be
deficient in the levels of amino acid arginine in comparison to other non-farming
ant genomes, pointing to the fact that the fungal cultivars provide the required
arginine thereby reducing the need for the particular pathway. This hypothesis is
supported by the evidence obtained from previous studies of the documented
compounds found in Atta colombica cultivar which showed presence of arginine
(Abe et al. 2000b; Johnson et al. 1981). The A. cephalotes genome was also found
devoid of the hexamerin gene responsible for amino acid sequestration during larval
development. Serine protease was another compound found in significantly low
amount, and as with arginine and hexamerin, is hypothesized to be provided by the
fungal cultivar (Abe et al. 2000b). These data indicate that over the years, having
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established a mutualistic relationship with fungi, leaf-cutter ants have lost their
ability and capacity to acquire nutrients on their own.
17.6
Fungus Cultivating Termite Species
Symbiotic relationship with a variety of organisms such as protists, methanogenic
Archaea, and bacteria have always been a major player in the evolution of termites
(Martin 1992). However, as on date only a single Termitidae sub-family,
Macrotermitinae is known to have evolved into a mutualistic association with
fungi of the genus Termitomyces (Abe et al. 2000c) and are a predominant component of the termite species in the African and Indomalayan region (Kambhampati
and Eggleton 2000). The fungus is grown on a specialized structure known as fungus
comb in the termite nest, maintained by the termites by the continual addition of new
predigested plant debris while concurrent degradation of the old material is carried
out (Kirk et al. 2001). The Macrotermitinae is divided into 11 genera with approximately 330 species and roughly about 40 species involved in Termitomyces symbiosis have been identified (Kambhampati and Eggleton 2000; Kirk et al. 2001).
They play a significant role in litter removal by rummaging through dead wood, dead
grass, and dung of herbivorous mammals. The workers collect substrate, chew them
into very small fragments, maintain constant conditions in the nest for growth of the
fungus cultivar, prevent the growth of potential competitors and thus in this way not
just disperses the fungal spores, but also ensures their healthy growth inside the nest
(Ausat et al. 1960).
The mature combs of Macrotermitinae which nests the cultivar is a firm intricate
structure housing sparse growth of mycelium and populous small white spores called
“mycotetes” that are accumulations of conidiophores and conidia, the asexual
reproductive fruiting bodies of fungi. The combs are constructed of plant material
that has been comminuted by chewing and passing through the workers gut. The cell
wall polysaccharides undergoes insignificant breakdown and transformation during
its transit through the digestive tract, as indicated by the presence of intact cells and
cell walls, high cellulose content of the comb, and negligible difference in the
cellulose-to-lignin ratio of the comb to the rummaged plant debris (Sands 1956).
The termite’s diet consists of both the fungus combs and the mycotetes, both
performing an integral role in providing a source of excellent nourishment to the
termites (Nygaard et al. 2011; Ausat et al. 1960) due to the presence of elevated
nitrogen content constituting a range of 5.7–7.9%. Additionally, the fungal enzymes
released from the mycotetes after ingestion combines with the enzymes present in
the insect gut rendering high catalytic activity that contributes to cellulose and
hemicellulose digestion of the plant material in the Macrotermes species (Sands
1956; Abo-Khatwa 1978; Martin and Martin 1978; Rouland et al. 1988). Due to the
availability of a better and richer source of nutrition, the termites no longer depend
on woodchips or filter paper to derive nutrition, unless in cases when they are
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destitute of all food obtained through the fungal symbiont (Martin 1987; Wood and
Thomas 1989).
It has been observed that the fruiting bodies of Termitomyces are always found in
association with the termite nests indicating that within the basidiomycetes
(Rouland-Lefèvre 2000; Rouland-Lefèvre and Bignell 2001) mutualistic symbiosis
traces back to a single evolutionary origin and no record of reverting back to the
nonsymbiotic way of living has been reported (Rouland-Lefevre et al. 2002;
Moncalvo et al. 2000, 2002). The patterns of cospeciation and specificity have
been found to be steady and undeviating with transmission of fungal symbionts
from host to host outside the vertical host lineage (Frank 1996). Horizontal symbiont
transmission is the pattern observed in many species of Macrotermitinae associated
with formation of fruiting bodies in its fungal symbiont. However, for Microtermes
and species of M.bellicosus, clonal uniparental transmission has been observed. The
female in Microtermes and the male in M.bellicosus takes up the task of transmitting
the fungus in the absence of sexual fruiting bodies (Darlington 1994).
17.7
Mutualistic Association of Beetle With Fungi
The bark and ambrosia beetles are either considered as two families Scolytidae and
Platypodidae within the weevils (Curculionoidea) or as sub-families of
Curculionoidea (Hsiau and Harrington 2003; Vega 2014). Ambrosia beetles are
derived from bark beetles that colonize and consume phloem, which is considered to
be more nutritious than wood and are mostly found in association with its fungal
symbiont Ascomycotan fungi, the level of association ranging from facultative to
obligative. Bark beetles, similar to ambrosia beetles, have been found mostly in
association with Ascomycotan and hardly ever in association with basidiomycotan
fungi. The variability of their association is reported to range from being facultative
to obligate mutualists (Harrington 2005; Vega 2014; Li et al. 2016).
Ambrosiodmus is a genus consisting of over 80 species within the largest group of
ambrosia beetles, Xyleborini (Wood 1982, 1992; Hopkins 1915; Batra 1985).
Ambrosia beetles are known to have evolved into symbiotic fungi culture after
at least 11 subsequent evolutionary patterns (Wood 1982). About 3200 species of
ambrosia beetles are known to be fungus farmers of the genus Ambrosia. The most
prominent ambrosia feeding genera include Xyleborus, Trypodendron,
Gnathotrichus, and Anisandrous, belonging to family Scolytidae and are widely
distributed across the temperate and tropical regions (Beaver et al. 1989). The fungus
cultivar, responsible for providing nutrition to the beetles during their period of
dormancy and inactiveness as well as during the stages of active growth and
development, is grown and sheltered by beetles in specialized storage organs
known as mycangia. Mycangia, also known as mycetangia are ectodermal glandular
pockets of beetles where ambrosia fungi are stored and where they grow and
multiply (Hulcr and Cognato 2010). Mycangia are known to have evolved in two
ways, as pocket like dilation of cuticle or as newly developed hollow glands from
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glands that previously produced defensive compounds and oils in order to prevent
excessive flow of sap (Vega and Blackwell 2005). Unlike other species of ambrosia
beetles that mostly colonize dead but relatively fresh wood tissues, ambrosia beetles
are capable of infesting wood throughout the period of its decay, including the final
stages of decay when the xylem is inhabited and colonized by other competitive
wood-rotting fungi (Beaver et al. 1989). In contrast to the fungus-farming behavior
of the Ambrosiodmus, its phylogenetically related sister Ambrosiophilus is known to
rely on mycoclepty (fungus theft) to obtain nourishment, having lost the ability to
culture their own fungal farms (Vega and Blackwell 2005).
The fungi inoculum carried inside mycangia gets discharged into the beetle
tunnels on the bark while burrowing or during oviposition. The nitrogenous waste
eliminated by the beetles serve as fertile manure providing nutrient-rich medium for
the fungus to grow and thrive. The beetle larvae in turn are dependent on the fungus
to derive nourishment. Prior to several days before the eggs are hatched, the fungus
weakens the wood elements aiding the larvae in excavation as well as broadening of
tunnels. Such a mutualistic interaction has its own importance in the ecosystem as
many nonspecific wood-decaying fungi in association with insects such as beetles
have been found to augment and accelerate the process of wood decaying, degradation, and nutrient recycling in the forest ecosystem (Beaver et al. 1989).
The mycangial secretions of ambrosia beetles are responsible for morphological
characteristics and biology of the ambrosia fungi. Conidia and other reproductive
spores of these fungi develop into the ambrosia stage within the mycangia of the
beetles. Mycangial secretions found to contain a rich store of oils and proteins, serve
as a source of nourishment and preservation of the fungal inoculum. Mycangial
contents of other species of ambrosia beetles have also reported to contain compounds such as amino acids, fat, and proteins, confirmed through thin layer chromatographic techniques (Hulcr and Cognato 2010).
17.8
Fungi and Insect Mutualistic Association
“Yeast” is used to refer to a fungal growth form consisting of only a single cell and
lacking specialized sex organs, thereby reproducing through the process of budding
via sexual spores from somatic cells, which are not enclosed within fruiting bodies
(Vega and Blackwell 2005; Vega et al. 2008; Kurtzman et al. 2011). This group is
considered to be omnipresent and found to occupy a variety of ecological niches in
both terrestrial and aquatic ecosystems (Kurtzman and Fell 2006). About 1500
species of yeasts have been identified, mostly which belong to the phyla Ascomycota
and Basidiomycota (Urubschurov and Janczyk 2011). Almost 700 species from
about 93 genera under the class Saccharomycetes of Ascomycota are referred to as
“true yeasts” (Batra 1979). Genera belonging to this class include Candida,
Kluyveromyces, Metschnikowia, Pichia, and Saccharomyces. Fellomyces, Tremella,
Ustilago, and Cystofilobasidium are some examples of fungi that belong to the class
17
Symbiotic Harmony Between Insects and Fungi: A Mutualistic Approach
279
Basidiomycota (Urubschurov and Janczyk 2011; Landell et al. 2009; Suh et al. 2004;
Fell et al. 1999; Gibson and Hunter 2005).
Most of the species of yeast discovered to be in mutualistic relationship with
insects is found to dwell within the gastrointestinal tract of the host and retrieved
from faeces, ovipositors, or other specialized organs of the insects, thus directing a
hypothesis of their facultative relationship with the host (Boekhout 2005).
A vast range of variability among the hosts and their habitats have led to the rise
of unknown number of new species of yeasts, that almost correspond to the total
number of organisms they are found to be in association with since each host is
assumed to carry their own particular and specific yeast partner. An example of this
is the wide range of yeast species inhabiting the guts of Erotylidae and
Tenebrionidae families of beetles, the number of yeast species being equal to the
number of beetle species under each family (Sung-Oui et al. 2005).
Although most often “true yeasts” are involved in symbiosis with insects, studies
report that a separate group of fungal endosymbionts known as “yeast like symbionts” (YLS) also exist in association with insects as shown in Fig. 17.2 (Suh et al.
2001, 2004). This group is supposedly believed to have evolved from ascocarpic
ascomycetes, especially from the subphylum Pezizomycotina (Gibson and Hunter
2010). Notwithstanding their ability to form beneficial association with insects, they
are known to be phylogenetically more related to Hypocreales than to
Saccharomycetales (true yeasts). This relation directs the attention to an interesting
area of evolutionary mutualism, since Hypocreales belong to the family of
Fig. 17.2 Taxonomic Classification of Yeast and Yeast-like Symbionts [adapted from Suh et al.
(2004) and (Suh et al. (2001)]
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S. Nagendran et al.
Clavicipitaceae which consists of entomopathogenic fungi making it quite evident
that YLS is comparatively more related to entomopathogenic fungi than to yeasts
(Fukatsu and Ishikawa 1996). This places an evidence of two separate pathways of
fungi evolution-mutualistic YLS having their source of origin in insect pathogenic
microbes whereas other related yeasts apparently having evolved from commensal
interactions (Noda and Kodama 1996). However, the variability in interactions
between YLS and insects are sparser than yeast-insect interactions. As per studies
and researches to date, examples of mutualistic insect-YLS interactions have been
found mostly in anobiid beetles and also in some planthopper and aphid species
(Sasaki et al. 1996; Douglas and Smith 1989; Engel and Moran 2013).
17.9
Services Offered and Benefits Gained
Though much speculation still exists on the benefits involved in insect–fungi
mutualism, studies till date have reported that the major benefits that fungi reap
out such a relationship include protection and dispersal of their spores as well as
provisions for outbreeding (Kurtzman et al. 2011; Coluccio et al. 2008).
Although insect digestive tracts are considered as one of the highly vulnerable
areas for microbial; colonization, few microbes have developed resistance to withstand the harsh gut environment and are successfully passed on to host congeners.
Although design of the digestive tract varies among different insects and insect
orders, the basic anatomy consists of three main regions: foregut, midgut, and
hindgut, each possessing their own specialized role in digestion. This variation in
turn facilitates distinct abilities in establishing symbiosis with microorganisms. It is
generally understood that symbionts are first achieved by ingestion or interaction
with the congeners and the environment followed by colonization of the guts and
subsequently released from insect molts and faeces for further dispersion of microbial cells. Though the survival and transfer mechanism of yeasts are not well known,
it is expected to have similar trends as that of the extensively explored mechanisms
of bacterial symbionts (Cory and Ericsson 2009; Carlile et al. 2001; Gonzalez 2014).
In contrast to the filamentous structures produced by certain fungi to aid the process
of dispersal of conidiospores and ascospores, yeasts are known to produce reproductive spores that are capable of withstanding even extreme conditions of stress
such as temperature, salt concentrations, and pH (Carlile et al. 2001; Reuter et al.
2007). The spore wall provides resistance from not just extremes of environmental
conditions but also against the stresses encountered in the digestive system, as
inferred from the study of survival of spores in the gut of Drosophila melanogaster
(Carlile et al. 2001). Thus the successful transfer of yeast spores is facilitated through
insect feeding and oviposition (Pulvirenti et al. 2002).
Outbreeding is another benefit gained yeast as an extension to the transit and
survival of sexual spores through the unwelcoming, harsh environment in the insect
gut. Outbreeding is an important concept in order to maintain genetic variation
among the descendants to facilitate adaptation and thus evolution. Unfavorable,
17
Symbiotic Harmony Between Insects and Fungi: A Mutualistic Approach
281
harsh conditions mostly contribute to the reproduction in yeasts through the formation of diploid vegetative cells that can undergo meiosis to give rise to a tetrad of
haploid spores that can germinate, mate, and restore the diploid state. Most often, it
has also come to notice that the four spores contained within the same capsule may
undergo selfing or inbreeding. This process is prevented by the gut enzymes that
break open the capsule to release the spores, thus inhibiting inbreeding and assisting
the phenomena of outbreeding (Guzmán et al. 2013; Lachance and Bowles 2002). A
study conducted by making use of genetically marked strains of S.cerevisiae divided
into two groups, one in contact with fruit flies and another in no contact with insects,
demonstrated a significant increase in the number of heterozygotes with insect
association than in yeast that was exposed to non-insect mediated mating (Guzmán
et al. 2013). An example of yeast receiving benefits in all three forms—protection,
facilitation outbreeding, and spore dispersal—is Metschnikowia species in association with pollinating insects of the orders Diptera, Coleoptera, and Hymenoptera.
The yeast is found particularly in the nectar of flowers thus explaining their association with insect pollinators (Lachance et al. 2001, 2003; Janson et al. 2008;
Bismanis 1976).
The benefits achieved by insects as a result of this interaction include nutrition
source, detoxication from harmful substances, protection from biotic stress as well as
an aid for chemical communication (Noda and Kodama 1996; Jurzitza 1970).
The role played by yeast mutualists in providing nourishment to insects is
interpreted from the fact that insect performance and development decreases in the
absence of their yeast associate (Kurtzman et al. 2011; Kurtzman and Fell 2006).
Yeast cells form an excellent source of nitrogen, containing about 7.5–8.5% of
nitrogen by weight, apart from other essential nutrients such as vitamin B3 and
B5, proteins, trace metals and amino acids which are broken down and absorbed by
simple digestion in the insect gut (96–97,108). Insects such as Pseodococcus citri,
and wood-boring cerambycids, Leptura, and Rhagium are reported to depend on
yeasts for their dietary source of nitrogen (Starmer and Aberdeen 1990; Noda and
Koizumi 2003). The rich source of nitrogen, lipids, and vitamins provide major
nutritional support to Drosophila flies especially during the stages of egg maturation
and larval development (Shen and Dowd 1991a). Similar to the nutritional role
played by yeasts, YLS are also reported to play a pivotal role in being nutritional hub
to their host. Symbiotaphrina and anobiid beetles are supplied by nutrients like
nitrogen, sterols, vitamins, and essential amino acids by YLS (Starmer and Aberdeen
1990; Noda and Koizumi 2003). Similarly important intermediate precursors for
ergosterol biosynthesis in rice planthoppers are also provided by the yeast-like
symbionts (Shen and Dowd 1991b).
The wide range of enzymes produced by insect associated yeasts includes
exoproteases and peptidases (involved in protein degradation), lipases (for digestion
of fatty acids), and hydrolytic enzymes involved in sugar degradation (Chararas et al.
1983). These enzymes play a role in conversion of complex molecules and polysaccharides to simple compounds like glucose or sugars that are easily and directly
absorbed by the insects. This role is however mainly attributed to the enzymes
produced by YLS, which release digestive enzymes into their surroundings for
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S. Nagendran et al.
colonization and spreading to new areas, in contrast to true yeasts, which are more
sessile and are not known to release digestive enzymes unless they are trapped in
their own erosion zones (Noda and Kodama 1996). An exception to this nature is
demonstrated by true yeasts belonging to the genus Candida which possess the
ability to degrade wood components such as cellulose, pectin, and glucosides
(Listemann 1988).
The process of detoxification by yeasts have proved that mutualism between
insects and fungi is a detail of much significance in herbivory. Detoxification in this
context relates to making certain nutrients available by neutralizing or decomposing
the toxins, thus aiding the process of digestion. In some cases, detoxification
converts these nutrients to more polar forms that can be easily removed from the
host’s digestive system (Kurtzman et al. 2011; Kurtzman and Fell 2006; Cory and
Ericsson 2009). An example of detoxification by yeasts is clearly elaborated in the
YLS, S.kochii which is found in association with the beetle L.serricorne (Kurtzman
et al. 2011; Kurtzman and Fell 2006). S.kochii is reported to have the potential to
detoxify a variety of plant allelochemicals, metal toxins, insecticides, and herbicides
in addition to producing detoxifying enzymes such as aromatic ester hydrolase,
glucosidase, phosphatase, and glutathione transferase that converts toxic chemicals
to important carbon sources (Chararas et al. 1983).
Biotic hazards faced by herbivores include competitors, parasites, predators and
plant chemical defenses. Plant diseases could have a negative impact on insects
therefore insect–yeast mutualism aides the availability of safe food sources as yeasts
have demonstrated various protection mechanism of plant tissues from infesting
pathogens (Pulvirenti et al. 2002; Listemann 1988). Yeasts could play a role in
limiting the presence of other fungi or microbes inside plant tissues thus favoring the
growth and development of associated insects. Metschnikowia species decrease the
prevalence of molds inside apples which indirectly correspond to lower mortality
and larval development time for Cydia pomonella (Witzgall et al. 2012). These
results are a clear illustration that yeasts not only performs the role as nutrition
provider to insects, but also ensures that opportunistic pathogens and microbes that
might hinder the development of both participants of symbiosis are kept at bay
(Pulvirenti et al. 2002).
17.10
Conclusion
Researches and studies conducted so far have pointed to the fact that fungus farmers
had not always been involved in mutualistic relations with fungi, but it is a phenomenon that has evolved over a period of nearly 50 million years due to variations
and adaptations amongst various species of organisms. It has manifested its effects
even at the genome level as in case of attine ants whose genome is deficient of genes
responsible for the production of several important enzymes since these are now
easily available in ready form from the fungi in association. Yeast and yeast-like
organisms have developed resistant mechanisms to survive in the harsh dynamic
17
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283
environment of insect gut which in turn facilitates spore disposal through feces and
insect oviposition. The mutualistic evolution has come a long a way in developing
mechanisms for the survival and development of both insects and fungi alike. It is a
demonstration of the potential of organisms to develop adaptations in the face of
unfavorable surroundings and environment giving rise to variations and modifications within different species.
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Chapter 18
Panorama of Metarhizium: Host Interaction
and Its Uses in Biocontrol and Plant Growth
Promotion
Srinivas Patil, Gargi Sarraf, and Amit C. Kharkwal
Abstract Vectors have been wreaking a fatal havoc on mankind by causing diseases in agriculturally important plants and humans. Not only diseases caused by
them are a hefty task to deal with, but their increasingly successful survival in human
settlements is also a rising concern. The entomopathogenic fungi are considered
amongst the first organisms for bio management of agriculturally important pests as
they are eco-friendly, economically sustainable, and effective. With this, the collateral need for biocontrol in human disease vectors is also being felt. The first
observation of fungi infecting insects was in as early as 900 AD, to the first data
published in 1726 about entomopathogenic fungi. Metarhizium is a widespread
fungus found all over the globe. More than 200 species of insects are infected by
the fungus thereby making it one of the most sought biocontrol agents. This chapter
gives an understanding of interaction between an arthropod host and
entomopathogenic fungi genera Metarhizium, description of the host and fungal
structure, what are some of the conventional and recent efforts done in order to
improve the application strategies and what could be some of the possible uses of
Metarhizium in enhancing plant health. Some of the plant pests and animal vectors
which have been explored as host for Metarhizium are also mentioned.
Keywords Entomopathogenic fungi · Metarhizium · Insect–fungus interaction ·
PGP · Arthropod
18.1
Introduction
Metarhizium is distributed very much uniformly across the globe, from arctic to
tropics and this feature gives it an edge over the other biocontrol agents. Being a
fungus it does not need to be necessarily ingested, mostly all it requires is contact
S. Patil · G. Sarraf · A. C. Kharkwal (*)
Amity Institute of Microbial Technology, Amity University Noida, Noida, Uttar Pradesh, India
e-mail: ackharkwal@amity.edu
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_18
289
290
S. Patil et al.
with the host cuticle (Kamareddine 2012) also it is very much prolific in terms of
mass production (Rasgon 2011). Metarhizium genus comprises of species that have
both
narrow-spectrum
entomopathogenecity
and
broad-spectrum
entomopathogenecity, however, one of the most sought after species is
Metarhizhium anisopliae (Aw and Hue 2017). M. anisopliae is a generalist that
has been known to infect more than seven orders (Aw and Hue 2017). Majorly the
earlier discovered species are M. anisopliae, M. truncatum, M. cicadinum,
M. brunneum, M. flavoviride, M. taii, M. cylindrosporum, and M. viridicolumnare
(Bischoff et al. 2009). However with advent of time several species and improved
strains were also added to the list. The fungus is target specific, their generation time
is short and it can survive in the environment for long when no host is available due
to its ability of resting stage (Sandhu et al. 2012). Entomopathogenic fungi can be
instilled in IPM (integrated pest management) as they show synergistic activity to
control pestiferous insects in addition to the use of natural predators and other
biocontrol agents like parasitoids and innovative approaches can be met in IPM
using genomic techniques (Chandler et al. 2011; Erler and Ates 2015).
This fungus has many reasons to be used some of which are: firstly, effective
broad-spectrum mortality, Secondly, fast and inexpensive mass production (Scholte
et al. 2004). The complexity of using fungal spores as biocontrol agent is that the
spores need optimal level of abiotic factors namely temperature, relative humidity,
salinity, sunlight, and UV light exposure to break dormancy. Also, it needs contact
with the host at all times to germinate which means it requires repeated applications
if there are no hosts present for prolonged time and when we use it as a vector control
agent it might be harmful to many nontarget insects (Scholte et al. 2004). The toxin
destruxin affects structural integrity of cell membrane of host thereby damaging host
tissue and it also causes fluid loss (Scholte et al. 2004)
18.2
Hosts
Certain strains of Metarhizium have successfully shown significant pathogenicity
toward certain human disease vectors and plant pests. However, there have been
some studies pertaining to some other strains which do not have a firm conclusion.
So all in all there are several hosts for different Metarhizium strains. But in this
chapter only selectively significant Metarhizium—host interactions have been mentioned (Table 18.1). The studies mentioning these interactions were aimed at the
possible use of Metarhizium as a biocontrol agent (Table 18.2).
18.3
Structure and Mechanism
Although the insect anatomical structure is highly detailed, in this chapter we have
elaborated certain portions of the arthropod anatomy, which is relevant in understanding the mode of infection. Metarhizium spore (conidia) generally germinates
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Panorama of Metarhizium: Host Interaction and Its Uses in Biocontrol and. . .
291
Table 18.1 Metarhizium interaction with human and animal disease vectors
Species/strain
Metarhizium
pingshaense (Met_S26
and Met_S10)
Parasitic on
Anopheles coluzzii
Disease
Malaria
Metarhizium anisopliae
ICIPE-30
M. anisopliae
Anopheles gambiae
Malaria
Aedes aegypti
M. anisopliae
Culex quinquefasciatus
M. anisopliae
Aedes albopictus
M. anisopliae
Rhipicephalus
sanguineus
Ixodes scapularis
Anocentor nitens
Yellow fever mosquito,
chikungunya, dengue, etc.
Wuchereria, West Nile
Virus, avian pox
Dengue fever, Chikungunya,
also capable of hosting Zika
virus and certain nematodes
Canine ehrlichiosis
M. anisopliae (Ma959,
MaE9 and Ma319)
M. anisopliae
M. anisopliae ICIPE 30
Metarhizium brunneum
ARSEF 4556 (with
Toxorhynchites
brevipalpis)
M. anisopliae
Ixodes scapularis
Amblyomma
americanum
Glossina fuscipes
fuscipes (controls parasite Trypanosoma
congolense)
Aedes aegypti
Rhodnius prolixus
Lyme disease
Tropical horse tick
Rocky Mountain spotted
fever (Rickettsia rickettsiae)
Trypanosomiasis
References
Liao et al.
(2017),
Bilgo et al.
(2018)
Mnyone
et al. (2011)
Carolino
et al. (2014)
Lacey et al.
(1988)
Scholte
et al. (2007)
Kirkland
et al. (2004)
Bittencourt
et al. (2000)
Kurtti and
Keyhani
(2008)
Wamiti
et al. (2018)
Yellow fever mosquito,
chikungunya, dengue, etc.
Alkhaibari
et al. (2018)
Chagas disease
Garcia et al.
(2016)
when it comes into contact with the host’s cuticle and then it outgrows the arthropod’s body by draining its nutrition, resulting in the death of the mosquito
(Fig. 18.1). The journey of the fungal spore from epicuticle (outermost interaction
site) to hemocoel (terminal) witnesses the upregulation and production of many
genes and proteins in both host and fungus. Prominent ones have been described in
pathogenesis section.
18.3.1 Host Structure
A well-known fact about the insects is that they are devoid of an endoskeleton. It has
only an exoskeleton. The integument is the outermost layer divided into epidermis
292
S. Patil et al.
Table 18.2 Metarhizium interaction with various plant pests
Species/strain
Metarhizium anisopliae
M. anisopliae (ESALQ1604)
M. anisopliae (Metschnikoff)
Sorokin variety anisopliae
M. brachyspermum sp. nov.
(Clavicipitaceae)
M. anisopliae
M. anisopliae
M. brunneum
M. anisopliae
M. anisopliae (ICIPE 69 and
ICIPE 18)
M. acridum
M. anisopliae
Parasitic on (common name)
Polyphylla fullo (june beetle)
Mahanarva (spittlebugs)
Tuta absoluta (tomato borer);
Aethina tumida (small hive beetle)
Elateridae (click beetles)
References
Erler and Ates (2015)
Iwanicki et al. (2019)
Muerrle et al. (2006),
Contreras et al. (2014)
Yamamoto et al. (2019)
Oryctes rhinoceros (coconut rhinoceros beetle)
Culicoides spp.
Bactrocera oleae (olive fly)
Bactrocera cucurbitae (melon fly)
Zeugodacus cucurbitae (melon fly)
Indriyanti et al. (2017)
Locusta migratoria manilensis (oriental migratory locust)
Thaumatotibia leucotreta (false
codling moth)
Zhang et al. (2015)
Narladkar et al. (2015)
Yousef et al. (2018)
Sookar et al. (2014)
Onsongo et al. (2019)
Mkiga et al. (2020)
and cuticle. The cuticle is a chitinous structure in which the host arthropod body is
enclosed. Protein makes 70% of the host cuticle (Charnley 2003). It can be
interpreted that the cuticle is segregated into the epicuticle and procuticle. The
procuticle is coated with a thin waxy and slimy layer known as the epicuticle. The
procuticle is comprised of exocuticle and endocuticle. Mesocuticle is sclerotized and
hardened region, which sometimes might be present in between them (Chapman
2012). It is mainly the pro cuticle, which is composed mainly of chitin and several
other proteins. As discussed above the pro cuticle has endo- and exocuticle. So the
endocuticle is a matrix of chitin intermixed with protein, providing a different
property to the pure chitin. The modification of chitin with the protein confers its
additional properties. While the exocuticle is mainly composed of sclerotin which
essentially is a cross-linked form of certain proteins (Pryor 1940; Li and Ortiz 2014).
Not only this sclerotin is present in exoskeleton, but it is distributed among the
mouthparts used for biting and the dorsal and ventral sides. The composition of
sclerotin varies among the insect hosts and also within the host sclerotin composition
varies among different body parts, also certain regions are less sclerotinised certain
regions are more sclerotinised (Russell et al. 2016). Beneath the exoskeleton lies the
body which is divided into head, thorax, and cuticle (Chapman 2012).
18.3.1.1
Hemolymph
Hemolymph, which is considered as blood of arthropods, flows through sinuses
referred to as a hemocoel. Hemocytes are the cells, which get circulated through
hemolymph. Hemolymph mainly comprises of water, chlorine, sodium, potassium,
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Panorama of Metarhizium: Host Interaction and Its Uses in Biocontrol and. . .
293
A
B
Epicuticle
Transmission of
spores
C
Procuticle
D
Epidermal
cells
E
Hemocoel
Fig. 18.1 The figure shows the path of fungal spore via the lateral cross-section of anatomical
layers of the host and its life cycle. (A) conidiospore adhesion, (B) appressorium formation and peg
penetration, (C) hyphal infection progressing in pro cuticle, (D) hyphae invading epidermal cells
and (E) hemocoel colonization blastospores formation. Reference: based on Constanza Mannino et
al. (2019)
calcium, magnesium, and biomolecules. Hemocyanin an oxygen-carrying protein is
also directly present in hemolymph (Sowers et al. 2006). Significant concentration of
free amino acids, presence of glucose, fructose, and sucrose are also found, although
concentrations of these substances might vary from stage to stage (Wyatt 1961). As
hemolymph is also the site for humoral defense responses for which many proteins
are present like antimicrobial peptides, enzymes (later mentioned pathogenesis
section), and also in some cases free amino acids like tyrosine aid in a process called
humoral encapsulation (Chapman 2012), which contains the foreign body into a
thick covering.
294
18.3.1.2
S. Patil et al.
Hemocoel
Arthropods have an open circulatory system and their body cavity is called hemocoel. The space is for open blood (hemolymph) circulation. It is divided into three
sinuses (pericardial, perineural and visceral sinus) and into compartments where
respective organs directly bathe in hemolymph (Chapman 2012; Theopold et al.
2004). In the end of infection process, the fungal spores through the hemolymph
reach hemocoel (Mondal et al. 2016) and subsequently all the organs.
18.3.1.3
Hemocytes
They can be called as the blood cells of the insect circulatory system and have an
integral role to play in insect defense mechanism. As mentioned by Chapman
(2012), they can be majorly classified as prohemocytes, plasmocytes, granulocytes,
adipohemocytes, oenocytoids, and spherule cells.
Prohemocytes are basically the progenitor for many other types of hemocytes.
Plasmocytes are present in large quantities mainly phagocytizing and encapsulating
the foreign bodies, for example, Beauveria an entomopathogenic fungus is phagocytized but it can suppress the immune response. Granulocytes -as the name suggests
contain large amounts of membrane-bound structures which are granules. These
granules are released as a part of defense mechanism.
Adipohemocytes are lipid containing hemocytes. Oenocytoids might not present
in all the orders, mainly present in lepidoptera (Chapman 2012). As such there is no
special function for this, but a study by (Wang and St Leger 2007) shows that
conversion of prophenoloxidase (which is stored into oenocytoids) into phenol
oxidase by eicosanoids plays a role in the defense response.
Spherule cells contain small spherical bodies which are called as spherules and
their function is unknown (Chapman 2012)
18.3.1.4
Fat Bodies
The biomolecules mostly present in the hemolymph are synthesized or stored at
some time in the fat bodies. Fat bodies are a tissue-like organization of trophocytes
or adipocytes which may be supplemented by the presence of urate cells, tracheal
cells. It synthesizes many hemolymph proteins, stores glycogen which is later
converted into trehalose a key sugar source in the hemolymph. It also serves as a
storage site for most lipids in insects (Chapman 2012).
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18.3.2 Fungal Spores
Metarhizium belongs to the Hypocreales order and the family Clavicipitaceae. The
peculiar characteristic is that they are widely used insect pathogens. The spores of
fungi are reproductive agents adapted for survival during unfavorable conditions and
proliferate in a conducive environment. Conidiospores are asexual, exogenic spores
formed mitotically. The hyphae are aseptate and the shape of each conidia is ovoid or
cylindrical. It forms chains that appear cylindrical column-like or prismatic. It
becomes conical at the apex and asci are arranged in dense hymenium (Money
2016; Sinha et al. 2016). The conidia grow on phialides which are whorls of
branches ramified from conidiophores (Sinha et al. 2016). Conidiophores which
bear conidiospores are translucent in appearance. The conidia can differ from species
to species. The conidia can be straight-sided and small as of M. anisopliae var
anisopliae or another var. majus can be large measuring up-to 18 μm. M. flavoviride
have swollen light green conidia with club-shaped phialides. The rate of growth of
conidia may differ too (Glare et al. 1996). The maximum temperature for most M.
anisopliae isolates is 37 C. However, there can be variability in thermotolerance
(Fernandes et al. 2010).
18.3.2.1
Appressorium as a Structure
Appressorium is a specialized invasive structure which is basically an extension of
germ tube to penetrate the host tissues which are intact. Appressorium is one of the
salient features of both plant and arthropod pathogenic fungi. A study on in vitro
appressorium production (Butt et al. 2016a) states that after germination as soon as
the germ tube comes into contact with a hard surface infection structures are
produced. As a plant defense response waxes may entrap and inhibit the germination
of fungal conidia (Butt et al. 2016a, b). From a general prospect, appressorium can
exist in unicellular, multicellular, or simply as a terminal swollen part of germ tube
or completely differentiated structure in certain plant pathogens (Liu et al. 2012).
Also as explained by Liu et al. (2012), in studies done on Magnaporthe oryzae
during the development of appressorium—after germination, the conidiospore
undergoes a set of events in the cell cycle and cell division. This is followed by
appearance of an actomyosin ring which partitions the cell and the structure which
will penetrate the host. Autophagy is then initiated in the spore cells which causes the
cellular contents to flow inside the appressorium which makes it turgid and enhance
its mechanical strength for penetration. Whereas in Metarhizium, MPL1 gene which
produces perilipin homolog, Ca+2, etc. maintains turgidity, actin cytoskeleton, chitin
and dihydroxynaphthalene maintains structural support (Leger et al. 1991b; Gauthier
and Keller 2013)
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18.3.3 Pathogenesis
Metarhizium spore (conidiospore) generally adheres and germinates when it comes
into contact with the host’s cuticle and then it outgrows the arthropod’s body by
draining its nutrition, resulting in the death of the mosquito. Initially it starts with the
development of appressorium, which penetrates into the host and then subsequently
forms an infection peg, and then when it enters hemolymph, the formation of hyphae
takes place, which releases toxins and then leads to death of the host (Scholte et al.
2004). The toxin destruxin affects the structural integrity of cell membrane of host
thereby damaging host tissue and it also causes fluid loss (Scholte et al. 2004).
As referred from Aw and Hue (2017), there are six stages in the pathogenesis of
Metarhizium (Aw and Hue 2017). These are given as below:
18.3.3.1
Adhesion
It is the initial event in which the asexual spores, which are conidia get attached to
the cuticle of the host. The Metarhizium conidia are surrounded by an outer layer of
rodlet cells. These rodlet cells consist of a protein “hydrophobin.”
Hydrophobins are cysteine-rich proteins which are present in majority of fungi.
These hydrophobins confer an amphipathic nature to rodlets aiding them in attachment to the hydrophobic epicuticle. Hydrophobins also play a major role in reducing
the spore wettability thus forming a water-resistant layer (Sunde et al. 2008). When
the spores are dispersed aerially they land on the epicuticle and the attachment of
spore is due to hydrophobic interactions, electrostatic forces, and interaction of
proteins (Aw and Hue 2017). Various external factors affect the attachment such
as water, oxygen, nutrients, pH, hydrophobicity of host surface, and environmental
conditions. The fungi can have specific requisites to infect restricted hosts (Sandhu
et al. 2012).
Another recent study states that Mad 1 and Mad 2 are responsible for anchorage
to insects as well as plants, respectively. Mad are Metarhizium adhesin like protein
(Wang and St Leger 2007).
As explained by Greenfield et al. (2014) the adhesion follows a two-step process.
Initially the superficial attachment occurs by electrostatic and hydrophobic forces or
by attachment via adhesion proteins. The next step is to release enzymes to facilitate
cuticle penetration. Also, the release of hydrolytic enzymes degrades fatty acids and
release nutrients, which might aid in germination. The adhesins protein Mad 1 and
2 further strengthen the attachment (Greenfield et al. 2014).
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18.3.3.2
297
Germination
The germination step is initiated by various non-specific exogenous nitrogen and
carbon sources (Sandhu et al. 2012; Aw and Hue 2017). A study done by Santi et al.
(2010) has reported different enzymes acting on the spore surface proteins. The
spore surface proteins have been found to undergo degradation by different
proteases.
These degrading enzymes comprise majorly of trehalase, seven different
chitinases, two lipolytic enzymes have been detected. The trehalase enzyme ensures
steady supply of glucose by breakdown of trehalose. Also phospholipase C which
cleaves phospholipids was detected in M. anisopliae spores. The spore surface
proteins not only have spore proteolytic activities but activities against reactive
oxygen species (Santi et al. 2010), which might be produced on the host cuticle as
defense response. Expression of Mest1 gene in M. robertsii helps aids in fast lipid
hydrolysis and germination, on the contrary, M. acridum is helped by broadened
host range expression of same gene. The expression of particular gene can be
specific to particular host (Wang et al. 2011).
During the spore germination the spore absorbs water and nutrition from the host
surface by osmosis and develops a germ tube which is an elongated structure
(R. Barkai-Golan 2001)
18.3.3.3
Formation of Appressorium
Apart from the general organization and development of appressorium explained
above, there are many specific molecular features in Metarhizium, which are
explained below. Although some of them might be common to certain other
entomopathogenic fungi. We can understand appressorium formation given below
by bifurcating it into two major points. First is differentiation of germ tube end into
appressorium and second maintaining of the turgor pressure for penetration into host
cuticle (Fig. 18.2). Expression of ODC1 gene aids in appressorium formation. It
encodes for ornithine decarboxylase. As it is known that ornithine decarboxylase
enzyme is essential for cell growth as it stabilizes the DNA structure which prevents
apoptosis (and supports the excess cell proliferation).The ornithine decarboxylase
causes decarboxylation of ornithine which in turn aids in production polyamines
which are directly involved in DNA structure stabilization (Pendeville et al. 2001).
The expression of this gene is increased during appressorium formation and germination (Pulido et al. 2011). Pmk1 MAP promotes the appressorium maturation
(Kershaw and Talbot 2009; Gauthier and Keller 2013). Chitin and
dihydroxynaphthalene melanin deposits in appressorium act as structural support
against the turgor pressure (Gauthier and Keller 2013). A study done by (Wang and
St Leger 2007) shows that Metarhizium produces a protein MPL1 which is similar to
a mammalian protein perilipin. The study reports that MPL1 confines the lipid
molecules into droplets by binding with them similar to perilipin. Phosphorylation
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Fig. 18.2 The figure above explains appressorium formation
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of this protein by cAMP dependent Protein Kinase A leads to release of the lipids
and fatty acids contained in the lipid droplet. This helps in maintaining the turgor
pressure, for example, in case of M. grisea a lipid breakdown product—glycerol acts
as a solute. Its accumulation increases the water uptake (Thines et al. 2000; Wang
and St Leger 2007). MAPK—mitogen activates protein kinase—plays a role in
appressorium differentiation, Thines et al. (2000) reported that a mutant of
M. grisea without MAPK did not had lipid mobilization. Aw and Hue (2017) stated
about MAPK’s intermediate role in adherence and lipid metabolism by regulating
their respective genes. Leger et al. (1991a) proposed that Ca+2 ions get disrupted in
the apical region of hyphae and get redirected to the region of cell enlargement in
appressorium. The role of Ca+2 is also for actin cytoskeleton maintenance, which
helps the appressorium to maintain its structure despite the turgor pressure (Leger
et al. 1991b). Zhang et al. mention that chitin synthase MaChsIII, MaChsV, and
MaChsVII are also involved in the disruption of host defense responses apart from
Appressorium development (Zhang et al. 2019).
18.3.3.4
Penetration
Penetration into the host body involves cuticle breakdown. As cuticle composition
varies from host to host as a result the amount and diversity of hydrolytic enzymes
released by the fungus also varies. So, different hosts have specificity for different
proteins and their concentration (Aw and Hue 2017). Studies by Leger et al. (1991a)
have shown the presence of cuticle degrading enzymes in ungerminated conidia of
Metarhizium
namely
esterase,
chymoelastase
protease,
and
Nacetylglucosaminidase. Also, the same study reported that the amount of these
enzymes was more on an infected host when compared to in vitro conditions.
Trypsins, subtilisins, carboxypeptidases, and chemotrypsins and other proteases
are secreted which degrades the protein part of procuticle of hosts. Pr1 and Pr2 are
spore surface proteins responsible for proteolytic activities (Santi et al. 2010). Pr1 is
a serine protease which degrades the cuticle by hydrolyzing proteins (Screen et al.
1997). Apart from proteases, chitinases are also involved.
Precisely chitin isoforms are secreted to limit its specificity to the host. Lipases
found on conidial surface which interact with the lipid-rich epicuticle release fatty
acids which leads to the enhanced hydrophobic interactions between conidia and
host (Beys da Silva et al. 2010; Aw and Hue 2017). Also free fatty acid may act as a
nutrition source.
18.3.3.5
Colonization
Host hemolymph is colonized next, after breaching the cuticle (Branine et al. 2019).
The hemolymph is the site for the host’s defense responses. The insect defense
system is divided into humoral and cellular responses. The humoral response
includes the antimicrobial peptides, reactive oxygen species, etc. The cellular
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response comprises of encapsulation and phagocytosis by the hemocytes (Lavine
and Strand 2002). To counteract the defense response when the encapsulated spores
release from the hemocytes, destruxin is released (Aw and Hue 2017).
Destruxins are the insecticidal secondary metabolites responsible for the fungi’s
virulence (Dornetshuber-Fleiss et al. 2013). The role of destruxins in being virulent
to the insect primarily includes (Golo et al. 2014)—suppressing the defense
response, hindering the fluid secretion by malpighian tubules thereby interfering
with the osmoregulation (James et al. 1993), they are also shown to block vacuolar
H+ ATPases (V-ATPases). V-ATPases via ATP hydrolysis pump the protons into
lysosomes, Golgi, late endosomes, and other membrane-bound compartments to
maintain the required acidic conditions (Toei et al. 2010) and it also possesses antifeedant properties (Amiri et al. 1999; Golo et al. 2014).
Presence of catalase, peroxidase (for breakdown of certain reactive oxygen
species), genes which code for proteins, which help in breakdown of antimicrobial
peptides, MaAC, and certain other genes protect the fungal cells from chemical and
physical stresses (Aw and Hue 2017) These stress might arise as a consequence of
host immune response. Trehalose makes up the major part of the insect hemolymph.
Instant energy to the insects and survival in abiotic stresses are its role. As it is a
disaccharide it proves to be the main carbon source for fungal spore growth when it
grows in insect hemolymph (Shukla et al. 2015).
Trehalase is also present to hydrolyze the trehalose in the hemolymph into
glucose, secreted as extracellular enzyme by the fungi (Xia et al. 2002), Presence
of MOS1 gene increases the fungal survival at high osmotic pressure (Wang et al.
2008). All these factors aid in the survival of the fungi in the harsh environment of
hemolymph. Inside the hemolymph, some fungal cells proliferate into protoplasts
which prevents their recognition by host defense system because the recognition
proteins may be present at the cell wall (Mondal et al. 2016).
18.3.3.6
Sporulation
After the fungi have proliferated into host hemolymph and Hemocoel, it feeds on
host’s nutrients, which leads to the death of the host insect. After this, upon receiving
suitable conditions the fungal spore germinates and the hyphae extrude out of the
corpse, although spores of M. anisopliae have shown to germinate internally in dried
corpses (Mondal et al. 2016). Spore formation is an important procedure for the
dissemination of fungal diseases. Metalloproteases Mrmep1 and Mrmep2 have been
shown to be responsible for the sporulation of M. robertsii, (Zhou et al. 2018). The
conidia of M. anisopliae were present before it fully colonized the host. Mycelium
growth is usually observed near the antennae base, on cervix, and its mouthparts
(Sun et al. 2002).
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Metarhizium Application Methods in Vector Control:
A Superfluity
As discussed in this text, it is quintessential need for the fungal spore to come into
direct contact with the host in order to infect it so the application strategies must be
wisely chosen. Use of Metarhizium in agricultural biocontrol procedures mostly
comprises of powder-like substances or wettable suspensions as a carrier material
but in case of vector biocontrol this entomopathogenic fungi can be inoculated into
the breeding grounds, stagnant waters and in some studies have also reported an
increase in mortality rate of Anopheles gambiae when use of mud panels, polyester
netting, and cotton cloth as a holding material was done for the fungus (Aw and Hue
2017). In another study, it was observed that Metarhizium was able to interfere with
the DDT and permethrin resistance genetically, due to which chances of susceptibility to these insecticides were increased (Farenhorst et al. 2009). Although
insecticide-treated net and residual sprays are most popularly used options but
apart from them plethora of setups and carrier materials have been experimented
with but here we shall discuss some important conventional and recent molecular
approaches.
18.4.1 Experimental Huts
A study was carried out in the malaria-endemic region of Tanzania (Mnyone et al.
2012). Local housing huts were designed to contain fungal suspension (which
comprised of B. bassiana and M. anisopliae) infected polyester nets, curtains, bed
net strips, panels, and baffles. The results showed a decrease in survival rate of
mosquitos and a significant percentage (68%–76%) of mosquitos had fungal growth
which shows increased contact surface (Mnyone et al. 2012). It also showed the
effect of this strategy on biting behavior and malaria transmission from the
mosquito.
18.4.2 Using Paper Substrates as a Resting Material
for Fungal Spores
In another study (Farenhorst and Knols 2010) smooth paper pieces were coated with
fungal formulations (Metarhizium anisopliae) made in low viscous material both
manually and mechanically and were used instead of sprays. A stainless steel bar
was used to apply this suspension. This method was very effective in standardizing
the amount of fungal biomass used and its exposure time to the mosquito.
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18.4.3 Water Storage Pots as a Carrier Material
for Metarhizium
In this project (Farenhorst et al. 2008) African water storage pots were used as wet
clay pots were an attractive breeding site for mosquitos. In this study, Anopheles
gambiae and Anopheles funestus, which are an evident malaria vectors, were used.
Oil formulated conidia were sprayed uniformly inside the surface of clay pots. The
dead mosquito cadavers after the application were studied for fungal growth. The
results inferred that clay pots were an attractive sight for mosquito breeding, also
these studies (Farenhorst et al. 2008) suggested that in future some other mosquito
attractants could also be combined in this clay pot approach.
18.4.4 Combination of Metarhizium
with Insecticide-Treated Nets
With the ease of availability and inexpensive nature, insecticide-treated nets (ITN)
have become the mainstay in vector control. But, while combining ITNs with a
fungal suspension major focus is to enhance the functional ability of ITNs. A study
was done (Hancock 2009) in order to check the performance of fungal suspension
intervened ITNs, in this various factors were considered like fungal infection,
gonotrophic feeding process, ITNs, etc. then a model was proposed for which
mathematical analysis was done. As a result (Hancock 2009) an extensive information was produce which had many suggesting reasons to use fungal biocontrol
integrated with ITNs such as high virulence, more probability of fungal infection
in host, prolonged period of fungal exposure, etc.
18.4.5 Metarhizium in Odor Bait Stations (OBS)
This was a study done by Lwetoijera et al. (2010) where an OBS was used against a
malaria vector Anopheles arabiensis. OBS are box-like structures made on a wooden
frame which is covered with a canvas. The entire device except the floor is covered
with a black cloth and inside this device there is a mosquito lure which mainly
consists of carboxylic acids, carbon dioxide, and ammonia (Okumu et al. 2010;
Lwetoijera et al. 2010). It contains a funnel-like opening and it also has Metarhizium
conidia treated baffles. The results showed an increase in infection rates and also it is
much anticipated because using OBS is much more safe than using fungal suspension in human dwellings as done in experimental huts (Mnyone et al. 2012) and
other such methods.
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18.4.6 Oil as a Carrier Material
18.4.6.1
Mineral Oil
A study done by (Bukhari et al. 2011) focused on comparing the efficiency of an
aqueous substance, a synthetic oil formulation (shellSol T) and dry carrier material
(wheat flour, white pepper, and fine bicarbonate particles) as a carrier material of
fungal spores in aquatic habitats. The results revealed that Shell Sol T a synthetic oil
is an effective spore carrier material in comparison to aqueous and the dry carrier
material in both laboratory and field trials. In fact the field trials witnessed a decrease
by 39–50 percent of the Anopheles gambiae population. In comparison to
non-formulated fungal spores where efficacy was very low, Shell Sol T formulated
spores showed a promising effect and increased persistence in water. This study
gives another perspective—usage of synthetic oils as a carrier material. Also, the oil
used here had minimal toxicity to the aquatic habitat as the quantity used here was
very less compared to that of the safe limit.
18.4.6.2
Vegetable Oil
This experiment focused to check the viability of M. flavoviride spores after storage
in different formulations and variable temperature. As a diluent, dedeorized kerosene
oil preserved the spores better than Shellsol K, apparently during short duration of
storage; however, mean conidial growth was found more in Shellsol K after 32
weeks. Vegetable oils were effective but its efficacy was improved with addition of
antioxidant in case of groundnut oil. It was also observed that lower temperature led
to increased germination of spores. The addition of silica gels, which aided by drying
out spores, showed significant results as well. Still there was requirement for further
studies to get the better understanding of storage techniques under variable conditions. (Moore et al. 1995).
18.4.7 Mosquito Landing Boxes (MLBs) for Metarhizium
MLBs are devices that have natural or synthetic human odor as a mosquito attractant.
It is a system based on odor baiting technology. These devices are basically a
wooden box with solar panels on the top which powers the odor dispenser. Particles
of the odor solution lands on the walls of the device which may have fungal spore
coating or some insecticide. In a study Lwetoijera et al. (2010) used this device and
the walls of the MLB were coated with spores of the entomopathogenic fungi
M. anisoplieae and vector targeted here was Anopheles arabiensis. This trial was
conducted in a semi-field system. Separate cups containing larvae were placed near
both MLBs and control system and it was supposed that if a mosquito captured from
the semi-field system is let into the cups containing larvae and the larvae is not able
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to survive, and then the mosquito has been contaminated with the fungal spores and
vice versa. Many factors like larval mortality, pupation, amount of hyphae growth on
cadavers, etc. were assessed to check the level of contamination of the mosquitos
with the fungi. The results revealed that 43% of mosquitos were contaminated with
the fungal spores compared to 0% in the control. In this study alternatively, a
chemical pyriproxyfen was also used in the system. This study inferred that MLBs
are an effective tool for delivering fungal biopesticides as a decrease in the survival
rate and direct killing of host-seeking mosquitos was witnessed.
18.4.8 Metarhizium in Combination with Phytochemicals
Use of neem oil as an insecticide is a very popular and old method but despite that
today we see very less use of such phytochemical in vector control. A study done by
Simone A. Gomes and coworkers (Gomes et al. 2015) focused on the use of neem oil
as an adjuvant for the entomopathogenic fungi Metarhizium. In their study, they used
two systems, one to check the effect of neem oil used solely on Aedes aegypti and
another one to check the effect of neem oil in combination with Metarhizium. Neem
oil of variable concentrations was used. Statistical analysis of the survival curve was
done. The results revealed that at the concentration of 1 108 conidia per ml, a very
low survival rate of 12% was observed. Also, later it was suggested that the addition of
neem enhanced virulence. This study suggested the use of adjuvants such as phytochemicals along with the fungal biopesticides. In future, many other potential phytochemicals can be used as an adjuvant to variety of fungal biopesticides.
18.4.9 Metarhizium for Chemical Resistant Vector Hosts
Insecticide resistance is a prominent and emerging problem in the area of vector
control. But the use of entomopathogenic fungi for insecticide-resistant vectors is
one of the ways to fight insecticide resistance. An interesting study by Blanford et al.
(2009), which was done on the vector Anopheles gambiae which was insecticide
resistant—has interacted with Metarhizium anisopliae and another entomopathogenic
fungi and results revealed increased susceptibility of the insecticide-resistant strain of
host. The resistant strain used here was Anopheles gambiae s.s. VKPER, which is a
pyrethroid-resistant strain. Also an insecticide susceptible strain Anopheles gambiae
SKK strain was treated with the same procedure. Various mechanisms were used to
deliver the fungal isolates to the mosquito-like formulations in synthetic oils
(Kamareddine 2012), dry conidia (Ondiaka et al. 2015), etc.
18.4.10
Delivery System in Agriculture Fields
Apart from the above-given methods, there are some specialized methods that can be
instilled for the delivery of fungal spores to the host. The formulations are designed
to increase the viability of spores and to expose them lucratively to the host.
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305
Kaolin Based
The spores of fungi are mixed with 80% kaolin. Prior to use, it is mixed with water
and wetting agent solution. Then solution is sprayed directly onto the plant (Goble
et al. 2016).
18.4.10.2
Patty Blend Formulation
Vegetable oil and sugar are mixed with pre-weighed conidiospores. Conidial viability is increased by the addition of Silwet and Saboraud maltose agar. Lactophenol
cotton blue is added to stop its germination. The insects are treated with the strip
containing serially diluted formulation (Kanga et al. 2010).
18.4.11
Molecular Approaches
The DNA technologies in the new age facilitate the addition of new gene into fungi
and perform gene manipulation to increase efficacy of the fungi. Due to various
stress condition their efficiency gets decreased but the DNA technology can be
useful to improve the ability of virus to sustain in unfavorable conditions and
more virulence. By expressing the endogenous proteins in the Metarhizium the
pathogenesis success rates increases by targeting the cuticle, physiology, and hormones (Lovett and St Leger 2018).
In an experiment performed by Leger et al. (1991b), a genetic modification was
done in Metarhizium in which more number of copies of Pr1 gene, which basically
codes for protease that degrades cuticle of host, was inserted. When newly
engineered Metarhizium was made to infect Manduca sexta there was
overproduction of gene and activation of phenoloxidase system. The results include
reduction in death timing, food consumption, and biological containment of the
fungi. One of the reasons for biological containment was due to the accelerated
melanization of the host cadaver which in turn provides insufficient substrate source
for fungal spores growth (St Leger et al. 1996). Phenoloxidase is activated by
prophenoloxidase cascade and provides immunity to the insects and polymerization
of the indole group of phenoloxidase leads to the formation of melanin which leads
to melanization precisely upon with infection with fungal spores (González-Santoyo
and Córdoba-Aguilar 2012; Carolino et al. 2014; Butt et al. 2016b; Zhang et al.
2017).
Peng et al. (2015) performed an experiment in which ATM1 gene was
overexpressed which codes for trehalase. Trehalase degrades trehalose which provides fungus with the carbon source in hemolymph of insect host. When results were
compared to the wild strain of Metarhizium the genetically engineered fungi showed
more degradation of trehalose and growth enhancement of fungi in host hemolymph.
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In an another experiment, scorpion toxin BjαIT was used for genetic manipulation of Metarhizium the results showed enhanced virulence by the fungi in host
infection. The growth of fungal spores on cadaver did not report any drop which
might not affect the transmission. Though the yield was reduced but germination and
formation of appressorium were the same as the wild-type strain and the lethal dose
and lethal time were less to (Peng and Xia 2015).
Transgenic M. pingshaense was used to control the insecticide-resistant Anopheline mosquitoes. The new genetically modified fungi were hybrid it had voltagegated calcium blocker with kappa hexatoxin Hv1a and calcium-activated potassium
genes. The results in labs showed that efficacy was increased and it was able to
control the resistant malaria vector. This can be used for on field application in the
future (Lovett et al. 2019).
In heat stress conditions hyphal cells may start producing Reactive oxygen
species (ROS). Pyruvate acts as ROS scavengers but the rate of formation of
pyruvate is slower than the formation of ROS. A transgenic Metarhizium was
designed so as there is overexpression of genes and therefore increased concentration of pyruvate kinase will be produced. During conidia formation, the pyruvate
kinase gets accumulated and this helps conidia to survive during heat stress
(Wu et al. 2019).
Genetic modification to create transgenic Metarhizium has been successful in
many of the cases as mentioned above and showed an increase in virulence as
performed by Peng et al. (2015), Lovett et al. (2019) and some (Wu et al. 2019)
showed considerable efficacy in lab as well which can further be implemented in
field and tested for the outcome. Extensive research in knowing the enzymes, genes,
and host immune system in addition to fungi evasion and invasion techniques can
help to genetically manipulate the fungus and increase its efficacy.
18.5
Plant Growth Promotion
Mycorrhizae are obligate biotrophs and endophytes aids in improving plant growth
and nutrients acquisition from soil to the plants (Karandashov and Bucher 2005;
Behie et al. 2017). Unlike mycorrhiza, Metarhizium as an endophyte shows no
obligatory nature as it can survive in soil freely, as entomopathogens or as saprophytes (Behie et al. 2017). Beyond the activity of being pathogenic to the insects,
there is one more benefit which is, plant growth promotion; although this is an area
that has not been extensively studied (Canassa et al. 2019). This improves the yield
In addition to the pest management of the plants. For sustainable agriculture plant
growth promotion coupled with pest inhibiting capabilities can help to discontinue
the use of heavy pesticides and fertilizers (Senthil Kumar et al. 2018). Metarhizium
can induce root hair development, nitrogen translocation, improved absorption of
iron, and auxin production (Behie and Bidochka 2014; Moonjely et al. 2019).
Colonization of plant tissue makes it an endophytic fungi, which enhance plant
biomass as well as it can increase nutrient mobilization and its transfer (Krell et al.
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2018). The soil fertility may affect fungal activity and nutrient metabolization. Many
studies have been performed but some of them lack a firm conclusion thus it requires
further research to be done.
18.5.1 Exchange of Nutrients and Endophytic Nature
Nitrogen is crucial for plant growth and the loss of nitrogen due to insect herbivory
leads to deprived nitrogen content available for plants (Behie and Bidochka 2014).
Several fungi in symbiotic association with plants transfer nitrogen (Wang et al.
2017a, b). Metarhizium has a wide host range and is pervasive worldwide (Hajek and
St. Leger 1994). Laccaria bicolor transfers the nitrogen derived from insects back to
the plant white pine. Metarhizium has shown similar results when experimented with
the insect Galleria mellonell (waxmoth) N15 labeled. This was performed on
Switchgrass and haricot beans. The Metarhizium spp. were able to increase the
plant productivity (Behie et al. 2012). In another experiment, five species of
Metarhizium were tested all expressed positive results. M. robertsii was tested on
field in natural conditions, showed significant results (Behie and Bidochka 2014). A
recent experiment conducted shows that MepC and Mep2 are two ammonium
permease genes which have been involved in nitrogen derived from insects and
also in colonization process (Moonjely et al. 2019). Research for finding genes
responsible for symbiosis of Insect pathogenic fungi can help to understand the
functioning elaborately.
A recent study conducted by Behie et al. (2017) gives evidence that Metarhizium
derives carbon from the plants as much as other endophytes. The carbon translocation can sustain fungi when insect host is absent. Reportedly, when the host was
present there was significant increase in carbon transfer to the fungi (Behie et al.
2017). This nature of reciprocation of nutrients helps both plants and fungi.
Plant photosynthates containing carbon were found in the roots of the plants and
in the rhizosphere which are utilized by the fungi as substrate. The 13C (CO2 given to
plants had 13C isotope) used was found to be incorporated in the fungi which had
been provided by the plants as a symbiotic relationship. In fungi, it was traced to
NAG and other Carbon-based components (Behie et al. 2017). The decomposing
cadaver when added with Metarhizium spores lead to increase in ammonium and
nitrate in the soil and spores were able to colonize plants as well that resulted in
better plant growth (Kryukov et al. 2019).
Metarhizium has evolved as an endophyte (Moonjely et al. 2016). In an experiment performed by Barelli et al. (2018), the analysis was made between how
extensively the Metarhizium colonizes the plant roots. For precise detection Plate
culture method (c.f.u count) and quantitative PCR, both were done. The results
showed that there was fungal colony in rhizosphere, rhizoplane, and within the roots
too. Another thing that was noticed that the population of fungi did vary with the
number of days post-inoculation (Barelli et al. 2018) but with this experiment it is
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evident that Metarhizium can colonize roots efficiently. Thus there is scope of further
investigation, whether this fungi can colonize phyllosphere or not.
18.5.2 Improved Iron Absorption on Calcareous Substrates
In an experiment performed by Raya-Diaz et al. (2017) on sorghum plants, the
relationship between plants, entomopathogenic fungi, and soil can be established
well. It concluded that the entomopathogenic fungi tested in case of calcareous soil,
M. brunneum turned out to be the most efficient fungus which lowered the pH of the
alkaline soil by releasing the organic acids. The plants suffering from iron chlorosis
are iron-deficient plants. This is common concern for the plants growing in calcareous soil either acidic or basic as it leads to the less iron availability in the form
plants requires for its uptake (Brown 1956). As tested in vitro, iron oxides changed to
dissolved iron forms. The Fe chlorosis symptoms were seen to be assuaged by
M. brunneum on the sorghum plants which were grown on the artificial substrate
that was calcareous. The best method was soil inoculation method (Raya-Diaz et al.
2017). In another experiment performed by Sánchez-Rodríguez et al. (2016) the
wheat and sorghum plant showed increase in growth and chlorophyll content by
improving the iron bioavailability in soil.
18.5.3 Auxin Formation for Plant Growth
Auxin is a plant growth hormone which influences plant physiology and developmental process to the stimuli sunlight and gravitropism (Bhattacharya 2019; Pandey
et al. 2019). In an experiment performed by Liao et al. (2017) vegetative growth of
corn plants has shown improvement and the yield was increased. Avirulent
Metarhizium strain (Δmcl1) contributed to the growth of plants as well proving
that the plant growth promotion activity is not influenced by entomopathogenic
activity. Auxin was produced by fungi, there was increase in formation of leaf collar,
foliage biomass, and the length of the stalk where the spores were able to colonize.
The culture filtrate even contained auxin which showed positive result (Liao et al.
2014). The growth of plants is due to combined effect of chemicals and auxin (IAA)
dependent pathways importantly which is produced by Metarhizium; promoting the
lateral root and root hair development (Liao et al. 2017).
18.5.4 Proliferation of Plant Cells and Disease Suppression
The Metarhizium species can be potential plant endophytes and can live inside plant
tissues. In an experiment performed it was noticed that their role can be in
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increasing the size of stalk, root length, and weight of the root (Mantzoukas et al.
2015; Greenfield et al. 2016). It can cause proliferation of lateral root hair for
enhanced plant growth (Sasan and Bidochka 2012). The growth rates differ with
respect to the strain used, duration, and the inoculation amount (Jaber and Enkerli
2017). In bean plants, there were significant results to show that it improved the
reproductive and vegetative growth also (Canassa et al. 2019).
Jaber (2018) performed an experiment whether M. brunneum can be effective for
suppression of disease-causing pathogens. The results showed significant decrease
in the occurrence of disease, its development, and intensity. The fungus was able to
promote shoot and root growth and the weights.
18.6
Conclusion: In the Light of Recent Advances
With the increased understanding of the molecular aspects of Metarhizium such as
the genes and proteins involved in pathogenesis and their upregulation, secondary
metabolites and small molecules, molecular aspects of host immunity, genome-wide
studies, etc. have made the scope of research on Metarhizium infinitely vast.
Donzelli and Krasnoff (2016) state that the recently available genome sequences
give many biosynthetic pathways and ability to produce secondary metabolites
which surpasses the current knowledge of chemistry. Basically, this study focuses
on details of genes involved in the production of secondary metabolites. Also, there
have been studies (Brancini et al. 2019) where light has shown to affect the gene
expression in Metarhizium, after periodic exposure to light upregualtion and down
regulation of certain proteins takes place. One such protein is photolyase which is
upregulated and is responsible for UV tolerance. The changes concluded in inference
that light is involved in stress and signaling. So light might be a factor in controlling
the efficacy of Metarhizium. Studies by Mukherjee and Vilcinskas (2018) and
Hussain (2018) discuss about changes in the gene expression. On recognition of
the fungal spore by the host immune system, the host increases the expression of
certain antifungal peptides in response to overcome the hostile environment in host,
the fungi also increases the expression of certain proteins that destroy these peptides
which are epigenetically controlled. This way of the counteracting molecular
responses in host and the fungi give further insights into the coevolution process.
Except from entomopathogenecity Metarhizium has certain effects on growth
promotion in plants. A recent review by Hu and Bidochka (2019) has mentioned that
species from Metarhizium genera are root endophytes and have a symbiotic relation
as they provide insect-derived nitrogen and get photosynthates in return. They have
further reviewed the factors governing the rhizospheric interactions. Also, there has
been a recent study on the host cadaver decomposition affecting plant growth
promotion (Kryukov et al. 2019). The decomposed cadavers contain more ammonia
and nitrogen compared to cadavers overgrown by fungus. It was concluded that
310
S. Patil et al.
fungi were unable to sporulate on decomposed cadavers and provided nitrogen faster
from the cadavers overgrown by fungus.
So all these facts and studies boil down to some important inferences:
1. Evolution of Metarhizium defense response—As discussed in the pathogenesis
section there are several lytic enzymes which, facilitate the entry of Metarhizium
into the host. As stated by Mukherjee and Vilcinskas (2018), host can recognize
and counteract these proteins by releasing antimicrobial compounds, proteinase
inhibitors, and antifungal compounds. Same study has shown that the expression
of chymotrypsin and metalloproteinases by Metarhizium can counteract the host
defense compounds. An in vitro increase in metalloprotease activity was
observed in response to the antimicrobial peptides (AMP) like metschnikowia,
lysozyme, and proteinase inhibitor. Another significant component of the insect
defense system is hemocytes. The presence of destruxin has been mentioned in
the text previously. This protein causes actin remodeling, pyknotic nuclei, and
blebbing in plasmocytes (Götz et al. 1997). Another toxin cytochalasin is also
involved but it is less toxic than destruxin. Both these toxins selectively regulate
the expression of IMPI and lysozyme, which are antimicrobial peptides. Also
induction of genes involved in epigenetic responses of histone acetylation and
deacetylation in M. robertsii against an AMP shows, how specific modification in
M. robertsii at transcriptional level is made to counteract host defense system.
This might suggest lesser chances of host gaining resistance against the fungi.
(Mukherjee and Vilcinskas 2018)
2. An ideal biocontrol strategy must focus on enhancing efficacy of both the
biological agent and its carrier—There have been several attempts in creating
a recombinant strain of Metarhizium, which is more effective by selecting genes
like toxin genes as a candidate some of which were discussed in this article. Also
carriers with improved efficacy are in a need to be developed considering certain
environmental factors which have shown to have a stressful impact on the growth
of Metarhizium. Study done by Wang et al. (2017a, b) on Galleria mellonella and
M. robertsii mentions an important role of DNA methyltransferases, which are
responsible for epigenetic or gene expression control. Here they have shown to
have a role in stress tolerance and virulence of the fungi. This shows how
transgenic fungi can be effective.
3. Metarhizium as a complete plant health package—With the well-established
entomopathogenic effects and some endophytic plant growth promotion activities, Metarhizium can be used as a holistic supplement. A suggestable effort could
be improving Metarhizium transgenically with plant growth promotion activity
besides its entomopathogenecity. Although all these have been proven experimentally, still there are requirements of field assays. Possessing the knowledge
about timely usage of biocontrol agents is very important to avoid emergency pest
mitigation; especially when using a fungus, because it is temperature and humidity dependent. All of these characteristics can be brought to better use by focusing
on synergistic approach like combining it with bio-fertilizers and using it with
other biopesticides. Not only nitrogen translocation but also other mineral utilization by plants can be improved with scientific studies. So as with time,
18
Panorama of Metarhizium: Host Interaction and Its Uses in Biocontrol and. . .
311
entomopathogenicty of Metarhizium is being explored in newer hosts we are
becoming more molecularly aware about it. The confluence of this scientific
awareness and industry requirements is where Metarhizium promises a vast
scope.
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Chapter 19
Arbuscular Mycorrhizal Fungi: Potential
Plant Protective Agent Against Herbivorous
Insect and Its Importance in Sustainable
Agriculture
Anandakumar Selvaraj and Kalaiselvi Thangavel
Abstract Wide use of fertilizers and chemicals for food grain production to feed the
world population with increasing demand leads to environmental pollution. Alternatively, the use of biological sources such as beneficial microbes to improve crop
production as a component of sustainable agriculture production and environmentally friendly. Among them, arbuscular mycorrhizal fungi (AMF) are well-known
soil microbe forms a symbiotic association with land plants including agricultural
important crops. This beneficial AM fungi improving plant growth, and it also fount
to improve resistance capacity of plants against diverse stresses, including herbivorous insect damage through altering the morphological and biochemical traits. In
response to herbivore stress, AMF augments plant defense in both constitutive and
inducible manner leads to reduce insect damage. AMF induced or primed plant
defense mechanisms against herbivorous insect damage have so far underestimated.
Therefore, we discuss here an overview of research findings related to AMF induced
or priming of immune response in plants against herbivore-induced stress. Acquired
mechanisms of plant associated with AMF to protect themselves from pests by
altering nutrient availability and physiology. AMF-mediated response of plants to
herbivore varied with host plants, AMF species, and degree of colonization, type of
pest, and crop management system. With these contexts AMF is could be a good
bioprotective agent against pest apart from improving plant growth and this is an
integral part of the integrated pest management system for sustainable agriculture
production.
Keywords Arbuscular mycorrhizal fungi · Pest · Defense · Sustainable agriculture
A. Selvaraj (*) · K. Thangavel
Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore,
India
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_19
319
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19.1
A. Selvaraj and K. Thangavel
Introduction
The word mycorrhizae derived from two Greek words, including “mycos” and
“rhizos” meaning fungus and root respectively. The successful colonization of
mycorrhizae on the roots of plant begins with the signaling from both the partners
plant, and arbuscular mycorrhizal fungi (AMF). AMF is a unique soil fungus
belongs to the phylum Glomeromycota which forms a symbiotic association with
almost 80% of land plants, including agriculturally important crops (Gupta et al.
2019). This plant-associated beneficial biotrophic fungi alters plant root system and
improves the uptake of immobile inorganic nutrients, primarily phosphate (P) and
other nutrients such as ammonia as well as micronutrients (Mg, Fe, Zn, Cu, Mn, and
Al) from the soil solution (Jung et al. 2012; Kumar 2018). Besides, AM fungal
hyphal network spread to meters and help the plants to absorb moisture from the
surrounding soil. In turn, plants provide around 20% of photosynthetically assimilated carbon (Smith and Read 2008). A plethora of reports confirmed that AMF
colonization improves plant growth by increasing the acquisition of nutrients and
water from their surrounding (Gill et al. 2013; Seguel et al. 2015; Frew 2019). The
effects of AMF colonization on plant growth varied depends on soil fertility, time of
AMF inoculation, and degree of fungal colonization on host plant roots (Hart et al.
2018). Many reports are confirmed that crop yield increased by AMF application
(Zhang et al. 2019) and it is recognized as an important component of sustainable
agriculture production (Ryan and Graham 2018; Rillig et al. 2019). AMF also
involved in diverse functions such as soil and plant health improvement (Kumar
et al. 2019).
Plants undergo numerous biotic and abiotic stresses and these effects are reduced
by AMF colonization through imparting plant defense and tolerance. The plethora of
research findings supported that AMF improves plant tolerance against stress like
salinity (Rodriguez and Redman 2008), phytopathogens (Slaughter et al. 2012),
drought (Ahanger et al. 2014), heavy metals (Salam et al. 2017) and temperature
(Schoenherr et al. 2019), etc. In addition, crops are also affected severely by the
herbivorous insect to causes transition economic loss in agricultural production.
There is a growing interest in developing plant protection measures that are
eco-friendly, cost-effective, and sustainable. In this context, it is essential to understand the plant defense mechanisms against insect attack (Gershenzon 2017). It is
equally important to understand the adaptation strategies of insects against these
plant defensive characteristics to develop sustainable management strategies (Hahn
et al. 2019). A research finding confirmed that AMF also found to improve plant
resistance or tolerance against herbivorous insect damage by altering morphological
and physiological characteristics (Kim and Felton 2013; Schoenherr et al. 2019). The
AMF provides tolerance to plants against herbivorous insects through various
mechanisms like increasing nutrient uptake, primary and secondary metabolites,
antioxidants, volatile organic compounds, and phytohormones production and
imparting faster regrowth of tissues (Korpita et al. 2014; Cabral et al. 2018). The
effect of AMF on the induction of plant defense against herbivore is studied in many
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plant species, including strawberry (Gange 2001), big bluestem grass (Miller et al.
2002), lotus (Nishida et al. 2010), ribwort plantain (Wang et al. 2015), faba bean
(Cabral et al. 2018), tomato (Formenti and Rasmann 2019), potato (Schoenherr et al.
2019), milkweeds (Meier and Hunter 2019), black gram (Selvaraj et al. 2020), and
maize (De Lange et al. 2020), etc. These effects varied on plant and AMF species,
degree of AMF colonization, insect pest type (generalist or specific) (Cabral et al.
2018). In this chapter, we discuss the role of AMF in imparting plant defense against
herbivorous insects and their importance in sustainable agriculture.
19.2
AMF Primed Plant Defense
AMF symbiosis modulates the plant system in many ways to improve growth and
defense against stress by altering plant physiology (Jung et al. 2012). Plants get
primed during the establishment of AMF symbiosis by modulating immune
responses of plants in both local and systemic manner, in which primed plants also
impart efficient activation of defense mechanisms against stress (Jung et al. 2012).
This AMF induced response is known as mycorrhiza-induced resistance (MIR),
which provides systemic protection against a wide range of attackers and shares
characteristics with systemic acquired resistance (SAR) and induced systemic resistance (ISR) (Schoenherr et al. 2019). The MIR protects plants from a wide range of
attackers, including pathogens, nematodes, and herbivorous arthropods through
SAR and ISR mediated priming by salicylic acid (SA) and jasmonic acid
(JA) dependent defenses (Cameron et al. 2013). Herbivore performance was affected
either positively (Gange et al. 1999) or negatively (Gange 2001) by AMF colonization, depending on both herbivore and fungal species. In general, mycorrhizal
colonization enhances plant resistance to root-feeding insects and generalist herbivores; but it may increase plant susceptibility to sucking insects and specialist
herbivores (Hartley and Gange 2009; Pineda et al. 2012). AMF symbioses role in
providing enhanced resistance and/or tolerance to plants against pests is remaining
elusive and not well studied.
19.3
AMF-Mediated Direct and Indirect Defense
Mechanisms of Plants Against Herbivorous Insect
Herbivorous insects fully depend on the energy fixed by plants. However, plants had
no chance to escape from attackers, so they must employ other strategies to defend
themselves from the deleterious effect of herbivores (Yan and Xie 2015). In response
to stress, plants exhibit both constitutive and induced defenses by recognizing stress
with a high degree to mount intracellular signaling into appropriate biochemical,
physiological, and cellular responses systemically throughout the plant system
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A. Selvaraj and K. Thangavel
(Karban 2020; Wilkinson et al. 2019). During plant–insect interaction they use
chemicals as a weapon to overcome each other’s ill effect and to bring morphological and biochemical changes in both the partners. Plants first protect themselves
from insect damages by bringing changes in the morphological related traits and
antioxidant production (Mithöfer and Boland 2012; War et al. 2013). The biochemical mediated defenses developed over a while against insect attack by producing
secondary or anti-digestive metabolites, toxic furanocoumarins and amino acids
(War et al. 2013) and other related toxic compounds in plants leads to a reduction
in feeding capacity (Chen et al. 2012), repelling of herbivorous insects and attraction
of insect parasitoids/predators. AMF association affects the development of insects
by modifying dietary specialization and feeding mode of the herbivores directly by
altering the quality of plants. AMF colonized plants exhibit accumulation of certain
defense compounds like phenolics, hormones, and reactive oxygen species
quenching enzymes during the interaction, which improves plant defense against
herbivore-induced stress (Koricheva et al. 2009; MacLean et al. 2017).
19.4
Direct Defense
19.4.1 AMF Induced Changes in Morphological
Characteristics of Plant Against Herbivorous Insect
In the plant system, the constitutive defense provides the first line of defense against
stress. Modified structures of plant like thorns, stings, sticky resins, and trichomes
provided defense against insects as a type of constitutive defense (Taggar and Gill
2012). All plant parts offer some sort of resistance against herbivory ranging from
tissue hardness to highly complex glandular trichomes and spines (Acharya and
Bhargava 2008). These special structures act as physical barriers that affect herbivore performance and reduces their severity further. Trichome is an important
special plant structure that negatively affects the feeding and ovipositional responses
of insect pests (Xing et al. 2017) directly by damaging the insect mechanically and
interfere with their movement, thereby reducing their access to leaf epidermis.
Glandular trichomes are yet another type of trichomes, which secrete secondary
metabolites including flavonoids, terpenoids, and alkaloids that can act as a poison,
repellent, or trap insects and other organisms, thus forming a combination of
structural and chemical defense (War et al. 2012). AMF colonization exhibited a
moderate increase in trichomes density of Solanum lycopersicum plants in response
to Spodoptera litura infestation (Formenti and Rasmann 2019).
Tissue hardiness is another mode of plant defense, which affects the penetration
ability of mouthparts of piercing-sucking insects on plant tissues, and also increases
mandibular wear in biting-chewing herbivores, thus preventing herbivore feeding
(Chaudhary et al. 2018a, b). Hardened leaves reduce the herbivore damage by
affecting the palatability and digestibility of the tissues (War et al. 2012). Enhanced
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leaf toughness by cell wall reinforcement with the deposition of chemicals such as
lignin, cellulose, suberin and callose, small organic molecules (phenolics), and even
inorganic silica particles provide mechanical resistance to insect feeding and penetration (Keeping and Kvedaras 2008). Kundu et al. (2018) reported that Spodoptera
litura infestation enhanced lignin content in leaves of Solanum lycopersicum. Lignin
and phenolic content in cucumber plants increased significantly upon inoculation
with the AMF (Chen et al. 2013). Manjarrez et al. (2009) reported that AMF
colonization improves the deposition of callose in tomato plants in response to biotic
stress.
19.4.2 AMF-Induced Plant Defense by Improving
the Nutrient Content
AMF indirectly influences the performance of herbivorous insect through changes in
the quality and quantity of plant nutrients, and even affect the behavior of natural
enemies of herbivores and plant pollinators (Cardoso Filho et al. 2017). Plant growth
and development depend on the available soil nutrient content, which enhances the
plant’s photosynthetic capacity (Smith and Read 2008), alteration in the production
of plant primary and secondary metabolites (Swamy et al. 2016). AMF colonization
increases nutrient acquisitions by the plants, thereby have great influences on plant
physiology, primary and secondary metabolism, and hormone balance (Cabral et al.
2018). A plethora of reports showed that AMF colonization improves plant growth
by enhancing nutrient uptake and by altering plant physiology (Gill et al. 2013;
Seguel et al. 2015; Frew 2019). Borowicz (1997) reported that AMF colonization
positively affected the growth of Glycine max under low phosphorus conditions,
which resulted in regulating the performance of Epilachna varivestis. These studies
support that AMF imparts plant to tolerate herbivore damage by supporting plant
growth with nitrogen and phosphorus, which are required to synthesize the defenserelated enzymes, secondary metabolites, and modified physical structures (Seguel
et al. 2015). Andrade et al. (2013) reported that nicotine contents of the leaves of
Nicotiana tabacum improved due to AMF colonization. Mycorrhiza colonized
Plantago lanceolata plants produced diverse carbon-based defensive compounds
such as aucubin and catalpol, which decreases feeding and growth characteristic of
Arctia caja insect (Gange and West 1994). This may evidence that AMF improves
plant photosynthetic rates (Miller et al. 2002) and increases the availability of
nutrients involved in the synthesis of defense related chemicals. Balestrini et al.
(2017) also reported that the AM fungus significantly altered the expression of
nutrient transport-related genes in grapevine roots. By contrast, healthy plants rich
in nutrients generally favor herbivore growth and get attracted by herbivores (Woods
et al. 2004). The AM symbiosis often benefits the host plant in terms of nutrient
acquisition and nutritional quality, which can benefit the insects feeding on it
(Koricheva et al. 2009); but an increase in nutrient acquisition can also mean greater
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investment in defenses (Pozo and Azcón-Aguilar 2007). Alterations in plant traits
directly affect the herbivory growth by reducing nutrient resources, increasing
secondary metabolites, and volatile production. AMF showed a significant impact
on plant defense by modulating plant traits directly through improving nutrient
requirements, which confers resistance or tolerance against insect damage.
19.4.3 The Antioxidant Activity of Plants Infested with AMF
and Herbivore
Plants produce reactive oxygen species (ROS) as a signaling molecule and causative
bioactive agent under environmental stress. Herbivore damaged plants accumulate
more amount of reactive oxygen species and hydrogen peroxide (H2O2) in tissues
(Jih et al. 2003) which leads to cellular damage by the degradation of biomolecules
like pigments, proteins, lipids, carbohydrates, and DNA. Plants provide the first line
of induced cellular defense against herbivore stress by producing antioxidants
(Gourlay and Constabel 2019). To mitigate the effect of ROS, plants evolved with
antioxidants mediated defense systems to eliminate or detoxify the excess production of reactive oxygen species. Antioxidant systems may be either enzymatic or
non- enzymatic mode (Das and Roychoudhury 2014). Enzymatic mode of antioxidants includes superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase
(GPX) and ascorbate peroxidase (APX). The SOD catalyzes the removal of O2• by
converting it into O2 and H2O2 (Das and Roychoudhury 2014). The peroxidases
(POD) and catalases (CAT) catalyze the decomposition of H2O2 into H2O and O2,
thereby eliminate the damage due to excess production of reactive oxygen species,
and increase the level of plant resistance to herbivore-induced stress (Ruley et al.
2004). AM fungi enhance the plant antioxidant enzyme activity like superoxide
dismutase (SOD), peroxidase (POD), and catalase (CAT) and SA concentration in
Lolium perenne infested with Claroideoglomus etunicatum than non-mycorrhizal
plants (Li et al. 2018).
Plants also exhibit a non-enzymatic mode of antioxidant system by producing
ascorbic acid (AA), carotenoids, phenolics, flavonoids, and proline. AA is the main
metabolite that protects the cell membrane from oxidative damage caused by ROS
(Kovalikova et al. 2019). AA is either oxidized into monodehydroascorbate reductase (MDHA) or it reacts with H2O2, OH•, O2• , and regenerates α-tocopherol from
tocopheroxyl radical thereby protecting the membranes from oxidative damage
(Shao et al. 2005). Carotenoids belong to the family of lipophilic antioxidants are
localized in the plastids of photosynthetic and non-photosynthetic plant tissues. It
exhibits antioxidant activity by protecting the photosynthetic machinery in any one
of the four ways, (a) reacting with lipid peroxidation (LPO) products to end the chain
reactions, (b) scavenging 1O2 and generating heat as a by-product, (c) preventing the
formation of 1O2 by reacting with 3Chl and excited chlorophyll (Chl), and
(d) dissipating the excess excitation energy, via the xanthophyll cycle (Das and
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325
Roychoudhury 2014). Flavonoids are considered as a secondary ROS scavenging
system in plants to scavenge the 1O2 and alleviate the damages caused to the outer
envelope of the chloroplast membrane (Agati et al. 2012). Proline is one of the
important osmolytes that act as a powerful antioxidant to reduce the effect of ROS by
scavenging OH• and 1O2 radicals and can inhibit the damages due to lipid peroxidation (Verbruggen and Hermans 2008). Phenolic compounds act as antioxidants by
inactivating lipid free radicals or inhibiting hydroperoxides to break down into free
radicals (Bhonwon et al. 2009). AMF colonization reported to enhance the production of flavonoids (Harrison and Dixon 1993), and apocarotenoids (Fester et al.
2002) in various plants.
In response to herbivore attack, plants produce an enhanced level of
lipoxygenases (LOXs), polyphenol oxidases (PPOs), peroxidases (PO), and phenylalanine ammonium lyases (PAL) involved in the synthesis of defense metabolites
against herbivore damage (Gourlay and Constabel 2019). LOXs are ubiquitous
enzymes that play important roles in plants, which involved in the production of
fatty acids, jasmonic acid, volatile aldehydes, and oxyacids (Babenko et al. 2017).
Phenylalanine ammonium lyase (PAL) is involved in the production of many
primary and secondary metabolites of higher plants and regulates lignification during
stress conditions (Chen et al. 2009). Polyphenol oxidases (PPOs) are anti-nutritive
enzymes that use molecular oxygen to oxidize common phenolic compounds to
highly reactive quinones. The quinones further react with amino acids of insects and
reduce the amino acid availability (Kundu et al. 2018). PO also plays a vital role in
the biosynthesis of lignin and imparting defense against biotic stress by degrading
indole acetic acid (IAA) and utilizing H2O2 in the process (Das and Roychoudhury
2014). Araji et al. (2014) proved that increase in insect resistance in tomato due to
overexpression of PPO genes compared to the mutant. Plants accumulate more of
cellulose, lignin, tannins, and silicates, which reduce the palatability or feeding
capacity of insects. AMF colonization has been reported to increase the phenolic
content and activity of key defense-related enzymes such as lipoxygenases (LOXs)
and phenylalanine ammonia lyase in Piper nigrum (da Trindade et al. 2019).
19.4.4 AMF Alters Primary Metabolite Production
and Allocation of It as Defense Response Against
Insect Damage
Synthesis of defense chemicals against herbivorous insect depends on the quantity
and quality of primary metabolites accumulation in plants. During defense response,
carbon-based materials are converted into nitrogen-based secondary metabolites
which affect the quality of feed, and the feeding performance of a herbivore (Pozo
and Azcón-Aguilar 2007). AMF colonization altered carbohydrate metabolism in
the root system of lychee was reported (Shu et al. 2016). They reported generally
increase in C: N ratio of plants during herbivorous, which implies that a reduction in
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protein, could have significant negative consequences for insects as they are typically limited by protein in their diet and reduction in the nutritional quality of the
host plant (Robinson et al. 2012). However, sucking insects which feed on phloem
are sometimes able to compensate for this reduction in sap quality, or even benefit
from changes in the phloem amino acid profile (Ryalls et al. 2017).
Reallocation of resources is yet another important defense mechanism of plants
against insect attack. Upon herbivore attack, plant nutrients are made unavailable to
the insect by reallocation of resources from insect infected area to uninfected plant
parts or roots (sink) or fungal hyphae in AMF plants. After herbivory passes on all
accumulated metabolites are reallocated back to whole plants from the sink. Schultz
et al. (2013) proved the translocation of nitrogen from shoots to roots of Centaurea
maculosa plant upon infection with Agapeta zoegana and reallocated back from the
root to shoot after removal of insect. Another example is the allocation of sugars
from infested green parts into the non-affected roots, as has been shown for
Manduca sexta infested with Nicotiana attenuata plants. Thus, at the necessary
time, all rescued material can easily be remobilized and used for building new
aboveground organs (Mithöfer and Boland 2012). Foliar arthropod pests might
also interact with AMF, as herbivory can alter the allocation of plant photosynthate
to the roots (Gange et al. 2007; Machado et al. 2013). Metabolic reprogramming like
increased biosynthesis of defense chemicals and relocating of primary metabolites in
tissues away from the site of feeding has been noticed in insect attacked plants to
affect herbivore performance and growth. Markkola et al. (2004) showed that the
increased sink strength of mycorrhizal fungi-colonized roots increased carbon limitation after defoliation. Mycorrhizae could increase shifts in resource allocation to
inaccessible tissues of herbivores (Song et al. 2013; Wang et al. 2015).
19.4.5 Herbivore Induced Synthesis of Secondary Metabolites
in AMF Associated Plants
AMF association affects the development of insects by modifying dietary specialization and feeding mode of the herbivores directly by altering the quality of plants.
AMF colonized plants exhibit accumulation of certain defense compounds like
phenolics, hormones, and reactive oxygen species quenching enzymes during the
interaction, which improves plant defense against herbivore-induced stress
(Koricheva et al. 2009; MacLean et al. 2017). Plants defend themselves by producing secondary metabolites along with primary metabolites, which are toxic to
herbivores and act as defense compounds (Wittstock and Gershenzon 2002).
These secondary metabolites provide direct defense against herbivorous insects by
affecting the growth and development of the pests (Chen et al. 2012; Brunetti et al.
2013). These are targeted against the nervous, digestive, and endocrine organs of
herbivores. Plant secondary metabolites can be divided into four chemically distinct
groups viz., terpenes, phenolics, nitrogen, and sulfur-containing compounds (Mazid
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Arbuscular Mycorrhizal Fungi: Potential Plant Protective Agent Against. . .
327
et al. 2011). AMF symbiosis helps to benefit plant health by modulating secondary
metabolism and thus potentially fortify both direct and indirect plant defense systems (Borowicz 2013; Jung et al. 2012).
Terpenes are important secondary metabolites that provides insecticidal activity
by acting as antifeedants, toxins, or as modifiers of insect development (Lackus et al.
2018). Bennett and Wallsgrove (1994) reported that Azadirachtin is the best insect
deterrents terpene and also inhibit egg maturation. Some important terpenoids like
gossypol, polygodial, glaucolide-A, pyrethroids, and cucurbitacins are reported as
deterrents and toxic to insects (Chaudhary et al. 2018a, b). Some secondary metabolites mainly volatiles provide indirect plant defense by recruiting of natural enemies
of insect pests (Yuan et al. 2008). AMF inoculated plants produce more of monoterpenes and sesquiterpenes, which were not found in the control plants (Shrivastava
et al. 2015).
Phenolics are another important defense compound in plants act as antioxidants
and insect deterrents. Some of the phenolics include coumarin, lignin, flavonoids,
isoflavonoids, quinones, and tannins. Quinones formed by oxidation of phenols bind
covalently to leaf proteins and inhibit protein digestion in herbivores (Bhonwon
et al. 2009). Chickpea infested with Helicocoverpa armigera showed the production
of a greater quantity of isoflavonoids, which deter larval feeding. Salicylates in salix
leaves reduce the feeding and growth of polyphagous larvae of Operophtera
brumata (Lattanzio et al. 2006). AMF (Gigaspora margarita) inoculated Lotus
japonicas plants strongly affects the oviposition of the spider mites by increasing
leaf phenolic content (Nishida et al. 2010).
19.4.6 The AMF Associated Plant Produced Anti-Nutritional/
Digestive Proteins Involved in Defense Against
Herbivore Performance
Plants can also defend themselves by producing proteins that reduce the nutritive
value to the attacking insect or causes physical damage to the insect digestive tract.
The major classes of such defense proteins are α-amylase inhibitor, chitinase,
proteinase inhibitor, and lectin: α-amylase inhibitor reduces the activity of
α-amylase, an enzyme that plays a role in the digestion of starch and glycogen in
insects (Sales et al. 2012). Triticale-α amylase inhibitor has a strong inhibitory
activity on Eurygaster integriceps gut α-amylase (Mehrabadi et al. 2010). Chitinases
enzyme degrades the chitin, which is the major component of the insect cuticle and
peritrophic membrane (Chandrasekaran et al. 2014). Sharma et al. (2003) reported
that the development of Colorado potato beetle is inhibited in transgenic tomato by
overexpression of the chitinase gene. Lectin is another entomotoxic protein bind
with carbohydrates and glycoproteins to make inhibiting the absorption of the
nutrients (Vandenborre et al. 2011). Proteinase inhibitor acts as anti-metabolic proteins, which interfere with the digestive process of insects by reducing the
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availability of amino acids necessary for growth and development (Habib and Fazili
2007). The AM fungus Glomus mosseae promoted expression of serine protease
inhibitors, oxygenase D, and allene oxide cyclase genes in tomato (Solanum
lycopersicum) infested by Helicoverpa armigera was reported by Song et al. (2013).
19.5
Indirect Plant Defense Against herbivorous Insect
Modulated by AM Fungi Association
In response to environmental stress, plants produce diverse volatile organic compounds (VOCs) as a part of plant protection and signaling molecules to communicate
among them. It is also involved in indirect plant defenses against biotic stress,
including herbivore attack to serve as an attractant of natural enemies of the attacking
insect (De Lange et al. 2020). These volatiles attract both parasitic and predatory
insects that are natural enemies of the insect herbivores (Paré and Tumlinson 1999).
Volatiles detected in different plants following attacks from different herbivores
share notable similarities even though the composition of these volatiles may differ
from case to case. Besides feeding, leaf injury caused by caterpillar movement and
insect oviposition can also increase volatile emission in plants (Arimura et al. 2005;
Hare 2011). The majority of volatiles are from the category of terpenoids, indole
(phenylpropanoid), fatty acid derivatives (green leaf volatiles), and nitrogen and
sulfur-containing compounds. AM fungal colonization also induces plant defense
through jasmonic and salicylic acid signaling pathways, enabling them to achieve
compatibility with the plant, and the cocktail of volatile organic compounds (VOCs)
released from the leaves (Jung et al. 2012).
Terpenes and terpenoids are the main components of plant volatiles play a
significant role in indirect defense by attracting natural enemies of herbivores in
various systems (Schuman et al. 2014; Böttger et al. 2018). Chen et al. (2011)
reported that terpenes and terpenoids play an important role in plant physiology
and defense. Overexpression of terpenes synthase tps10 gene in Arabidopsis results
in higher emission of sesquiterpenes, and the transgenic Arabidopsis plants are more
preferred by the parasitic wasps than the wild type (Kappers et al. 2005). AMF also
imparts defense by affecting volatile organic compounds (VOCs) emitted by plants
(Asensio et al. 2012); these might act as a cue for attracting natural enemies of
herbivores (Hunter 2002). The plants colonized with Glomus intraradices reported
attracting more natural enemies of aphid (Volpe et al. 2018). AMF colonization
reported enhanced the production of triterpenoids in various plants (Kapoor et al.
2017). AMF colonization influences plant VOC production influences plant defense
against herbivores by attracting predator and parasitoid of insect pests (Meier and
Hunter 2019). Maize roots release (E)-β-caryophyllene in response to the attack by
the larvae of Diabrotica virgifera for attracting Heterorhabditis megidis nematodes
that feed on the beetle larvae (Howe and Jander 2008). Kessler and Heil (2011)
reported that maize seedlings attacked by Spodoptera exigua attract parasitoid
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329
Cotesia marginiventris. Plants colonized with Funneliformis mosseae showed indirect defense responses through the release of volatiles like β-ocimene and
β-caryophyllene, which increased spider mite predation rates in Phaseolus vulgaris
(Schausberger et al. 2012).
Indole and indole-alkaloid molecules contain nitrogen to attract the natural
enemies of herbivorous insects. The effect of indoles on natural enemies of herbivorous insects is very specific to different species under different conditions. Indoles
have been detected after an insect attack in many plant species, including cotton
(McCall et al. 1994), gerbera (Krips et al. 1999), maize (Zhuang et al. 2012), and
lima bean (Maurya et al. 2020). Indoles have also been detected in plants treated with
the elicitor volicitin (Frey et al. 2000). Turlings et al. (1995) reported that the blend
of indoles and terpenoids released by plants after attack by lepidopteran larvae is
attractive to the Cotesia marginiventris. In addition to their roles in attracting natural
enemies directly indoles also play roles in regulating the emission of other volatiles.
In maize, indole emission precedes the release of other volatiles and the release of
indoles is essential for priming and to the synthesis of mono- and homoterpenes in
the attacked plants, suggesting that indoles may act as potent aerial priming agents
that prepare other tissues and neighboring plants for incoming insect attacks (Erb
et al. 2015). Mycorrhizal Solanum lycopersicum plants are more attractive to the
Aphidius ervi (parasitoid) than non-mycorrhizal plants (Guerrieri et al. 2004).
Fatty acid derivatives are another common volatiles emitted by plants and they
are often referred to as green leaf volatiles because they impart the typical odor of
green leaves (Clavijo Mccormick et al. 2014). Multiple pathways can lead to the
production of green leaf volatiles and are emitted at elevated levels when leaf tissues
are disrupted by the herbivore. Linoleic acid and linolenic acids are unsaturated fatty
acids released from the plasma membrane due to cell damage, are oxidized and
decarboxylated by hydroperoxide lyases, resulting in the production of volatiles
C6-aldehydes, C6-alcohols, and esters such as (Z )-3-hexen-1-yl acetate (Feussner
and Wasternack 2002). Green leaf volatiles can also be produced by lipoxygenases
activity via the oxylipin pathway in many plants (Vincenti et al. 2019). The
application of Manduca sexta oral secretions to the wounds of wild-type Nicotiana
attenuate plants leads to a remarkable change in composition resulting in increased
foraging efficiency of predators in nature (Allmann and Baldwin 2010). Unlike other
HIPVs, green leaf volatiles are released immediately upon herbivore damage. Earlier
emission of green leaf volatiles in herbivore-attacked plants can induce the emission
of other herbivore-induced plant volatiles and therefore may play a potential role in
intra- and interplant signaling (Allmann and Baldwin 2010). Exposure of plants to
synthetic green leaf volatiles (GLVs) induces the rapid production of JA and
emission of sesquiterpene in maize (Ton et al. 2007), and triggers the emission of
local and systemic terpenes in tomato (Farag and Pare 2002). AMF colonized
Leucanthemum vulgare plants infected with Chromatomyia syngenesiae (leafminer) attract Diglyphus isaea parasitoid (Gange et al. 2003). The HIPV’s produced
vary according to the AM fungal, plant and herbivore species, the developmental
stage and condition of the plants, and the herbivores. These factors also affect the
composition of the VOCs emitted from the leaves (Schausberger et al. 2012) that
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render the infested plant less attractive or even repellent to subsequent herbivores,
and attractive to natural enemies of these herbivores, such as parasitoids. It is
through these induced changes in plant health and defense responses that mycorrhizal fungi and insect herbivores interact with each other indirectly.
Nitrogen (N) compounds such as nitriles and oximes are among many other
volatile compounds that are commonly emitted from herbivore damaged plants
(Irmisch et al. 2014). In contrast to the abundant and widespread compounds such
as monoterpenes, sesquiterpenes, and green leaf volatiles, nitrogenous volatiles are
emitted in minor amounts. However, nitrogenous volatiles do play crucial roles as
active gradients of cues for carnivore attraction. The proportion of nitrogenous
compounds in a volatile blend can contribute to the specificity of the volatile mix
for different plant–carnivore systems (Clavijo Mccormick et al. 2014). In plants,
nitrogen-containing volatiles is synthesized from secondary metabolites of
glucosinolates (Hopkins et al. 2009) and amino acid derivatives (Irmisch et al.
2014). Some of the nitrogen-containing compounds include aldoximes, nitriles,
and benzyl cyanides, which have been detected in the volatile blends of Populus
sp. in response to Lymantria dispar attack (Irmisch et al. 2014). Electrophysiological
and behavioral experiments suggest that nitrogenous compounds are key attractants
for parasitic Hymenopterans (Clavijo Mccormick et al. 2014). The effect of AMF
induced variation in parasitoid behavior fully depends on plant size or volatile
emission (Schausberger et al. 2012).
The plant produced volatile compounds prime the plants to resist forthcoming
herbivore attack apart from the indirect defense. Defense priming is defined as the
enhanced readiness of defense responses (Conrath et al. 2006; Kim and Felton
2013). Primed plants display faster and/or stronger activation of various cellular
defense responses to forthcoming stress (Ton et al. 2007; Jung et al. 2009; Slaughter
et al. 2012). Plants primed by neighboring plants volatiles and display faster or
stronger defense activation and enhanced insect resistance following herbivore
attack (Kessler et al. 2006). Anti-herbivore defense response often is induced with
greater efficiency in plants that have previously experienced with insect attacks
(Karban and Baldwin 1997). Recent studies demonstrate that the primed state of
Arabidopsis thaliana plants can be transferred to their progeny, conferring better
protection from pathogen attack as compared to the descendants of unprimed plants
(Slaughter et al. 2012). The primed state in the plant also can be provoked by various
natural and synthetic compounds, such as jasmonic acid (JA), salicylic acid (SA),
and β-aminobutyric acid (BABA) (Worrall et al. 2012). Tomato plants are grown
from JA-treated and BABA treated seeds showed increased resistance against
herbivory by spider mites, caterpillars, and aphids, and fungal pathogens (Worrall
et al. 2012). Even though many reports displayed that AMF could prime the plant
defense against stress in several plants, the underlying mechanisms remain elusive?. In order to feed the growing world population and to achieve global food
security under climate change and environmental stress, we need to have a sustainable agriculture that is less impacted by the environmental factors. A sustainable
agriculture refers to the key components of agriculture conservation that practices
the use of continuous cover crops, no tilling, crop rotations, and environmental
friendly. To make use of mycorrhizal technologies for a greener future we need to
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Arbuscular Mycorrhizal Fungi: Potential Plant Protective Agent Against. . .
331
have a clear understanding of the contribution of mycorrhiza to the causal metabolic
pathways of above ground plant parts (Plant–insect interaction) that influence the
interaction with rhizosphere for maintaining a sustainable agriculture.
19.6
Conclusion and Future Thrust
AMF symbiosis has an important impact on plant growth by enhancing nutrient
availability and also aided in sustaining plant health during herbivore infestation.
Mycorrhiza-induced resistance (MIR) in aboveground tissues seems effective in
generalist chewing insects. Experimental evidence confirms that this protection is
based not only on improved nutrition or local changes within the roots and rhizosphere but that priming of plant immunity plays a major role in MIR. AMF
colonization improved the tolerance against insect infestation by modulating morphological and physiological characteristics of the plant by producing defenserelated compounds like antioxidant enzymes and metabolites, phytoalexins,
herbivore-induced plant volatiles, green leaf volatiles, primary and secondary metabolites, cell wall components, and also improved photosynthetic efficiency of the
plants. The vast research indicates that there are changes in the morphological and
biochemical behavior of plants in response to insect attack. These changes have been
modulated positively to provide defense against insect attack in AM infected plants.
It was also understood that insect cues accountable for eliciting defense response in
plants vary with the insects, plants, and AMF colonization. These insect cues and
plant responses have not been understood fully. Further, the mechanisms of plant
defense against insect attacks are only in selected plant species. Although the
molecular basis for the regulation of plant defenses and the priming of the plant
immune system during mycorrhization remains mostly unknown, a prominent role
of jasmonate signaling has been confirmed. Thus, further studies are required to
elucidate the insect cues, responses of AMF plants to different insect attack, and
mechanisms of resistance or counter adaptation developed by insects. This chapter
indicated that AM fungi may be recommended as a potential bioprotective agent
against pests during the cultivation of field crops. Moreover, we need to develop
strategies to investigate different parameters influencing the effectiveness of mycorrhizal colonization in plants- insect interaction along with the implementation of
systemic approaches for crop improvement and integrated pest management (IPM).
This approach will be a further step towards sustainable agricultural production.
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Chapter 20
Eradication of Malaria by the Mutualistic
Interaction Between Wickerhamomyces
anomalus and Anopheles sp.
Arpit Gupta, Arpita Balakrishnan, and Amit C. Kharkwal
Abstract The fatality of malaria is quite prominent worldwide, which is majorly
affecting developing nations having subtropical to tropical climate. The disease
burden is quite hefty and its eradication is of utmost importance for the economic
development of nations. Many chemical control methods have been used in the past
decades, but these methods have been turned obsolete due to the development of
resistance of the parasite Plasmodium and the vector Anopheles sp. A new potent
method has been developed which focuses on the elimination of the parasite, using
the mutualistic interactions between the naturally present microbiota in the mosquito
midgut and the mosquito itself; which is termed as symbiotic control. Here we focus
on the yeast Wickerhamomyces anomalus and elucidate its mutualistic interactions
with Anopheles sp. which can help eradicate the fatal malarial infection.
Keywords Symbiotic control · Wickerhamomyces anomalus · Pichia anomala ·
Anopheles sp. · Plasmodium sp.
20.1
Introduction
Malaria, a devastatingly fatal epidemic affects millions annually all across the globe.
According to the WHO in 2017 itself, nearly half of the world’s population was at
the risk of malaria, the numbers of the affected were approximately 219 million
(World Health Organization 2019). Malaria is quite prominent all around the world
but certain hotspots are the hubs for this malicious disease. These hubs are usually
confined to tropical and subtropical regions (World Health Organization 2019).
Another major factor that affects this disease is the economic development of the
region; countries that are underdeveloped or are developing nations are quite prone
A. Gupta · A. Balakrishnan · A. C. Kharkwal (*)
Amity Institute of Microbial Technology, Amity University Uttar Pradesh, Noida, Uttar
Pradesh, India
e-mail: ackharkwal@amity.edu
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_20
339
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to the intensive fatality of malaria and the developed countries are usually out of
bounds (Gallup and Sachs 2001).
International discussions usually have malaria as a focal point due to its health
burden on the social and economic development of nations. Thus, constant steps
toward the global eradication of the disease are being taken place, be it preventive
measures or treatment options (Alonso et al. 2011). Governments of the affected
nations are spreading the word of awareness regarding the precautions that the
citizens can follow to minimize the acquirement of the disease. Even these small
steps help in decreasing the fatality of the disease. If we shift our prime focus to
India, the statistical evidence shows a decrease in the disease burden between 2016
and 2017 with 3 million fewer cases (World Health Organization 2018a). This
evidence shows that certain steps toward the prevention of malaria may have helped
to invoke this kind of result.
To eliminate the disease, local transmission of the parasite needs to be interrupted
and continued control measures need to be implemented to prevent the
re-establishment of the disease (World Health Organization 2018b). Eradication,
on the other hand, is the complete removal of the overall worldwide incidence of the
disease, once this has been achieved intervention methods are no longer required
(World Health Organization 2018b).
In 1955, WHO launched the Global Malaria Eradication Programme which
utilized two major tools: chloroquine for prophylaxis and prevention and dichlorodiphenyl-trichloroethane (DDT) for vector control (Greenwood et al. 2008). The
implementation of these tools had a substantial influence in some areas, which had
relatively low transmission rates, such as Sri Lanka and India (Greenwood et al.
2008). Regardless of these achievements, the campaign was eventually unsuccessful
due to the emergence of chloroquine-resistant Plasmodium strains and
DDT-resistant Anopheles mosquitoes; another factor which caused the campaign
to become unsuccessful was that the campaign never attempted to eradicate malaria
in most parts of Africa, where the malarial transmission is intense (Greenwood et al.
2008).
More potent methods and new tools have been deployed and one of the most
important ones is being highlighted in this chapter, which is by using microflora
present in the mosquito midgut naturally (Drexler et al. 2008), as they are present in
the same compartment where the most vulnerable stages of plasmodium development occur (Wang et al. 2012). Microbes present naturally may offer opportunities
to successfully manipulate the vector competence to reduce their abilities to transmit
the human pathogens (Ricci et al. 2012a). Furthermore, there is a growing interest of
symbionts in mosquito disease vectors since their manipulation may offer novel
control methods that are uniformly defined as Symbiotic Control (Ricci et al. 2012a),
which can disrupt mosquito-transmitted pathogens.
A plethora of microorganisms inhabits the midgut that has been known to show
symbiotic interactions; such as bacterium Enterobacter agglomerans (Ricci et al.
2012b), yeast Wickerhamomyces anomalus (Cappelli et al. 2014), fungus
Metarhizium anisopliae (Fang et al. 2011), and even virus Densovirus (Ren et al.
2008). This chapter deals with the symbiotic control using the mutualistic
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Eradication of Malaria by the Mutualistic Interaction Between. . .
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interactions between Wickerhamomyces anomalus and Anopheles stephensi to eliminate the burden of the fatal disease—Malaria.
20.2
Malaria and Its Mechanisms
Malaria is a widely spread and very significant health problem all around the globe
(Mawson 2013). Even though it affects millions around the world, the core regions
where it is predominating are places having the necessary climatic conditions
required for that disease to thrive (Gallup and Sachs 2001).
Malaria is a mosquito-borne infectious disease that affects a majority of vertebrates and not only human beings. Inside the host body, the target cell (organ) of the
causative agent is the liver (hepatic cells), which it reaches through the bloodstream.
The causative agent is a parasite that belongs to the Plasmodium group. This parasite
requires two hosts to complete its life cycle—vertebrate and female Anopheles
mosquito (Arrow et al. 2004). It usually infects the mosquitos and is transmitted to
the vertebrate via the bite of the infected mosquito. Other transmission methods may
also involve blood transfusions, in which Plasmodium-infected blood products are
transferred to a healthy host (Arrow et al. 2004).
Particularly in the human host, the clinical manifestations in the beginning stages
of the infection are nonspecific and mimic a flu-like syndrome (Bartoloni and
Zammarchi 2012). Flu-like syndromes usually elicit fever, headache, and many
more common symptoms. An extremely diverse range of severity is observed,
which ranges from mild headaches to serious complications which may even lead
to death (Bartoloni and Zammarchi 2012). In other vertebrates like mice, the
commonly observed symptoms were behavioral and neurological (Basir et al. 2012).
Progression to these complications can be quite rapid, thus patients who are
suspected of malaria must be assessed and treated swiftly (Bartoloni and Zammarchi
2012). Severe malaria is a life-threatening disease but is also a curable disease
(Bartoloni and Zammarchi 2012). Nonspecific clinical findings are quite common
in malaria, this often leads to a false diagnosis of the disease (Bartoloni and
Zammarchi 2012). Malaria is sometimes falsely detected as influenza, dengue,
typhoid fever, viral hepatitis, gastroenteritis, or encephalitis (Bartoloni and
Zammarchi 2012) due to the presence of nonspecific symptoms. This can be quite
detrimental for the patient and this may allow the parasite to thrive for a much longer
time and may lead to a deadly outcome.
Thus, immediate diagnosis and appropriate treatment are of utmost importance to
prevent morbidity and this may also help in averting fatal consequences (Bartoloni
and Zammarchi 2012).
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20.2.1 The Causative Agent: Plasmodium
Plasmodium is an obligate parasitic protozoan. It is from the phylum Apicomplexa,
which is a large group of parasitic eukaryotes (Zilversmit and Perkins 2008). It
differentiates into a series of morphologically distinct forms in the vertebrate and
mosquito hosts (Zilversmit and Perkins 2008) which is highlighted in Fig. 20.1. It
alternates between invasive stages (sporozoite, merozoite, and ookinete) and replicative stages (pre-erythrocytic, erythrocytic-schizont, and oocyst) interposed by a
single phase of sexual development that facilitates the transmission from the human
host to the anopheline vector (Hall et al. 2005).
In the Plasmodium group, the main species involved in causing malaria are
mentioned below:
• P. falciparum is found in tropical and subtropical areas, predominately found in
Africa (Global Health 2018). P. falciparum can cause severe malaria because it
multiplies rapidly in the blood, which may lead to anemia. Infected parasites can
also clog small blood vessels (Global Health 2018). Cerebral malaria can occur if
the parasite reaches the brain, and as a result, the complications that follow can be
lethal (Global Health 2018). The incubation period is approximately—2 weeks
(Bartoloni and Zammarchi 2012). This malignant species is known for its high
fatality. It mainly infects humans among other vertebrates.
• P. vivax is found mostly in Asia, Latin America, and in some parts of Africa. It is
the most prevalent species of Plasmodium (Global Health 2018). P. vivax has the
same incubation period as P. falciparum (Bartoloni and Zammarchi 2012).
P. vivax has dormant liver stages called hypnozoites that can activate and invade
the blood several months or years after the infection (Global Health 2018).
Fig. 20.1 Lifecycle of Plasmodium sp.
20
•
•
•
•
•
Eradication of Malaria by the Mutualistic Interaction Between. . .
343
P. vivax is also rather malignant has come to light by many studies and research in
the same matter is being conducted (Bartoloni and Zammarchi 2012).
P. ovale is found mostly in Africa and the islands of the western Pacific. It is
biologically and morphologically very similar to P. vivax (Global Health 2018). It
is rarely very severe, but respiratory distresses have been observed in this case
(Lee and Maguire 1999).
P. malariae is found worldwide and is the only human malarial parasitic species
that has a three-day cycle (Global Health 2018). P. malariae causes chronic
infections that can even last a lifetime if left untreated (Global Health 2018). In
some chronically infected patients, P. malariae can cause grave complications
such as nephrotic syndrome (Global Health 2018). It is known to cause the
mildest but the most persistent form of malaria (Harinasuta and Bunnang 1988).
P. knowlesi is found throughout Southeast Asia and it has been known to be a
significant cause of zoonotic malaria in that region (Global Health 2018). It was
also identified as an infectious agent in long-tailed and pig-tailed macaques
(Garnham 1966; Singh et al. 2004). P. knowlesi has a 24-h replication cycle
and so can rapidly progress from an uncomplicated to a severe infection with a
high fatality rate (Global Health 2018).
P. berghei is a parasite that causes infection in rodents and it has been noticed in
African murine rats, it is the most commonly used model organism for studying
severe malaria cases in humans caused by P. falciparum (Franke-Fayard et al.
2010). The symptoms shown by this species are to a certain degree comparable to
P. falciparum infection (Franke-Fayard et al. 2010).
P. chabaudi is a rodent malaria species that cause uncomplicated disease (Wilson
et al. 2016). It is usually located in the central regions of Africa (Landau and
Killick-Kendrick 1966).
20.2.2 Lifecycle of Plasmodium
The malaria parasite, Plasmodium develops in both vertebrate and the female
Anopheles mosquitoes. It requires two hosts to complete its lifecycle. It changes
through several stages with different morphological characteristics in both of the
hosts (Fig. 20.1).
It starts when an infected female Anopheles mosquito bites a healthy vertebrate,
injecting Plasmodium parasites in the form of sporozoites (Siciliano and Alano
2015) into the bloodstream. The sporozoites move to liver hepatocytes where they
divide asexually over the next 7–10 days, causing no symptoms (Siciliano and Alano
2015). After the maturation of the cells in the hepatocytes, they are released from the
liver in the form of vesicles into the bloodstream, they are called merozoites
(Siciliano and Alano 2015). These merozoites invade red blood cells (erythrocytes)
and multiply until lysis of the cell occurs, here asexual reproduction also occurs
(Siciliano and Alano 2015). This cycle is repeated and many more blood cells are
invaded; Some of the infected blood cells leave the cycle of asexual multiplication
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and instead multiply into sexual forms of the parasite, called gametocytes (Siciliano
and Alano 2015), and these then circulate in the bloodstream.
When a healthy mosquito bites an infected vertebrate, the parasite is transmitted
from the vertebrate host to the mosquito. The mosquito ingests these gametocytes,
which develop into mature sex cells called gametes (Siciliano and Alano 2015). This
maturation occurs in the midgut of the mosquitos, where the fertilization of female
gamete takes place and they form oocysts (Siciliano and Alano 2015). Inside the
oocyst, thousands of active sporozoites develop, and when it eventually bursts, the
release of sporozoites ensues into the body cavity that travels to the mosquito’s
salivary glands (Siciliano and Alano 2015). And from the salivary gland, the
sporozoites get transferred to the vertebrate host (Siciliano and Alano 2015) when
this infected mosquito bites, and then the cycle of infection begins again.
20.2.3 The Vector: Anopheles Mosquito
The description of the Anopheles genus of mosquito was provided in the year 1818
by J.W. Meigen (Meigen 1818). Around 40 species of this Anopheles genus are
vectors in the malarial infection (Tiwari 2017). They derive blood meal from
vertebrates for nurturing their eggs, and sometimes it occurs that the blood being
derived is from an infected host and thus the plasmodium present in the bloodstream
is ingested by the mosquito and hence it becomes a carrier of the disease (Tiwari
2017). The blood meal ingested by the mosquito is the primary link between the
vertebrate and the mosquito vector in the parasite’s lifecycle (Tiwari 2017).
In Amazon, many species of the Anopheles mosquito are present and three
species are considered as the primary vectors of malaria, they are namely,
A. darlingi, A. albitarsis s.l., and A. aquasalis (Pimenta et al. 2015). In South
America, the major vector is A. darlingi and in the Amazonian regions of French
Guiana, Bolivia, Guyana, Columbia, Peru, Suriname, and Venezuela the same has
been known to be associated with the dynamics of malaria transmission
(Zimmerman 1992; Hiwat et al. 2010; Pimenta et al. 2015). In Venezuela
A. albitarsis s.l. is found (Rubio-Palis et al. 1992; Pimenta et al. 2015) and
A. aquasalisi is found in Trinidad (Chadee and Kitron 1999; Pimenta et al. 2015)
and Venezuela as well (Berti et al. 1993; Pimenta et al. 2015).
Other Anopheles species can be occasional malarial vectors due to their geographic distributions, natural infectivity, and their population density (Deane 1986;
Zimmerman 1992; Sinka et al. 2010, 2012; Pimenta et al. 2015). A. nuneztovari s.l.
and A. triannulatus s.l. are commonly observed in the Amazon by researchers and
they are known to be infected with P. vivax and P. falciparum, but their role as
malaria vectors has yet to be elucidated (de Arruda et al. 1986; de Oliveira-Ferreira
et al. 1990; Klein et al. 1991; Tadei and Dutary Thatcher 2000; da Silva-Vasconcelos
et al. 2002; Povoa et al. 2003; dos Santos et al. 2005; Póvoa et al. 2006; Galardo et al.
2007; da Rocha et al. 2008; Santos et al. 2009; Pimenta et al. 2015).
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In the Anopheles mosquito, there are certain defenses against the parasite, the first
line of defense or the physical barriers along with the innate immune system poses a
significant challenge for parasite development (Pimenta et al. 2015). The limiting of
the infection in the salivary glands is majorly done by the innate immune system
(Pimenta et al. 2015). For the successful completion of the Plasmodium life cycle, it
is necessary for the transmission to occur and for the parasite to survive in nature.
Thus, there has been enormous pressure on the parasite to evolve means to escape
the mosquito’s immune defenses (Pimenta et al. 2015). Much more research on this
matter needs to be conducted for a better understanding of the role of mosquito’s
immune response against Plasmodium (Pimenta et al. 2015).
20.3
Wickerhamomyces anomalus
Wickerhamomyces anomalus is a yeast, which is a robust microorganism and is
known for its environmental, industrial, and medical aspects (Walker 2011). A
distinguished metabolic and physiological diversity is exhibited by this microorganism (Walker 2011). From an application viewpoint, its role in biotechnology has
been of much significance and future research on the same is quite beneficial for
better understanding (Walker 2011).
20.3.1 Basic Characteristics and Morphology
W. anomalus is a killer fungus, which is identified by many other names viz. Pichia
anomala, Hansenula anomala, Candida beverwijkiae, Candida pelliculosa, and
commonly termed as non-Saccharomyces wine yeast (Ricci et al. 2011a; Riley
et al. 2016). It is a heterothallic yeast, that reproduces by forming one to four
hat-shaped ascospores, morphologically it creates highly textured white colored
colonies and is classified as a biosafety level-1 organism (Kurtzman 2011; Ricci
et al. 2011a; Satora et al. 2014; Landis 2018).
20.3.1.1
Niche
They are found in diverse environments and have also been isolated from various
alternate sources exempli gratia soil, plants, cereal grains, maize silage, fruit skin,
fruit juices, wine, and food products (like dairy, baked, fermented, high-sugar, and
salted foods) (Pfaller et al. 2009; Cappelli et al. 2014; Satora et al. 2014; Riley et al.
2016). These killer toxin-producing yeasts have also been weeded out from contaminated oil, marine environment, wastewater, and also from immunocompromised
patients (Cappelli et al. 2014; Riley et al. 2016). They even have been isolated
(in trace amounts) from microflora of human skin, throat, and alimentary canal;
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likewise, in the gut of insects like beetles, mosquitoes, and flies (Pfaller et al. 2009;
Cappelli et al. 2014).
20.3.1.2
Abiding Environment
W. anomalus can survive in highly intolerable (by other microbes) environmental
stress and is adaptive to inexcusable growth conditions like varying temperature
(3–37 C), high osmotic pressure, a wide range of pH (2–12), low water activity;
simultaneously it possesses the ability to endure in very little or no oxygen environment that is anaerobic conditions, all these qualities make it highly adaptable to a
plethora of environments which makes them highly competitive (Cappelli et al.
2014; Satora et al. 2014; Riley et al. 2016).
20.3.1.3
Compounds Secreted and Its Uses
W. anomalus can produce a substance called Killer toxin, which can kill other molds
and yeasts, therefore, it can be used as a biocontrol agent and bio preservative in the
agro-food sectors (recognized by European Food Safety Authority) (Cappelli et al.
2014; Riley et al. 2016). Other than killer toxin production, it secrets 2-phenyl
ethanol, which prevents spore formation in human pathogenic fungus Aspergillus
flavus and reduces the biosynthesis of aflatoxins (Pretscher et al. 2018).
Moreover, it is one of the strong producers of volatile compounds including
Isoamyl acetate (which has a redolence of artificial banana), acetic acid, and ethyl
acetate in pure culture (Satora et al. 2014; Landis 2018). Apart from its cytocidal
activity, it is used in biofuel production, it is used in wine fermentation due to its
ability to produces volatile compounds that contribute to the aroma of wine and
some of its molecular products are also used in medicines for humans (Cappelli et al.
2014; Riley et al. 2016).
20.3.2 Killer Toxin
The ability of W. anomalus to produce killer toxin is known, but incidences of
various types of killer toxins being produced have also been observed, which can be
accounted to post-translational modifications; these modifications render peculiar
differences in molecular weight among the different types of Killer Toxins (Cecarini
et al. 2019). Those yeasts which are known to produce Killer Toxins are immune to
their own Killer Toxins but they might be susceptible to toxins produced by other
yeasts (Cecarini et al. 2019). Killer Toxins are potent to kill various microbes but
what makes them fatal is a prevailing question. These killer toxins are a group of
glycoproteins consisting of a major globular core with extensive polar surfaces, 12%
Beta sheets, and 32% Helix (4% 3(10) helix and 28% Alpha-helix) (Cappelli et al.
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2014; Cecarini et al. 2019). There are different strains of W. anomalus which are
known for their killer toxin production, some of them are depicted as follows:
W. anomalus HN1-2 isolated from the mangrove ecosystem produces a killer
toxin that can kill cells of Metschnikowia bicuspidata, Candida albicans,
Kluyveromyces aestuarii, Pichia guilliermondii, Lodderomyces elongisporus,
Yarrowia lipolytica, and Saccharomyces cerevisiae, these are present in natural
marine environments (Sun et al. 2012).
W. anomalus-killer toxin a and W. anomalus-killer toxin b secreted by
W. anomalus VKM Y-159 are reported to show cytocidal effects, W. anomaluskiller toxin a shows the effect on pathogen Candida spp. and several other yeasts
while W. anomalus-killer toxin b exhibits killer activity on Candida norvegica and
Candida alai (Farkas et al. 2012).
Panomycocin, a potent antifungal agent, is a fatal protein secreted by
W. anomalus NCYC 343 (Satora et al. 2014).
W. anomalus F17.12 exerts strong anti-plasmodial activities on Plasmodium
berghei (Cecarini et al. 2019).
20.3.2.1
Conditions for Secretion
Killer phenotype’s killer toxin secretion gets enhanced or activated due to stress in
the environment which can be experienced due to competition with other microbes
for the acquisition of resources; but host body serves as a niche, where yeast can
proliferate and utilize a good amount of food thus the secretion of the killer toxin
may be hindered (Cappelli et al. 2019). Therefore, in general terms, killer toxin
production gets triggered, in the presence of environmental stress and competition.
20.3.2.2
Mechanism of Action
Killer Toxins exert cytocidal effects via a two-step mechanism (Cecarini et al. 2019).
The first step involves the binding of Killer toxins with primary receptors, which are
usually cell wall carbohydrates (mainly β-1,3 glucans), and these glucans are
hydrolyzed by the β-1,3 glucanase activity of the killer toxins into glucose. Later
the second step proceeds by translocation of Killer Toxins to the secondary receptors
of the plasma membrane, where it ultimately performs cell lysis which leads to death
(Cappelli et al. 2014, 2019; Satora et al. 2014; Cecarini et al. 2019) as demonstrated
in Fig. 20.2.
20.3.2.3
Validation of the Mechanism of Action
EXGI and EXG2 are the genes coding for β-glucanase synthesis, which are found in
the genome of W. anomalus and the lack of the W. anomalus antimicrobial activity
was found to be correlated with coupled or single silencing of these genes (Valzano
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Fig 20.2 Mechanism of action of Killer Toxins
et al. 2016). To confirm that the cytocidal effect of Killer Toxins is mediated by
β-glucanase activity, Castanospermine (a β-glucanase inhibitor) was induced with
Killer Toxins to observe its effect against a parasite and as a consequence, the killer
activity of the toxin was reduced to 46.2% from 79.7% in case of Killer Toxins
isolated from W. anomalus F17.12 strain and in case of the Killer toxin isolated from
W. anomalus ATCC 96603 strain it was reduced to 49.5% from 88.5% (Valzano
et al. 2016). So, castanospermine confirmed the Killer Toxins’ β-glucanase mediated
activity (Valzano et al. 2016).
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20.3.3 The Mutualism of W. anomalus and Anopheles
Mosquito
W. anomalus a microorganism with high potential is known to form symbiotic
relationships with many organisms (Ricci et al. 2011c). Within the insect
populations, it has been located within the inner body of Drosophila sp. (Ricci
et al. 2011c), it has also been identified as a killer yeast, acting against pathogenic
fungi in the crab Portunus trituberculatus (Wang et al. 2007) and its presence has
been observed in many mosquito species (Ricci et al. 2012b) as well.
W. anomalus has been known to form an association with specific species of
mosquito hosts (Ricci et al. 2012a); They were detected at all the developmental
stages of both malaria (A. stephensi, A. gambiae) and dengue mosquito vector
species (Aedes aegypti, Aedes albopictus) where it localizes in the midgut and
reproductive organs (Ricci et al. 2011a, c). These mutualistic interactions shine a
light on the possibility that this particular yeast strain is pre-adapted to colonize the
mosquito host body not only in the GI tract but it is intimately associated with the
mosquito’s reproductive systems (Ricci et al. 2011a). This suggests that some
beneficial biological properties are present in the yeast to aid its locomotion to
different body compartments while escaping the host immune barriers (Ricci et al.
2011a). These host-barrier evasion mechanisms, are yet to be explored.
The combination of the niche preference by W. anomalus, together with its
capacity to retain growth under variable environmental conditions, are major factors
involved in its survival in the midgut and gonads (Ricci et al. 2011a). The presence
of W. anomalus has been identified through transmission electron microscopic
analysis of the laboratory-reared colony of A. stephensi (Ricci et al. 2011a). This
proved the presence of the yeast in the mosquito species but to confirm symbiotic
association much more evidence was collected, which is mentioned as follows:
• W. anomalus was identified by molecular and cultivation-based methods.
• W. anomalus was PCR-detected in mosquitoes from all development stages.
• W. anomalus was detected by using specific fluorescence in-situ hybridization
probes in both male and female guts and reproductive systems (Ricci et al.
2011a).
This association between the organisms is quite favorable to both the counterparts; W. anomalus is located in certain vital organs, which is a great source of
nutrition for the yeast required for its growth and development (Ricci et al. 2011a).
Their presence in the gonads could indicate that they are colonizing there to increase
fecundity (Ricci et al. 2011c). Interestingly, the insect gonads are characterized by a
thick system of tracheal trunks that could represent a means for meeting the oxygen
demands (Gibson and Hunter 2005). The localization of W. anomalus in the reproductive organs suggests a vertical transmission route through generations; Such a
possibility is supported by the occurrence of W. anomalus in all life stages of
A. stephensi (Ricci et al. 2011a). W. anomalus-Killer toxin signals were also
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A. Gupta et al.
observed in progeny of mosquitoes fed with W. anomalus F17.12 strain, which
suggested it could be a consequence of vertical transmission (Cappelli et al. 2014).
On the other hand, A. stephensi is attracted to yeasts due to its high CO2
production levels and the fact that there is a vital requirement of CO2 in the mosquito
body (Smallegange et al. 2010; Blumberg et al. 2015). Yeasts could also take part in
driving some behavioral traits of the insects such as oviposition site selection and
host-seeking (Phelan and Lin 1991; Ricci et al. 2011c). More possible functional
roles of W. anomalus are still to be assessed, but potential benefits can be hypothesized. We assume that a certain, protective role against pathogenic fungi is being
provided to mosquitoes through the killer toxin production activity of the
W. anomalus (Polonelli 2000; Passoth et al. 2006). Considering that the marine
strains of W. anomalus have already been proposed to protect the host crab against
pathogens (Wang et al. 2007), such a possibility should be also considered for
A. stephensi.
In recent years, the relationship between symbionts and mosquitoes has attracted
a great deal of attention, mainly for the perspective of exploiting the symbionts for
blocking the transmission of parasites, through the production of antagonistic factors
(Favia et al. 2008; Moreira et al. 2009). This can help improve the future potential of
vector-borne disease control (Bian et al. 2013; Koehler and Kaltenpoth 2013). The
novel finding of the yeast from A. stephensi displays the presence of a killer strain,
which supports the hypothesis of a protective function (Cappelli et al. 2014) and
could make the Plasmodium vulnerable to the antagonistic actions of the killer toxins
(Abraham and Jacobs-Lorena 2004).
20.3.4 Competition
Now, we are aware that W. anomalus is present inside the mosquito, not as a
transient commensal, but as a symbiont (Ricci et al. 2011a; Wang and JacobsLorena 2013) and that they secrete a Killer Toxin which has been reported to
show anti-Plasmodium activity on different developmental stages of Plasmodium
spp. (Cappelli et al. 2019; Cecarini et al. 2019). Thus, serving as a competition to the
Plasmodium spp. in the mosquito midgut; there are many in vivo and in vitro,
experimental researches performed to gather more shreds of evidence to support
this hypothesis.
20.3.4.1
In Vivo
Evidence proved that Killer Toxins are secreted by W. anomalus F17.12 and
W. anomalus ATCC 96603 strains in the mosquito’s body and its presence was
confirmed by a fluorescent signal produced by staining with a monoclonal antibody
(mAb) KT4 on the free yeast’s cell surface (this result was obtained using immunofluorescence assay), while W. anomalus UM3 strain did not reflect any such signals
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(Cappelli et al. 2014). By using immunofluorescence assay, it was revealed that
Killer Toxin signals were found on the 10th day after the introduction of the yeast,
exclusively in the gut but after a time they produced a comparable signal in the
gonads as well (Cappelli et al. 2014).
When activated W. anomalus F17.12 strain was introduced to A. stephensi
through diet, it showed up in its midgut interfering with ookinete development of
P. berghei, while in mice, reduction in parasitemia was observed due to inhibitory
action of purified W. anomalus F17.12-killer toxin on P. berghei’s erythrocytic
stages (Cappelli et al. 2019).
Inhibitory effect of W. anomalus F17.12 strain on the development of
P. berghei’s early sporogonic stages in A. stephensi was found to be 65.21%,
while W. anomalus UM3 strain, which is a non-killer toxin-producing strain of
W. anomalus, exerted 38.02% effect on parasites (Cappelli et al. 2019).
20.3.4.2
In Vitro
Killer Toxins isolated from W. anomalus F17.12 strain (obtained from An. stephensi)
unveiled strong anti-plasmodial activity on P. berghei’s early sporogonic stages and
found to be lethal due to membrane damage (Cappelli et al. 2019). In another
experiment, Killer Toxins’ effect in inhibiting plasmodial development was
exhibited to be ~90%, it exerted a strong anti-plasmodial response against sporogonic stages of berghei species of Plasmodium (Adnani et al. 2017). W. anomalus
F17.12 strain demonstrated the prevention of ookinete development to 40%
(Valzano et al. 2016; Cappelli et al. 2019).
It was observed that Killer toxin tends to follow dose-dependent activity, as Killer
toxin obtained from both W. anomalus ATCC 96603 and W. anomalus F17.12
strains exhibited highest inhibition percentage (92.3 and 87.5%, respectively)
against the development of P. berghei’s sporogonic stages at 100 μg/ml (Valzano
et al. 2016). While Lethal Concentration 50 (it is the concentration of Killer toxin at
which half of the parasite population is killed) values for W. anomalus F17.12 and
W. anomalus ATCC 96603 strains were found to be 64.4 and 61.3 μg/ml, respectively, which could be the possible equivalent activity of Killer Toxin (Valzano et al.
2016).
20.3.4.3
Impact
After studies, it was found that Killer Toxin strongly inhibits the development of
P. berghei’s gametocyte stage to ookinete, as the consequence of its interaction with
β-glucans as discussed earlier (Valzano et al. 2016). Microscopic observation threw
a limelight on some structural/morphological alterations in post-zygotic stages due
to its interaction with Killer toxin, these changes include—jagged cell borders,
irregular cell-shape, lack of crystalloid assembly, cytoplasmic region’s feeble
staining and less-defined cytoplasmic granule (Valzano et al. 2016). Using Giemsa
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A. Gupta et al.
staining it was revealed that W. anomalus F17.12 strain’s Killer Toxins strategy is to
hinder the mature ookinete development in the Plasmodium’s sporogonic stages
(Valzano et al. 2016).
20.4
Malaria Eradication
Eradication of malaria is a topmost priority for any developing nation, due to the
fatality that the disease poses on the health and welfare of the citizens. Malaria is a
disease that is known to cause a significant health burden on individuals (Alonso
et al. 2011) and the need for treatment is required to be promptly provided (Bartoloni
and Zammarchi 2012) otherwise if the disease is not dealt with it may showcase
some highly complicated clinical manifestations (Bartoloni and Zammarchi 2012).
Certain measures can be taken to limit the pathogenic effect of the disease.
Vitamin A supplementation seems to provide a rather positive impact on the disease;
a trial conducted on children in the malaria-endemic area of Papua New Guinea led
to a 68% decrease in parasite density in those consuming the Vitamin A supplement
(Shankar et al. 1999; Mawson 2013).
Global strategies against malaria eradication have been deployed, by the means of
rapid application of Dichloro-diphenyl-trichloroethane (DDT) to ensue interruption
in transmission around the world regardless of the geographic conditions (World
Health Organization 1956). This approach was quite successful in the beginning, but
later on, the mosquito species evolved to resist the impact of DDT, rendering it
unusable for malaria elimination. Malaria eradication through chemicals has a large
impact but constant evolution through time has made the mosquito vectors resistant
to the chemicals.
So, an alternative for chemicals was the need of the hour, which could be more
potent in the eradication of mosquitos. A much more lethal and effective substitute
could be Symbiotic control (Ricci et al. 2012b). Symbiotic Control also referred to as
symbiont-based control (Douglas 2007) is a multifaceted approach that employs
symbiotic microorganisms, to control insect pests or to render the vector incompetent (Ricci et al. 2012b). This approach was originally termed as paratransgenesis,
defined as the modification of the insect phenotype by genetic transformation of its
associated microorganisms (Ashburner 1998).
Symbiotic control of Malaria is possible by many microorganisms, like Pantoea
agglomerans (Bisi and Lampe 2011), P. berghei (Cappelli et al. 2014), etc. A proper
investigative study of these microorganisms on model organisms, like mice, can aid
us to better understand how to curb malaria.
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20.4.1 Symbiotic Control of Malaria by Mutualism Between
W. anomalus and Anopheles sp.
Numerous studies and experimental data indicate that W. anomalus is found as a
symbiont in the gut of Anopheles sp. and Aedes sp. mosquitoes, while various
species of W. anomalus are found in reproductive organs of A.stephensi and A.
gambiae (Ignatova et al. 1996; Ricci et al. 2011a, b; Crotti et al. 2012; Wang and
Jacobs-Lorena 2013).
Studies indicate that killer toxin produced, elicits antimicrobial properties which
are mediated by β-glucanase activity that targets cell wall of yeasts, protozoa, and
bacteria; moreover, it is attested to be lethal for some marine microbes as well (Sun
et al. 2012; Cappelli et al. 2019).
W. anomalus F17.12 strain is coming into the limelight due to its antimicrobial
properties, mainly for inhibition of early sporogonic developmental stages
P. berghei therefore, it is deemed as a potent symbiotic control of malaria (Cappelli
et al. 2014, 2019). W. anomalus is a quality presumption of safety status
recommended biological agent (Koutsoumanis et al. 2019) but at the same time, it
is also known to produce killer toxin which is quite ironical (Sundh and Melin 2011;
Cappelli et al. 2019).
Some notable experimental evidence that indicates how W. anomalus can be
effectively used against malaria in the form of Symbiotic control
• The killer toxin produced by W. anomalus F17.12 strain elicited good response
against sporogonic stages of P. berghei (Cappelli et al. 2019).
• The smell produced by yeast’s fermentation acted as a bait for mosquitoes
(St Laurent et al. 2016; Cappelli et al. 2019).
• When tested on Murine cell lines, the killer toxin produced by W. anomalus
F17.12 strains exhibited no harm to vertebrate cells (Cappelli et al. 2019).
• Interaction between W. anomalus F17.12 strain and carbohydrates of the parasite
cell wall is more productive in the lumen of the midgut in mosquitoes (Cappelli
et al. 2019).
An important point to be noted here is, as per Cappelli and colleagues suggestion
W. anomalus can be easily released in mosquito feeding sites, which implies that it
can be ingested by mosquitoes and get into their gut (Cappelli et al. 2019).
20.4.2 Symbiotic Control of Malaria by Other Mutualistic
Examples
Many methods of symbiotic control for malaria have been researched and developed
in recent years. Other than W. anomalus, many other robust microorganisms are also
active components in symbiotic control of malaria. The bacteria Enterobacter
agglomerans present in the midgut of mosquitoes is known to survive in mosquito
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bodies (Ricci et al. 2012b). E. agglomerans is non-pathogenic and it is branded as an
excellent candidate for a paratransgenic malaria control strategy (Ricci et al. 2012b).
Moreover, Pantoea agglomerans, a bacterial symbiont of Anopheles mosquitoes,
has been engineered to secrete anti-Plasmodium (Bisi and Lampe 2011) effector
proteins, thus can also be a potentially useful tool for malaria paratransgenic control
(Ricci et al. 2012b). This has been demonstrated by a study that used these
engineered Pantoea agglomerans strains to see the effect it would have on Plasmodium, it gave a result of 98% inhibition of P. falciparum in vivo (Hertig and Wolbach
1924).
Densoviruses, capable of infecting and disseminating in A. gambiae, has been
proposed as a paratransgenic tool for malaria control strategies (Ren et al. 2008).
Studies describe the use of the transgenic Metarhizium anisopliae fungus to inhibit
malaria transmission, which abolishes the parasite development within the mosquito
(Fang et al. 2011).
Attempts to modify Asaia to produce strains able to express and secrete antiPlasmodium effector molecules to be used in ‘malaria transmission-blocking’ experiments are being conducted (Ricci et al. 2012b).
20.5
Conclusion
In the current world scenario, when globalization and urbanization are the topmost
priorities across the nations, people are enticed toward synthetic or chemical-based
drugs, which are quite fast and effective in treating illnesses, but their lifethreatening side-effects should not be ignored. Other than this, parasites and even
vectors get resistant to it and may cause a situation of quagmire.
To prevent this situation, we should roll toward, more natural ways to combat
deadly diseases. There was a time when chloroquine and DDT were the most
effective weapon for combating malaria worldwide but after a few years, both the
parasite and mosquito vector got resistant to it. Therefore, an alternative against
malaria could be W. anomalus, a mutualistic yeast which lives in mutual harmony
inside the Anopheles mosquitoes has unveiled a strong anti-plasmodial activity on
P. berghei’s sporogonic stages and from gametocyte to ookinete stages via the
production of killer toxin.
In our suggestion, spraying of W. anomalus in water bodies and environments
where the mosquito resides can be beneficial. It could be a wise alternative because it
can survive in a highly intolerable environment where many other microbes cannot;
and as it is a fermenting yeast the smell produced by it, can act as a bait for the
mosquitoes. Furthermore, when it comes to safety, W. anomalus is a Biosafety
level—1 microorganism, which can kill other yeasts and fungi but not impose any
harmful effects on human beings. Due to this, it has been thought to be used as a
biocontrol agent in agro-food industries. It has already been found in many organisms such as Drosophila sp., and crab Portunus trituberculatus where it showed no
harm and rather killed the pathogen present in them. When Killer toxin was tested on
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murine cell lines, it exhibited no harm to vertebrate cells. Also, W. anomalus was
found to be transmitted vertically in male as well as female mosquitoes of all ages. In
our opinion, these reasons are more than satisfactory for W. anomalus to be sprayed
in water bodies and environments where the mosquito resides to control malarial
infections worldwide.
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Part IV
Microbial Symbiosis in Disease and Stress
Management
Chapter 21
Halophyte–Endophyte Interactions:
Linking Microbiome Community
Distribution and Functionality to Salinity
Bliss Ursula Furtado and Katarzyna Hrynkiewicz
Abstract Many plants are unable to adapt to rapid environmental changes (e.g.,
salinity, drought, or limited nutrients) and may acquire assistance from microbes that
have the capacity to increase tolerance of host-plants in stress conditions. By having
the right microbes, the plants are more resilient! Such microbes include endophytes
that inhabit inner tissues of the plant without causing symptoms of disease in their
host. However, this plant–endophytic association exists only when chemical equilibrium is maintained between both, therefore making this mutualistic interaction
even more unique. Therefore it is interesting to decode the endophytic community
composition in halophytes specifically in the most salt-tolerant halophyte species
Salicornia europaea, and further determine the factors that could affect this association. Moreover, understanding the endophytes potential plant growth-promoting
activities in association with host (S. europaea) and non-host plant (non-halophytes)
are the focus of this chapter.
Keywords Salicornia europaea · Non-mycorrhizal plant · Salt tolerance · Bacteria ·
Fungi · Proteobacteria · Ascomycota · Soil salinity · Culture dependent ·
Metagenomic · Enzyme activity
21.1
Introduction
Soil salinity is among the major abiotic stresses affecting crop production today. It is
caused due to climate change, low precipitation, high surface evaporation,
weathering of native rocks, improper irrigation management on landscapes, salts
used for deicing roads, and poor agricultural practices (Litalien and Zeeb 2019). Soil
salinization is characterized by a high concentration of soluble salts, when the
B. U. Furtado · K. Hrynkiewicz (*)
Department of Microbiology, Faculty of Biological and Veterinary Science, Nicolaus
Copernicus University, Torun, Poland
e-mail: hrynk@umk.pl
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_21
363
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B. U. Furtado and K. Hrynkiewicz
1. Organ level:
• Salt compartmentation in organs
• Salt excretion
• Leaf drop or senescence
• Salt glands or bladders
• Succulence
• Stomatal density
2. Cellular level
• Compartmentation of Na+ and C1• Synthesis of compatible solutes
• Na+/ K+ replacement
Halophytes
3. Molecular level
• Detoxification mechanisms
• Synthesis of osmolytes
Glycophytes
Fig. 21.1 Response of halophytic and glycophytic plants under salinity stress at the (1) organ,
(2) cellular, and (3) molecular levels. Halophytes possess all of the mechanisms to combat salinity
stress while glycophytes have minimal tolerance to stress via organ and cellular responses only and
no mechanisms at the molecular level
electrical conductivity (ECe) in the root zone exceeds 4 dS m 1 (approximately
40 mM NaCl) at 25 C and has exchangeable sodium of 15% and osmotic pressure of
approximately 0.2 MPa (reviewed by Munns et al. 2020). The yield of most crop
plants is reduced at this ECe, though many crops exhibit yield reduction at lower ECe
(reviewed by Munns et al. 2020). Salt stress damages various physiological and
metabolic processes in plants by inducing osmotic stress and increasing ion toxicity,
nutrient deficiency, membrane disruption, and inhibition of metabolic activities, as
well as changes in gene expression (Shahzad et al. 2019).
Nevertheless, there are some plants that show no evidence of inhibition in
extreme salinity, and normally exhibit a pronounced salt requirement for optimal
plant growth (Nikalje et al. 2019). These plants are called halophytes, which are
representative vegetation of saline habitats ranging from coastal regions, salt
marshes and mudflats, saline depressions, inland deserts and sand dunes and rocky
coasts (Nikalje et al. 2019). Most of the halophytes belong to family Amaranthaceae,
Plumbaginaceae, Plantaginaceae, Aizoaceae, Poaceae, Brassicaceae, with the
Chenopodiaceae being dominant (Slama et al. 2015). The database “eHALOPH”
currently identified more than 1500 halophyte species reported from different parts
of the world. Clearly, halophytes have evolved a number of adaptive traits expressed
at various levels of organization (Fig. 21.1) that includes adjustment of their internal
water relations through salt exclusion, succulence, salt-secreting glands and bladders, ion compartmentation in cell vacuoles and accumulation of compatible organic
solutes and are distinguishable from glycophytes (salt-sensitive plants) (reviewed by
Fan 2020).
It is inevitable that no organism thrives alone, which questions the probable
existence and role of the closely associated microbiome present in halophytes and
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Halophyte–Endophyte Interactions: Linking Microbiome Community Distribution. . .
365
their contribution toward the halophytes’ high salt tolerance ability. Endophytic
microorganisms ubiquitously occur in all plant species and are important components of the biological diversity. The term “endophyte” can be defined as all
organisms that at some point in their life cycle colonize internal plant tissues without
causing harm or symptoms of disease in their plant host (Petrini 1991). These
include bacteria, actinomycetes, or fungi that colonize the internal plant tissues.
Endophytes are particularly fascinating because of their multifaceted lifestyle, i.e.,
they may exist as either free-living soil microbes or saprobes or pathogens (Tadych
et al. 2009). They exhibit variations in their mechanisms of transmission from one
plant host to another including strict vertical transmission from maternal plants to
seeds, infectious transmission from one host plant to another, or infection by spores
from environmental sources like wind, rain, soil, and leaf litter (Tadych et al. 2009).
Endophytes are known to share a symbiotic relationship with their plant host,
wherein they use the internal environment of the plant as an ecological niche, and
in return protect their host from the negative effects of the adversely changing
environment (Petrini 1991). Alternatively, some endophytic species could be neutral
and do not offer any benefits or neither harm their hosts (Backman and Sikora 2008).
With this background, this chapter discusses on the diversity of endophytic bacterial
and fungal communities in halophyte Salicornia europaea found in different salinity
environments. It is a well-known fact, that the true endophyte state in the host plant is
established only when the chemical equilibrium between the host and microbe is
achieved during their long-term association. During this process of endophyte
stabilization in host plant, many environmental factors such as abiotic (e.g., temperature, salinity, soil composition) and biotic (e.g., competition with microbes already
present in the host) could alter or redefine the metabolic capacity of endophytes.
Hence, we assess on the possibility of these endophytes possessing beneficial traits
or properties related to plant growth promotion or stress tolerance in their host
further on their resilience when inoculated in other non-host plants. Further, we
underlie the importance of understanding the endophytic microbiome in halophytes
which will likely pave a new avenue in engineering endophyte-mediated stress
tolerance in plants.
21.2
Salicornia a Potential Halophytic Crop Plant
The halophyte chosen for this discussion, i.e., Salicornia europaea
L. (Amaranthaceae) is a non-mycorrhizal plant (Sonjak et al. 2009) and one of the
most salt-accumulating halophyte known. It is a “pioneering plant” in both coastal
and inland saline sites, which has generated significant interest as a multi-purpose
halophyte (Nikalje et al. 2018). S. europaea grows up to a height of 35 cm and is
fairly richly branched. They are dark green becoming yellow-green and ultimately
flushed pink or red (Ventura and Sagi 2013). They are succulent annuals with
extremely reduced leaves and a spike-like terminal inflorescence (Ventura and
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B. U. Furtado and K. Hrynkiewicz
Sagi 2013). This genus is widely dispersed in Eurasia, North America, and
South Africa, presently comprises around 25–30 species (Ventura and Sagi 2013).
S. europaea is a crop with great commercial value and ecological importance. It is
suitable for cultivation as a vegetable in highly saline environments (Ventura and
Sagi 2013) and as a source of valuable secondary compounds (Singh et al. 2014).
Salicornia spp. has been successfully grown in aquaculture systems (Singh et al.
2014). As a salt-accumulating halophyte, as much as 50% of the dry weight of
S. europaea may be salt ions (Ushakova et al. 2005; Furtado et al. 2019a). This was
also observed in the results of Furtado et al. (2019a) where high accumulation of Na+
in shoots, whereas K+ and Ca2+ levels were higher in roots throughout the sampling
period (spring and autumn) in all S. europaea samples collected from the two saltaffected sites. Therefore, this species is promising for soil desalination, which is
required for the development of agriculture on salty soils and beaches. Moreover,
research on halophyte plants is of particular interest today not only for their high salt
tolerance and agronomic value but also because it is a non-mycorrhizal plant. This
fact makes it interesting to discuss their associated microorganisms (endophytes)
that could compensate for the missing symbiotic protection and increase plant
growth and fitness particularly under unfavorable conditions. S. europaea is especially known to produce secondary compounds (e.g., alkaloids, triterpenoid saponins, and flavonoids, among others) (Isca et al. 2014). Many of these compounds are
used as energy sources by endophytic bacteria and fungi suggesting that fluctuations
in the proportions and type of compounds during the plant development play an
active role in selection for specific bacteria and/or fungi.
21.3
Biodiversity of Endophytic Microbiome in S. europaea
The endophytic community in halophytes may be different from those in other plants
because salinity acts as an environmental filter. Thus, allowing the survival of
selected taxa that can withstand extreme environments. To date, endophyte community diversity investigated in S. europaea have been analyzed via cultureindependent approaches, e.g., sequencing of the 16S rRNA gene and/or the internal
transcribed spacer regions (ITS1 and ITS2), 454 pyrosequencing (e.g., Shi et al.
2015; Szymańska et al. 2016; Zhao et al. 2016a; Szymańska et al. 2018; Yamamoto
et al. 2018; Hrynkiewicz et al. 2019; Furtado et al. 2019a) or culture-dependent
approaches involving procedures for surface sterilization of plant tissue followed by
fragmentation and culture of the fragments onto specific agar plates (e.g., Potato
Dextrose Agar, R2A media, Czapek dox agar, etc.) amended with antibiotics (e.g.,
tetracycline, Nystatin, etc.) (e.g., You et al. 2014; Okane and Nakagiri 2015; Park
et al. 2016; Zhao et al. 2016b; Furtado et al. 2019b).
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367
21.3.1 Salicornia Bacterial Diversity
Prokaryotic endophytes are considered to be diverse comprising of phyla bacteria
and a small portion of Archaea. Overall, the bacterial endophytes are found across
many phyla, generally the most abundant community is Proteobacteria (group of
Gram-negative bacteria), including α, β, and γ-Proteobacteria classes followed by
Firmicutes, Actinobacteria, and Bacteriodetes. Using the culture-independent
approach, the bacterial community in the rhizosphere soil and endophytes of
S. europaea growing at the Fukang Desert Ecosystem Observation and Experimental
Station (FDEOES) in Xinjiang Province, China were investigated (Shi et al. 2015).
Proteobacteria was most dominant phylum in all the samples followed by other large
phyla Firmicutes, Bacteroidetes, and Actinobacteria. This study is consistent with
results obtained in Fukang, China (Zhao et al. 2016a), Ciechocinek and Inowroclaw,
Poland (Szymańska et al. 2018), Lake Notoro in the eastern part of Hokkaido, Japan
(Yamamoto et al. 2018), Ciechocinek and Inowroclaw, Poland (Furtado et al.
2019a). Szymańska et al. (2018) suggested that the endophytes representing phyla
Proteobacteria and Bacteroidetes predominate in saline environments regardless of
the level of salinity in the root zone soil and plant roots. It was also found that
bacteria representing Actinobacteria exhibited lower tolerance to salt stress and this
phylum exists more frequently in environments with lower levels of salinity (e.g.,
Shi et al. 2015; Szymańska et al. 2018; Yamamoto et al. 2018).
This is contrary to the results obtained via culture-dependent methods in which
phylum Firmicutes and Actinobacteria dominate in many reports. Zhao et al. (2016b)
isolated endophytic bacteria of S. europaea growing at Gurbantünggüt Desert,
China. These isolates belonged to phylum Firmicutes and Actinobacteria with
13 different bacterial genera Arthrobacter, Bacillus, Brachybacterium,
Brevibacterium, Glycomyces, Isoptericola, Kocuria, Mesorhizobium, Pseudomonas,
Phyllobacterium, Planococcus, Streptomyces, and Variovorax. Another showed
Gram-positive bacteria (phylum Firmicutes and Actinobacteria) dominating in all
experimental variants including genera Bacillus sp., Streptomyces sp., and
Microbacterium sp. (Szymańska et al. 2016).
Some microbes are ubiquitous and can exist as free-living soil microbes, as
epiphytes or as endophytes. For instance, Bacillus sp., Salinicola sp., Serratia sp.,
Streptomyces sp., Microbacterium sp. and Rhodococcus sp. were reported as endophytes in S. europaea shoot and roots collected from two sites in Poland (Furtado
et al. 2019b) and similar bacterial diversity was previously obtained in the rhizosphere samples at the same investigated sites (Szymańska et al. 2016). Although
another study showed no significant differences in bacterial diversity and richness
between the bulk soil, rhizosphere, and the root endosphere for S. europaea but the
differences were observed at the genera level with the most abundant root endophytes including Sulfurimonas, Coleofasciculus, and Aestuariispira while,
Roseovarius, and Halochromatium were highly abundant in the rhizosphere and
seven genera were dominant the S. europaea bulk soil: Thiogranum, SEEP-SRB1,
Caldithrix, Ignavibacterium, Sva008 sediment group, Candidatus Thiobios, and
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Spirochaeta (Yamamoto et al. 2018). Overall, the bacterial endophytic community in
S. europaea is much greater in comparison to the fungal community according to
results obtained by Szymańska et al. (2016) and Furtado et al. (2019b).
21.3.2 Salicornia Fungal Diversity
Although the endophytic microbiome may comprise a small portion of fungi (compared to bacteria) their contribution can be essential in the plants development.
Notably, class 2 and class 4 fungal endophytes are commonly found in halophytes,
are capable of extensive tissue colonization and are relevant for plant survival in
stress habitats (Flowers and Colmer 2015). Moreover, the class 2 fungal endophytes
can establish habitat-adapted symbiosis and confer specific stress tolerance to their
host and non-host plants in extreme habitats (Flowers and Colmer 2015).
Furtado et al. (2019a) performed metagenomic analysis of S. europaea shoots and
roots obtained from two different high-salinity environments in Poland and showed
that 95% of the fungal reads belonged to phyla Ascomycota. In the next study, the
culturable endophytic fungal diversity was investigated by Furtado et al. (2019a)
which isolated 320 fungal strains mainly represented phylum Ascomycota (96% of
the isolates) from the roots and shoots of S. europaea in the same sites. The
endophytic fungal strains mainly consisted of the orders: Pleosporales (dominated
by Alternaria sp. and Stemphylium sp.), Eurotiales (mainly Aspergillus sp. and
Penicillium sp.) and Hypocreales (only Fusarium sp. and Trichoderma sp.). The
remaining genera represented the order Dothideales, Incertae sedis, Capnodiales,
Sordariales, Botryosphaeriales, and Chaetothyriales. Okane and Nakagiri (2015)
found Pleospora sp. and Alternaria alternata were the major endophytes of
S. europaea roots in the eastern Hokkaido and the Seto Inland Sea (Setouchi) regions
in Japan. Endophytic fungal isolates belonging to 9 genera: Aspergillus, Penicillium,
and Fusarium were dominantly distributed genera in roots of S. europaea native to
saltern of the Korea, followed by Aureobasidium, Cladosporium, Gibberella,
Macrophoma, Phoma, Stemphylium, and unidentified (Pleosporales), respectively
(Park et al. 2016). Alike, Booth et al. (1988) reported a high frequency of Alternaria
species isolated from S. europaea in southern Manitoba and Saskatchewan, Canada.
Furthermore, the dominance of Alternaria sp. found in S. europaea was also
previously reported in Canada (Muhsin and Booth 1987), South Korea (You et al.
2014), Japan (Okane and Nakagiri 2015) and Poland (Furtado et al. 2019b). To date,
most of the fungal strains (e.g., Epicoccum sp., Alternaria sp., Phoma sp., Fusarium
sp., Cladosporium sp., Penicillium sp., Acremonium sp., Lewia sp., Pleosporales
sp., Stemphylium sp. and Aspergillus, etc.) reported in S. europaea belong to a group
of common plant pathogens and saprobes found in other plants (Okane and Nakagiri
2015; Park et al. 2016; Furtado et al. 2019b). However, colonization of these
endophytes in healthy halophyte plant tissue indicates they are not pathogenic and
we propose that these fungi may have “co-evolved” in their host. Few studies have
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369
discussed the abilities of fungi to switch lifestyles between the endophyte-pathogen
(Hyde and Soytong 2008) and endophyte-saprotroph (Promputtha et al. 2010).
In general, very few endophytic fungal strains belonging to the Phylum
Basidiomycota have been reported in S. europaea, e.g., a study showed 4% of the
isolated diversity were categorized in Basidiomycota into four orders; Polyporales
(most representative genus being Peniophora) and the other strains corresponded to
Russulales, Agaricales, and Cantharellales (Furtado et al. 2019b). These findings
were parallel to studies using culture-independent techniques (e.g., Furtado et al.
2019a).
21.4
Factors Shaping S. europaea–Endophyte Association
The halophyte–endophyte association is dependent on many factors, mainly the host
plant lifestyle (i.e., obligate halophyte S. europaea in this discussion) and soil
characteristics (e.g., salinity) can have a strong effect on the rate of endophyte
colonization (Szymańska et al. 2014; Shi et al. 2015; Zhao et al. 2016a; Szymańska
et al. 2018; Furtado et al. 2019a, b). Most endophytic communities are strongly
influenced by the soil micro-ecological environment as endophytes are recruited
from the soil which may act as a filter for microbial species (e.g., Szymańska et al.
2014; Shi et al. 2015). Research has shown that plants growing in soils with high salt
content harbor endophytic assemblages that differ significantly from those in other
environments. Two saline sites characterized by different salinity and ion composition (anthropogenic salinity site: lower ECe with Ca2+ and natural salinity site:
higher ECe with Na+) showed the greatest abundance of bacteria in the rhizosphere
samples than that observed in plant roots (Szymańska et al. 2016). They found
majority of rhizosphere bacteria belonged to Firmicutes however; this proportion
was lower compared to the endophytes (consisted of 50% Firmicutes). This is in line
with previous observations by Shi et al. (2015) where a higher total bacterial biomass
was measured in soil, followed by roots of S. europaea and the bacterial diversity in
the endosphere of S. europaea was lower than that in the rhizosphere of S. europaea
(Shi et al. 2015).
In a second survey, Szymańska et al. (2018) analyzed the endophytic bacterial
community in S. europaea roots from the same test sites using metagenomic
approaches. The results revealed that the higher levels of soil salinity did not reduce
the composition of endophytic bacterial diversity in roots. However, a distinct
taxonomic composition was observed at the two sites which were attributed to the
distinct adaptation of halotolerant microorganisms. This was also found in the
culturable diversity study where the highest abundance of bacterial endophytes
was isolated from the natural saline site compared to the anthropogenic site and
frequency of isolation in the culture medium increased with increasing NaCl concentrations (Szymańska et al. 2016). Thus, indicating the presence of a significant
number of halophilic bacteria known to survive at high salt concentrations. This
difference indicates that the local environment has a complex effect on the bacterial
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community. Supporting this view, a clear influence of soil properties on the existence of unique bacterial and fungal endophytes of S. europaea, specific to the
geographical regions in different countries, such as Japan (Okane and Nakagiri
2015), Canada (Muhsin and Booth 1987), China (Zhao et al. 2016a, b), South
Korea (You et al. 2014) and Poland (Furtado et al. 2019a, b) was demonstrated.
The importance of acclimatization time in adaptation of microorganisms to
salinity was emphasized by Szymańska et al. (2016, 2018) and Furtado et al.
(2019a, b) when they found that the site exposed to salinity for a long time had a
greater diversity of endophytic bacteria and fungi in native S. europaea. They
propose that time necessary for shaping the bacterial community at the saline sites
was longer in the naturally saline area which existed much longer as compared to the
anthropogenic salinity area. A study by Furtado et al. (2019a) investigated the
composition of endophytic community in S. europaea and observed some bacteria
were specific in some samples. Bacterial class Sphingobacteriia were found only in
the high-salinity site, while Epsilonproteobacteria were characteristic for the site
with lower salinity. Some endophytic genera: Halomonas, Levinella, Vibrio,
Pseudoalteromonas, and Leuweenhoekiella were found exclusively at highsalinity site.
Yamamoto et al. (2018) studied the bacterial diversity and community structure
of rhizosphere, root endosphere, and bulk control soil samples in two halophytic
plants: Glaux maritima and Salicornia europaea. Among the G. maritima samples,
the richness and diversity of bacteria in the rhizosphere were higher than in the root
endosphere but were lower in comparison to the bulk soil. In contrast to S. europaea,
the bulk soil, the rhizosphere, and the root endosphere had similar bacterial richness
and diversity (Yamamoto et al. 2018). Another study by Shi et al. (2015) showed the
diversity of bacteria was abundant in the rhizosphere soil, while the endophytic
diversity was poor in S. europaea samples. In conclusion, the variation in endophyte
frequencies could be due to differences in host preference rather than environmental
factors as the halophyte hosts in the abovementioned studies were at the same
location and were investigated simultaneously. This also confirms high endophyte
species specificity of S. europaea, which depends on the halophytic plant species
and the sampling site.
Previous data revealed that bacterial communities in the rhizosphere exhibit
greater richness than endophytes in the organs of halophytes. In addition, Momonoki
and Kamimura (1994) reported that during the growth period of wild S. europaea
found around Lake Notoro, the pH and osmotic pressure of the plants increased from
7.6 to 8.8, and from 650 to 2000–2600 mOsm/kg (1 mOsm/kg ¼ 17.02 mm Hg),
respectively. This fact is clear that the internal environments of halophytes such as
S. europaea are likely to be stressful to microbes. Much of the endophytic diversity
research is limited to the roots of S. europaea. However, some studies have indicated
plant organ specificity among endophytes in S. europaea, where different bacterial
and fungal species inhabited tissues and a few species were overlapping between
shoots and roots (Furtado et al. 2019a). At the family level, Halomonadaceae were
much more abundant in shoots, whereas Alteromonadaceae, Cellvibrionaceae,
Flammeovirgaceae, Rhodobacteraceae, and Saccharospirillaceae were characteristic
21
Halophyte–Endophyte Interactions: Linking Microbiome Community Distribution. . .
371
for roots. Kushneria sp. was abundant genus in shoots while Saccharospirillum was
significantly more common in the roots. In case of fungi, roots at the high-salinity
soils were strongly dominated by Pleosporaceae. Family Leptosphaeriaceae,
Teratosphaeriaceae, and Didymosphaeriaceae were found exclusively in shoots,
and Paradendryphiella arenariae was the only species present in all sample types
(Furtado et al. 2019a). Moreover, Paradendryphiella arenariae (Nicot)
Woudenberg and Crous, was previously found in S. europaea in Hokkaido
(Okane and Nakagiri 2015) and in Canada (as Dendryphiella arenariae Nicot)
(Booth et al. 1988). In general, the endophytic community (both bacteria and
fungi) in S. europaea shoots was found to be less diverse than in roots
(S. europaea). One of the reasons being that the endophyte colonization is dependent
on the plant host, that is, imposed by a large salt concentration in this organ
(Salicornia shoot accumulate salts). Secondly, these differences in endophytic
assemblages in different tissue types might be the preferences of individual dominating taxa and may reflect on their capacity for utilizing or surviving within a
specific substrate.
Zhao et al. (2016a) observed a marked difference in endophytic bacterial communities from different stages of plant growth. The richest endophytic bacteria
diversity of S. europaea was detected at the seedling stage, and thereafter, variety
of endophytic bacteria declined during flower and fruit setting stage. Phylum
Gammaproteobacteria increased during the growing period while Betaproteobacteria
decreased. Five genera Serpens, Halomonas, Pseudomonas, Azomonas, and
Pantoea were observed during all growth phases which were suggested as the
core-microbiome of S. europaea. The fungal diversity in S. europaea reported by
Furtado et al. (2019b) changed from one growing season to another. The fungal
strain from two investigated sites was more diverse (based on genera) in spring
(young plants) than in autumn (mature plants) sampling. Certain genera (e.g.,
Aureobasidium, Cladosporium, Epicoccum, and Talaromyces) occurred only in
the autumn, while Neocamarosporium, Ascochyta, and Acremonium in spring. The
ease of colonization in the young host plant stage and the microorganism’s potency
to compete for survival in their host may account for this effect.
21.5
S. europaea as a Reservoir of Specialized Endophytic
Diversity
Soil salinity not only affects microbial community composition and abundance but
also affects microbial functions, i.e., enzymatic and metabolic processes. High
salinity can reduce the level of respiration, biomass, and activity of microorganisms
(Szymańska et al. 2014). For example, the activity of endophytes was found to be
higher in sites with lower salinity than at the site with high salinity. Further the
endophytes isolated from different samples (different sites and plant organ) although
in the same genera Epiccocum sp., Arthrinium sp., and Trichoderma sp. displayed
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different metabolic activities (Furtado et al. 2019b). Moreover, different microbial
species have specific salt requirements and possess varying tolerance levels. Based
on their ability to grow in the saline environments, microbes can be grouped as
halotolerant (tolerate up to 25% NaCl) and halophilic (require salt for growth)
(Sultanpuram and Mothe 2019). Bacterial and fungal endophytes of S. europaea
are classified halotolerant as experimentally tested by Szymańska et al. (2016, 2018)
and Furtado et al. (2019b). The halotolerant microbiome can positively affect the
halophyte by providing nutrients (e.g., atmospheric nitrogen fixation, phosphorus
solubilization), producing hormones (e.g., 1-aminocyclopropane-1-carboxylic acid
(ACC) deaminase and indole-3-acetic acid (IAA)), regulating antioxidant response,
synthesizing exopolysaccharides (EPS), maintaining plant defense against biotic
stress (e.g., production of antibiotics, competition with pathogens for nutrients,
and induction of systemic resistance), accumulating organic solutes such as proline
and betaine and increasing soil aggregation (Zhao et al. 2016b; Szymańska et al.
2014, 2016, 2018; Piernik et al. 2017; Hrynkiewicz et al. 2019; Furtado et al.
2019b).
Secretion of phytohormones, particularly indole-3-acetic acid (IAA), increases
plant salt tolerance by stimulating root proliferation. IAA-secreting endophytes have
been isolated from S. europaea, e.g., Szymańska et al. (2016) observed a higher
frequency of endophytic bacteria Serratia marcescens, Kushneria marisflavi,
Microbacterium sp., Hymenobacter psychrotolerans capable of IAA synthesis as
compared to rhizosphere bacteria reflecting on direct interaction between the
S. europaea and endophytes. The endophytic fungi from the roots of S. europaea
synthesized more IAA compare to endophytes from the shoots (Furtado et al.
2019b).
Bacterial ACC deaminase (1-aminocyclopropane-1-carboxylic acid (ACC)deaminase) reduces plant ethylene levels and involved in nitrogen fixation, both
are considered important mechanisms in bacteria that can promote plant growth,
especially under stress conditions (del Carmen Orozco-Mosqueda et al. 2020). ACC
deaminase-producing root endophytes isolated from S. europaea: Pseudomonas
sp. I-S-E-12 and Rhodococcus erythropolis I-S-E-16 strains showed the presence
of a gene encoding dinitrogenase reductase (Szymańska et al. 2016). Similarly,
S. europaea endophytes with ACC deaminase activity belonging to the genera
Arthrobacter, Bacillus, Planococcus, and Variovorax enhanced in vitro
S. europaea seedlings growth in the presence of increasing concentrations of
NaCl. In addition, these strains were also able to solubilize phosphate and produce
IAA, which stimulated seed germination and increased the rate of root development
(Zhao et al. 2016b).
Plant growth-promoting endophytes are able to release siderophores that are
involved in biocontrol mechanisms thus preventing the growth of pathogenic microorganisms in plant hosts (Johnson 2008). High synthesis of siderophores was
obtained for bacteria Streptomyces sp., S. griseoplanus, and Bacillus thuringiensis
(Szymańska et al. 2016). Siderophores production by endophytic fungi was the most
active function reported in S. europaea (Furtado et al. 2019b).
Hrynkiewicz et al. (2019) analyzed the endophytic diazotrophs of S. europaea
which represented a broad range of N2-fixing bacteria with Actinobacteria
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Halophyte–Endophyte Interactions: Linking Microbiome Community Distribution. . .
373
dominating at the site characterized by higher salinity, and Proteobacteria at lower
salinity. Most of the isolates from Actinobacteria belonged to the genus
Curtobacterium (Curtobacterium sp., C. flaccumfaciens, C. herbarum) and
Microbacterium (Microbacterium sp., M. kitamiense, M. oxydans), and some of
them to Rhodococcus, Mycobacterium, Cellulomonas, Sanguibacter, Clavibacter,
Cryocola/Labedella, Frigoribacterium, Agreia, Herbiconiux, and Plantibacter. The
presence of the nifH gene was also identified in S. europaea endophytic bacteria:
Pseudomonas sp. ISE12 and R. erythropolis ISE16 (Szymańska et al. 2016).
Phosphate solubilizers, e.g., Bacillus endophyticus, B. tequilensis, Planococcus
rifietoensis, Variovorax paradoxus, and Arthrobacter agilis were identified in
S. europaea roots (Zhao et al. 2016b). No activity for phosphate solubilization
was observed in fungal endophytes of S. europaea (Furtado et al. 2019b).
All fungi possess pathways to biosynthesize polyamines, which are important in
restoring cellular homeostasis under stressful conditions (Nikolaou et al. 2009).
Furtado et al. (2019b) investigated the fungal culturable diversity and found nearly
all of the strains possessed the ability to produce polyamines (90% of isolated strains
from shoots and 83% from roots). However, the strains isolated from the S. europaea
shoots actively produced polyamines that were correlated with the hyperaccumulation of salts in Salicornia shoots, which can be more stressful for fungal
colonization.
The sulfur-oxidizing genera Sulfurimonas and Halochromatium were significantly abundant in the root endosphere and rhizosphere in S. europaea (Yamamoto
et al. 2018). According to a previous report, Sulfurimonas is involved in host
detoxification by oxidizing sulfide and producing sulfate as an end product,
suggesting that the accumulation of these bacteria around the rhizosphere might be
critical for the host tolerance of coastal environments (Fahimipour et al. 2017).
Most of the endophytic fungal strains isolated from two saline sites in Poland
displayed proteolytic, lipolytic, and chitinolytic activity (Furtado et al. 2019b). On
comparing the two sites, the fungal strains obtained from higher salinity site possessed higher cellulolytic, proteolytic, and amylolytic activities. While the strains
isolated from lower salinity sites possessed proteolytic, lipolytic, and chitinolytic
activities. Fungal strains Aureobasidium pullulans and Sarocladium sp. displayed
high cellulolytic activity (Furtado et al. 2019a, b). Endophytic bacteria Bacillus
baekryungensis, Thalassospira permensis, and Xanthomonadales sp. from the same
sites showed high activity for hydrolysis of cellulose (Szymańska et al. 2016). High
proteolytic activity was exhibited by few fungal strains, e.g., Sarocladium sp.,
Stereum gausapatum, Epicoccum nigrum, Epicoccum sp., Porostereum spadiceum,
and Stemphylium sp. (Furtado et al. 2019b). Endophtyic fungi belonging to
Aureobasidium sp. tested positive for all the enzymatic activities, while some strains
were negative for most of the activities, e.g., Coprinellus domesticus, C. ellisii, and
Arthrinium arundinis (Furtado et al. 2019b).
Many pigmented spore- and/or mycelia-forming fungi black fungi or
dematiaceous fungi such as the genera Alternaria, Phoma, Cladosporium, Lewia,
Pleospora, Epicoccum, Stemphylium, Ascochyta, Plenodomus, Neocamarosporium,
Dematiopleospora, Aspergillus, Penicillium, Eurotium, Talaromyces, Fusarium,
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and Aureobasidium are frequently isolated from S. europaea in salt marsh and desert
areas (Okane and Nakagiri 2015; Furtado et al. 2019b). This suggests that pigmented
dematiaceous fungi universally inhabit this halophyte and can play an important
ecological role in plant stress resistance as they possess some protective substances
and are capable of tolerating extreme temperatures, desiccation, and saline environments (Gostinčar et al. 2009).
Endophytes can have myriad effects on host plant fitness, with the outcome of
interactions ranging from beneficial to antagonistic. They exhibit a range of symbiotic relationships with their host plant and are well known to contribute to plant
fitness, which helps the host to better adapt in stress conditions (Gopi and
Jayaprakashvel 2017; Hrynkiewicz et al. 2019; del Carmen Orozco-Mosqueda
et al. 2020). Therefore, the application of endophytes in crop productivity has gained
importance today. Few studies on the compatibility and role of S. europaea endophytes have been established experimentally. For instance, five S. europaea strains,
namely Bacillus endophyticus, Bacillus tequilensis, Planococcus rifietoensis,
Variovorax paradoxus, and Arthrobacter agilis inoculated in S. europaea seeds
significantly enhanced seed germination percentage, seedling growth, shoot and
root length under salt stress condition (approx. 500 mM NaCl) (Zhao et al.
2016b). A pot experiment by Piernik and co-workers (2017) provided evidence
that endophytic bacteria (Xanthomonadales sp. CSE-34 and Pseudomonas
sp. ISE-12) isolated from S. europaea shoot and root stimulated the growth of
Beta vulgaris (cv. Zentaur poly) under salinity conditions (approx. 300 mM
NaCl). Szymańska et al. (2019) showed that halotolerant plant growth-promoting
endophytic bacteria may have a beneficial effect on the growth and development of
Brassica napus L. cultivated under salt stress conditions. Psuedomonas stutzeri
ISE12 stimulated the elongation of roots, hypocotyls, and stems of B. napus and
decreased the level of oxidative damage to cellular membranes exposed to salt stress.
Another study re-inoculated Staphylococcus sp. (isolated from S. europaea) in
S. europaea seedlings and found that this strain promoted plant growth and alleviated the negative effects of salinity at 200 mM NaCl in comparison to the
non-inoculated plants (Komaresofla et al. 2019). Further, fungal endophytes isolated
from S. europaea have also demonstrated plant growth-promoting effects in Lolium
perenne (Furtado et al. 2019b). These fungal strains were selected based on specific
properties (positive metabolic activity for IAA, siderophores, polyamines, and
enzyme activity for cellulase, protease).
21.6
Conclusions
Through this chapter, we provide an overview of the community diversity and role of
the closely associated microbiome, i.e., the endophytes present in halophyte
S. europaea. The most abundant endophytic community among bacteria is the
phylum Proteobacteria, while the majority of the fungal endophytes mainly belong
to Ascomycota. Some of the factors responsible for shaping the endophytic diversity
21
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375
in S. europaea include soil salinity, acclimatization time for microbes, halophytic
plant species, plant organ, and stages of plant growth. The halophytic plant host, e.g.,
S. europaea, filters the specific microbiome. The saline soil harbors a higher
microbial diversity in comparison to the endophytic diversity in roots (closer to
the soil and the main entry point for endophytes), while the diversity in shoots is very
low (the shoot accumulates salt that can be detrimental to the endophytes). Overall,
we can infer that increasing salinization may eventually disturb the plant–endophytic
association, regardless of the plant host having robust mechanisms to cope with salt
stress. Moreover, much of the research on S. europaea endophytic microbiome
found they are mainly halotolerant and possess traits that contribute toward the
host and non-host plant growth and salt tolerance. Application of these endophytes
for commercial use requires optimization of endophyte inocula, as questions on their
association in natural field conditions, the effect of climate change, and the microbial
diversity present in the new site that may affect the endophyte functioning in new
host needs investigation.
Conflict of Interests The authors of this chapter declare no conflict of interests.
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Chapter 22
Root Endophytic Microbes and Their
Potential Applications in Crop Disease
Management
Alka Tripathi, Ajit Varma, and Swati Tripathi
Abstract As the increasing incidence of plant yield loss due to pathogen infection
as well as abiotic stress is increasing, the need to reduce loss is required more and
more. The use of chemical pesticides, insecticide, and fertilizers does improve plant
growth and reduce the losses, but the harm posed to the biosphere cannot be
neglected and a new problem is posed through bioaccumulation and
biomagnification of these chemicals. Thus, plant growth-promoting rhizobacteria
(PGPR) and endophytes are studied more concerning their role as a bio protector.
These microorganisms alleviate the disease symptoms in many plants and minimize
the loss. Their mechanism of action has now been well-studied and their role has
been well established. The utilization of endophytes for sustainable agriculture
has been looked upon as an alternative. In this chapter, we will see how the yield
of crop plant is challenged due to pathogens and the role of endophytes in controlling
pathogens as well as growth promotion.
Keywords PGPR · Endophytes · Biological control · Sustainable agriculture · Crop
plants
22.1
Introduction
The word Agriculture is originated from Greek αγρóς, and cultūra meaning “cultivation” or “growing.” Agricultural practices are believed to be originated in SouthEast Asia and eventually reaching the far east with evolving cropping techniques
used by Sumerians and Romans like monocropping, organized irrigation, and traderelated farming (Agriculture 2019). During the middle age, North Africa and near
east regions develop better irrigation technologies based on hydraulic conductivity
and hydrostatic pressure, building dams and reservoirs, location-specific cropping
A. Tripathi · A. Varma · S. Tripathi (*)
Amity Institute of Microbial Technology, Amity University, Uttar Pradesh, Noida, India
e-mail: stripathi2@amity.edu
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_22
379
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A. Tripathi et al.
manuals, and crop-rotation also lead to increased agriculture productivity (Greatest
engineering achievements of the twentieth century 2007). Agriculture in modern
days is less dependent on human efficiency and has become more efficient with the
rapid rise of modern agricultural tools (World Agricultural Production 2020).
Global production of food grains has reached 2666.5 million metric tons on
57 million square miles arable land being utilized for agriculture (Tracking Productivity: The GAP Index 2018). The USA Department of Agriculture’s Economic
Research Service (USDA ERS) estimates that TFP growth globally has been rising
by an average annual rate of only 1.51% since 2010. According to GHI, global
agricultural productivity must increase by a rate of 1.75% to meet productivity
demand (U.S. Code 2007). For this purpose, sustainable agriculture must be incorporated in year-round agricultural practices, which can
1. satisfy human food and fiber needs
2. enhance environmental quality and the natural resource based upon which the
agricultural economy depends
3. make the most efficient use of non-renewable resources and on-farm resources
and integrate, where appropriate, natural biological cycles and controls
4. sustain the economic viability of farm operations and
5. enhance the quality of life for farmers and society as a whole (Barra-Bucarei et al.
2020). Around 375 million people in the world are vegetarians and are entirely
dependent on plant-derived nourishment, advocating the adoption of the sustainable agriculture practices.
22.2
Concerns of Economically Important Plants
Majority of world population is dependent on plant-based food products exclusively,
while the rest of the population still incorporates plant-based food in their regular
diet. Although the productivity of crop plants has been improved significantly with
crop management practices and modern mechanized tools, it is not sufficient to meet
the demand of splurging population. Biotic stress has been documented as a major
constraint for crop production worldwide (Teng and Krupa 1980) and frequency of
both biotic and abiotic stress incidences have been increasing at unprecedented scale
due to changing climate in past few decades. The direct yield loss in global
agricultural productivity caused by pathogens alone ranges from 20 to 40%
(Martínez et al. 2003) hampering gross agricultural productivity by more than
10% (Egel and Martyn 2007).
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Root Endophytic Microbes and Their Potential Applications in Crop Disease. . .
22.3
381
Crop Pathogens
Despite several morphological and biochemical barriers, which prevents invasion of
pathogen in plants, a variety of crop-specific and nonspecific pathogens cause
disease across the crop species. For example, Botrytis cinerea affects members of
the solanaceae family and this pathogen is difficult to control as it invades plants
through pruning cuts, flower, and fruit as well as it has several hosts and infection
strategies (Mittler 2002). Cucurbitaceae family is affected by a variety of fungus and
bacterial pathogen, in which major fungal diseases are Fusarium wilt caused by
Fusarium oxysporum, damping-off by Fusarium spp., Alternaria blight by
Alternaria cucumerina, powdery mildew by Erysiphe cichoracearum, anthracanose
by Colletotrichum orbiculare, downy mildew by Pseudoperonospora cubensis,
scab, or gummosis by Cladosporium cucumerinum, damping-off of seeds by
Pythium spp., charcoal rot by Macrophomina phaseolina, vine decline by
Monosporascus cannonballus, Phytophthora blight by Phytophthora capsica and
major bacterial diseases are angular leaf spot by Pseudomonas syringae
pv. lachrymans, bacterial leaf spot by Xanthomonas campestris pv. cucurbitae,
bacterial fruit blotch by Acidovorax avenae subsp. citrulli, bacterial rind necrosis
by Erwinia carnegieana (Winther and Friedman 2008). The crop plants belonging to
the family cucurbitaceae are used differently in culinary like salad, cooked food,
pickle, etc. and they have a good nutritious value but the loss of crops due to biotic
disease is alarming, Fusarium spp., alone causes a loss above 70% of the yield in
India (Tenberge 1999). Overall, pathogens affect plant fitness, reduce growth, and
competitive ability of plants (Burdon et al. 2006). Conventional approaches to
reduce disease incidences in agricultural crops include use of different set of
pesticides, which are effective to control pathogen spread and contain yield losses.
However, indiscriminate use of pesticides has emerged as a greater challenge for
ecosystem and human health. Thus, a sustainable approach is warranted to improve
crop productivity without using harmful levels of pesticides. One of the promising
alternatives that has been documented across the studies is use of certain plant
growth-promoting rhizobia (PGPR) and endophytes, which can augment plant
defense response to a broad range of pathogens as well as improve growth and
productivity of crop plants.
22.4
Endophytes and Their Mechanism of Action
The term “endophyte” is derived from “endon” meaning within, and “phyton”
meaning plant. The name was coined by De Bary in 1866 for the organism living
inside the plant body, however, the term was precisely defined in terms of their type
into bacterial, fungi and actinomyces, or algae (Li et al. 2008). Endophytes reside
inside plant tissues without damaging and causing any negative impact resulting in a
disease. By assisting in the process like nitrogen fixation, phosphate solubilization,
chelation, and production of phytohormones these microbes directly or indirectly
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A. Tripathi et al.
Fig. 22.1 Mechanism of action of endophytes associated with plant (Vendan et al. 2010)
improve plant performance under various biotic and abiotic stresses (Backman and
Sikora 2008) (Fig. 22.1). Endophytic fungi and bacteria are found intimately associated with plant rhizospheric region and over 129 different bacterial species have
been reported to be isolated from crop plants (Xu et al. 2012; Ryan et al. 2008).
22.4.1 Phytohormone Production
Phytohormones are small organic compounds produced in small quantities in plants
that have a role in all aspects of plant growth and development ranging from
embryogenesis, reproduction to maturity. Some of these hormones are also known
to play a role in disease resistance and prevention. Phytohormones such as auxin,
cytokinin, and gibberellic acid are known to induce germination, promote growth,
and have roles in various developmental stages of plant (Glick et al. 1998). Conversely, abscisic acid, salicylic acid, jasmonic acid, and ethylene are important
hormones that regulate stress-induced responses in plants (Asgher et al. 2015).
Endosymbionts, when present in symbiotic association with plants have been
shown to enhance various phytohormones and many hormones are produced by
these microorganisms, which regulate tolerance to biotic and abiotic stresses.
Firmicutes isolated from stem of ginseng are the pronounced producer of IAA
22
Root Endophytic Microbes and Their Potential Applications in Crop Disease. . .
383
(Vendan et al. 2010). Also, Acetobacter diazotrophicus and Herbaspirillum
seropedicae are known to produce gibberellins on their own through a pathway
which is yet to be discovered (Bömke and Tudzynski 2009). Besides phytohormones, there is certain category of the enzymes that are produced by endosymbionts,
for example, Cryptocin, which is produced by Cryptosporiopsis quercina. This
enzyme is a unique tetramic acid that inhibits pathogens like Pyricularia oryzae
and other plant pathogenic fungi. Other enzymes documented are β-1,3-glucanases,
chitinases, and cellulases. These enzymes could function directly against plant
pathogens and hydrolyze the cell wall of fungal pathogens and oomycetes.
These enzyme are induced in the presence of endosymbionts which act as a
bioprotectant for the plants (Li et al. 2018; Sharma et al. 2013). Likewise, endosymbionts stimulate enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR), which play a vital
role for plants to cope up biotic and abiotic stress (Abdelshafy Mohamad et al. 2020).
Helmut Baltruschat et al. reported increase in ascorbate enzyme activity in salt
sensitive barley co-inoculated with P. indica; thus, this endosymbiont directly acts
to neutralise oxygen free radicals (Baltruschat et al. 2008).
22.4.2 Nitrogen Fixation
Nitrogen is an essential component of many bio-molecules like amino acid, protein,
nucleic acid, enzymes, and hormones. Atmospheric nitrogen cannot be readily
utilized by the organisms as such, it has to be converted into ammonia by
rhizobacteria, a process known as nitrogen fixation. Nitrogen-fixing bacteria move
toward the plant root by chemotaxis and colonize the roots followed by infection as
seen in the Rhizobium-cereal model (Khare et al. 2016). The bacteria are mostly
endosymbiotic associating with both leguminous and non-leguminous plants.
Frankia is one of the bacteria, which is well-studied model for the association for
actinorhizal plants (Narayan et al. 2017). Besides, Parasponia andersonii is the only
non-legume that shows nitrogen fixation symbiosis (Waller et al. 2005). Moreover,
Azospirillum has been shown to improve growth and fitness of crops such as wheat,
maize, and rice (Chenniappana et al. 2018).
22.4.3 Phosphate Solubilization
Phosphorous is an inorganic chemical element found in the various form on earth’s
surface mainly on rocks. The weathering of rocks causes phosphorous leaching to
water bodies and this, in turn, is taken directly by plants and indirectly by animals by
feeding on plants. Phosphorous forms the component of nucleic acids and various
proteins in the form of the disulfide bond. Phosphorous is an essential nutrient for
plants for their growth and development. Phosphorous quantity is insufficient in soils
of the various area thus farmers have to supply them externally in the form of
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phosphate fertilizers. The fertilizer is available in the form of superphosphate, which
is prepared by the reaction of sulfuric acid with rock phosphate. Conversely,
phosphate solubilizing bacteria and fungus such as Bacillus, Pseudomonas, Rhizobium, Penicillium, and Aspergillus have been documented to solubilize insoluble
inorganic phosphate in soil and make it available for plants in the rhizosphere (Yanni
et al. 2016). Through phosphate solubilization, endophytes also provide resistance
towards different environmental stresses such as biotic stresses. (Li et al. 2018;
Salama and Mishricky 1973).
22.4.4 Siderophore Production
Siderophores are low molecular chelator, which assist in transport of iron from the
soil microbiome to the rhizobacteria. Strains that have more beneficial traits
belonged to the genera Bacillus, Enterobacter, Pseudomonas, Klebsiella, and
Microbacterium are documented to produce 54% of siderophores (Ingram
2011). It has been reported that arbuscular mycorrhizal fungi significantly enhance
iron uptake rate in the associated host plants (Lee and George 2005). Pantoea
agglomerans, P. ananatis, P. stewartia, Enterobacter ludwigii, Ralstonia sp., and
Pseudomonas thivervalensis are known to be involved in uptake of copper and
siderophore production (Zhang et al. 2011). These strains stimulate plant biomass
and assist in phytoextraction of metals like Ni, Zn, and Cr. Various studies report that
many siderophore producing endophytes exhibit antagonistic activity as reported in
case of strains of the genera Pseudomonas and Burkholderia and two species of
Pantoea (P. ananatis and P. agglomerans) (Yang et al. 2008). Thus, siderophore
production is a part of defence strategy against other pathogen as well as mechanism
to increase the uptake of mineral like iron.
22.4.5 ACC Deaminase Production
The enzyme 1-Aminocyclopropane-1-carboxylase deaminase was first characterized
by Honma and Shimomura in the year 1978 in a soil microorganism and it was
known to metabolize ACC into a form which could be metabolized by another
microorganism (Hardoim et al. 2008). ACC is an immediate precursor compound in
the biosynthesis of ethylene and plays an important role in growth physiology and
stress (Ali et al. 2012). The plant growth promoting bacteria employ ACC deaminase
to convert ACC into α-ketobutyrate and ammonia, thus reducing the abnormal
increase in ethylene level during the stress condition, therefore protecting plants
during different biotic and abiotic stresses (Steenhoudt and Vanderleyden 2000).
22
Root Endophytic Microbes and Their Potential Applications in Crop Disease. . .
22.5
385
Importance in Sustainable Agriculture
Endophytes have different modes of inoculation and transmission. They infect plants
directly through roots or transmitted via seeds as reported in maize (Smil 2000) and
systemically infect the plant following seed germination or by parasitic competition
for infection site as seen in celery yellow disease caused by F. oxysporum f.sp. apii
(Igarashi et al. 2000). Maize plants have also known to be infected by air and rain
splash and through insect damage (Dutta et al. 2014). The endophytes may remain
dormant or sporulate to benefit the host soon after the infection (Li et al. 2000).
Some of the endophytes and role played by them in agronomically important
crops are listed below.
22.5.1 Cereals
Endophytes play an important part in sustainable agriculture by reducing biotic and
abiotic stresses and by lessening the harm posed by chemical alternatives. Cereals
form a major diet for the majority of world population. Cereal production has to be
increased enough to meet the demand of growing population. However, both biotic
and abiotic stresses are major constraints causing significant yield losses in cereal
crops. Endophytes have been reported to act as bioprotectant in biotic stress, for
example, maize seeds when coated with conidia of entomopathogenic fungi offers
better protection against Costelytra giveni and F. graminearum (Quecine et al.
2012). In another example, P. indica-inoculated barley plants are more resistant to
abiotic and biotic stress (Moreno et al. 2009; Bunbury-Blanchette and Walker 2019).
Streptomyces sp. Strain DEF09 obtained from wheat root has been documented to
act as bioprotectant against Fusarium sp. in crops (Egel and Martyn 2007).
22.5.2 Pulses
Pulses are a rich source of protein and nutrients. Pulses increase soil fertility and
utilize less water than any other crop plants. Endophytic fungi like P. indica decrease
disease severity of chickpea by inhibiting its pathogen B. cinerea and also promotes
healthier growth (Moreno et al. 2009).
22.5.3 Vegetables
Extracts from rhizobacteria like Streptomyces sp. suppress the infection of
Alternaria brassicicola on chinese cabbage seedlings (Gao et al. 2010). Similarly,
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many antagonistic strains of Trichoderma have been isolated which alleviate the
disease symptoms as seen in the effect of T. atroviride and T. harzianum in foot base
rot of onion (Feng et al. 2017).
22.5.4 Fruits
Tomato is an economically important crop and is used in various forms in the diet
like ketchup, sauce, vegetable gravy, and as a salad. This crop is harmed by a variety
of pathogens of which Fusarium spp., harms it the most. Endophytes like
Trichoderma, Beauveria bassiana and P. indica are used as bioprotectant against
different races of Fusarium spp. (Dong et al. 1994; Sefloo et al. 2019).
22.5.5 Sugar and Starches
Sugarcane is prone to many diseases due to its delicate rind and rich source of the
nutrient. An endophytic bacterium was seen to colonize and promote the growth of
sugarcane as studied by Quecine by inducing synthesis of chitinase and
endoglucanase enzymes which protects the plant (Lee and George 2005).
Acetobacter, an N2-fixing endophyte colonizes the intercellular spaces of sugarcane
and contributes to high content of N, approximately 180 kg of N required by the crop
each season (Vendan et al. 2010).
22.5.6 Spices and Condiments
The PGPR enhance the rhizome yield and curcumin content in turmeric and act as a
biocontrol agent against rhizome rot disease of turmeric (Rivas-Franco et al. 2019).
22.6
Conclusion
Endophytic bacteria and fungi are promising to control disease and protection
of crops during both biotic and abiotic stress. Agricultural practices involving the
use of endophytes is warranted particularly when the world is already facing the
impact of changing global climate and ill effects of indiscriminate uses of pesticide,
insecticide, and chemical fertilizers. Several endophytic fungal species are now
successfully tested and recommended as potential biocontrol agents against a variety
of plant pathogens. Conversely, a positive impact of these endophytes in plant
growth promotion and improving yield advocate their need in sustainable
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Root Endophytic Microbes and Their Potential Applications in Crop Disease. . .
387
agricultural practices. On the other hand, some endophytes assist in the process of
phytoremediation and bioremediation, thus improving not only agricultural crop
production, but also the soil and environment (Fig. 22.1).
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Chapter 23
Do Mycorrhizal Fungi Enable Plants
to Cope with Abiotic Stresses by
Overcoming the Detrimental Effects
of Salinity and Improving Drought
Tolerance?
I. Ortas, M. Rafique, and F. Ö. Çekiç
Abstract Soil salinization and drought are major and growing ecological problems.
They limit the productivity of crop plants cultivated on more than 20% of total
agricultural lands worldwide. Global climate changes and sequences of agriculturerelated management practices would induce salinity to more than 50% of the arable
land by 2050. Excess salt in soil impedes plant photosynthetic processes, seed
germination, and root uptake of water and nutrients such as K+. Under the same
soil and climate conditions, water deficiency is also one of the serious limiting
factors for plant growth and food security. Application of biological processes
such as mycorrhizal fungi as inoculants provide a cost-effective long-term solution
for coping with saline and drought conditions. Inoculation of mycorrhizal fungi
along with certain microbial strains in salt and drought-affected soils increase root
infection. Arbuscular mycorrhizal fungi (AMF) are renowned for effective scavengers of free radicals in soil thereby increasing soil parameters optimal for plant
growth. The mechanism to cope with drought stress involves in AMF-enhance
drought and salt tolerance through direct water and nutrient uptake via extraradical
hyphae, better root system architecture, enhancement of antioxidant defense systems, and greater osmotic adjustment. Mycorrhizal colonization upregulates the
expression of chloroplast genes in leaves, and genes encoding membrane transport
I. Ortas (*)
Faculty of Agriculture, Department of Soil Science and Plant Nutrition, University of Cukurova,
Adana, Turkey
e-mail: iortas@cu.edu.tr
M. Rafique
Department of Soil Science, The University of Haripur, Haripur, Khyber Pakhtunkhwa,
Pakistan
F. Ö. Çekiç
Faculty of Science and Literature, Aksaray University, Aksaray, Turkey
e-mail: ozlemcekic@aksaray.edu.tr
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_23
391
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I. Ortas et al.
proteins involved in K+/Na+ homeostasis in roots. Mycorrhizal inoculated seedlings
exhibit high root salicylic acid concentrations and lower leaf and root jasmonic acid
concentrations under salt stress. The AMF improve root hydraulic conductivity as
well as the plant water status and tolerance under drought stress. Essential nutrients
are also taken up through mycorrhizal hyphae and differences in P and K acquisition,
transpiration, and stomatal conductance are related to mycorrhizal efficiencies of
different fungi. Indigenous microorganisms may be a promising biological technology to improve plant performance and development and to alleviate salt stress
damage.
Keywords Salinity · Drought · Mycorrhizal fungi · Genes · Root architecture ·
Abiotic stress
23.1
Introduction
Abiotic stresses such as salinity and drought seriously threat the agricultural productivity and food security (Wang et al. 2003). Especially under semiarid and arid
climatic conditions climate change increases soil salinization. Currently, over 7% of
the Earth’s land area is estimated to have saline soils (Ruiz-Lozano et al. 2001).
Nearly 20% of the cultivated world’s land and half of the irrigated land are affected
by high salt concentrations Sudhir and Murthy (2004). Wang et al. (2003) and Porcel
et al. (2012) reported that increased salinization of arable land is expected to have
devastating global effects, resulting in up to 50% soil salinization by the year of
2050. At present, nearly 5% (77 million hectares) of total cultivated arable land is
affected by salinity (Sheng et al. 2008). In the same climatic regions, water scarcity
as well as salinity problem poses a serious threat to food security. Especially under
arid and semiarid regions, water stress has limited crop productivity (Maggio et al.
2000). Under such semiarid and arid soil regions, salinity problem leads to major
constraints on agricultural production. The salt tolerance of a plant is affected by
soil, water, plant, and environmental conditions. Plant roots, soil nutrients availability, nutrient absorption capacity, and soil microbial activity especially mycorrhizal
fungi significantly affect the plant tolerance to salt and drought stresses.
Arbuscular mycorrhizal fungi (AMF) are present in all kind of ecosystems,
regardless of soil type, vegetation, or growing conditions (Mosse et al. 1981). This
may lead to indirect effects of the AM association on the plant nutrients availability
and uptake (Smith and Read 2008). Their early establishment in the growth process
of plants is important. Mycorrhizae have a significant contribution on soil stabilization, as well. If soil erosion is pronounced, the scarcity of microbial propagules in
such ecosystems may be a serious handicap to plant establishment and survival. In
such cases it may be necessary to inoculate with indigenous fungal species or
augment the natural AMF already present within the rhizosphere of leguminous
plants.
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According to Chitarra et al. (2016), Ruíz-Sánchez et al. (2011) mycorrhizal fungi
play an instrumental role in the protection against abiotic stresses such as drought
and salt stresses (Naher et al. 2013). Since salt and drought stress factors are serious
for food security and sustainable agriculture; it is sound to use plant rhizosphere
mechanisms to address the problems. Soil and environmental stress factors affect the
efficiency and establishment of mycorrhizae. Mycorrhizae also have many advantages on stress tolerance (Barea et al. 1996) and abiotic stress factors (Swaty et al.
2004). For example, salinity suppressed the growth of AM. Salinity stress significantly reduced the root, stem and leaf dry matter and leaf area due to the direct effects
of ion toxicity on plant. However, it has been shown that mycorrhizal colonization
significantly improved plant chlorophyll concentration in comparison to the
non-salinized and salinized plants (Latef and Chaoxing 2011).
The hyphal networks of AM fungi improve soil particle aggregation thereby they
improve the resistance of plant to stress factors, as well. Lehnert et al. (2018)
indicated that worldwide in the majority of wheat-growing areas, the incidence of
drought stress has increased significantly resulting in a negative impact on plant
development and grain yield. In several pot cultures, it has been tested the effects of
AM symbiosis on the improvement of drought stress tolerance of wheat plants
(Lehnert et al. 2018; Al-Karaki et al. 2004; Al-Karaki 1998; Al-Karaki and Clark
1999). Mycorrhizae can be a strong supporter to help symbiosis needed plantlets.
Mycorrhizal fungi seem to act in three ways:
1. Help the plants to attain its best performance
2. Buffering the stress during acclimatization
3. Improve overall plant and soil health
It has been indicated that AMF can promote many aspects of plant life such as
plant growth improvement, nutrients uptake, and stress tolerance (Chen et al. 2018).
Also, AMF inoculation can increase resistance potential of plant against salt and
drought stresses. Mycorrhizal association increases plant tolerance to drought stress
as well (Wu et al. 2013). The work of Latef and He (2011) has shown that under
several levels of salt application, concentrations of P and K were higher in
Rhizophagus mosseae inoculated tomato (Lycopersicon esculentum L. cv.
Zhongzha105) plants when compared with non-AM plants grown under non-saline
and saline soil conditions. Usually, under salt stress conditions, AM inoculation
reduces the tissue Na+ concentration. Previously it has been shown that mycorrhizal
inoculation significantly affects plant biochemical defense system by enhancement
of antioxidant enzyme activities such as superoxide dismutase (SOD), catalase
(CAT), peroxidase (POD) and ascorbate peroxidase (APX) in leaves of both saltaffected and non-affected plants. The results of Latef and Chaoxing (2011) have
shown that AMF may protect tomato plants against salinity by alleviating saltinduced oxidative stress. Fan and Liu (2011)’s results also have shown that under
drought, G. mosseae inoculated Poncirus trifoliata seedlings exhibited higher level
of proline and activities of two antioxidant enzymes, superoxide dismutase (SOD)
and peroxidase (POD) as compared to non-inoculated plants. Several studies showed
that mycorrhizal inoculation can protect plants against salinity by alleviating the
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salt-induced oxidative stress. Lower oxidative damage in the mycorrhizal colonized
plants may help plants to survive and grow properly. Wu et al. (2010) have shown
that R. mosseae inoculated trifoliate orange seedlings significantly alleviated the
growth reduction of salinity. It seems that mycorrhizae-inoculated citrus seedlings
exhibited a more efficient antioxidant defense system, which may provide better
protection against salt damage.
Salinity and AMF also had a significant effect on the concentration of phenols
and ascorbic acid in the fruits (Grimaldo-Pantoja et al. 2017). Phenols and ascorbic
acid may have direct and/or indirect effects on P nutrition (Smith and Read 2008). It
has been estimated that approximately 90% of the land plant species roots have
symbiosis with mycorrhizal fungi (Gadkar et al. 2001). Despite the low mycorrhizal
affinity of the halophytes (Brundrett 1991) mycorrhizae occur under natural saline
environmental conditions (Yamato et al. 2008).
It has been found that AMF inoculation improved water relations and alleviated
the salt stress of many plants. Also, AMF inoculation provide high resistance to
drought through enhanced water uptake (Ruiz-Lozano et al. 2001; Ruiz-Lozano
et al. 2006). Under water deficiency since AMF associated plant roots enhanced
mineral nutrients especially P, crop productivity is high (Al-Karaki et al. 1998;
Marschner and Dell 1994). Also, mycorrhizae-inoculated plants have higher water
uptake due to hyphal extraction of soil water and higher root hydraulic conductivity
than non-mycorrhizal plants (Auge 2004; Ortuno et al. 2018). All such results
suggest that mycorrhization brought biochemical changes helpful in mitigating
different stresses experienced by drought and salt factors. The AMF (Smith and
Read 2008) and rhizosphere organisms colonization may alleviate drought, salt, and
metal stress of plants showing capability in binding heavy metals and mitigate the
stress tension.
23.2
Effects of Mycorrhizal Inoculation on Salt
and Drought Tolerance
Since the increase in human population has negative effects on land use for food
security, soils are under stress. Recently poor soil quality and crop management have
negative impact on salt stress, nutrient deficiency, and heavy metal pollution. Many
researchers reported that mycorrhizae-inoculated plant species are more tolerant to
stress factors such as nutrients concentrations (Zrnic and Siric 2017). Soil and
environmental stress factors affect the efficiency and establishment of mycorrhizae.
For example, salinity suppressed the growth of AMF. Salinity stress significantly
reduced the root, stem and leaf dry matter and leaf area due to direct effects of ion
toxicity on plant. However, it has been shown that mycorrhizal colonization significantly improved plant chlorophyll concentration in comparison to the non-salinized
and salinized plants (Latef and Chaoxing 2011). The AM fungi also decrease
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395
nutrient leaching from the soil, consequently contributing to the retention of nutrients in the soil for saving the chemical fertilizers.
Various ecological approaches such as AM fungi, PGPR, and entophytic bacteria
have been conducted. It is well known that a wide range of soil microbes including
AM fungi and interaction with endophytic bacteria are able to alleviate soil stresses
by (1) enhancing the availability of soil nutrients and water, (2) production of plant
hormones, (3) controlling pathogens by producing antibiotics, (4) adjusting and
regulating the concentrations of toxic ions, and (5) production of different biochemical compounds to increase plant defense systems (Miransari 2016; Hamilton III and
Frank 2001; Miransari 2014). Miransari (2016) indicates that especially under stress,
the right combination of AM fungi and the host plant may result in the highest level
of efficiency. It is possible under stress conditions that the growth and activity of
both AM fungi, other microorganism, and the plant can be adversely affected. It is
well known that salt has detrimental effects on spore germination of AMF. For better
management of stress factors such as salt on plant growth and plant health, it is
possible to isolate the tolerant species of mycorrhizal fungi from the saline soils.
Rivero et al. (2018) reported that AMF isolated from the stressful environment was
the most effective approach in improving plant tolerance to salt stress. In many
Central European soils, AMF spores were isolated from different sodic soils and
results showed that up to 80% of all spores from different sites gave single
PCR-pattern which closely matched to R. geosporum (Bothe 2012).
23.3
Effects of Salinity and Water Stress on Soil Properties
and Plant Growth
It is estimated that more than 33% world’s irrigated arable land is affected by
salinity. Salt stress causes decrease in plant productivity by disrupting the photosynthesis mechanism. Hokmabadi et al. (2005) indicated that relative growth rate
(RGR), net assimilation rate (NARw) decreased with increasing salinity level with
time for all treatments and rootstocks of pistachio. In general, salt stress is due to
accumulation of Na+ of The intracellular accumulation of Na+ ions under salt stress
conditions alters the ratio of K: Na, which affect the bioenergetic processes of
photosynthesis (Sudhir and Murthy 2004). High concentrations of Na+ and Cl
accumulation in the root and root cells produce extreme rations of Na+/Ca2+, Na+/
K+, Ca2+/Mg2+, and Cl /NO3 which are depressed nutrient–ion activities (Grattan
and Grieve 1998). As a result, of nutritional disorders, plants undergo stress conditions. Since mycorrhizae have affected the absorption of other nutrients, they may
dilute the effect of Na+ and Cl ions in the root medium. In a screened work with
29 different citrus genotypes and rootstocks, it has been postulated that high
concentration of Cl and/or Na+ in the leaves of citrus has been related to disturbances in mineral nutrients, CO2 gas exchange and water relations (Cimen and
Yesiloglu 2016). Navarro et al. (2014) result indicated that response of mycorrhizal
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inoculation to salt stress was related to the nutritional status and their findings
confirm that AM fungi can alter host responses to salinity stress, improving P, K,
Fe, and Cu plant nutrition in Cleopatra mandarin plant. Under saline treatments,
mycorrhizal inoculation increased root Mg concentration. More Mg concentration
may dilute Na+ concentration in the root medium. The results of Latef and Chaoxing
(2011) under nonsaline and saline conditions, Na+ concentration was lower in AM
than non-AM tomato plants. Also, the concentrations of P and K were higher in AM
when compared with non-AM tomato plants. According to results of Porras-Soriano
et al. (2009a) R. mosseae was the most efficient inoculum in reducing the detrimental
effects of salinity; it increased shoot growth by 163% and root growth by 295% in
the nonsaline medium, and by 239% (shoot) and by 468% (root) under the saline
conditions in olive plantlets. Porras-Soriano et al. (2009a) have shown that K content
was enhanced under salt conditions by 6.4-fold with R. mosseae, 3.4-fold with
R. intraradices, and 3.7-fold with R. claroideum inoculated olive plantlets under
nursery conditions.
Mycorrhizal inoculation also decreased the plant shoot/root dry weight ratio as
well (Porras-Soriano et al. 2009a). The results of Yang et al. (2014) show that
mycorrhizal inoculation significantly increased the root length colonization of
mycorrhizal apple plants under high degrees of salinity levels as compared to
non-mycorrhizal plants. The work of Al-Khaliel (2010) has shown that under
salinity and P deficient soil conditions, mycorrhiza inoculated peanut plants chlorophyll content and leaf water content increased significantly and also salinity tolerance significantly increased. In another pot experiment, Al-Karaki (1998) used two
durum wheat genotypes (drought-sensitive and drought-tolerant) under waterstressed and well-watered conditions and the results showed that AM inoculated
plants had high shoot and root dry matters under water stress than non-inoculated
plants.
23.3.1 Mycorrhizal Fungi: Role in Soil Property
Improvement Under Stress Conditions
In order to rehabilitate land sources suffering from soil salinization, useful reclamation programs are undertaken. The AM fungi is suggested as a useful strategy for
saline soils (Zhang et al. 2019a). Mycorrhizae can influence soil aggregation by
improving structure of the soil (Rillig and Mummey 2006), and they have a direct
impact on soil aggregation (Ji et al. 2019). The AM fungi can enhance soil stability
by producing hyphal network and glycoproteins (Trouvelot et al. 2014). They can
provide a direct link between soil and roots (Lenoir et al. 2016). Therefore, the
relationship and feedback between soil structure and mycorrhizae are of special
interest. The AM fungi can affect N metabolism of the soil by enhancing the
proportion of soil macroaggregate in saline soil. The increase in soil NH4+-N by
AM fungi can regulate the hyphal growth and AM fungal hyphae can cause a
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decrease in salt concentration in the hyphosphere (Zhang et al. 2019a). Therefore,
AM fungi can be suggested as an important bioindicator for soil quality or soil
pollution and a potential application for restoring the degraded ecosystems (Lenoir
et al. 2016).
The AM fungi regulate glomalin-related soil protein which is produced by spores
and hyphae of AM fungi (Chi et al. 2018; Lovelock et al. 2004). Glomalin and
glomalin-related soil protein (GRSP) can lead to an interesting response between soil
structure and fungal growth. In poorly aggregated soil, GRSP can be produced in
considerable concentrations (Rillig and Mummey 2006; Jones et al. 1997). In a
previous study, it was mentioned that spore density, GRSP content, and hyphal
length were significantly enhanced by AM fungi under both drought and wellwatered conditions, and they strongly suggest dominant role of AM fungi in the
management of soil water-stable aggregates by improving soil aggregate stability
especially under drought stress (Ji et al. 2019). Likewise, AM fungi can display
strongly positive feedback to the conditions by stimulating aggregated soils (Rillig
and Mummey 2006).
The GRSP released from mycorrhiza influences the properties of rhizosphere.
Previous studies indicate that exogenously applied GRSP could strongly stimulate
root morphology and plant growth under drought stress. The GRSP can also
modulate the phytohormones especially auxin (IAA), abscisic acid (ABA), and
methyl jasmonate (MeJA) concentrations under drought stress. Therefore, the exogenous treatment of GRSPs is suggested as a plant growth regulator for improving
drought tolerance (Chi et al. 2018). However, sodic soil can cause a decrease in
GRSP concentration. It can be related to the adaptation of the plant-fungi interaction
to various environmental conditions (Zhang et al. 2017). The AM fungi symbiosis
can be used as an adaptation strategy by enhancing water use efficiency of the plant
and it can eliminate the deleterious effects of water stress (Pavithra and Yapa 2018).
Moreover, different species of mycorrhizae can stimulate the aggregation of soil to
different degrees (Rillig and Mummey 2006). In addition, the physical and chemical
properties of the soil effect AM fungi colonization such as low nutrients level and
clay concentration are the key factors for colonization (Coutinho et al. 2019).
Pesticide residues such as glyphosate in the soil or the plant can cause an inhibition
in the mycorrhizal colonization, and a reduction in mycorrhizal symbiosis can be
strongly dependent on the soil history (Helander et al. 2018). Therefore, the interaction between AM fungi and plant species should be well evaluated to improve
success.
23.3.2 Effects of Salt and Drought Condition on Arbuscular
Mycorrhiza Development
The interaction between plants and AM fungi is strongly correlated with the soil
properties, and it depends on environmental and atmospheric conditions (Bitterlich
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et al. 2018; Mirzaei and Moradi 2017). Drought and salinity affect mycorrhizal
symbiosis (Füzy et al. 2008). Spore production and hyphal development of AM
fungi are negatively affected by water availability. Le Pioufle and Declerck (2018)
reported that polyethylene glycol which stimulates osmotic stress in plants decreased
AM fungi development. Hyphal growth and spore germination of AM fungi can be
reduced in saline environments (Evelin et al. 2009; Juniper and Abbott 2006). The
colonization of some AM fungal species is reduced because NaCl impacts directly
on AM fungi. The extension of hyphae and spore germination can be inhibited in the
presence of NaCl. However, various AM fungal species have different germination
abilities under salinity. Spores of some AM fungal species can germinate even under
300 mM NaCl (Juniper and Abbott 2006). Bencherif et al. (2015) demonstrated that
number of spores of some AM fungal species isolated from various saline soils could
increase under salinity, and this adaptation of AM fungi could be used to restore
saline arid lands. Carvalho et al. (2004) mentioned that some AM fungal species
could adapt to salt marsh soils. The AM fungi diversity and fungal spore density are
also strongly affected by soil nutrient availability and land use practices and agricultural soils (Soka and Ritchie 2018). Therefore, environmental conditions should
be well evaluated before the application of AM fungi.
23.3.3 Mycorrhizal Fungi for Salinity Stress Remediation
Mycorrhizae remediation is an important on-site remediation strategy which uses
microorganisms and plants for cleaning heavy metals from contaminated environments. As mycorrhizae remediation is a relative low-cost, natural method, it is
suggested as a solution for environmental problems. In the mycorrhizae remediation
process fungi can sequester or degrade the contaminants because of their mycelium
morphology supplying highly extensive and reactive surface (Aroca et al. 2017;
Barun Kumar Manjhi et al. 2016). The AM fungi can reduce the transport of heavy
metals from roots to shoots (Zhang et al. 2005). Khan (2006) mentioned that AM
fungi can act as a bioprotectant, biofertilizer, and biodegrader. The AM fungi can
have a significant role in phytoremediation (Wang et al. 2005) and in contaminated
soils, phytoremediation can be enhanced by AM fungi inoculation to crops (Khan
et al. 2014). The AM fungi have also beneficial role in phytoremediation under
drought and salt stresses (Liu et al. 2018a).
Cadmium (Cd) and Nickel (Ni) are important heavy metals, which can enter to
food chain via contaminated agricultural products or drinking water. These metals
have deleterious effects on human health (Barun Kumar Manjhi et al. 2016). Several
studies indicate that heavy metals such as Zn, Cd, As, and Se can be taken up by AM
fungi from the environment (Aroca et al. 2017; Giasson et al. 2006). The AM fungi
can help to immobilize heavy metals such as Cu, Pb, Zn, and Cd in the roots and
alleviate the toxicity of heavy metals (Zhang et al. 2005). The useful effects of AM
fungal colonization under metal toxicity are the improved P uptake and decreased
Cd, As and Cu concentrations in the shoots. In another study, it was reported that
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AM fungi could induce the expression of metallothionein genes under Ni stress and
help to alleviate the negative effects of Ni stress (Shabani and Sabzalian 2016).
Therefore, AM fungi could have a beneficial impact on the ecological restoration of
metalliferous mine areas (Chen et al. 2007).
Basidiomycetes can also degrade persistent organic pollutants, recalcitrant hydrocarbons, such as polyaromatic hydrocarbons (PAHs), aromatic hydrocarbons, halogenated hydrocarbons, and phenols, explosives and dyes. Basidiomycete species can
degrade various kinds of hydrocarbons by their essential enzymes (Treu and
Falandysz 2017). Boldt-Burisch et al. (2018) mentioned that AM fungal colonization
in the roots are not affected by soil hydrocarbons. Therefore; the use of AM fungi
symbiosis is suggested in contaminated soils with high efficiency (Zaefarian et al.
2013). However, heavy metal translocation can be affected by the interaction
between host plants and different AM fungi isolates (Liang et al. 2009) and the
interaction of heavy metal with other metals (Giasson et al. 2006). Therefore, the
selection of the species used for bioremediation and the potential interactions with
other soil organisms should be well evaluated (Treu and Falandysz 2017).
Barun Kumar Manjhi (Barun Kumar Manjhi et al. 2016) mentioned that AM
fungi can be suggested as a filter and AM fungi can inhibit the transport of heavy
metals to the plants. The AM fungi can protect the host plants from heavy metals
such as Zn, Cd, and Pb toxicity in contaminated soils. The AM fungi can inhibit
heavy metals uptake in high concentrations, therefore; they could enhance the plant
growth (Liang et al. 2009). In addition, AM fungi can induce heavy metal accumulation in some plant species. The AM fungi may enhance stress tolerance in the
contaminated soils via trapping heavy metals in their extraradical hyphae and plant
root systems (Carvalho et al. 2006). Roy et al. (2018) reported that AM fungi could
be used in the remediation of toxic fly ash by phyto-bio-rhizo-mycoremediation
application. The AM fungal species such as R. tenue, R. mosseae, and Gigaspora
spp. can defend plants against the deleterious effects of heavy metals (Lal 2002).
Giasson et al. (2006) mentioned that plants infected with R. intradices can sequester
Cd, Zn, As, and Se more than non-mycorrhizal plants. In another study, it was
mentioned that R. mosseae could lead to high tolerance to heavy metal toxicity
(Zhang et al. 2005).
Gai et al. (2018) reported that some species such as Claroideoglomus claroideum
and R. etunicatum tolerated Cd in soil. R. claroideum was identified as more tolerant
to the toxicity of Cd by measuring root colonization and total extraradical mycelium
length. In another study, it was mentioned that R. intraradices increased plant
growth in Cd contaminated areas (Redon et al. 2008). The AM fungi can also
increase As uptake via active arsenite-translocating ATPase. However, the translocation can be altered via AM fungi (Giasson et al. 2006). Glomalin has an important
impact on the achievement of mycorrhizoremediation in heavy metal tolerance.
Glomalin can isolate heavy metals (Khan 2005). The AM fungi can have an
important impact on the accumulation of glomalin-related soil protein, soil organic
matter and soil organic carbon and influence the particle-size distribution and
aggregate formation in heavy metal contaminated areas (Li et al. 2017). Therefore,
the AM fungi are suggested for the recovery of contaminated soils (Abu-Elsaoud
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et al. 2017; Yang et al. 2017). (Loha et al. 2018) mentioned that a cyclin (SiPHO80)
in the protein family could play important roles in homeostasis of inorganic phosphate and regulate the tolerance to heavy metal stress in Serendipita indica an
osmotolerant AM fungal specie. Therefore, S. indica is suggested as a potential
biofertilizer. We can also benefit from AM fungi by their role on heavy metal
phytoextraction (Wang et al. 2005). Among other metals, Se can be extracted
more easily by AM fungi inoculated plants (Giasson et al. 2006). In another study,
R. intraradices caused an increase in available Cd and reduced Cd contents in
leachates. Therefore, AM fungi were suggested as a good approach for the
phytoextraction process (Redon et al. 2008).
23.4
Mycorrhizal Inoculation: Effects on Plant Shoot
and Root Growth Under Salt Conditions
Salinity affects the plant root and shoot growth. Number of studies have been
conducted to evaluate the counter-effect of AM fungi in salinity tolerance implacable
to plant growth attributes which include root and shoot growth, chlorophyll content,
stomatal conductance, inter and intracellular CO2 concentration in the plant leaves.
Elhindi et al. (2017) conducted a study in sweet basil (Ocimum basilicum) plants at
three salinity levels which were nonsaline (EC ¼ 0.64 dS m 1), low saline
(EC ¼ 5 dS m 1), and highly saline (EC ¼ 10 dS m 1). The AM fungi used in
the study were R. deserticola. Observed data showed that AM fungi significantly
increased dry biomass of plant as a whole but shoot height and their branches were
not enhanced significantly. Similarly, another study was conducted on rice plants
under salinity stress in the presence of AM fungi (C. etunicatum) by Porcel et al.
(2016). The study was performed at two salinity levels (75 mM and 150 mM NaCl)
where results showed that plants inoculated with AM fungi had more growth and
shoot dry weight was increased by 40–62% under nonsaline (75 mM NaCl) conditions. Only increase of 51% was observed under 150 mM NaCl in the presence of
AM fungi. It was decreased only by 10% in AM fungi inoculated plants at 150 mM
NaCl. Besides that, a 17% reduction in dry weight was observed at 75 mM NaCl
non-AM fungi inoculated plants.
In another study, Garg and Bhandari (2016) used silicon nutrition and AM fungi
(F. mosseae) inoculation to evaluate plant biomass, root to shoot ratio and yield of
Cicer arietinum L. genotypes under saline (0, 60, 80, and 100 mM NaCl) conditions.
Observations showed that salinity significantly reduced the plant dry matter and
declined the plant growth in all genotypes. Roots were observed more prone to
salinity than leaves resulted in decrease of R/S ratio. Study also concluded that
individual application of Si and AM fungi mitigate salinity effects on plant and
induce significantly positive changes in plant growth. Moreover, when Si was
applied with F. mosseae, in saline conditions, plant biomass significantly increased
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in comparison to Si fertilization only. Additionally, AM fungi directly facilitate in
root biomass development and it enhances in comparison to Si application only.
23.5
Mycorrhizal Symbiosis and Mineral Uptake Under
Salt and Drought Stress Factors
Several studies have been performed to evaluate the contribution of AM fungi in
mitigating salinity and drought effect for better nutrient uptake and plant growth
(Hammer et al. 2011; Ruiz-Lozano et al. 2012; Evelin et al. 2012). Studies suggest
the mechanism of selective nutrients uptake through AM fungal hyphae into the
plant roots which eliminate toxic effects of salt. Moreover, studies showed an
increase in K+ and a decrease in Na+ concentrations in AM fungi inoculated plants
(Evelin et al. 2012; Garg and Manchanda 2009).
Elhindi et al. (2017) grew sweet basil plants in nonsaline (EC ¼ 0.64 dS m 1),
low saline (EC ¼ 5 dS m 1), and high saline soils (EC ¼ 10 dS m 1) where observed
the mineral nutrients concentration in plant leaves. Results showed that K+, P, and
Ca2+ were higher in G. deserticola inoculated plants under non-stressed conditions.
The content of Na+ and Cl were exceptionally low in sweet basil. Similarly,
significantly high amount of K+, P, and Ca2+ was recorded in the sweet basil leaves
of AM fungi inoculated plants either NaCl treatment was there or not. Overall
increase in NaCl concentration decreased the nutrient content with exception for
Na+ and Cl content. The AM fungi significantly enhance leaf-K+, P, and Ca2+
content under salinity stress conditions and reduce Na+ and Cl content. Salinity had
significant reducing effect on the ratio between K+ and Na+ and between Ca2+ and
Na+. There was a significant difference between K+/Na+ ratio and Ca2+ and Na+ in
AM fungi inoculated plants. In comparison to non-AM fungi inoculated plants, K+/
Na+ ratio was higher regardless of the salinity strength. Therefore, AM fungi have
the capability to reduce the imbalance of ions and their ratios such as Na+ and Ca2+
uptake and K+/Na+, Ca2+/Na+ ratios in saline soil (Kaya et al. 2009).
Contribution of AM fungi in coping with abiotic stress to the associated plant is
related to the alterations in hormonal homeostasis where ABA signaling is thoroughly studied (Ruiz-Lozano et al. 2012; Osakabe et al. 2014) (Fig. 23.1). The ABA
is a stress hormone, and its production is triggered during environmental stresses
such as salinity and drought (Osakabe et al. 2014). Plants adjust their ABA level
according to physiological changes, environmental stress, and symbiotic relation.
Aroca et al. (2013) endorsed the increase in ABA content when the plant is under
stress condition. İn presence of AM fungi, stress is mitigated and ABA level
decreases which may suggest that AM fungi inoculated plants are less stressed
than non-AM fungi inoculated plants. Moreover, plant growth parameters and
plant yield also prove plant fitness.
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Fig. 23.1 Summary of the main processes by which AM fungi symbiosis can regulate the
integrated physiological plant response in order to improve tolerance to salinity. The exchange
flux of water, minerals (M), and carbon compounds (C) between the plant and the fungus is also
represented. Minerals include nutrients and salt ions present in the soil solution. Adapted from RuizLozano et al. (2012)
23.5.1 Phosphate Uptake Assisted by the AM Symbiosis
Under Salt Stress Conditions
Phosphorus (P) is a major nutrient of interest in symbiotic relationship of plant-AM
fungi. It derives basic cellular functions in bioenergetics, metabolites activation, and
enzymes regulation during structural formation as nucleic acids (Bucher 2007).
Besides that, P is a limiting nutrient for plant productivity because of its immobile
nature in soil. Porcel et al. (2016) conducted a study to evaluate the salinity effect on
rice plant with non-AM fungi and AM fungi (C. etunicatum) application. Results
revealed that P concentration was high in root and shoot of AM fungi inoculated
plants regardless of the salt concentration. In roots, 175% increase in P concentration
on average was recorded, whereas in the shoots, it approached 460% under
nonsaline conditions and 190% in highly saline conditions. Indeed, the shoot P
concentration decreased in AM plants due to salinity applied, although these plants
always maintained higher shoot P concentration than non-AM plants.
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23.5.2 Nitrogen Uptake and Transfer at the Mycorrhizal
Interface Under Salt Stress Conditions
Nitrogen is a major nutrient required to the plants for their biomass (1–5% of dry
weight), biochemical processes, and plant yield. As a significant amount of N
requirement to the plant, it is available in the soil as two inorganic forms such as
NO3– in upland soil and NH4+ in flooded soil (Bücking and Kafle 2015). İn AM
fungi inoculated soils, N is taken up by three forms such as NH4+ (Frey and Schüepp
1993), NO3- (Tobar et al. 1994a), and amino acids (Cliquet et al. 1997). Besides that,
AM fungi prefer to uptake NH4+ for the plant as it need extra energy to reduce NO3–
into NH4+ before conversion into organic compounds (Courty et al. 2015; Nakano
et al. 2001) (Fig. 23.2). This mechanism was further supported molecularly, and
three mycorrhizal fungal ammonium transporter (AMT) genes such as GintAMT1, 2,
and 3 have been identified in R. irregularis (Pérez-Tienda et al. 2011; Calabrese et al.
2016). Among them, GintAMT1 has high affinity for NH4+ transporter expressed in
cortical cells with arbuscules and extraradical mycelium (ERM). The transcripts of
GintAMT1 could up-regulate in low supply of NH4+ particularly in acidic soils
(López-Pedrosa et al. 2006). GintAMT2 expresses in high-P soils and it suggests
that more NH4+ is transferred (Calabrese et al. 2016). In anaerobic soils, NO3–
uptake to the plant root is supported by mycorrhizal hyphae coupled to H+-symport
dependent process (Bago et al. 1996). Molecular evidence of NO3– absorption was
confirmed as NO3 transporter, GiNT, from R. irregularis was found which is
usually present in ERM (Koegel et al. 2015). The fungal GiNT represses in surplus
supply of NH4+ which shows that upregulation of GiNT and GintAMT is dependent
on NH4+ to NO3– ratio in soil.
Besides inorganic form of N, AM fungi can take up certain organic forms of
N. Several studies have been conducted with labeled 13C and 15N from organic
sources (amino acids) to evaluate the uptake capability of AM fungi. Results showed
that only 15N was taken up (Atul-Nayyar et al. 2009; Hodge 2001). Hodge, Campbell (Hodge et al. 2001) noticed 72% capturing from glycine source. An amino acid
permease, GmosAAP1, involved in transporting amino acid across fungal membrane
has been identified in the AM fungus Funnelliformis mosseae (Cappellazzo et al.
2008). Multiple transporters are involved in the uptake of amino acids through ERM.
Another transporter RiPTR2 has been identified in R. irregularis which is responsible for the transportation of dipeptides (Belmondo et al. 2014).
23.5.3 Water and Potassium Relationship in AM Colonized
Plant Under Salt and Drought Conditions
The AM fungi are widely distributed in saline environment. In the presence of NaCl,
AM fungi inoculated plant leaves showed high relative water content, moreover,
high efficiency of water usage, and reduced water saturation (Sheng et al. 2008). The
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Fig. 23.2 Current knowledge about inorganic N transfer mechanisms in arbuscular and ectomycorrhiza and the rhizobium-legume symbiosis. Ammonium and nitrate are exchanged across
the different compartments (soil, fungal/bacterial cells, apoplast, plant cells) by several membrane
transporters that are not yet fully characterized. The different abbreviations are: NRT, nitrate
transporter; AMT, ammonium transporter; LIV, leucine, isoleucine, valine; AA; amino acid; Asn,
Asparagine; UPS, ureide permease; VG, voltage gated; Am, Amanita muscaria; Gint, Glomus
intraradices (recently renamed Rhizophagus irregularis); Gm, Glycine max; Hc, Hebeloma
cylindrosporum; Lb: Laccaria bicolor; Le, Lycopersicum esculentum; Lj, Lotus japonicus; Mt,
Medicago truncatula; Pta, Populus tremula x alba; Ptt, Populus trichocarpa; Pp, Pinus pinaster;
Pv, Phaseolus vulgaris; Rl, Rhizobium leguminosarum; Tb, Tuber borchii. Adapted from Courty
et al. (2015)
improvement in water status through AM fungi linked to enhanced water uptake
mainly because of AM fungal hyphae penetration (Jiang and Huang 2003). As water
status is severely disturbed by the salinity stress, key role of AM fungi is to save the
host plant from dehydration and thus aid the host plant to enable maximum
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absorption of water via deep fungal hyphal network system (Porcel et al. 2012). But
the K concentration and its accumulation in plant under the saline environment did
not affect significantly the AM fungi colonization. Minimum uptake of K was
reported in tomato plant by AM fungi root colonization under salt stress
(Al-Karaki 2000). Reports showed that maximum salt stress-tolerant mycorrhizal
plants showed the highest concentration of K in their shoot which is correlated with
the stomatal K regulation (Porras-Soriano et al. 2009b). The root colonizing fungi
R. intraradices in olive plants under the medium saline condition showed the K
acquisition by 3.4 fold as compared to nonmycorrhizal root colonized plants, and in
turn reaching up to 6.4 fold in plants colonized by R. mosseae (Thomas et al. 2003).
Under salt stress conditions, maximum effect on the K uptake was only noted upon
the inoculation of AM fungi and thus both shoot and root accumulated more K in
AM fungi inoculated plant in comparison to uninoculated plant under salt stress
condition. In this way, A. nilotica inoculated with mycorrhiza showed high amount
of K in shoot and root under all of the salt treatments (Giri et al. 2007). Some
previous reports showed similar increase in K concentration and hence noted that
high K+:Na+ was maintained by the mycorrhizal plant under the salinity stress
condition by accumulating more K+ (Mohammad et al. 2003). Furthermore, at
medium and high levels of salinity stress; there is a minor effect of AM fungi root
colonization on shoot K concentration (Mardukhi et al. 2011). Similarly, AM fungi
symbiotic association has the potential to improve the plant tolerance against water
deficit condition thereby maintaining the plant water relation (Stevens et al. 2011).
Both under water stress and well water conditions the water relations were
prominent for mycorrhizal plants (Asrar et al. 2012). The adaptation of AM fungi
to drought stress condition is quick and hence deliberate the fruitful effects on the
host plant under water deficit stress conditions (Nasim 2010). The uptake and
transportation of water and nutrients from the soil to host plant roots is among the
key functions of extraradical hyphae of AM fungi (Peterson and Massicotte 2004).
As compared to saturated soil conditions, the hyphal network transport more water
under the dried soils. Similarly, the leaf water potential also enhanced by symbiotic
association under drought stress condition (Gholamhoseini et al. 2013). The uptake
of water is due to hydrophilic nature of hyphal tips, which absorb water from soil and
transport via either cytoplasmic pathway or inner layer of wall from a single soil
pore, alongside the AM hypha toward the cells of cortex (Allen 2007). When the soil
dehydrates severely, the extraradical AM hyphae with a diameter of 2–5 m penetrate
through soil pores and provide the mycorrhizal root more access to water zone
(Wu et al. 2013). Some previous reports also showed that AM fungal colonization
improved plant water relationship (Zhang et al. 2011). The AM symbiosis improved
K tissue concentration which is an important physiological attribute to regulate the
root water uptake potential of plant (Benlloch-González et al. 2009). The high
potassium uptake by mycorrhizal application under drought stress conditions
showed that AM symbiosis has the potential of reducing drought stress and reconstruction of ecosystems (Wu et al. 2011).
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23.5.4 Mycorrhizal Fungi: Effects on Macro
and Micronutrients Uptake Under Salt and Drought
Stress Conditions
Generally, under salinity stress conditions, due to imbalance in nutrient composition
such as excessive Na+ and Cl ions uptake caused the excessive toxicity thereby
leading to a reduction in osmotic potential of plants, disruption of cell organelles and
their metabolism and ultimately salt affect plant growth and reduce the yield. The
nutrient imbalance in the plant cells caused by nutrient uptake and/or transport to the
shoot leading to ion deficiencies (Adiku et al. 2001). The AM fungi inoculation
significantly increased the content of P and ascorbic acid of the pepper plant and also
salinity and AM fungi had a significant effect on the concentration of phenols and
ascorbic acid in the fruit (Grimaldo-Pantoja et al. 2017).
The AM fungi have the potential to assist plant for nutrients uptake under
different stresses including salinity and drought (Al-Karaki 2000). The performance
of mycorrhizal resistant species under salinity condition is determined by the uptake
of nutrients (Daei et al. 2009). In mycorrhizal plants, the uptake of N, P, K, Ca, and
Mg was significantly enhanced as compared to non-mycorrhizal plants (Heidari and
Karami 2014; Ortaş and Rafique 2017). The prominent species of AM fungi such as
R. mosseae and R. intraradices increased Zn and Mn concentration significantly in
pistachio plants, regardless of the soil moisture conditions (Bagheri et al. 2012).
Similarly Al-Karaki et al. (2001) demonstrated that the uptake of Cu, Fe, and Zn
significantly increased for mycorrhizal plants with respect to non-mycorrhizal plants
under salinity stress (Al-Karaki et al. 2001). According to Daei et al. (2009),
mycorrhizae absorbed maximum amount of Zn and produced high quantity of root
and yield under saline stress condition. Various nutrients such as N, Ca, Mg, Fe, Cu,
and Mn were absorbed by different cultivars of wheat under saline soil inoculated
with AM fungi species including R. etunicatum, R. mosseae, and R. intraradices
(Mardukhi et al. 2011). Plants inoculated with AM fungi showed significant uptake
of micronutrients as compared to uninoculated control in lettuce plant whereas other
studies showed that rhizospheric bacteria with AM fungi inoculated wheat plants had
more efficiency against Se (Durán et al. 2016). The study of Mohammad et al. (2003)
showed that under salinity stress conditions, the uptake of micronutrients improved
upon the inoculation of AM fungi, similarly the indigenouos AM fungi inoculation
enhanced the uptake of Fe, Mn, and Cu. While the Zn uptake was promoted by AM
fungi under all the treatment of salinity stress.
There is an increase in AM fungi response by decreasing fertility of soil and
furthermore along with increasing the severity of drought stress (García et al. 2008).
Reports showed that AM fungi application significantly reduced the drought stress
and drought-induced deficient nutrients such as Fe and Zn (Gholamhoseini et al.
2013). The drought stress was reduced in pistachio cultivars assisted by the inoculation of AM fungi which improve the uptake of slowly diffusing mineral ions, i.e.,
PO42 and Zn2+ (Bagheri et al. 2012). The higher mineral nutrients content such as
Fe and Zn were observed in plants inoculated with AM fungi as compared to
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non-mycorrhizal plants under drought and well water conditions (Amiri et al. 2017).
Furthermore, the inoculation of F. mosseae and R. intraradices incremented the Zn
content under severe drought conditions (Shirani et al. 2018). The inoculation of AM
fungi species such as R. mosseae and R. intraradices enhanced the Zn uptake up to
4.14% and 3.95%, Cu concentration up to 12.72% and 11.72% with respect to
uninoculated treatment under drought stress (Askari et al. 2018).
23.6
Effects of Mycorrhizal Inoculation and Biochar
Application to Reduce the Salt Effects on Nutrient
Uptake and Plant Growth
Conversion of the barren saline land into cultivated field is a solution to meet current
challenges of world global food security (Biswas and Biswas 2014). The emerging
knowledge of biochar addition has shown that it improves the physiochemical and
biological properties of salt-affected soils (Dahlawi et al. 2018; Rafique et al. 2017).
The increased uptake of P in salt-affected soil by biochar is due to its direct action of
P source facility and indirectly biochar promotes the growth medium condition
particularly soil organic carbon (Lashari et al. 2013). The higher K concentration
in salt-affected soil induced by biochar addition is considered as one of the most
significant mechanisms for biochar to enhanced the growth of plants under salt stress
(Abbas et al. 2018). The nutrient status of plants affected by biochar ability of
increasing nutrient retention, decreased leaching, and gaseous losses through
improving surface properties of soil (Mukherjee and Zimmerman 2013). By promoting the root biochar interaction, the application of biochar increment nutrients
procurement potential of plants in saline soils (Olmo et al. 2016). The addition of
biochar in field and Laboratory condition in saline soil might mitigate its adverse
effects and plant growth is augmented by releasing essential macro- and
micronutrients including Ca, K, N, P, and Zn (Kim et al. 2016). The combined
application of biochar and AM fungi improve plant yield under saline soil with
respect to individual application of AM fungi and biochar. The enhanced yield was
credited by increasing P and Mn and moreover the Na+/K+ ratio in plant grown under
salt stress condition inoculated with AM fungi and biochar (Hammer et al. 2015).
The green house study showed a significant synergistic application of biochar and
AM fungi on the growth parameters and nutrient uptake in seedlings (Budi and
Setyaningsih 2013). A generally better plant nutrition status may help to overcome
salinity stress. Not only Na+ levels were reduced, but also K and Mg concentrations
and total K and Mg uptake were clearly increased in plants that received biochar
and/or AM fungi addition (Hammer et al. 2015).
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Effects of Mycorrhizal Inoculation on Water Uptake
The influence of AM fungi mainly depends on the uptake and transportation of water
and nutrients, which enhances the hydration of plant tissue for securing physiological sustainability and improvement in growth (Abdel-Salam et al. 2018). The AM
fungi inoculation significantly enhanced plant water relation under drought stress
conditions (Borde et al. 2012). Therefore, improved water relation in plant inoculated with mycorrhiza also augmented the nutrient status which in turn exclusively
extract the moisture contents of soil (Subramanian et al. 2006). The application of
AM fungi regulates activity and expression of aquaporin both in fungal species and
host plant in order to tolerate the drought stress conditions (Li et al. 2013). By
activating the antioxidant defense system and stabilizing water table of soil aggravates the AM fungi boost the plant tolerance to water deficit conditions (Bedini et al.
2009). The AM fungi symbiosis significantly influenced the water uptake from soil.
Regarding water uptake from the soil, effectiveness of different fungal species vary
among themselves. This effectiveness in plant water uptake by AM fungi is mainly
related to the external mycelium produced by individual AM fungi species and also
related with the root colonization rate for alive and active structure (Baum et al.
2015). The increase in water and several macro- and micronutrients uptake was due
to AM fungal hyphae that can extend to explorable surface area almost up to
50 times (Berruti et al. 2014). Therefore the host plant along with AM fungal species
inoculation showed positive impact on water use efficiency (Bernardo et al. 2019).
The AM fungal species alter the rate of translocation of water, hydration of tissue,
and thus improved the physiological and water status of host plants (Liu et al. 2015).
Through the plant symbiosis with AM fungi, the plant can be beneficial for transportation of water and thus helping the plant to possess stomata opening (Zhu et al.
2012). Mycorrhizal seedling enhanced more uptake of water than non-mycorrhizal
seedling under water deficit conditions (Gong et al. 2013). This is due to the
expansion of fungal hyphae in the absorption region in host plant thereby enhancing
the water absorption through root (Liu et al. 2015).
23.8
Mechanisms of Mycorrhizae on Salt Tolerance in Soil
and Inside the Host Plant
Salinity is a major problem for plant growth and yield (Porcel et al. 2012). The
toxicity of Na+ and Cl ions causes an imbalance in the nutrient composition and a
decrease in plants osmotic potential, therefore salt stress induces physiological
drought in plants (Evelin et al. 2009). Under salinity, AM fungi can improve water
content and enhance beneficial nutrient uptake such as P, N, Mg, and Ca. The AM
fungi lead various biochemical, physiological, and molecular changes in plants. The
AM fungi induce photosynthetic efficiency, nitrogen fixation, and the accumulation
of various osmolytes, polyamines, prolines, betaines, and carbohydrates and
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enhance leaf, stem, and root biomass and K+:Na+ ratios in leaves. It is also strongly
mentioned that antioxidant system is enhanced by AM fungi (Porcel et al. 2012;
Evelin et al. 2009; Cekic et al. 2012; Chang et al. 2018). Therefore, AM fungi
symbiosis is suggested as a promising method for utilization of salt-alkaline lands
(Chang et al. 2018). And mycorrhizal inoculation could be used in order to develop
salt-tolerant crop plants (Porcel et al. 2012; Evelin et al. 2009).
The AM fungi can induce the expression of aquaporin and stress-related genes
like absciisic acid (Lsnced), late embryogenesis abundant protein (LsLea), Δ1pyrroline-carboxylate synthetase (LsP5CS) and PIP, Na+/H+ antiporters. The expression of these genes protects mycorrhizal plants from the detrimental effects of salt
stress(Porcel et al. 2012; Evelin et al. 2009). The AM fungi can also maintain water
use efficiency and stomatal behavior by regulating the key genes in the ABA
pathway (14-3-3 genes), therefore AM fungi can improve drought tolerance
(Xu et al. 2018). Under water deficiency it was reported that AM fungi and
N-fixing bacteria could cause an increase in grain protein content and benefits to
agricultural production (Oliveira et al. 2017).
Mycorrhizal fungi enhance plant growth, nutrition acquisition, antioxidant system, and siderophore production under adverse conditions. Therefore, they are
suggested instead of the use of pesticides and inorganic fertilizers in agricultural
applications and for developing sustainable and safer agricultural productions. As it
is a biological process, it is essential for sustainable agriculture and it can be replaced
by conventional agriculture applications (Kumar and Verma 2018). Hence, AM
symbiosis can be a potential answer for conservation of some plant species in their
natural ecosystems (Zarik et al. 2016). However, further studies should be done
focusing on AM fungal salt-tolerant strains, cyclic nucleotide-gated channels, and
cation proton antiporters to develop of salt-tolerant inoculums and for successful
environmental and agricultural managements (Kumar et al. 2015).
23.8.1 Mycorrhizal Effectiveness for Hormonal Process
and Signaling Under Salt Stress
The symbiosis is older than 450 million years, and it is environmentally friendly.
The alleviation of detrimental effects of stress conditions is known to be related to
phytohormones, secondary metabolites, and signaling molecules (Lopez-Raez
2016). Phytohormones have vital roles in plant metabolism. They act as stimulators
in plant defense response under various environmental stresses. Phytohormones can
also be produced by AM fungi which can be used for inducing the host tolerance
under various conditions such as salinity, drought, heavy metal stress, and nutrient
deficiency (Egamberdieva et al. 2017). The AM fungi can mediate with the phytohormone balance in the host plants, therefore AM fungi have important impact on
the plant development by influencing as bioregulator and enhancing tolerance
against environmental stresses as bio protector. By selecting the appropriate
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combinations of plant and fungus, maximum benefits can be achieved for farming. In
addition, AM fungi can lead to a reduction in biocides and chemical fertilizers
(Rouphael et al. 2015). Therefore, identification of interactions between host,
microbe, and stress should be well evaluated (Egamberdieva et al. 2017).
Plant-associated microbes have beneficial impacts on the stimulation of phytohormones such as cytokinins, gibberellins, auxins, ABA, and salicylic acid in plant
tissues (Lopez-Raez 2016). Among the phytohormones, AM fungi and salicylic acid
mediate carbohydrate metabolism and ion homeostasis in crop plants and they can
eliminate the deleterious effects of salt stress. Salicylic acid can enhance number of
arbuscules and vesicles and cause an increase in the root colonization under salt
stress. Therefore, seed priming with salicylic acid improves AM symbiosis and it is
suggested as a potential approach in sustainable agriculture production under salinity
(Liu et al. 2018b). Salicylic acid can also modulate water conductivity by regulating
the root aquaporins. In addition, there is a strong network between aquaporins and
phytohormones, especially salicylic acid, abscisic acid, and jasmonic acid in the
controlling of the water transport in the roots (Quiroga et al. 2018). In addition, AM
fungi can induce auxin synthesis and lead to high root hair growth under drought
stress, therefore AM fungi can help to stimulate the deleterious effects of osmotic
stress (Liu et al. 2018b).
The ABA normally regulates stomatal closure under drought stress, however in
mycorrhizal plants (Ouledali et al. 2019) demonstrated that ABA was not the key
factor in controlling the stomata behavior, AM fungi also control the stomata
regulation. The AM fungi can also cause an increase in expressing the jasmonic
acid gene in the roots under drought stress and this increase could help to respond to
water stress (Duc et al. 2018). Under nutrient deficiency strigolactones (SLs), a plant
hormone which modulates the coordinated plant development, can act as signals for
the establishment of AM fungal symbiosis. The SLs can help host plant to alleviate
the symptoms of stresses. Because of the beneficial effects of strigolactones, they are
suggested as sustainable and innovative strategy for modern agricultural processes
(Lopez-Raez 2016). The AM symbiosis can mediate various plant hormones and
plant growth regulators; therefore, this symbiosis can have beneficial effects on plant
metabolism under normal and stress conditions.
23.9
Alleviation of Salt and Drought Stresses by Arbuscular
Mycorrhizal (AM) Fungi
Soil salinity and drought are among the most harmful stresses which affect the plant
growth by reducing water uptake and cause osmotic stress (Santander et al. 2017).
Both salinity and drought effect negatively on the distribution of mineral nutrients
balance. Salinity causes imbalances of nutrients because of the deficiencies or the
competitions of Na+ and Cl– ions with the essential nutrients. In this response, K+
concentration is important in order to maintain turgor pressure under stress
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conditions. It is mentioned that high ratios of K+:Na+ is important for the improvement of the plant resistance to salinity. The Ca2+ can also regulate the plant
resistance as a signal under salinity and drought (Hu and Schmidhalter 2005). Sun
et al. (2017) mentioned that AM fungi formation could alleviate the deleterious
effects of drought stress and eliminate growth retardation, therefore AM fungi
increase plants yield in semiarid and/or arid environments. In a previous study, it
was reported that AM fungi caused an increase of P uptake in dry soil (Neumann and
George 2004). The AM fungal species such as Septoglomus constrictum can have a
positive effect on the plant tolerance to drought stress by expression of some genes in
the roots and it was mentioned that inoculation with S. constrictum could have
higher stress tolerance to drought than non-mycorrhizal plants (Duc et al. 2018). In
another study, it was reported that AM fungi improved photosynthetic efficiency,
and proline, protein concentrations, and leaf gas exchanges. The symbiosis enhanced
C sequestration in drought and salinization affected regions and increased the
resistance of plants to drought and salinity (Zhang et al. 2018). Combination of
AM fungal species could also increase the tolerance to drought stress in addition to
abiotic stress tolerance (Oyewole et al. 2017). Therefore, AM fungi can be suggested
as a promising biological application for the alleviation of salt and drought stresses
(Zhang et al. 2018).
23.9.1 Arbuscular Mycorrhizal Fungi Increase Tolerance
to Salinity in Plant Species
The AM fungi can make symbiosis with various vegetable crops. This symbiosis can
lead to profitable and commercial agricultural and horticultural products. The success of the inoculation is strongly related to the properties of soil and genotypes of
AM fungi and host plants. Moreover, environmental conditions such as water supply
and nutrient content affect significantly AM fungi efficiency on their host plants.
Optimum combinations of AM fungi and crop plants should be well evaluated
according to soil properties and inoculation methods (Baum et al. 2015). The AM
fungi can have a potential to enhance sustainability and profitability of salt tolerance
of plants (Ashok Aggarwal et al. 2012). Hashem et al. (2018) indicated that AM
fungi inoculation could ameliorate the deleterious effects of salt stress by enhancing
biomass and pigment, phenols, proline contents, jasmonic acid, salicylic acid contents and antioxidant enzyme activities (Ashok Aggarwal et al. 2012).
Some families such as Apiaceae, Amaryllidaceae, Cucurbitaceae, Asteraceae,
Solanaceae, and Fabaceae have high mycorrhizal dependency. The AM fungi can
induce growth promotion and product quality of these host plants (Baum et al. 2015).
The AM fungi affect secondary metabolites and enhance the nutraceutical compounds in the host plants. In order to supply the global food demand, high sustainable horticultural products should be well developed. In this response, the AM fungi
is a promising environment friendly strategy (Garg and Bharti 2018). In addition to
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I. Ortas et al.
the applications of biofertilizers and biopesticides, AM fungi inoculation is a
promising strategy for future applications (Baum et al. 2015). Amanifar et al.
(2019) mentioned that AM fungi induced genes expression which have important
role in triterpene saponin glycyrrhizin biosynthesis and pharmaceutical contents
quality under salt stress and also enhanced growth, P and K uptake, higher K+/Na+
ratio, proline content, and membrane integrity. Therefore, AM fungi can eliminate
the deleterious effects of salt stress, and it can be used as a practical application for
exploiting the salinity in soils.
23.9.2 Crop Tolerance to Salinity and Drought and Relation
with Mycorrhizal Dependency
Drought and salt stress are usually developed at the same time and in the same area.
It is well known that plants use some root-rhizosphere mechanisms against drought
and salt stress. For instance, mycorrhizal fungi cooperation with the plant roots to
reduce the severity of salt and drought stresses. Especially plant genotypes which
depend on mycorrhizae have more tolerance than other genotypes. Under pot
culture, soil culture 94 bread wheat genotypes tolerance to water stress was tested
and it has been shown that drought stress tolerance of wheat was significantly
increased in the presence of mycorrhizae compared to drought stress tolerance in
the absence of mycorrhizae (Lehnert et al. 2018). Zrnic and Siric (2017) reported that
mycorrhiza inoculated plants are more tolerant to nutrients and water stress, soil
salinity, and heavy metals concentrations. It has been indicated that plant drought
tolerance is under the genes control. Fan and Liu (2011) reported that mRNA
abundance of four genes involved in reactive oxygen species homeostasis and
oxidative stress battling was higher in the AM plants when compared with the
non-AM plants. They indicated that possible drought-induced genes may enhance
the tolerance of AM plants to water deficit (Fan and Liu 2011).
23.9.2.1
Selective Interactions Between Different Species of Mycorrhizal
Fungi and Plant for Salt and Drought Tolerance
Salinity stress also causes water deficiency of plant tissue, and under low water
potential reduces growth by inhibiting cell division and cell expansion (Hasegawa
et al. 2000a). Under salinity stress plants can develop several mechanisms such as
(1) increasing the plant membrane thickness and enhancing the cell wall thickness
(Miransari 2016). (2) increasing the number of vesicles in plant cells. (3) mycorrhizal
infected unit can increase against salt damage to reduce the deleterious effects.
(4) Plant roots and mycorrhizae increase water efficiency and uptake. Miransari
(2016) indicates that especially under stress, the right combination of AM fungi and
the host plant may result in the highest level of efficiency. The AM fungi can
23
Do Mycorrhizal Fungi Enable Plants to Cope with Abiotic Stresses by Overcoming. . .
413
alleviate salt stress in mycorrhizal inoculated plant species through several mechanisms. Evelin et al. (2009) indicated the mechanisms of AM fungi which can employ
to enhance the salt tolerance of host plants such as enhanced nutrient acquisition
(P, N, Mg, and Ca), maintenance of the K+:Na+ ratio, biochemical changes (accumulation of proline, betaines, polyamines, carbohydrates, and antioxidants), physiological changes (photosynthetic efficiency, relative permeability, water status,
ABA accumulation, nodulation, and nitrogen fixation), molecular changes (the
expression of genes: PIP, Na+/H+ antiporters, Lsnced, Lslea, and LsP5CS) and
ultrastructural changes. Mycorrhizal infection seems that significantly control
many plant physiological and biochemical mechanisms. Under water stress conditions Wu and Xia (2006) shown that Citrus tangerine leaves and root parts have
higher K+ and Ca2+ than non-inoculated plants. Ortuno et al. (2018) reported that
when the substrate (silt and compost) was well-watered mycorrhizal inoculation
reduced the Na and increased phosphorus uptake of Cistus albidus plants.
It seems that mycorrhiza species have different effects on reducing salt effects on
plant growth. Estrada et al. (2013) treated three native AM fungi inoculation on
maize plant and the results showed a significant increase of K+ and reduced Na+
accumulation as compared to non-mycorrhizal plants, concomitantly with higher K+/
Na+ ratios in their tissues. The work of Estrada et al. (2013) has shown that when
native AM fungi isolates are used mycorrhizal benefits could be enhanced. One pot
experiment was conducted under drought stress conditions by Liu et al. (2018c) and
their results indicated that mycorrhizal inoculation stimulated greater root hair
growth of trifoliate orange that is independent on AM fungi species related with
mycorrhiza-modulated auxin synthesis and transport, which benefits the host plant to
enhance drought tolerance.
The study of (2006) suggests that R. versiforme mycorrhizal inoculation helps in
increments of enzymatic and non-enzymatic antioxidant productions which in turn
help plants to enhance drought tolerance. The AM fungi inoculated plants activities
of SOD, guaiacol peroxidase (G-POD) and glutathione reductase (GR), catalase
(CAT) and ascorbate peroxidase (APX) were significantly higher than in non-AM
roots and those higher enzymatic and non-enzymatic antioxidant productions would
partly alleviate oxidative damage (Wu et al. 2006). In another work, Lehnert et al.
(2018) have shown that genotypes differed in their response to mycorrhizae under
drought stress conditions. In many work AM fungi and PGPB dual inoculation have
a significant role in stimulation of plants growth, induce tolerance to drought, and
salinity (Tobar et al. 1994b). The PGPB (Pseudomonas aurantiaca, P.
extremorientalis) and R. irregularis inoculated wheat seed germination is better,
seedling growth and root elongation is better, salinity tolerance is high
(Egamberdieva and Kucharova 2009). Ruiz-Lozano et al. (2018) reported that a
symbiotic association with AM fungi resulted in salinity tolerance, CO2 utilization,
and enhanced growth in rice. Elhindi et al. (2017) have also documented the role of
AM fungi in reducing salt stress in sweet basil.
The AM fungi inoculation help pistachio growth in several ways such as growth,
success of grifting and water and nutrient uptake (Abbaspour et al. 2012). Also
Abbaspour et al. (2012)’results have shown that AM formation enhanced the
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drought tolerance of pistachio plants. On the other hand, the results of Bagheri et al.
(2011) have shown that the adverse effects of water stress were significantly reduced
by AM inoculation. Ferguson et al. (1998) indicated that mycorrhizal growth
promotion is generally observed in more stressful conditions. The results of
Shamshiri and Fattahi (2016) showed the depressing effect of salt stress on
mycorrhization extent and showed that the effect of salinity on colonization rate is
completely under the influence of host plant of pistachio (Pistacia vera) rootstocks.
Mycorrhiza species especially indigenous species have significant effects on salt
tolerance. The results of Paymaneh et al. (2019) shown that one of the indigenous
AMF communities from low-salinity soils conferred a significant tolerance of
pistachio to salinity in terms of maintaining its phosphorus acquisition upon the
stress.
23.9.3 Effects of AMF-Colonization on Survival Rate
of Horticultural Plants After Transplantation
to the Field Conditions
According to Rouphael et al. (2015) the AM fungi interfere with the phytohormone
balance of host plants, thereby influencing plant development (bioregulators) and
inducing tolerance to soil and environmental stresses (bioprotector) factors. In
general, under salinity and water stress conditions plant photosynthesis rate
decreases. Salinity also indirectly affect plant growth by affecting photosynthesis,
turgor, and enzyme activities of plant (Hasegawa et al. 2000b). Mycorrhizal inoculation is expected to stimulate the photosynthesis. Irrigation with saline water and
mycorrhizal inoculation in cucumber plants increased fresh and dry weight, proline,
electrolyte leakage, photosynthesis, and stomatal conductance (Haghighi et al.
2017). In many works, it has been shown that mycorrhiza inoculation increases
citrus, pistachio, maize, tomato, wheat, clover, lettuce plants tolerances to salinity
stress (Al-Karaki et al. 1998; Al-Khaliel 2010; Paymaneh et al. 2019; Feng et al.
2002; Satir et al. 2016). Mycorrhizal inoculated trifoliate orange seedlings displayed
significantly lower polyamine oxidase activity and diamine oxidase activity in leaves
and roots, irrespective of NaCl concentration (Wang et al. 2016). Also, in that work,
they have reported that mycorrhizal inoculated seedlings showed significantly higher
soluble protein concentration, ornithine decarboxylase, arginine decarboxylase, and
superoxide dismutase activity in leaves and roots.
23
Do Mycorrhizal Fungi Enable Plants to Cope with Abiotic Stresses by Overcoming. . .
415
23.9.4 Effect of Biochar and Mycorrhizae on Alleviation
of Salt and Drought
Biochar is an important multifunctional carbon material that can have effect biological, chemical, and physical properties of soil and improve the soil quality (Yu et al.
2019). Biochar is a pyrolyzed organic material as a soil amendment. The effects of
biochar on soil properties mainly depend on feedstock and pyrolysis conditions, pH,
nutrient contents, and ion exchange capacities of biochar (Hammer et al. 2015). The
longevity of biochar in the soil presents can act as bioremediation in comparison to
other organic materials such as compost and animal manure that more quickly break
down. It has shown that the application of biochar has a potential improvement on
the soil's physical and chemical properties and also on plant growth under abiotic
stress factors such as heat, drought, and salinity. Under salt-affected and waterstressed soil conditions biochar addition usually ameliorated the soil physicochemical and biological properties and also enhanced the plant physiological performance
as well as plant growth, yield, and nutrients uptake (Ali et al. 2017). Through this
way plants’ growth is better than non-biochar amendment control treatments
(Farhangi-Abriz and Torabian 2017). Farhangi-Abriz and Torabian (2017)
conducted, a pot experiment, under salt added soil conditions biochar amendment
reduced osmotic substances and oxidative stress of common bean plant.
It is a potential source of nutrient especially P recycling from the agricultural
wastes to enrich the soil fertility and quality. Biochar production and its soil
application as an amendment achieved promising results for crop production
(Dickinson et al. 2015; Ortas 2016), soil chemical and physical properties improvement and biochemical properties enhancement to facilitate soil biota (Puga et al.
2015), mitigation of climate change effects in a long-term experiment (Smith 2016)
and disposal of large scale waste biomass such as sludge wastes (Jeffery et al. 2015).
Also, biochar can be used for reducing the abiotic factors such as salt effects. Biochar
addition widely increases soil porosity and accordingly which can enhance the
potential of soil to boost plant growth (Mollinedo et al. 2016). Co-application of
biochar and mycorrhizae that promote plant growth and reduce the salt and drought
stress. In a pot experiment it has been demonstrated that biochar application together
with AM fungal inoculation resulted in an additional yield increase in Lactuca sativa
compared to each alone under nonsaline conditions (Hammer et al. 2015). A field
experiment was conducted by Thomas et al. (2013) that their results showed that
hardwood sawdust biochar addition significantly adsorbed great amounts of added
salt from the soil. And they reported that biochar application alone increased
biomass of P. vulgaris, with a 50% increase relative to untreated control plants.
And their results also indicated that biochar can ameliorate salt stress effects on
plants through salt absorption. Through biochar large surface and mycorrhizal
hyphae can be novel dual applications to mitigate the effects of salinization in
agricultural, urban, and contaminated soils.
Drought stress can cause a decline in colonization of AM fungi; however, biochar
amendment can enhance nutrient uptake, chlorophyll content, and photosynthesis
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I. Ortas et al.
efficiency and ameliorate significantly the deleterious effects of drought stress by
enhancing mycelium, arbuscule, and spore numbers, therefore the colonization can
increase under water deficiency (Hashem et al. 2019). Biochar treatments in the soils
can increase AM fungi colonization and improve the interaction between roots and
AM fungi (Yu et al. 2019). Biochar application can have beneficial effects on the
nutrient especially P uptake and enhance plant growth (Shen et al. 2016). In saline
soils biochar treatment may have benefits, the application can cause an increase in
AM fungi growth and inhibit the negative effects of salt stress (Hammer et al. 2015).
Zhang et al. (2019b) reported that combination of biochar amendment and AM fungi
inoculation could have positive effects on both nutrient uptake and decrease the
deleterious effects of heavy metal stress in polluted soils. The AM fungal spores
were isolated from different sodic soils in Central Europe results are indicating that
up to 80 % of all spores from the different sites gave one single PCR-pattern which
closely matched that of R. geosporum (Bothe 2012).
23.10
Conclusion
All literature records show that AM colonization may alleviate and compensate the
growth limitations imposed by salt and water deficiency stress conditions. It seems
that AM symbiosis improve plant nutrition by allowing the cells to regulate ions
more effectively. Also, inoculation can improve mineral nutrient uptake by availability or transport of mycorrhizal hyphae, thus enhancing salt tolerance. Under AM
inoculation conditions higher absorption of P, Zn, Cu, K, Ca, and Mg in plants under
saline conditions may improve growth rate, salt tolerance and suppress the adverse
effects of the salinity stress. Under saline conditions, there may be a displacement of
membrane-associated Ca by Na in roots membranes. Potassium also can have a
similar displacement with excess Na. Since AM inoculated plants have high Ca and
Mg, Ca may help to keep membrane integrity and protect host plants against salt
damage (Läuchli and Epstein 1990). Under salinity conditions, lower Na uptake and
the higher Mg absorption by mycorrhizal inoculation might be an important saltalleviation mechanism for salt tolerance of plant species. In salinity affected soils,
AM inoculation may also alleviate some of physiological mechanisms of plants.
Mycorrhiza inoculated plants may exudate more carbohydrate like cytokinin to
rhizosphere to enhance chloroplast development and increase the chlorophyll levels
in order to increase the photosynthesis values.
23
Do Mycorrhizal Fungi Enable Plants to Cope with Abiotic Stresses by Overcoming. . .
417
PNW 601-E • November 2007. D.A. Horneck, J.W. Ellsworth, B.G. Hopkins, D.M. Sullivan, and
R.G. Stevens. Managing salt-affected soils for crop production (Horneck et al. 2007)
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Chapter 24
Combined Use of Beneficial Bacteria
and Arbuscular Mycorrhizal Fungi
for the Biocontrol of Plant Cryptogamic
Diseases: Evidence, Methodology,
and Limits
Yuko Krzyzaniak, Maryline Magnin-Robert, Béatrice Randoux,
Joël Fontaine, and Anissa Lounès-Hadj Sahraoui
Abstract Keeping up agricultural production, while reducing chemical inputs,
under biotic and abiotic stresses, falls within a considerable challenge. Among the
solutions, developing more sustainable strategies to protect crops by using biocontrol agents is a promising alternative. Several beneficial microorganisms can limit
pathogen development by direct (antibiosis, competition) and/or indirect effects
(plant growth promotion and resistance induction) thanks to nutritional and hormonal balance regulations. A great deal of research articles have already reported the
beneficial roles of rhizobacteria or arbuscular mycorrhizal fungi (AMF) on reducing the development of fungal pathogens when applied alone, or on plant nutrition
when co-inoculated; while fewer of them reviewed the co-inoculation as a mean to
protect against fungal pathogens. Hence, this review aims to present recent developments on the effectiveness of the co-inoculation with beneficial bacteria and AMF
for fungal disease management. Firstly, main mechanisms beneath the beneficial
effects of bacterial or AMF inoculation alone on plants are recalled. Secondly,
results relating their combined inoculation on disease severity are presented and
sorted according to their outcomes (synergistic, neutral, or antagonistic interactions),
along with the possible underlying mechanisms. Finally, we gathered the main
methodologies employed in tripartite interaction experiment and brought to light
future challenges for practical use.
Keywords Arbuscular mycorrhizal fungi · Plant growth-promoting rhizobacteria ·
Mycorrhiza helper bacteria · Tripartite interactions · Fungal diseases · Biocontrol
Y. Krzyzaniak · M. Magnin-Robert · B. Randoux · J. Fontaine · A. Lounès-Hadj Sahraoui (*)
Université du Littoral Côte d’Opale, Unité de Chimie Environnementale et Interactions sur le
Vivant (UCEIV) UR 4492, Calais Cedex, France
e-mail: lounes@univ-littoral.fr
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_24
429
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24.1
Y. Krzyzaniak et al.
Introduction
Protecting agricultural crops from cryptogamic diseases generally requires a massive
use of phytosanitary products. However, unintentional negative impacts on the
environment and human health (Gill and Garg 2014) have been building up, along
with the increasing number of fungicide resistant strains of pathogens (Rupp et al.
2017), making crop management more and more challengeable (Brauer et al. 2019).
This led several actors to think out complementary strategies to keep on protecting
crops by creating new forms of agroecosystems that would both respect biological
processes, while maintaining a sufficient income to the farmers (Gianinazzi et al.
2010). Along with a more restrictive regulatory frame such as the European Union
directive 2009/128/EC on the sustainable use of pesticide in pest management,
Member States are now compelled to emphasize the use of alternative
(non-synthetic) products in pest control, by launching national programs which
implement the principles of Integrated Pest Management (IPM) for instance. Indeed,
according to the Food and Agriculture Organization of the United Nations : “IPM
means the careful consideration of all available pest control techniques and subsequent integration of appropriate measures that limit the development of pest
populations and keep pesticides and other technical interventions to levels that are
economically justified and reduce or minimize risks to human health and the
environment. IPM emphasizes the growth of a healthy crop with the least possible
disruption to agro-ecosystems and encourages natural pest control mechanisms”
(FAO 2016).
One means among many, is the use of biological control agents. According to
Heimpel and Mill, biological control (also called biocontrol) is “simultaneously a
natural phenomenon, a pest management strategy and a scientific discipline”
(Heimpel and Mills 2017). It allows controlling pests and diseases caused by
fungi, bacteria, or viruses, with the help of other organisms or their derivative
products. Biocontrol relies on natural mechanisms occurring during plant-microbe
interactions (Flint et al. 1998; Alabouvette et al. 2006; Barratt et al. 2018). Based on
predation, parasitism, herbivory, antibiosis, competition, or other natural mechanisms, it can maintain the pest population below the economic threshold level
(Benjamin and Wesseler 2016). More specifically in the case of fungal disease
management, biocontrol consists in using living fungal (Perazzolli et al. 2011;
Mustafa et al. 2017) or bacterial (Bach et al. 2016) microorganisms; but also
microorganism- (Li et al. 2019) or macroorganism-derived compounds such as
polysaccharides from plants, algae, or crustaceans (Trouvelot et al. 2014; El Guilli
et al. 2016; De Bona et al. 2019). Among soil microorganisms, Arbuscular Mycorrhiza Fungi (AMF) and the beneficial rhizospheric bacteria such as the Plant GrowthPromoting Rhizobacteria (PGPR) attract much interest as typical models used in
laboratories for this past century, with the hope to improve crop health.
As several articles report successful disease severity reduction in response to a
singly inoculated microorganism, the combination with two of them is also expected
to be more effective than individual application, via a synergistic or additive effect
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(Saldajeno and Hyakumachi 2011a). Indeed, on the contrary to sterilized substrates
used in laboratories, plant roots associated with mycorrhizal fungi in field conditions
or their natural environments encounter many other organisms, such as bacteria.
These latter can induce neutral, positive, or negative interactions toward mycorrhizal
fungi (Bonfante and Anca 2009). However, it was pointed out that mycorrhizal fungi
would rather attract more bacterial allies than enemies (Frey-Klett and Garbaye
2005), comforting the idea that synergistic interaction is likely to happen. Evidence
reporting positive outcomes from the combined inoculation of AMF and beneficial
rhizosphere microbiome are growing, and have been well reviewed. For instance,
some bacteria are able to stimulate AMF growth and mycorrhizal colonization rate:
this is the case of some bacteria called Mycorrhiza Helper Bacteria, or MHB
(Garbaye 1994; Frey-Klett et al. 2007).
Thus, assessing the effects of a tripartite interaction, between AMF, bacteria, and
host plant, is a recent but a very interest-drawing issue. The underlying mechanisms
in this triangle remain still uncovered, yet some elements have been brought to light
(Deveau and Labbé 2016). The combined inoculation with AMF and bacteria was
reported to have numerous positive effects, leading to enhanced plant nutritional
status and growth as described in many reviews (Artursson et al. 2006; Nadeem et al.
2014); and also on disease protection, but to a lesser extent (Baysal and Silme 2019).
However, reviews that focus essentially on the effects on plant disease protection in
response to such interaction are still scarce. Thus, in the present review, we will
target the bibliography on two types of soil beneficial microorganisms, the AMF,
and the beneficial rhizospheric bacteria, and report the state-of-the-art knowledge
about the potentials of the combined use of these latter in plant protection, with
recent results concerning aerial or soil borne fungal diseases. More particularly, we
aimed to provide a comparative study of the methodology employed to set up an
experimental design, with the view to make available a practical “starters’ guide” for
those who initiate research projects on tripartite interactions. A more critical section
is dedicated to highlight difficulties and future perspectives linked to the development of such a strategy in field experiments.
24.2
Beneficial Microorganisms in Plant Health
Plants are complex hubs of microscopic life and may suffer from pathogen attacks,
as well as host several communities without any harm nor benefits, by actively
gathering and selecting their own microbiome (Jones et al. 2019). It is now largely
accepted that the plant physiology and performance can be affected by the hostassociated microbial community; and more widely, plant ecology cannot be considered on its own anymore, but rather understood as a whole (with its associated
organisms), called the holobiont (Vandenkoornhuyse et al. 2015; Agler et al. 2016).
Among the microorganisms living in, or on the different compartments of the host,
some maintain a mutualistic relationship, providing benefits for the plant. These
include a wide variety of microorganisms- bacteria or fungi- and special attention
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was given for those living in the rhizosphere in the past century (Desai et al. 2016).
They are referred as Plant Growth-Promoting Microorganisms/Rhizobacteria/Fungi,
or PGPM/R/F (Lugtenberg and Kamilova 2009; Kundan et al. 2015). Yet the
denomination is not always adapted, because, over time and discoveries, some of
them turn out to both be able to stimulate plant growth and protect them from
diseases; therefore, the term Plant-Health Promoting Microorganism (PHPM) is also
interestingly used by some authors (Hayat et al. 2010).
Among the fungi, the upmost studied ones for their protective effect are, for
example, some species of Trichoderma, Aspergillus, Pythium, Fusarium,
Coniothyrium (Singh et al. 2018); but also the ectomycorrhizal fungi (Mohan et al.
2015), or the AMF (Comby et al. 2017), which will be detailed in this review.
Among the bacteria, the most reported in the literature are mainly the strains of
Bacillus, Pseudomonas, or symbiotic N2 fixing bacteria (Rhizobia species), the freeliving nitrogen-fixing PGPR such as some species of Azospirillum, Azotobacter, or
Burkholderia (Desai et al. 2016).
24.2.1 Arbuscular Mycorrhizal Fungi (AMF)
24.2.1.1
Main Beneficial Effects of AMF on Plants
Among beneficial associations existing between plants and microorganisms, the
arbuscular mycorrhizal symbiosis is probably the oldest relationship, back to more
than 400 millions years ago (Karandashov and Bucher 2005), and the most widespread spatially and phylogenetically, throughout the plant kingdom (Wang and Qiu
2006). AMF are obligate biotrophic fungi from the phylum Glomeromycota, which
are able to establish a symbiotic relation with over 80% of land plant species
(Gianinazzi et al. 2010). This mutualistic interaction is based on nutrient exchanges
between the plant and the fungus. While the host plant provides carbohydrates up to
20% of its fixed carbon (Parniske 2008), the AMF facilitate the uptake of water, and
some major elements such as nitrogen (N), phosphorus (P), potassium (K), sulfur (S),
or trace elements like iron, copper, and zinc (Smith and Read 2008; Lehmann and
Rillig 2015; Wipf et al. 2019).
This interaction results in various beneficial outcomes at different scales and these
latter were already widely reviewed by many authors (Miransari 2010; Gianinazzi
et al. 2010; Rouphael et al. 2015). Briefly, these include, among many others: a
better plant growth and development, a better stabilization of soil aggregates against
erosion (Rillig and Mummey 2006; Bedini et al. 2009), an increased tolerance to
abiotic stress (Lenoir et al. 2016; Begum et al. 2019), such as salt (Sannazzaro et al.
2006), drought (Doubková et al. 2013), or pollutants (Hildebrandt et al. 2007). AMF
inoculation can also confer a better resistance to biotic stresses (Comby et al. 2017).
Indeed, more specifically, the effectiveness of plant protection in response to AMF
inoculation against fungal pathogens has been increasingly more reported, along
with the interests to develop new alternatives to reduce chemical inputs.
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In some cases, the inoculation with AMF alone can improve host tolerance to
pathogens (Wehner et al. 2010), but studies about plant protection by AMF target
predominantly soil-borne fungal pathogens. Classically, several direct or indirect
modes of action are reported in many studies (Azcón-Aguilar et al. 2002; Vierheilig
et al. 2008).
24.2.1.2
24.2.1.2.1
Underlying Mechanisms in Plant Protection by AMF
Direct Modes of Action Involved in Reducing Pathogen
Development
There are several modes of action described in the literature, and some of them are
specific for soil-borne pathogens. Indeed, in that case, the two protagonists may
share the same space around the rhizosphere, and can therefore directly interact.
Concerning direct mechanisms, competition for infection sites can occur. For example, a Phytophthora was unable to penetrate into the roots that were already
containing Funneliformis mosseae arbuscules, seat of nutrient exchanges between
plant and fungal partners, in the cortex cells, leading to a reduced disease severity.
Authors concluded that effects on numbers of infection loci are one of the mechanisms leading to the protection of tomato against this pathogen (Vigo et al. 2000).
On the contrary, it was shown that Rhizophagus irregularis inoculation could not
reduce the development of Ilyonectria liriodendra, as AMF inoculation increased
the abundance of the pathogen within grapevine rootstocks. Reciprocally, the presence of the pathogen resulted in increased AMF abundance compared to AMF
inoculation alone (Holland et al. 2019). Authors hypothesized that plants with
AMF alone may not have been placed in stressful conditions enough for
mycorrhization, while the infection with the pathogen could have triggered a sufficient stress level to allow AMF colonization.
Competition for nutrient sources such as plant photosynthates can also be
involved (Lerat et al. 2003), as both AMF and soil-borne pathogenic fungi may
depend on the same carbon sources; however, limited data support this mechanism
(Smith and Read 2008).
Other studies typically mention direct inhibition as a potential mode of action, but
reports on the production of antimicrobial compounds by AMF are not common.
Some works report unidentified antimicrobial substances produced by the
extraradical mycelium of an R. irregularis, reducing conidial germination of Fusarium oxysporum f. sp. chrysanthemi (Filion et al. 1999). Antibiosis is often cited, but
more supposed than demonstrated in literature (Cameron et al. 2013; Schouteden
et al. 2015).
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Y. Krzyzaniak et al.
Indirect Modes of Action Involved in Reducing Pathogen
Development
Still in the context of soil-borne pathogen—AMF interaction, indirect modes of
action that are mediated via the host plant can also explain the reduction of pathogen
development. Morphological changes of the root in response to AMF are indeed
likely to be involved, but with contrasting results. On the one hand, authors observed
that an increased root lignification induced by AMF could slow down the penetration
by the pathogen (Dugassa et al. 1996); while on the other hand, increased rootbranching induced by AMF provided new infection sites for the pathogen (Norman
et al. 1996).
Also, main reviews suggest that a better protection is correlated with a compensation of damages by an increased biomass, thanks to an improved nutrient status
(Azcón-Aguilar et al. 2002). Research articles putting specifically this mechanism
under light in plant–fungal pathogens context are actually scarce, on the contrary to
those in the context of herbivory. Indeed, compensation regrowth was, for example,
evidenced especially for mycorrhizal tallgrass prairie plants, in response to grasshopper defoliation (Kula et al. 2005). However, this hypothesis was invalidated in
some cases, where the enhancement of Astragalus adsurgens biomass by AMF was
also shown to increase susceptibility against powdery mildew (Liu et al. 2018).
Another indirect mechanism could occur through the activation of plant defenses,
which can interestingly lead to resistance against foliar fungal pathogens. The
induction of plant defense mechanisms upon AMF colonization causes significant
transcriptional and hormonal changes (Pieterse et al. 2014), leading to MycorrhizaInduced Resistance or MIR (Jung et al. 2012). This establishes a local or systemic
resistance, in the roots and/or on the aerial parts (Liu et al. 2007). As an example,
an inoculation of F. mosseae on tomato caused a local accumulation of phenolic
compounds in roots in response to Phytophthora nicotianae var. parasitica attack
revealed by a strong autofluorescence; leading to a reduced disease severity compared to control (Cordier et al. 1996). Song and collaborators showed that mycorrhizal inoculation alone did not impact the accumulation of transcripts of most
defense-related genes tested in tomato plants. However, upon Alternaria solani
attack on AMF-inoculated plants, authors noted a strong induction of three genes
encoding pathogenesis-related proteins, PR1, PR2, and PR3, as well as other
defense-related genes encoding lipoxygenases (LOX), allene oxide cyclase (AOC),
and phenylalanine ammonia-lyase (PAL) in leaves. The induction of defense
responses in AMF pre-inoculated plants was much higher and more rapid than that
in un-inoculated plants in response to fungal challenge (Song et al. 2015), implying
that systemic resistance was associated with a priming of plant defenses (Conrath
et al. 2015). Another study also reported a systemic resistance of wheat against
powdery mildew in AMF-inoculated plants, along with the upregulation of genes
encoding a PAL, a chitinase, and a peroxidase, however only in the absence of the
pathogen, suggesting that it was not related to a primed state of wheat leaves
(Mustafa et al. 2017).
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Most importantly, AMF-mediated protection can result from either one or a
combination of different mechanisms cited above (Whipps 2004; Pozo et al.
2013). Multiple modes of actions can be an advantage in terms of crop protection,
since if one mode of action is ineffective under specific conditions, the others can
take over and maintain host protection. However, it also implies that protection
efficacy can be inconsistent over time, since complex factors affect the pathosystem
(Vierheilig et al. 2008).
24.2.2 Beneficial Bacteria: Definition Boundaries and Modes
of Action
24.2.2.1
How to Define a Beneficial Bacteria?
On the contrary to AMF, there are no phylogenetic levels to gather all beneficial
bacterial in one group; and terms to define them are prolific. The different names
given for special bacteria aim to emphasize one feature of their life, such as (1) the
spheres where they live, which is the case for free-living bacteria, root-associated
ecto- or endophytes bacteria (Ryan et al. 2008), or even for AMF spore-associated
bacteria (Bonfante 2003); (2) their beneficial functions that are known so far, such as
the PGPR, able to stimulate plant growth (Compant et al. 2010), or Induced Systemic
Resistance (ISR) bacteria able to stimulate plant resistance against pathogens and
pests (Pieterse et al. 2014), or MHB able to facilitate mycorrhiza establishment
(Deveau and Labbé 2016); or even bacteria with very specific abilities, like the case
of phosphate-solubilizing bacteria (Kalayu 2019). Therefore, the more research is
carried out, the more features are discovered, making sometimes overlay
between them.
In the light of these facts, amalgams can rapidly be made, for example, by
automatically associating endophytes as beneficial bacteria, however, the definition
of endophytes does not specify their functional relationship, and they can exist also
as latent pathogens, saprotrophs, or mutualistic associations (Fesel and Zuccaro
2016; Khare et al. 2018). Hence, in order to be as inclusive as possible within this
review, we will define as “beneficial bacteria,” not only bacteria positively impacting
plant growth, but rather all those who have positive outcomes in any aspects within
the tripartite interaction, i.e., on plant (growth and defense), and AMF (growth and
function).
24.2.2.2
Underlying Mechanisms in Plant Protection by Beneficial
Bacteria
It is possible to class bacteria by their positive effects observed on plants
(Lugtenberg and Kamilova 2009). Authors distinguish “direct” PGPR, which
directly promotes growth in noninfectious context; and the “indirect” PGPR,
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which can result in growth promotion because they are able to reduce harm caused
by phytopathogens, acting therefore as biocontrol agents. Among direct PGPR,
many categories of bacteria were then defined, including “Biofertilizers,”
“Phytostimulators,” “Stress Controllers,” “Rhizoremediators,” etc. (Lugtenberg
and Kamilova 2009). Nonetheless, some bacteria can have multiple roles (Martínez-Viveros et al. 2010). Indeed, some bacteria such as Azotobacter paspali are well
known to both fix N2 and to produce plant growth factors like indole-3-acetic acid
(IAA) (Abbass and Okon 1993); while others are able to both inhibit soil-borne
pathogens development and to stimulate plant growth as it was shown for Pseudomonas oryzihabitans in rice seedlings (Verma et al. 2018). Therefore, from one
bacterial activity (such as metabolite production or nutrient uptake), many effects
can be observed; and conversely, one effect can result from an association of many
activities, making the classification difficult. Regarding plant protection issues
against fungal pathogens only, we will focus on the mechanisms to direct and
indirect effects toward the pathogen.
24.2.2.2.1
Direct Modes of Action Involved in Reducing Pathogen
Development
Bacteria are able to produce metabolites, which are small molecular weight molecules (Davies 2013). There is a vast chemical diversity, including amino acids,
vitamins, pigments, hormones, organic acids, volatile organic compounds, etc.
(Kanchiswamy et al. 2015; Singh et al. 2017). Among these metabolites, some
turn out to have bioactive properties with direct antimicrobial activities and therefore
can have antagonistic effects towards a pathogen. Classical compounds cited in the
literature are, for example, hydrogen cyanide, phenazines, or pyrrolnitrin (Compant
et al. 2010; Hayat et al. 2010). More recently, bacterial rhamnolipids or cyclic
lipopeptides attracted interest because they were found to have multiple roles, such
as both antimicrobial and plant defense eliciting effects (Borah et al. 2016; Mejri
et al. 2018). Some bacteria also synthesize enzymes such as cellulases, chitinases
or protein and lipid degrading enzymes that have the potential to lyse the cell walls of
fungal pathogens (Kundan et al. 2015).
Another direct effect involves competition for nutrient sources and ecological
niches, similarly to AMF modes of action cited above. Indeed, as bacteria can act as
biofertilizers, they can directly provide readily available nutrients for the plant
(Fuentes-Ramirez and Caballero-Mellado 2006). This is, for example, the case of
Rhizobia nodules in leguminous plant roots such as pea, peanut, alfalfa. Atmospheric N2 is converted by bacterial nitrogenases into ammonia, which can be used
by the plant as a nitrogen source, in contrast to N2 (Van Rhijn and Vanderleyden
1995). Also, low levels of phosphate can limit plant development, but some bacteria
are able to solubilize phosphate from inorganic or organic bound phosphate with
bacterial phosphatases and phytases, releasing an assimilable form of phosphorus for
the plant (Rodríguez and Fraga 1999; Kalayu 2019). Therefore, uptake of nutrients
of great biological interest can constitute a source of competition toward pathogens,
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in particular for those which require exogenous nutrients to germinate, such as amino
acids for Botrytis cinerea (Yoder and Whalen 1975). The competition is not only for
nitrogen but also for transition metals such as iron, zinc, or manganese (Fones and
Preston 2013). Strains of Pseudomonas can produce siderophores like pyoverdine or
pseudobactin that bind to iron and facilitate its assimilation. It may therefore limit
iron availability for pathogens such as F. oxysporum sp., resulting in pathogen
growth reduction (Duijff et al. 1994; Arya et al. 2018). In parallel, bacteria engage
physical interactions with their close environment, which have several consequences
in the context of pathogen growth limitation. Along the competition for nutrients,
competition for niches also occurs around root colonization sites, and is globally
known as CNN, as for “competition for nutrients and niches” (Latha et al. 2019).
Pliego and collaborators demonstrated that two similar Pseudomonas alcaligenes
strains, selected by their ability to colonize avocado roots, use strongly different root
colonization strategies. This suggested that in addition to the total bacterial root
colonization level, the sites occupied on the root are important for their protective
level against avocado root rot caused by Rosellinia necatrix (Pliego et al. 2008).
Physical interactions also include modes of action such as hyperparasitism. It is
the case of a special direct interaction between two organisms, in which one of them
is gaining nutrients from the other. If the latter is considered as a parasite or a
pathogen, the interaction is then defined as hyperparasitism or predation (Köhl et al.
2019). Bacterial hyperparasitism has not been widely documented. Indeed, references report mainly confrontation against pathogenic bacteria (McNeely et al. 2017)
or parasitic nematodes, but it is not excluded that pathogenic fungi can also be
targets.
24.2.2.2.2
Indirect Modes of Action Involved in Reducing Pathogen
Development
Bacterial inoculation can also limit pathogen development by other indirect mechanisms, mediated by the host plant defense regulation. In some cases, interaction of
bacteria or their metabolites with plant roots can lead to ISR (Pieterse et al. 2014).
ISR-inducing bacteria produce elicitors, or Microbial-Associated Molecular Patterns
(MAMPs), that involve a vast chemical diversity, including lipopolysaccharides,
flagellins; but also metabolites such as 2,4-diacetylphlotoglucinol, pyocyanin (also
having antimicrobial effects), N-acyl homoserine lactones, iron-regulated
siderophores and biosurfactants (as reviewed in De Vleesschauwer and Höfte
2009). Moreover, volatile organic compounds are also able to elicit ISR, as it was
previously reported for the volatiles 2R,3R-butanediol produced by a Bacillus
subtilis, and a C13 volatile liberated by Paenibacillus polymyxa in Arabidopsis
(Ryu et al. 2004; Lee et al. 2012).
Pathogen development can also be limited by other indirect mechanisms, involving plant growth and metabolism regulation. Many rhizospheric bacteria are known
to produce hormones, such as auxins (in particular, IAA), ethylene, cytokinins,
gibberellins, abscisic acid, salicylic acid (SA) and jasmonic acid (JA). These are
438
Y. Krzyzaniak et al.
either excreted for root uptake or hormonal balance manipulation in the host plant, to
stimulate growth and alleviate stress responses (Tsukanova et al. 2017). Many of
those hormones are involved in root/shoot growth, production of root exudates, but
also in defense-related pathways (Rosier et al. 2018). As an example, Paenibacillus
lentimorbus B-30488 was shown to produce aminocyclopropane-1-carboxylase
(ACC) deaminase -an enzyme involved in reducing ethylene production in
plants leading to an enhanced tolerance against southern blight disease in tomato
caused by Scelerotium rolfsii. The inoculated plants showed a modulation of the
ethylene pathway and antioxidant enzyme activities; while systemic tolerance could
be confirmed by overexpression of defense-related genes such as PR1, PR2, PR4, or
PR7 (Dixit et al. 2016). However, hormonal changes induced by PGPR is not
consistently correlated to an increased resistance toward a pathogen, as antagonistic
signaling molecules such as JA, SA, and IAA are part of multiple crossroads.
Although not a PGPR, but rather in the context of hormonal change inductors, the
biotrophic pathogen Pseudomonas syringae pv. tomato, which induces SA-mediated
defense, made Arabidopsis more susceptible to the necrotrophic pathogen,
Alternaria brassicicola, by suppressing the JA signaling pathway (Spoel et al.
2007). These data suggest the possibility that a prior inoculation with a microorganism with one lifestyle might modulate, or even compromise the ability of the plant to
defend itself against pathogens with another lifestyle, due to hormonal antagonism
(Walters et al. 2013).
Finally, another indirect mechanism would be for the case of interactions between
soil microorganisms and their impact on plant health. There is increasingly more
evidence for bacterial strains reported as promoters of AMF symbiosis, called MHB
(Garbaye 1994; Duponnois 2006; Frey-Klett et al. 2007). MHB promote the establishment of mycorrhizal symbiosis by several means, including: (1) the improvement
of root receptiveness of the fungus; (2) the interference in plant-fungus recognition
and symbiosis establishment; (3) the promotion of AMF propagule germination, as
well as spore survival and mycelium growth; (4) the modification of soil chemical
properties for better conduciveness to the fungus (for review: Deveau and Labbé
2016). So, MHB can have two levels of impacts: on the AMF itself, and on its
biological activities, such as its protection abilities against plant diseases.
24.3
Plant Protection Against Fungal Diseases Using AMF
and Bacteria Co-Inoculation: Several Scenarios
and Possible Mechanisms
The concept of co-inoculation of plants with AMF and bacteria was first reviewed
about 30 years ago (Garbaye 1994), and some authors have overviewed these
experiments since then (Frey-Klett et al. 2007; Deveau and Labbé 2016). The
cited literature was mostly focused on evaluating the impact of the co-inoculation
on (1) the main stages of AMF life cycle in vitro (spore germination, hyphal
24
Combined Use of Beneficial Bacteria and Arbuscular Mycorrhizal Fungi for the. . .
439
elongation, root colonization) and (2) plant growth and development parameters
such as aerial and root biomass, yield, mineral content (Saldajeno and Hyakumachi
2011b). Indeed, the literature showing results of protection efficacy against diseases
in response to the combined inoculation of AMF and bacteria is still in short supply,
and the underlying mechanisms are often suggested more than brought out, undoubtedly due to the complexity to lead these kinds of multifactorial complex
experiments.
24.3.1 Common Base Grounds Between Studies
After gathering data from the last 30 years, studies mostly report protection assays of
co-inoculation against soil-borne diseases. They represent more than 80% of
research content cited so far in this review, against less than 20% focused on
aerial-borne pathogens. Among these, more than half of the content is reporting
efficacies against root rots or wilts, caused essentially by Fusarium or Rhizoctonia
species, and to a lesser extent, against Verticillium, Pythium, or Phytophthora
species. The AMF species frequently tested are F. mosseae (previously known as
Glomus mosseae), R. irregularis (previously known as Glomus intraradices), Glomus fasciculatum, and some Gigaspora sp., as commercial inoculants or produced
by the research laboratories themself. The bacterial inoculants are either sampled
from the corresponding AMF spores, host plant or simply formulated as a commercial inoculant. The main bacteria which are used as inoculant belong to Pseudomonas, Rhizobium, or Bacillus genus. The main studies were gathered in Table 24.1 and
were reported according to the efficacy of protection obtained in response to (1) the
combined inoculation; compared to (2) the AMF alone and (3) to the bacteria alone.
In this way, it is possible to understand which microorganism contributes the most to
the resulting protection; or if the co-inoculation leads to a synergistic, or perhaps
antagonistic type of interaction.
First, all of the articles found have in common that the dual inoculation protects
significantly against the tested pathogen, compared to the double negative control
(without any beneficial microorganism). Few cases of “negative” interactions are
found in the literature, maybe and especially because these data are not accepted, or
unfortunately not submitted due to complexity and variability of these interactions.
Also, these articles have also in common that authors used at least already one wellprotective microorganism (AMF or bacteria) in the interaction. But their combined
level of protection resulted in different ways. Hence, with these different possibilities, we could categorize the studies according to three types of outcomes so far. For
further sections, we defined as a protection “gain” when the co-inoculation resulted
in better protection as compared to one of a single inoculant (AMF or bacteria).
Scenario
“Full-gain”
Host plant
Soybean (Glycine max L.)
AMF
Funneliformis mosseae
Bacteria
Bradyrhizobium
sp. BXYD3
AMF
+
Bacteria
+
AMF
+ Bacteria
+++
Common bean
(Phaseolus
vulgaris L.)
Coleus (Coleus
forskohlii Briq.)
Glomus sinuosum,
Gigaspora albida
Pseudomonas
fluorescens
+
+
+++
Neeraj and
Singh (2011)
Glomus fasciculatum
Pseudomonas
monteilii
+
+
+++
Singh et al.
(2013)
G. fasciculatum
P. fluorescens
strain VuPf1
+
+
+++
Basu and
Santhaguru
(2009)
Fusarium wilt
(F. oxysporum f. sp.
Cubense)
Mung bean
(Vigna radiata
L. cv. Wilczek
VA02)
Banana (Musa
acuminata Colla
cv. Grand Naine)
25 different isolates
+
+
+++
Sumathi and
Thangavelu
(2016)
Fusarium wilt
(F. oxysporum
FOPV001)
Papaya (Carica
papaya L. cv.
Maradol)
+
++
+++
HernándezMontiel et al.
(2013)
Fusarium root rot
(Fusarium solani)
Geranium (Pelargonium
graveolens
L’hér)
AMF complex (MTZ01) :
Rhizophagus irregularis,
F. mosseae, Glomus
etunicatum, G. albida
F. mosseae
29 isolates of
AMF sporeassociated
bacteria
Pseudomonas
sp. (PPV3)
+
++
+++
Haggag et al.
(2001)
Disease (causal agent)
Red crown rot
(Cylindrocladium
parasiticum)
Root rot disease (Rhizoctonia solani)
Root rot and wilt
(Fusarium
chlamydosporum and
Ralstonia
solanacearum)
Fusarium wilt (Fusarium oxysporum) root
rot disease (R. solani)
References
Gao et al.
(2012)
Y. Krzyzaniak et al.
Bacillus subtilis
440
Table 24.1 Table summarizing the main studies reported in the literature concerning plant co-inoculation with arbuscular mycorrhizal fungi (AMF) and
beneficial bacteria for the control of fungal pathogens
Fusarium wilt (F.
oxysporum f. sp.
lycopersici)
“No gain,
no loss”
Spring black stem and
leaf spot (Phoma
medicaginis)
Basal stem rot
(Ganoderma
boninense)
Pythium root
damping-off (Pythium
ultimum)
Verticillium wilt
(Verticillium dahliae)
Fusarium root rot
(F. solani)
Geranium
(P. graveolens
L’hér.)
Tomato
(Lycopersicon
esculentum
Mill.)
Tomato
(Lycopersicon
esculentum Mill.
cv Pant Tomato3)
Lucerne
(Medicago
sativa L.)
Oil palm (Elaeis
guineensis Jacq.)
F. mosseae
B. subtilis
+
++
+++
Haggag et al.
(2001)
F.mosseae
Paenibacillus
sp. strain B2
++
+
+++
Budi et al.
(1999)
R. irregularis
P.fluorescens
++
+
+++
Srivastava
et al. (2010)
F.mosseae
Sinorhizobium
medicae
++
+
+++
Gao et al.
(2018)
R. irregularis UT126, Glomus clarum BR152B
Pseudomonas
aeruginosa U
+
Not
tested
+++
Sundram
et al. (2015)
Cucumber
(Cucumeris
sativus L.)
Strawberry
(Fragaria x
ananassa
cv. Selva)
Common bean
(Phaseolus
vulgaris L.)
R. irregularis
Burkholderia
cepacia
+
+
+
Larsen et al.
(2003)
Vaminoc (commercial mix)
: Glomus caledonium,
G. fasciculatum, F. mossae
Commercial
inoculum :
B. subtilis
FZB24
Rhizobium
leguminosarum
pv. phaseoli
+
+
+
Tahmatsidou
et al. (2006)
++
+
++
Hassan Dar
et al. (1997)
F. mosseae
(continued)
Combined Use of Beneficial Bacteria and Arbuscular Mycorrhizal Fungi for the. . .
Charcoal rot
(Macrophomina
phaseolina)
Buckeye rot
(Phytophthora
parasitica)
24
“Full-gain”
(continued)
441
Scenario
“Partial
loss”
442
Table 24.1 (continued)
Disease (causal agent)
Fusarium wilt
(F. oxysporum f. sp.
lycopersici)
Host plant
Tomato
(Lycopersicon
esculentum
Mill.)
AMF
R. irregularis
Powdery mildew
(Sphaerotheca
macularis)
Strawberry
(Fragaria vesca
L. cv Elvira)
Vaminoc®:G. caledonium,
G. fasciculatum,
F. mosseae
Bacteria
P. fluorescens,
Pseudomonas
putida,
Enterobacter
cloacae
Commercial
inoculum :
B. subtilis
FZB24
AMF
+
Bacteria
++
AMF
+ Bacteria
+
+
++
+
References
Akköprü and
Demir (2005)
Lowe et al.
(2012)
The studies are classified according to several protection gain scenarios: “full-gain,” “no loss, no gain,” “partial loss.” Protection rates assigned with different
signs “+, ++ or +++” should be compared within the same study. A variable number of “+” signs suggests that protection rates are different between the
conditions tested within the same case study: AMF alone or bacteria alone and AMF/bacteria co-inoculation. For the co-inoculation column, protection rates are
assigned according to the gain over those obtained with simple inoculants: “+++” means a net gain in protection over those obtained compared to simple
inoculation; “++” means a comparable level of protection, as the most protective of the two inoculants; “+” means a comparable level of protection, as the least
protective of the two inoculants
Y. Krzyzaniak et al.
24
Combined Use of Beneficial Bacteria and Arbuscular Mycorrhizal Fungi for the. . .
443
24.3.2 “Full-Gain” Scenario
24.3.2.1
Evidence for Protection Gain with a Dual Inoculation
In this case, AMF or bacteria alone protects partially the plant against the disease, at
the same or different levels, and the combined inoculation leads to a better protection
level (slight or strong). Many authors used F. mosseae as AMF inoculant: (1) combined with a Bacillus subtilis, the protection was better against Fusarium solani or
Macrophomina phaseolina of geraniums than with single inoculants (Haggag et al.
2001); (2) with a Paenibacillus sp. strain B2, the protection level against
Phytophtora parasitica on tomato plants was improved (Budi et al. 1999);
(3) with Sinorhizobium medicae, Phoma medicaginis development was better
reduced on Medicago sativa compared to AMF or bacteria alone (Gao et al. 2018).
The combination of P. fluorescens with R. irregularis leads to a stronger protection of tomato against F. oxysporum f. sp. lycopersici than the microorganisms used
alone (Srivastava et al. 2010). Similarly, Singh and collaborators observed that the
combination of Pseudomonas monteilii with Glomus fasciculatum reduced the
severity of root rot and wilt (Fusarium chlamydosporum and Ralstonia
solanacearum) by 63% on Coleus forskohlii, while the AMF alone could already
reduce by 56% (Singh et al. 2013). The same AMF in combination with Pseudomonas fluorescens strain VuPf1 also reduced disease severity caused by
F. oxysporum or Rhizoctonia solani on mung bean plants, by increasing the plant
vigour index by two times higher than the AMF alone (Basu and Santhaguru 2009).
Other studies tested a mix of different AMF inoculants with a bacteria.
Co-inoculation of Glomus sinuosum and Gigaspora albida with P. fluorescens could
better reduce root rot disease caused by R. solani on common bean plants, compared to
microbial agents alone (Neeraj and Singh 2011). An AMF complex (MTZ01) composed of three species of Glomus and one Gigaspora combined with a Pseudomonas
sp. (PPV3) could better protect against fusarium wilt on papaya plants (F. oxysporum),
than AMF or bacteria used singly (Hernández-Montiel et al. 2013).
Another extensive work studied 25 different isolates of AMF and 29 AMF sporeassociated bacteria against fusarium wilt of banana (F. oxysporum f. sp. cubense): to
give an insight of their work, they observed for instance that Glomus etunicatum and
Pseudomonas aeruginosa together could reduce disease severity by 60%, while
inoculants alone reduce by 40 and 45%, respectively (Sumathi and Thangavelu 2016).
It is noteworthy that the protection efficacy is not always visible in any circumstances, because many factors may influence the interaction, such as fertirrigation
and thus, nutrient bioavailability. Indeed, co-inoculation of a Rhizobium with
F. mosseae in soybean in low P condition could reduce red crown
rot (Cylindrocladium parasiticum) incidence by 89% compared to the double
negative control, while the AMF or bacteria reduced it by 50% (Gao et al. 2012),
thus representing more than 40% gain in protection level. Interestingly, this differential was not as strong in higher P condition. The authors suggest that high P
availability has been demonstrated to play opposite roles in nodulation and
444
Y. Krzyzaniak et al.
mycorrhization as indicated by enhanced nodulation but suppressed mycorrhization
with increasing P availability. They also found that P addition significantly
decreased AMF colonization rate, implying that the increased disease incidence
might be partly due to the decrease of AMF colonization rate. However, the link
between AMF colonization rate and protection level might not always be correlated.
24.3.2.2
Possible Mechanisms Explaining a “Full-Gain” Scenario
In the scenario where protection rate is higher with co-inoculation than with one
microorganism alone, it can be explained by these following hypotheses. On the one
hand, bacteria and AMF may work independently from each other and the plant. In
this case, bacteria can protect the plant in its way and AMF can add up supplemental
protection rate in parallel. As it was shown in Sects. 24.2.1.2.1 and 24.2.2.2.1
beneficial microorganisms can exhibit direct and indirect effects against pathogens,
and may, therefore, work independently from the presence of each other (Fig. 24.1).
It is possible that two direct effects are involved (e.g., antimicrobial compounds of
bacteria, combined with the competition for infections sites and nutrients by the
AMF, against the soil-borne pathogen, see mechanisms in red Fig. 24.1); or that two
indirect effects are additive (e.g., ISR and MIR building up stronger plant defense
responses, see mechanisms in blue Fig. 24.1). However, few studies pointed out
specifically independent and adding modes of action between microbial inoculants
to explain a protection gain when co-inoculated, and these explanations still remain
speculative and need further research to support this hypothesis. On the other hand,
beneficial microorganisms can act in synergy with each other. As mentioned in Sect.
24.2.2.1, some bacteria are able to facilitate mycorrhizal establishment as MHB (see
mechanism in purple, Fig. 24.1) and therefore, set up mutual beneficial interactions.
The study mentioned earlier (Gao et al. 2018) showed that improved plant growth of
lucerne resulted from a higher P and N uptake via the symbiotic F. mosseae and
S. medicae, and a mutual promotion of arbuscular mycorrhizal fungal and rhizobial
colonizations. AMF or rhizobium inoculation in the roots reduced the adverse effects
of the pathogen Phoma medicaginis, which was correlated with enhanced defenserelated enzymatic activities such as chitinase, β-1,3-glucanase or PAL, and with
higher lignin or JA contents.
Alternatively, the co-inoculation with Glomus species and P. aeruginosa were
reported to reduce the disease severity of basal stem rot disease (Ganoderma
boninense) of palm seedlings by 80% (compared to negative control), while Glomus
alone reduced it by 68%; but no difference in mycorrhizal colonization rate was
observed with or without the bacteria (Sundram et al. 2015). So on the contrary to the
previous study case, AMF colonization rate and the resulting protection are not
always correlated, and alternative mechanisms can be involved.
In the context of possible indirect synergistic effects via plant metabolic regulations (see mechanisms in green Fig. 24.1), it is well known that the role of
nitrogen (N) and its metabolism in plant immune defenses are fundamental (Fagard
et al. 2014). It was reported that N starvation of tomato plants increased plant
susceptibility to the necrotrophic pathogen B. cinerea, by partially impairing MIR
24
Combined Use of Beneficial Bacteria and Arbuscular Mycorrhizal Fungi for the. . .
445
Aerial-borne pathogen
Induced Systemic Resistance
Mycorrhiza –Induced Resistance
Beneficial rhizobacteria
Direct biofertilization
Plant growth regulation (siderophores, phytohormones)
Abiotic stress alleviation
AMF
Mycorrhiza
Helper Bacteria
Direct biofertilization
Modification of root architecture
Abiotic stress alleviation
Competition for niches and nutrients
Competition for niches and nutrients
Antagonism (antibiotics, predation)
Individual direct mechanism
against the pathogen
Soil-borne pathogen
Individual indirect mechanism
via host plant growth regulation
Individual indirect mechanism
via host plant defense regulation
Synergistic indirect mechanism
between microbial inoculants
Fig. 24.1 Main mechanisms involved in reducing aerial- or soil-borne fungal pathogen developments, in response to a single or a combined inoculation of plants with arbuscular mycorrhizal fungi
(AMF) and beneficial bacteria. For soil-borne pathogens, beneficial microorganisms can reduce
pathogen development, thanks to their direct effect (mechanisms identified in red), such as
competition for niches and nutrients, or antagonism. They also limit pathogen development via
indirect mechanisms, that are mediated by the plant: either, through the regulation of the host plant
growth (identified in green), or through the activation of plant defenses (identified in blue). In this
latter case, mechanisms can be effective for both aerial- or soil-borne pathogens. Microbial
inoculants can also interact with each other, as the example of bacteria called Mycorrhiza Helper
Bacteria, able to facilitate mycorrhization (identified in purple). Co-inoculation of both beneficial
microorganisms may result in controlling pathogen development via additive effect of direct or
indirect individual mechanisms of each beneficial agent or synergistic effect of indirect mechanisms
mediated by both agents
(Sanchez-Bel et al. 2016). Therefore, the combination with a N2 fixing bacteria or a
Rhizobium, may also explain how an enhanced nitrogen uptake would contribute to
stronger MIR, leading to an overall improved resistance to pathogens.
Finally, root colonization by AMF is known to change the quality and the
quantity of root exudates released in soil. And since these latter affect microbial
populations in soil (Hage-Ahmed et al. 2013), it is not excluded that it can both
inhibit the pathogen and also profit to beneficial bacteria at the same time. For
example, on the one hand, it was shown that AMF-inoculated corn releases a
benzoxazinoid, the 2,4-dihydroxy-7-methoxy-2 H-1,4-benzoxazin-3(4 H)-one (DI
MBOA) in root exudates, enhancing the resistance to sheath blight caused by
R. solani (Song et al. 2011). In parallel, this secondary metabolite with antimicrobial
446
Y. Krzyzaniak et al.
Table 24.2 Table summarizing the different methods used to study the tripartite interactions
reported in the literature. This compilation of methods represent a “methodological guide” in
order to optimize future studies on tripartite interactions, with the aim to improve plant protection
by a better understanding of the system
Parameters
Culture conditions
Method options
In vitro
Growth chamber
Greenhouse
Field
AMF
Inoculation
method
Adding solid inoculum in the
sowing/transplanting hole
Adding solid inoculum and
mixing to the whole substrate
Adding liquid mycorrhizal spore
suspension in sowing/planting
spot
References
Duponnois and Plenchette
(2003)
Fester et al. (1999), Berta et al.
(2005), Akköprü and Demir
(2005), Hashem et al. (2016),
Battini et al. (2017)
Hassan Dar et al. (1997), AbdelFattah and Mohamedin (2000),
Jaizme-Vega et al. (2006),
Akhtar and Siddiqui (2008),
Kohler et al. (2009), Lowe et al.
(2012), Gao et al. (2012, 2018),
Hernández-Montiel et al. (2013),
Flor-Peregrín et al. (2014), Xun
et al. (2015), Pérez-de-Luque
et al. (2017), Todeschini et al.
(2018), Ma et al. (2019)
Tahmatsidou et al. (2006), Khan
and ZAIDI (2007), Neeraj and
Singh (2011), Singh et al.
(2013), Cely et al. (2016), Bona
et al. (2017), Espidkar et al.
(2017), Aalipour et al. (2019),
Jaffuel et al. (2019)
Gao et al. (2018), HernándezMontiel et al. (2013), Singh et al.
(2013), Tahmatsidou et al.
(2006), Abdel-Fattah and
Mohamedin (2000), Akköprü
and Demir (2005), Cely et al.
(2016), Duponnois and
Plenchette (2003), Khan and
Zaidi (2007), Hassan Dar et al.
(1997), Lowe et al. (2012),
Neeraj and Singh (2011), Akhtar
and Siddiqui (2008), Espidkar
et al. (2017)
Gao et al. (2012), Sumathi and
Thangavelu (2016), Battini et al.
(2017), Fester et al. (1999),
Hashem et al. (2016), JaizmeVega et al. (2006), Kohler et al.
(2009), Todeschini et al. (2018),
Xun et al. (2015), Berta et al.
(2005), Aalipour et al. (2019)
Pérez-de-Luque et al. (2017),
Jaffuel et al. (2019)
(continued)
24
Combined Use of Beneficial Bacteria and Arbuscular Mycorrhizal Fungi for the. . .
447
Table 24.2 (continued)
Parameters
Control
Method options
Adding liquid mycorrhizal spore
suspension at planting + at
transplanting to field
References
Bona et al. (2017)
Adding inoculum with a seed
coating material
Adding solid inoculum in holes at
sowing + at transplanting
Adding autoclaved AMF+
corresponding washing filtrate of
the inoculum
Adding washing filtrate of
inoculum
Adding autoclaved AMF
Ma et al. (2019)
Adding matching volume of distilled water
Adding non-mycorrhizal pieces
of roots of host plant used for
propagation
Adding matching volume of
substrate
Adding the seed coating material
without inoculum
No specific addition or
unprecised
Detection
Bacteria
Inoculation
method
Flor-Peregrin et al. (2014)
Abdel-Fattah and Mohamedin
(2000), Battini et al. (2017),
Kohler et al. (2009)
Aalipour et al. (2019)
Xun et al. (2015), Jaffuel et al.
(2019)
Pérez-de-Luque et al. (2017)
Duponnois and Plenchette
(2003)
Flor-Peregrin et al. (2014),
Jaffuel et al. (2019)
Ma et al. (2019)
Khan and Zaidi (2007), Hassan
Dar et al. (1997), Lowe et al.
(2012), Neeraj and Singh
(2011), Akhtar and Siddiqui
(2008), Fester et al. (1999),
Hashem et al. (2016), JaizmeVega et al. (2006), Todeschini
et al. (2018), Berta et al. (2005),
Espidkar et al. (2017), Bona
et al. (2017)
Trypan blue staining (Phillips and Hayman 1970) or chitin staining
(Koske and Gemma 1989) + observations according to Trouvelot
et al. (1986), Giovanetti and Mosse (1980) or McGonigle
et al. (1990).
Gao et al. (2018, 2012),
Adding once the bacterial suspension in the transplanting spot Hernández-Montiel et al. (2013),
Sumathi and Thangavelu
(2016), Duponnois and
Plenchette (2003), Hassan Dar
et al. (1997), Pérez-de-Luque
et al. (2017), Akhtar and
Siddiqui (2008), Flor-Peregrin
et al. (2014), Aalipour et al.
(2019), Jaffuel et al. (2019),
Bona et al. (2017), Ma et al.
(2019)
(continued)
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Y. Krzyzaniak et al.
Table 24.2 (continued)
Parameters
Method options
Immersing rooted seedlings in
bacterial suspension before
transplanting
References
Akköpü and Demir (2005),
Tahmatsidou et al. (2006),
Hashem et al. (2016), Lowe
et al. (2012), Berta et al. (2005)
Adding bacterial suspension at
multiple times
Battini et al. (2017); JaizmeVega et al. (2006); Kohler et al.
(2009); Todeschini et al. (2018)
Fester et al. (1999), Khan and
Zaidi (2007), Xun et al. (2015),
Espidkar et al. (2017)
Singh et al. (2013)
Incubating seeds in bacterial suspension before sowing
Resuspension
solution for
inoculation
Immersing stem cuttings in bacterial suspension before planting
Incubating seeds in bacterial suspension before sowing + adding
suspension after planting
Mixing thoroughly a volume of
bacterial suspension with
substrate
Saline solution
Culture medium
Distilled water
Unprecised
Control
Distilled water
Culture medium
Saline solution
Cely et al. (2016)
Neeraj and Singh (2011)
Cely et al. (2016), Duponnois
and Plenchette (2003), Hashem
et al. (2016), Jaizme-Vega et al.
(2006), Pérez-de-Luque et al.
(2017), Todeschini et al. (2018),
Berta et al. (2005), Bona et al.
(2017), Ma et al. (2019)
Akköprü and Demir (2005),
Sumathi and Thangavelu
(2016), Khan and Zaidi (2007),
Akhtar and Siddiqui (2008),
Flor-Peregrin et al. (2014)
Tahmatsidou et al. (2006),
Kohler et al. (2009), Lowe et al.
(2012), Xun et al. (2015), Jaffuel
et al. (2019), Espidkar et al.
(2017)
Fester et al. (1999), Aalipour
et al. (2019)
Abdel-Fattah and Mohamedin
(2000), Akhtar and Siddiqui
(2008), Aalipour et al. (2019),
Jaffuel et al. (2019)
Khan and Zaidi (2007), Neeraj
and Singh (2011), Flor-Peregrin
et al. (2014)
Duponnois and Plenchette
(2003), Pérez-de-Luque et al.
(2017), Todeschini et al. (2018),
Ma et al. (2019)
(continued)
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Table 24.2 (continued)
Parameters
Detection
Method options
No specific addition or
unprecised
References
Hassan Dar et al. (1997), Kohler
et al. (2009), Lowe et al. (2012),
Neeraj and Singh (2011), Fester
et al. (1999), Hashem et al.
(2016), Jaizme-Vega et al.
(2006), Xun et al. (2015), Berta
et al. (2005), Espidkar et al.
(2017), Bona et al. (2017), Cely
et al. (2016)
Counting CFUs after plating on
antibiotics supplemented media
Berta et al. (2005), Hernandezmontiel et al. (2013), AbdelFattah and Mohamedin (2000)
Khan and Zaidi (2007)
P-solubilizing strains : Enrichment culture on NBRIP growth
medium + counting CFUs with
clear halo of P solubilization
Rhizobium strains : Counting
nodules numbers
GFP-tagged/fluorescent strains:
CFUs counting after plating on
media under UV
DNA extraction + gel migration
with PCR products (targeting
16S rRNA)
No assessment or unprecised
Gao et al. (2018, 2012)
Pérez-de-Luque et al. (2017)
Ma et al. (2019)
Xun et al. (2015), Cely et al.
(2016), Jaizme-Vega et al.
(2006), Kohler et al. (2009),
Duponnois and Plenchette
(2003), Hashem et al. (2016),
Lowe et al. (2012), Todeschini
et al. (2018), Akhtar and
Siddiqui (2008), Flor-Peregrin
et al. (2014)
properties was also shown to be involved in semiochemical recruitment of a beneficial bacteria Pseudomonas putida (Neal et al. 2012). Taken all together, these
studies support the potential synergies between AMF and attraction of beneficial
bacteria through changes in root exudates.
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24.3.3 “Partial-Loss” Scenario
24.3.3.1
Evidence for a Partial Protection Loss with a Dual Inoculation
In this case, AMF or bacteria protects at same or different levels, but the protection
level of their combination becomes lower than the greatest of the two (although still
better than the double negative control). A commercial bacterial (B. subtilis FZB24)
inoculation alone reduced powdery mildew (Sphaerotheca aphanis) severity by 56%
on strawberry plants, while the commercial mix of Glomus (Vaminoc®) inoculation
alone reduced disease severity by 13%. Co-inoculation did not improve protection
rate and was similar to AMF alone (Lowe et al. 2012). Similarly, Akköpru and
Demir observed that R. irregularis inoculation alone reduced fusarium
wilt (F. oxysporum f. sp. lycopersici) on tomato plants by 17%, and P. putida by
58%, while the combination resulted in 30% of protection efficacy (Akköprü and
Demir 2005). Apart from the practical viewpoint of losing disease suppression
potential, this is a worthy outcome to know, as it might reveal antagonistic relationships between the AMF and the bacteria, which are usually less readily published.
24.3.3.2
Possible Mechanisms Explaining a “Partial-Loss” Scenario
A partial loss in protection with the co-inoculation, compared to the most effective
microbial inoculant, can be explained by several ways. Firstly, in the case where a
partial loss in protection is observed along with a partial decrease in AMF colonization rate in dual inoculation compared to AMF alone, this would probably suggest
that a direct or indirect antagonistic effect from the bacteria against the AMF can be
suspected. If a bacteria produces an antimicrobial compound able to limit a fungal
pathogen development or is more competitive for root niches, it is not excluded that
it can also have antagonistic effect against AMF, via its diffusible or volatile
metabolites (Xavier and Germida 2003). Indeed, Akköpru and Demir (2005)
observed that the combination of a P. putida with R. irregularis ended up in partial
loss of protection efficacy compared to single inoculations, as it was correlated to a
decrease of AMF root colonization rate in presence of this bacteria, with a 12% less
colonization rate compared to AMF alone. Authors suggested that the inhibiting
effects are thought to be related to a hypothetic secretion of antimicrobial substances
toward AMF, although no analysis revealing the presence of such substances could
unfortunately be pursued. However, they did demonstrated that their tested bacterial
strain had a potential direct inhibitory effect against F. oxysporum in vitro, that was
not associated to a siderophore effect: hence, we could question that, if this strain
could exhibit an antagonistic activity toward a fungal pathogen, would it also be
possible to exert it to another fungus, such as an AMF?
Another explanation is that some PGPR can lead to changes in the chemical
composition and therefore structural properties of root cell walls (Vacheron et al.
2013). For example, the biocontrol agent Bacillus pumilus INR-7 is able to enhance
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451
lignin deposition in pearl millet epidermal tissues. These cell wall modifications
induced by PGPR have been reported to limit pathogen development, by stimulating
ISR plant defense responses, such as enhanced lignin synthesis and callose apposition (García-Gutiérrez et al. 2013). Therefore, if the bacteria colonizes the root cells
faster than the AMF, it may induce root structural changes that would limit the
penetration of other microorganisms, AMF included. However few data are available to support this hypothesis, but further studies would be worth carried out to
examine such cases of antagonism. Conversely, if a partial loss in protection is
observed with dual inoculation, but no decrease in AMF colonization rate is
observed, it would either mean that (1) AMF limits the bacteria to exert its mode
of action, thus lessening its efficacy against the pathogen; and/or (2) protection rate is
not linearly correlated with AMF colonization rate. As stated above for bacteria,
causes of such incompatibility may also find the source in root exudates or structural
changes, as induced by AMF, but negatively impacting the inoculated bacteria.
Otherwise, microbial inoculants may act indirectly, via the metabolic regulation
of the host plant (see mechanisms listed in green, Fig. 24.1). If the two microbial
inoculants induce contradictory metabolic pathways, the biochemical antagonism
may trouble defense signalization and response toward the pathogen. Lowe (2012)
suggested that inoculation of Glomus species with B. subtilis may induce conflicting
hormonal pathways (ex: SA vs. JA) in controlling powdery mildew on strawberry
plant, because defense responses to biotrophic pathogens are generally associated
with SA-hormonal pathway (Glazebrook 2005).
Indeed, as PGPR may promote plant growth on the one hand, while AMF can
activate plant defenses (or vice-versa) on the other hand, this raises a fundamental
question regarding the plant reactions to these stimuli. If a plant allocates its energy
to its defense activation in response to a microbial inoculation, how can it ensure the
supply of other biological functions, such as growth? This balance, or “trade-off,”
between growth and defense is an ongoing issue in plant immunity and physiology
(Heil 2001; Bolton 2009). Many studies more focused on plant–pathogen interactions, then evidenced that the allocation of energetic resources for defense reactions
are taking place at the expense of their “fitness”. This latter, also called selective
value, is described in evolutionary biology as the ability of an individual of a certain
genotype to reproduce; and can be measured in many ways such as in number of
fruits, or growth rate. Visible or not, systematic or not, the impacts of defense
activation (by MIR or ISR) exist, as there are common metabolic pathways between
growth and defense, mediated by several phytohormones such as SA, JA or IAA
(Pieterse et al. 2009). And since some AMF can transiently lead to the accumulation
of SA (García-Garrido and Ocampo 2002), it might act against the action of a
bacteria, that was meant to stimulate IAA accumulation (Vacheron et al. 2013).
Indeed, one typical example of hormonal crosstalks is between SA and IAA signaling (Wang and Wang 2014). It was shown in Arabidopsis plants, that SA does not
directly affect auxin synthesis, but instead, inhibits plant responses regulated by
auxin-dependant gene expression. Without SA, auxin is supposed to be perceived
and linked to the auxin receptor TIR1 F-box protein. This complex is able to fix and
degrade a class of repressors, called the auxin/indole-3-acetic-acid proteins
452
Y. Krzyzaniak et al.
(AUX/IAA), that is meant to repress the expression of auxin-dependant genes.
Therefore, its degradation frees the expression of auxin-dependant genes, leading
to typical auxin responses. However with SA, the expression of TIR1 F-box is
repressed: auxin cannot bind to its receptor, and no complex can degrade Aux/IAA
proteins, which are then able to exert their role of repressor of auxin-dependant
genetic transcription. At the end, no auxin-like responses can be observed in the
presence of SA, independently from auxin concentration (Wang et al. 2007; Wang
and Wang 2014; Huot et al. 2014).
24.3.4 “No-Gain, No-Loss” Scenario
24.3.4.1
Evidence for No Protection Gain with a Dual Inoculation
In that case, AMF or bacteria alone protects the plant against the disease at the same
or different level, but the combined inoculation leads neither to an improvement,
neither to a lower protection than the greatest of the two inoculants. The protection
efficacy of a dual inoculation is therefore at the same level compared to the best
obtained by one of the two microbial inoculants. This case was observed by Larsen
and collaborators, where R. irregularis or Burkholderia cepacia could singly reduce
Pythium ultimum population density at similar efficacy level, but the combined use
did not significantly improve the pathogen control on cucumber plants (Larsen et al.
2003). Similarly, F. solani propagules number was reduced in response to
F. mosseae inoculation, but was not more lowered in the presence of Rhizobium
leguminosarum bv. phaseoli on common beans (Hassan Dar et al. 1997) and disease
index was barely improved (0,04 unit less) by the addition of the bacteria.
Tahmatsidou and collaborators (2006) demonstrated that the protection level
against Verticillium wilt on strawberry runners was so strong that, in any case, it
was not possible to improve it any higher, as no pathogen could develop, in presence
of the commercial inoculants Vaminoc® (AMF), B. subtilis FZB24, alone or in
combination (Tahmatsidou et al. 2006).
24.3.4.2
Possible Mechanisms Explaining a “No-Gain, No-Loss”
Scenario
No changes in protection level by adding a supplemental microbial inoculant is a
complex scenario to explain. First of all, the above-cited studies reported that one of
the microbial inoculants, particularly AMF, was already very effective in reducing
pathogen development (Tahmatsidou et al. 2006), placing, therefore, the
pathosystem in a context where there was only a tight scope for improvement.
Indeed a total protection ensured by one of the microorganisms would mask a
potential gain conferrable by the other one. In this context, it would be interesting
to carry out preliminary tests to evaluate which conditions are optimal to obtain an
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453
intermediate protection rate (by exposing the plants to more stressful conditions,
such as abiotic stress or higher pathogen pressure), and test an additional inoculation
thereafter.
If no or slight changes are observed in terms of protection while an increased
colonization rate of AMF or bacterial population is reported in co-inoculation, as it
was reported by Hassan Dar and collaborators, it supports again that protection rate
may not be always positively correlated to a quantitative presence of the microorganism (Hassan Dar et al. 1997). Similarly, within a tripartite interaction context, but
apart from a plant–pathogen context-, no changes in date palm seedlings physiological parameters (root and leaf dry weight) was measured with a phosphatesolubilizing bacteria strain such as Pseudomonas oryzihabitans with F. mosseae,
while the P content or the mycorrhizal colonization rate were enhanced compared to
AMF alone (Boutheina et al. 2019). Another study also reported that papaya plant
roots were better mycorrhized with Glomus species in presence of B. coagulans than
Glomus alone, but no improvement was observed on plant height, fruits per plant,
density of fruits (Mamatha et al. 2002). Then, it is not because an MHB increases
mycorrhizal colonization rate, that the repercussion at phenotype scale will be an
obligatory issue in response to mutualistic interactions at microscopic scale.
Finally, it also occurs that no change in microbial population density or colonization rates is observed, along without any gain in protection rates. In that case, one
can suspect whether the bacterial inoculation was effective or not: is the mode of
inoculation or the culture system suitable for the beneficial microorganism lifestyle?
Did the latter survive along the experiment? If so, are the conditions favorable
enough to reveal the best potential of microbial biological activity? Or on the
contrary, are the conditions stressful enough to reveal differentials, as suggested
by Nadeem and collaborators (Nadeem et al. 2014)? Indeed, several studies show
that both individual biological activity and microbial interactions are submitted to
the environmental factors such as pH or soil characteristics (Rousk et al. 2009;
Ratzke and Gore 2018). It legitimately raises the issues on the factors influencing
such interactions, and how to cope with them by testing the most suitable experimental design and methods.
24.4
Methodological Guide: How to Start Working
on Tripartite Interactions ?
The aim of this following synthesis is to produce an overview of the main methods
employed to study tripartite interactions, in order to facilitate the first step of
experiment designing that would best answer to working hypotheses. This does
not aim to compare the methods between them in order to rule out one method
among others, which would surely ensure a high protection efficacy, as it is believed
that there is no “ideal” method because every system is unique in such complexity.
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Y. Krzyzaniak et al.
We rather aim to compile what is possible to do, but also where are the lines of
improvement, which would be interesting to explore for future research.
The different methods are summarized in Table 24.2 and are organized according
to three main entries: cultivation design, AMF, and bacterial inoculation methods.
For the last two sections, we decided to emphasize on the different reported modes of
inoculation (frequency, time, mode of application), as well as which negative
controls were used, and which protocols were applied to check microbial colonization or their presence at the end of the experiment.
Among the thirty references dealing with the combined inoculation of AMF and
bacteria, half of them carried out the experiments in greenhouse conditions, one third
in field conditions, one-sixth in growth chamber, and only one in vitro conditions, as
far as we found.
24.4.1 Microbial Inoculation Methods
Concerning AMF inoculation, six principal means to bring the AMF inoculum to the
system were identified. Equivalent number of publications reported adding solid
inoculum, either by putting it right in the sowing/transplanting hole or by mixing it to
the whole substrate. More recently, some research teams brought the inoculum by
pouring spore suspension in the sowing or transplanting spot (Pérez-de-Luque et al.
2017; Jaffuel et al. 2019) or even by applying a specific coating material to the seeds
in order to fix AMF inoculum thereafter (Ma et al. 2019). The volumes of inoculum
brought to the soil are very variable between studies, and the concentration in
propagule numbers constituting it are not always precise.
Concerning bacteria-related methods, seven principal means were identified. The
vast majority reported adding a bacterial suspension in the transplanting or sowing
hole at the beginning of the experiment (Hernández-Montiel et al. 2013; Gao et al.
2018). Other methods reported also immersing rooted seedlings in bacterial suspension before transplanting (Lowe et al. 2012; Hashem et al. 2016); incubating seeds in
bacterial suspension before sowing (Xun et al. 2015; Espidkar et al. 2017); immersing stem cuttings in bacterial suspension before planting (Singh et al. 2013); or
mixing thoroughly a volume of bacterial suspension with the substrate (Neeraj and
Singh 2011). Finally, others bring bacterial suspension twice during the experiment,
with a combination of some of the methods cited above, such as incubating the seeds
in bacterial suspension and adding again at planting time (Cely et al. 2016).
In both cases, for AMF or bacterial inoculation, most of the studies reported the
addition of the inoculum only once during the whole experiment (at sowing or
transplanting), while a minority add it twice (at sowing, then at transplanting, for
example).
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24.4.2 Choosing the “Mock-Inoculum”
Consequently, the technique of microbial inoculation questions which negative
control (or “mock”-inoculum) to use. There does not seem to have one kind of
control used universally, especially for AMF mock inoculation.
Concerning AMF-associated controls, several types of controls were found in the
framework of this review. Most predominantly, authors either (1) do mention that
their negative control conditions consist of adding matching volume of substrate; or
(2) do not mention anything specifically. Other kinds of more specific “mockinoculum” were used: authors mentioned the addition of autoclaved AMF propagules with the view to provide same amount of organic matter, but with the
inactivated AMF (Jaffuel et al. 2019), or of the washing filtrate of the inoculum in
order to simulate equal microflora (Aalipour et al. 2019), or both (Battini et al. 2017).
Authors also added a matching volume of distilled water, in correspondence to the
AMF spore suspension prepared in water (Pérez-de-Luque et al. 2017), or by adding
non-mycorrhizal pieces of roots of the host plant used for propagation, in correspondence to in vitro-produced mycorrhized roots placed in sowing holes (Duponnois
and Plenchette 2003).
Concerning bacterial-associated controls, the options are less varied. Indeed, it is
associated with which kind of solution was used to prepare bacterial suspension. In
this way, if bacterial cells were resuspended in saline solution (usually 0.85% NaCl
or 0.1 M MgSO4) or fresh nutrient liquid medium, then mock-inoculated plants
received the same volume of the corresponding saline solution (Todeschini et al.
2018) or liquid medium (Flor-Peregrín et al. 2014). However, it sometimes occurs
that even if the resuspension method is described, the corresponding mock inoculation may not be explicitly mentioned.
It is possible that multiple controls (substrate only, AMF, and bacterial mocks)
cannot be all carried out for obvious material reasons. However, preliminary experiments can be carried out in order to check whether the disease develops at the same
level in every mock-condition. In that case, controls could be considered as equivalent in terms of disease protection, and the following experiments may include
fewer “control” conditions for saving reasons.
24.4.3 Colonization or Bacterial Viability Assessment
Methods
In some cases, protection rates are not always improved with a dual inoculation,
compared to AMF or bacteria alone, especially results exhibit great variability
between biological repetitions. The first checks made concern the microbial inoculation effectiveness, i.e., are AMF or bacteria alive, and did they successfully
colonize the roots (or other compartments in cases of migration by bacteria) at the
end of the experiment?
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Y. Krzyzaniak et al.
To check AMF colonization in roots, protocols are basically using a staining
procedure, in the articles studying tripartite interactions such as Trypan blue staining
(Phillips and Hayman 1970), followed by microscopic observations of fungal
stuctures and counting (Giovanetti and Mosse 1980; McGonigle et al. 1990).
However, apart from tripartite interactions, there are several other options to track
and quantify AMF root colonization. A few of the following methods have been
developed: AMF species-specific isoenzymes (Tisserant et al. 1998), genus or
species-specific antibodies (Treseder and Allen 2002) and various nuclear (Van
Tuinen et al. 1998; Lee et al. 2008) or mitochondrial DNA-based (Sarma et al.
2017) or RNA-based molecular methods such as the fragments of a RNA polymerase II gene (Thioye et al. 2019).
However, tracking and monitoring the presence of the bacteria in the culture
system (plant organs or rhizospheric soil) is more difficult and challenging. Tracking
refers to phenotypic or genotypic detection, quantification, and localization of
inoculated PGPB strains, whereas monitoring includes tracking as well as determining the physiological activity of the inoculated PGPB strains, such as N-fixation,
P-solubilization, and phytohormone production, among others.
Antibiotic-resistant strains have the advantage to be identified by dilution-plating
of root buffer extracts, onto solid media supplemented with an antibiotic like
rifampicin (Berta et al., 2005). Also, rhizobium strains on leguminous plants have
the advantage to establish nodules, so that a simple visual counting gives a good
information about successful bacterial colonization and the set up of the symbiosis
(Gao et al. 2018, 2012). An extensive review was recently published to gather and
sort the main methods for tracking and monitoring of bacteria into three main
categories: (1) reporter genes-based methods, (2) immunological methods, and
(3) nucleic acid-based methods (Rilling et al. 2019). The reporter gene is a gene
attached to a regulatory sequence of a target gene that can be used to detect the
presence and/or expression of the target. The reporter genes are usually identified by
color, luminescence, or fluorescence, such as green fluorescent protein marking
(Zhao et al. 2011).
24.5
Future Challenges Regarding Current Limits
for Practical Use
Biocontrol, and more specifically, the use of microbial inoculants could constitute
one of the ways to set up a more sustainable agriculture. They possess several
advantages when compared with chemical agricultural inputs (mineral fertilizers,
conventional pesticides). Indeed, along with a pre-selection of the most efficient
microorganism, there is a potential reduced risk on environmental and human health.
They are safer to apply, with potential multiple modes of actions, and also are able to
multiply if inoculated in appropriate conditions and may survive to the next season
(Berg 2009). There is strong evidence that microorganisms play a central role in
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plant health. However, studying several living organisms at the same time, which
can be affected each differently by a slight change, may be challenging, even in
controlled conditions. In this section, we will identify the different factors that can
influence tripartite interactions: this will therefore reveal the limits of their use, but
more importantly, list out the potential research axis to work on for their better use.
Microbial inoculant efficacy in plant protection depends on quite a number of
factors, including: (1) biotic factors such as disease pressure, crop type/plant species/
genotype (Zhao et al. 2016), trophic lifestyle of the pathogen (Ojiambo and Scherm
2006), AMF species in relation to their aggressiveness toward plant colonization
(Martinez-Medina et al. 2009); (2) abiotic factors, including climatic parameters
such as temperature and relative humidity (De Curtis et al. 2012), seasonal variations
(Escudero and Mendoza 2005), soil types or nutrient bioavailability (Baar et al.
2011); (3) agronomical or application-related factors included, for example, crop
management, formulations of inoculants, mode and timing of applications of the
tested strains (Fedele et al. 2020; Tabassum et al. 2017); (4) and finally industrial and
socioeconomic situations, such as cost and profitability balance, biocontrol market,
national or EU regulation for authorizations (Nicot et al. 2012).
Indeed, we should note that most of the studies were carried out in pots under
controlled conditions, on artificial substrates in order to simplify the model with the
view to unravel the mechanisms and the part of protection rate conferred by each of
the microorganisms. But in natural field conditions, roots are confronted with a
plethora of microorganisms, varying with the seasons and geographic locations. The
applied inoculants may compete with naturally occurring microbial populations and
be placed under complete unfavorable conditions to thrive and deploy all the
mechanisms that could have been observed in laboratory. Therefore, it is legitimate
to wonder how to fill the gap between the lab and the field, as under certain cases, the
results obtained in field are not similar to those of laboratory (Smyth et al. 2011). The
probability of working in compatible conditions can be very thin, to obtain a
favorable context that matches for each component of the system (plant, inoculant
1, inoculant 2, pathogen, physicochemical characteristics of soil).
To cope with these issues, it would be interesting to isolate from the targeted soil,
indigenous microbial strains which would be the most adapted to pedoclimatic
conditions, or naturally the most competitive ones as compared to other native
strains, or to exogenous strains (Khalid et al. 2004; Mahmoudi et al. 2017). Then,
after isolation and study of their potential beneficial effects, authors underline the
importance of the development process for further marketable products. Indeed,
some Gram-positive bacteria are potential efficient biocontrol strains, but they are
difficult to formulate because they do not produce spores of long shelf life: the
process of dessication for the formulation is then compromising their practical use.
Due to their persistence, Gram-negative bacteria can be preferred over and can last
and stabilize more efficiently within a formulation (Tabassum et al. 2017).
As a conclusion, co-inoculation of AMF and beneficial bacteria is visibly worth
using in some studies. Taken all the cited literature, one of the most studied or
successful cases involve AMF and Rhizobia associations on legume plants, through
their mutual biological stimulation in their interactions. Main technical locks reside
in how to deliver viable, easily formulable microbial inoculants, that are both
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compatible with themselves, and with the environmental conditions in which there
are brought to. Hence, robustness of results and transferability of the tests to the field
can be hoped to improve, by a better understanding of the specific needs of each
inoculant, alone and combined, with the plant. In the context of the upcoming
of more and more variable and extreme abiotic stresses linked to climate change,
priority in crop managements should be to promote plant health, by restoring soil
health. Helping plants to cope with the stresses by the use of beneficial microorganisms is then, one of the many drivers for a more sustainable agriculture, along with
integrated pest and diseases management strategies.
Acknowledgments The authors wish to thank the “Université du Littoral Côte d’Opale (ULCO)”
for providing financial supports for Y. Krzyzaniak post-doctoral fellowship. This work has been
carried out in the framework of TRIPLET project which was supported by the partnership A2U
(Artois, UPJV, ULCO) and in the framework of CPER ALIBIOTECH project which was financed
by European Union, French State and the French Region of Hauts-de-France.
Conflict of Interests The authors declare no conflict of interest.
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Chapter 25
Remediation of Toxic Metal-Contaminated
Soil and Its Revitalisation with Arbuscular
Mycorrhizal Fungi
Irena Maček
Abstract Soil is a finite resource and its preservation is essential for food security
and our sustainable future. Soil contamination caused by the large amounts of
pollutants is becoming a major problem on a global scale. In particular, toxic
(heavy) metals are a big concern as they are non-degradable, persist in soil and
accumulate in food webs. Soil that is heavily contaminated with toxic metals is
referred to as a hazardous waste. As the world human population rise also urban
agriculture is increasing and fulfilling diverse functions including food production.
However, samples from urban areas are frequently exceeding safety standards for
toxic metals concentration, which is particularly concerning for children as the most
sensitive group to metal toxicity impacts. Thus it is of great importance to find good
solutions for efficient restoration of degraded areas and toxic metal-contaminated
soil. Recently, a novel and efficient chelant-based (EDTA—ethylenediamine
tetraacetate) soil washing technology for remediation of toxic metal-contaminated
soils has been developed. The innovative procedure is well suited for cleaning
contaminated soils and has a potential to return the valuable resource—fertile
soil—back into function. However, the harsh treatment of soil EDTA washing
eliminates most of the biota in the remediated soil and significantly reduces its
biological activity. It has been shown to be particularly harmful to soil filamentous
fungi, including plant symbiotic arbuscular mycorrhizal fungi. After the treatment
practically no arbuscular mycorrhizal fungal colonisation has been detectable in
plant roots growing in remediated soil several months after the soil remediation
treatment procedure, however latter functional mycorrhiza is established. The same
was true for the molecular signal (arbuscular mycorrhizal fungal DNA) in the plants
growing in remediated soil immediately after the treatment. Currently, there is very
little knowledge on the composition, succession, function, and dynamics of
I. Maček (*)
Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
Department of Biodiversity, Faculty of Mathematics, Natural Sciences and Information
Technologies (FAMNIT), University of Primorska, Koper, Slovenia
e-mail: Irena.Macek@bf.uni-lj.si
© The Editor(s) (if applicable) and The Author(s), under exclusive licence to
Springer Nature Switzerland AG 2021
N. Shrivastava et al. (eds.), Symbiotic Soil Microorganisms, Soil Biology 60,
https://doi.org/10.1007/978-3-030-51916-2_25
469
470
I. Maček
microbial communities in the soil after the EDTA based remediation treatment. This
chapter focuses on the importance of the integration of the role of microbial ecology
in environmental sciences along with the application of the modern molecular
techniques to improve our understanding of the succession of soil microbial communities and determinants of the survival of introduced microbial inocula in the
remediated soil. Particular stress is put on the ubiquitous arbuscular mycorrhizal
fungi, potential for the development of functional microbial communities, and
following plant–soil feedbacks, including mycorrhiza, after the remediation
procedure.
Keywords Abiotic stress · Arbuscular mycorrhizal fungi · Biodiversity ·
Contaminated soil · Heavy metals · Pollution · Remediation · Revitalisation · Soil ·
Soil ecology · Toxic metals
25.1
Introduction
Contamination of soils with toxic metals is a serious issue with soil being a finite
resource, meaning its loss and degradation is not recoverable within a human
lifespan. New soil formation is a slow process taking millennia for a few cm to
form, thus it is of great importance to find good solutions for efficient restoration of
degraded areas, including toxic (heavy) metals contaminated soils. In most
industrialised countries as well as developing countries land contamination exists
and is a growing problem. Waste disposal and treatment, together with industrial and
commercial activities often result in local soil contamination. Toxic metals (As, Cd,
Cr, Cu, Ni, Pb, Zn) are one of the major causes of concern as they are difficult to
remove, are non-degradable, persist in environment for a long time and accumulate
in food webs. Toxic metals are known to cause severe toxicity and represent a health
hazard for people and animals, with children being the most endangered group (Jez
and Lestan 2015). In the recent document (EEA 2020, The EU Environment, State
and Outlook) EU Environmental Agency reports that in the EU-281, potentially
polluting activities took place on an estimated 2.8 million sites, but only 24% of the
sites are inventoried. Currently, only 28% of the registered sites are investigated, a
prerequisite to deciding whether remediation is needed or not (Payá Pérez and
Rodríguez Eugenio 2018). Considering the estimated extent of past and current
pollution, and the uncertainties of reliable estimates, little progress has been made
in the assessment and management of contaminated sites (EEA 2020, The EU
Environment, State and Outlook). In a large part of these sites, toxic metals are the
most important contaminants (EU Environmental Agency Report No. 1/2007). Soil
1
EU-28: The European Union (EU) currently counts 27 EU countries. The UK withdrew from the
European Union on 31 January 2020. The EEA (EU Environmental Agency) 2020 report still
includes contaminated sites in the United Kingdom.
25
Remediation of Toxic Metal-Contaminated Soil and Its Revitalisation with. . .
471
containing dangerous substances—including high concentrations of toxic metals—
is considered a hazardous waste (EU Waste Catalogue & Hazardous Waste List,
EPA 2002). EU is implementing the Landfill Directive (1999/31/EC) with heavy
restriction of hazardous waste disposal which must be subject to remediation
treatments.
Thirty of the 39 countries surveyed in the ‘Progress in management of contaminated sites’ (European Environment Agency 2014) maintain comprehensive inventories for contaminated sites. Contaminated soil continues to be commonly managed
using ‘traditional’ techniques, e.g. excavation and off-site disposal, which accounts
for about one-third of management practices, however, also new technologies are
evolving (e.g. Lestan 2017). Due to the severity of the heavy metal contamination
problem several soil remediation processes have been developed. One possibility is
EDTA (ethylenediaminetetraacetic acid) chelating agent extraction of the contaminants. The recently developed and modified procedure of EDTA soil washing has
shown good results in metal removal from contaminated soil (e.g. Finzgar and
Lestan 2007; Pociecha and Lestan 2012; Voglar and Lestan 2013) and therefore
could be a cost-efficient and sustainable strategy for reclamation of contaminated
urban gardens and farmlands. This procedure however has strong negative impact on
soil microbial communities, with the filamentous arbuscular mycorrhizal fungi
appearing to represent one of the most sensitive groups of the soil microbiota to
both, chemical and mechanical disturbance during the remediation procedure
(Maček et al. 2016b).
The objective of this chapter is to summarise the current knowledge on the subject
of soil fungal diversity and ecology in metal-polluted and EDTA-washed soils
before and after remediation. The emphasis is on a ubiquitous soil fungal group
that forms arbuscular mycorrhiza, an ancient symbiosis between plants and
arbuscular mycorrhizal fungi. Arbuscular mycorrhiza is present in roots of the
large majority of terrestrial plant species in a wide range of ecosystems, including
urban soils and agroecosystems. Within the chapter soil remediation using EDTA
washing is presented along with its negative impacts on soil biota (Maček et al.
2016b). The importance of the new methodological development in the field of
molecular ecology with its possible applications in monitoring of the succession and
development of microbial communities and revitalisation of the soil after the remediation treatment is emphasised. Finally, future directions in microbial ecology
research in these specific systems and advances in the use of the new highthroughput molecular techniques are presented, with an acknowledgement of the
possibilities for widening the scope of research, which could include bioprospecting
toxic metal-contaminated sites for industrially important microbes and research into
potential toxic metal-tolerant fungal taxa (Maček et al. 2016a).
472
25.2
I. Maček
Soil Remediation Using EDTA (Ethylenediamine
Tetraacetate) Soil Washing
In selecting appropriate remediation methods for a specific polluted site, characteristics of the soil and contaminants need to be considered (Lestan et al. 2008; Lestan
2017). The method of soil washing by EDTA (ethylenediamine tetraacetic acid)
chelating agent and extraction of the contaminants has shown to result in high
multimetal (Pb, Zn, Cd) removal efficiency (e.g. Finzgar and Lestan 2007; Pociecha
and Lestan 2012; Voglar and Lestan 2013) especially from bioavailable and labile
fractions (Jelusic and Lestan 2014), see Fig. 25.1. This innovative procedure promises a cost-efficient and sustainable strategy for the reclamation of contaminated
urban areas and farmlands, also on a larger scale (Lestan 2017). No wastewater is
generated and solid wastes are efficiently bitumen stabilised before disposal (Voglar
and Lestan 2013, 2014).
The procedure successfully removes available forms of toxic metals and thus
lowers the human and environmental hazards of the remediated soil, however, it
also significantly diminishes soil microbial activity (Jelusic and Lestan 2014; Maček
et al. 2016b). This has been shown by enzymatic tests (Jelusic and Lestan 2014;
Kaurin et al. 2018; Kaurin and Lestan 2018), has been studied on soil fauna (Tica et al.
2013; Udovic and Lestan 2010), and in a pot experiment also on plant symbiotic
arbuscular mycorrhizal fungi (Maček et al. 2016b). Thus far no trends (succession) of
the remediated soil recovering its microbiological properties have been followed for a
longer time-period and mainly short-term experiments have been done using
remediated substrate (Maček et al. 2016b; Kaurin et al. 2018; Kaurin and Lestan
2018). It is known, however, that in addition to physical and chemical properties
ecosystem services of soils largely depend on the diversity and activity of soil
microbes (e.g. Jeffries et al. 2003), thus further research of this aspect is needed.
25.3
Arbuscular Mycorrhizal Fungi in Metal
Contaminated Soil
Microbes represent the largest portion of biodiversity and biomass in soils. Healthy
soils have high biodiversity that regulates ecosystem functions and processes, which
lead to a great variety of ecosystem services. Understanding the mechanisms regulating the diversity and structure of microbial communities is urgently required for
predicting the ecological impacts of rapidly changing environments. To be able to
observe the effect remediation treatment has on the microbial communities, we first
need to characterise the communities in the contaminated sites (original soil). Those
communities however represent an interesting research object themselves. It is
important to know how soil microorganisms respond to disturbance or environmental change (Griffiths and Philippot 2013) and at the extreme end of that is also soil
that has been exposed to a long-term toxic metals contamination. Soils that are
25
Remediation of Toxic Metal-Contaminated Soil and Its Revitalisation with. . .
TOXIC METAL
CONTAMINATED SOIL
EDTA REAGENT
RECYCLING
REMEDIATION
PROCEDURE - EDTA
REAGENT WASHING
REMOVING EDTA MOBILIZED TOXIC
METALS FROM SOIL
GRINDING &
SIEVING OF THE SOIL
473
CONTROLLED
DISPOSAL
REMEDIATED SOIL
WITH ARTIFICIAL
AGGREGATES
AMENDMENTS ADDITION
(FERTILIZER, BIOCHAR...)
SOIL REVITALISATION
POTENTIAL ADDITION
OF MYCORRHIZAL
INOCULA, COMPOST
REMEDIATED SOIL
AS A PLANT
SUBSTRATE
Fig. 25.1 A simplified scheme of the toxic metal-contaminated soil remediation procedure using
EDTA (ethylenediamine tetraacetic acid) chelating agent soil washing and extraction of the
contaminants. EDTA recycling loop is shown, returning used EDTA reagent solution in the
remediation process. The soil remediation procedure has been fully described in Lestan (2017).
Different amendments (e.g. compost, fertilizer, soil, mycorrhizal inocula, biochar) have been tested
for better performance of the remediated soil to serve as a substrate for plant growth and for its
revitalisation with microbiota (e.g. Jelusic et al. 2014; Maček et al. 2016b; Kaurin et al. 2018).
Revitalisation of the EDTA-washed remediated soil with arbuscular mycorrhizal fungi has been
tested for commercial inocula [Symbivit Remed, Symbiom Ltd., Czech Republic (See footnote 1)]
and indigenous inoculum (rhizosphere soil with root particles) in Maček et al. (2016b), however
further research on succession of the microbial communities in the newly formed soil substrate is
needed
severely contaminated with toxic (heavy) metals still harbour very diverse, microbial
communities including organisms that can support plant growth and development
like symbiotic arbuscular mycorrhizal fungi. Some of the organisms inhabiting those
soils may also have specific adaptations to high levels of contaminants in the
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environment and may be suitable for isolation and application in biotechnology and
agriculture (see Fig. 25.4).
25.3.1 Arbuscular Mycorrhiza
About 80% of all vascular plant species form arbuscular mycorrhiza, which is an
underground symbiotic association between plants and arbuscular mycorrhizal fungi
(Smith and Read 2008) (see Figs. 25.2 and 25.3). This functionally important group
of soil fungi is involved in many terrestrial ecosystem processes (Fitter 2005). The
symbiosis is ancient, over 450 million years old, and was significant in enabling the
colonisation of land by plants (Redecker et al. 2002; Hoysted et al. 2018; Field and
Pressel 2018). Arbuscular mycorrhizal fungi, along with other fungal groups
(Hoysted et al. 2018; Field and Pressel 2018), were accompanying plants in their
transition from water to land from the very beginning and have been evolving in a
range of diverse terrestrial ecosystems. For plants, there are several benefits of being
mycorrhizal and among the best known are increasing the soil volume for the
acquisition of mineral nutrients and increased stress tolerance (e.g. drought, pollution, pathogen attacks). An important feature of mycorrhizal fungal presence in soil
is also the impact these organisms have on the formation and stabilisation of soil
aggregates and soil structure (Rillig and Mummey 2006; Rillig et al. 2017; Lehmann
et al. 2020). Therefore, arbuscular mycorrhizal fungi are common in many stressed
environments and have been shown to increase plant survival and vitality in such
ecosystems along with the positive impact they have on soil. Arbuscular mycorrhizal
fungi acquire all their carbon from the host plants and have central roles (e.g. nutrient
cycling) in many habitats. Several indicators exist that the benefits provided to plants
by arbuscular mycorrhizal fungi will become even more important due to increased
Fig. 25.2 Arbuscular mycorrhizal fungal colonisation of carrot roots (Daucus carota L.).
Arbuscules can be seen as darker spots connected with intraradicular hyphae (a) in the root cortex.
A longitudinal root view with arbuscular mycorrhizal fungal hyphae and spores visible around the
root and fungal colonisation with arbuscules in the root cortex (b). Root associated fungal structures
were stained with trypan blue dye. Photos were taken with Olympus Provis AX70 microscope and
digital camera Olympus DP70
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Fig. 25.3 Arbuscular mycorrhizal fungal spores of several species, representing three families
Glomeraceae (a, b), Acaulosporaceae (c), and Gigasporaceae (d). A crushed spore, mounted in
PVLG (polyvinyl alcohol-lactic acid-glycerol) with visible cell wall layers and lipid reserves—oil
droplets (d). Morphology of the cell wall is used for taxonomical identification and differentiation
among the arbuscular mycorrhizal fungal species. Photos were taken by Olympus Provis AX70
microscope and a digital camera
abiotic stresses caused by global change in the future (e.g. Hanson and Welzin
2000).
25.3.2 Use of Molecular Methods in Community Ecology
of Arbuscular Mycorrhizal Fungi
Understanding arbuscular mycorrhizal fungal ecology and identification of the main
predictors of their community-level processes is still difficult and it applies to a wide
range of habitats. By delivering to the plant a range of benefits, arbuscular mycorrhizal fungi have a profound effect on plant community dynamics and diversity,
highlighting the central role they have in terrestrial ecosystem processes (Fitter 2005;
Rosendahl 2008). Recent molecular studies have shown that communities of
arbuscular mycorrhizal fungi in nature are more diverse than originally thought on
the basis of spore morphology (Fig. 25.3). Arbuscular mycorrhizal fungal spores still
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serve as the main taxonomic characteristic since they cannot be identified morphologically in roots (Merryweather and Fitter 1998) (Fig. 25.2). Since the morphological features of the structures of arbuscular mycorrhizal fungi in plant roots only
allow low levels of identification of these fungi (Fitter and Moyersoen 1996),
molecular approaches are needed for a more detailed description of their communities. The majority of the ecological studies on arbuscular mycorrhizal fungi are
constructed on DNA-based techniques that have been developed to quantify
arbuscular mycorrhizal fungi in field-collected soil and plant roots since the 1990s
(e.g. Helgason et al. 1998). Although over 300 morpho-species of this fungal group
have been described (Walker and Trappe 1993; Schüßler 2008, http://www.amfphylogeny.com/amphylo_species.html), molecular data show that significantly more
arbuscular mycorrhizal fungal taxa exist, however, those are currently known solely
by their environmental sequences (e.g. Helgason et al. 2002; Öpik et al. 2013, 2014).
With improved DNA-based identification methods, such as next generation sequencing (e.g. Roesch et al. 2007; Öpik et al. 2009; Schloss 2009; Lemos et al. 2011;
Dumbrell et al. 2011, 2016; Maček et al. 2019), our ability to study soil microbial
diversity has started to increase, allowing the characterisation of important mechanisms structuring natural communities and tracking their seasonal dynamics
(e.g. Dumbrell et al. 2011, 2016; Maček et al. 2019). The heterogeneous and
dynamic nature of soil ecosystems, however, still makes it challenging to study the
effect of the soil environment on natural microbial communities in situ.
In the last decade, the next generations of sequencing approaches (e.g. Dumbrell
et al. 2011, 2016; Maček et al. 2019) have revealed a much higher diversity than
reported before the wide application of these technologies, mainly by reported more
rare taxa, which are usually found only with the use of high-throughput technology.
The latter allows also more intense sampling and is increasing the number of the
analysed sequences and sequencing depth for each analysed sample. In the sampling
sites, a combination of different approaches and expertise is needed. In particular,
uniting species- (taxonomy) (Figs. 25.3 and 25.4) and community-oriented (ecology) approaches would be a major advantage in studying arbuscular mycorrhizal
fungal community ecology and global diversity patterns (Öpik and Davison 2016).
Moreover, extreme environments (including toxic metal-contaminated soils) can
serve as systems to examine how long-term abiotic selection pressures drive natural
communities and their evolution and possibly result in new specialists and
extremophilic taxa. Potential new taxa could be isolated, characterised, and stored
in international collections (Maček 2017) (Fig. 25.4).
Typically in temperate ecosystems with well-developed arbuscular mycorrhizal
associations like semi-natural grasslands >50 arbuscular mycorrhizal fungal taxa
(Operational Taxonomic Units - OTUs) from a range of different arbuscular mycorrhizal fungal families are reported using next generation of sequencing methods and
sampling over several seasons, based on 18S rRNA SSU (small subunit ribosomal
ribonucleic acid) marker genes. For example, in an old-growth (>100 years) seminatural grassland ecosystem (Giessen free-air carbon dioxide enrichment experiment, Germany) a total of 55 arbuscular mycorrhizal fungal taxa (OTUs) from eight
arbuscular mycorrhizal fungal families has been reported colonising mixed root
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Fig. 25.4 Technique of establishing single-spore arbuscular mycorrhizal fungal cultures in pipette
tips (a) with the goal to grow monospecific cultures of arbuscular mycorrhizal fungi established
from a single spore isolated from environment (toxic metal-polluted site). Each individual plantspore combination is later transferred to a bigger pot (b) where additional plant host of different
species (e.g. Plantago lanceolata L., Lolium perenne L.) can be planted in order to initiate trap
cultures and grow mycorrhizal fungi with their plant hosts to produce enough arbuscular mycorrhizal fungal spores of sufficient quality to enable taxonomical and functional studies on the
potentially new (adapted) species of fungi
samples within one year vegetation season (Maček et al. 2019). This estimate of
arbuscular mycorrhizal fungal OTU richness is as expected from grassland systems,
given both the number and nature (i.e. mixed roots) of samples examined, and the
next generation of sequencing methods used (Dumbrell et al. 2011; Hiiesalu et al.
2014; Moora et al. 2014).
Moreover, recent molecular studies on arbuscular fungal ecology from environmental samples (e.g. semi-natural temperate grassland) show that ecological studies
should avoid relying only on broad-scale community-level responses of soil
microbes including mycorrhizal fungi (for example, like commonly used community
composition visualised using nonmetric multidimensional scaling—NMDS) (Maček
et al. 2019). Likely, at a local scale, subtle changes in the relative abundances of
specific arbuscular fungal populations are often driven by environmental factors and
also stochastic processes (e.g. Dumbrell et al. 2010; Maček et al. 2019) and this is
not necessarily reflected in the presence of a novel community that is entirely
compositionally distinct from some other investigated or control community. A
novel approach includes a more detailed examination of specific taxa. For example,
methods, relying on modelling taxa (OTU—operational taxonomic units) abundances using multivariate generalised linear models (MV-GLMs) (Wang et al.
2012) or bias reduction binomial generalised linear models (BR-GLMs, Firth
1993), give a good insight into details of the population-level dynamics of specific
taxa as shown in studies on the arbuscular mycorrhizal fungal community dynamics
from environmental samples (e.g. Maček et al. 2019). In addition, snapshot data
observed on single sampling time-point are not sufficient for a full investigation on
the soil community composition. Therefore, temporal sampling throughout several
seasons and at several time-points and years should be done. Such approaches can
show us the susceptibility of the functionally important soil microbes to global
change, including land-use change and soil pollution, with responses evident across
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both population and community levels. This raises critical questions and highlights
the need for far greater research into the temporal response of soil microbes
including arbuscular mycorrhizal fungi to environmental change; the functional
differentiation observed across arbuscular mycorrhizal fungal taxa means any
changes in their temporal dynamics has the potential to resonate throughout associated plant communities, changing aboveground competition dynamics and thus
future ecosystem productivity in currently unpredictable ways (Maček et al. 2019).
25.3.3 Arbuscular Mycorrhizal Fungal Diversity in Toxic
Metal Contaminated Soil
By using molecular methods, fungal communities have been described also in toxic
(heavy) metal-contaminated sites (e.g. Zarei et al. 2008, 2010; Hassan et al. 2011;
Maček et al. 2016b). Among other abiotic factors toxic metal concentration and
biological availability in soils has been shown to impact the composition of
arbuscular mycorrhizal fungal communities (e.g. Zarei et al. 2008, 2010, Hassan
et al. 2011). Many reports indicate a reduction of arbuscular mycorrhizal fungal
diversity in heavy metal-contaminated areas, based both on spore morphology
(e.g. Griffioen 1994; Pawlowska et al. 1996; Leyval et al. 1997; del Val et al.
1999) and molecular data (e.g. Zarei et al. 2008, 2010; Hassan et al. 2011). A
predominance of the taxa within the genus Glomus (old nomenclature before the
major modifications published in the years 2010 by Schüßler and Walker, and 2011
by Oehl et al., see also Öpik et al. 2013) has been reported in most of the studied
areas with severe toxic metal disturbance (e.g. Whitfield et al. 2004; Vallino et al.
2006; Zarei et al. 2008; Sonjak et al. 2009; Hassan et al. 2011), as well as other
anthropogenic environments, such as agricultural sites, phosphate-contaminated
sites (Daniell et al. 2001; Renker et al. 2005), and sites with fungicide treatments
(Helgason et al. 2007). Zarei et al. (2008) analysed the diversity of arbuscular
mycorrhizal fungal associated to Veronica rechingeri growing in the heavy metalcontaminated soil of the Anguran Zn and Pb mining region in Iran. Three species
could be separated morphologically, while phylogenetic analyses revealed seven
different arbuscular mycorrhizal fungal MOTUs (molecular operational taxonomic
units) in plant roots, all within the genus Glomus. Some MOTUs were only found at
sites with the highest and lowest soil toxic metal concentrations and some in both,
which is a pattern also observed in other studies (e.g. Zarei et al. 2010; Hassan et al.
2011). Thus, the patterns of new taxa identifications are also showing in extreme
environments that entirely originate from human-impacted pollution.
In most of the reported cases, arbuscular mycorrhizal fungal communities have
not been sampled to saturation, and more intensive sampling and a higher depth of
sequencing (for example, using next generation of sequencing) might result in
detecting additional taxa and would allow for a more realistic description of the
patterns in the community ecology of this group of organisms. Importantly,
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questions on long-term (press) related changes in soil microbial communities are
relevant to many human drivers with long-term nature, including climate change,
nutrient input, land-use change, soil contamination, and others (Maček et al. 2016a).
However, in particular questions about the stability against press perturbations have
received relatively little attention so far (Ives and Carpenter 2007). The introduction
of the next generation of sequencing into ecology can largely change this. These
tools are allowing us to obtain an exceptional amount of data (DNA sequences for
different molecular markers) in a short time. This is a key condition to be fulfilled in
order to understand the complex temporal and spatial patterns in soil microbial
communities along with their environmental drivers, including soil pollutants.
Moreover, the same molecular techniques and data analyses protocols could also
be used for following development and succession of the microbial populations and
communities in soil and rhizosphere after the contaminated soil remediation
treatment.
25.4
Microbial Communities in Soils After Soil
Remediation with EDTA
Remediation technologies often concentrate only on metal removal efficiency and
overlook the treated soil’s overall health, functioning, and potential use after remediation. The negative side of EDTA soil washing is its impact on soil biota and in
addition other soil properties (e.g. soil structure) (Jelusic et al. 2014; Jelusic and
Lestan 2014). It significantly diminishes soil microbial activity (Jelusic and Lestan
2014) and diversity (Maček et al. 2016b). This has been shown by using enzymatic
tests (Jelusic and Lestan 2014), lately also by Kaurin et al. (2018), Kaurin and Lestan
(2018), and has partially been studied on soil fauna (Tica et al. 2013; Udovic and
Lestan 2010). Jelusic et al. (2014) have shown that soil remediation reduces the toxic
metal concentrations in plants grown in experimental plots but the biomass of tested
plants significantly diminished. Presumably, micronutrients were removed along
with the toxic metals due to the nonselective nature of EDTA chelation. Effective
means of revitalisation are needed to restore health and reclaim the remediated soil as
a fertile plant substrate, including fertilisation to restore the soil nutrient pool (Jelusic
et al. 2014) (see also Fig. 25.1). In addition, different amendments (e.g. hydrogel,
vermiculite), each carrying a specific function, may help to improve the properties of
the soil (e.g. Tica et al. 2013). Moreover, the addition of microbial inocula might
help the soil to restore functional microbial communities (Fig. 25.1).
The harsh remediation conditions destroy the majority of life in the remediated
soil, though direct measurements of biodiversity and community structure change
have only been done for plant symbiotic arbuscular mycorrhizal fungi (Maček et al.
2016b). The results from this study show that after revitalisation of soil that was
subjected to remediation treatment functional arbuscular mycorrhizal symbiosis can
establish, either by commercial or indigenous inoculum addition to the soil substrate
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I. Maček
with higher diversity of arbuscular mycorrhizal fungi resulting from the soil inoculation with the indigenous inoculum (Maček et al. 2016b). Soil diversity is important
for ecosystem stability and function thus following diversity loss after soil remediation treatment should be a part of the procedure integrated into the remediation plan
(see Fig. 25.1 for the EDTA washing soil remediation procedure scheme).
25.4.1 Importance of Soil Biodiversity for Ecosystem Stability
According to the insurance hypothesis (Stuart and Pimm 1984), one of the proposed
consequences of biodiversity loss is a reduction in the ecosystem stability in the
sense of resistance (ability of the system to withstand the disturbance) and resilience
(the speed by which the system returns to its pre-disturbance state) (e.g. Stuart and
Pimm 1984). This hypothesis is based on the idea that the probability of finding
species able to adapt to changing conditions and allowing ecosystem functioning is
greater in a more diverse ecosystem. Several attempts have been made to test this
hypothesis, for example, one of the approaches is to experimentally build up
different levels of biodiversity. Assemblages of up to 43 species of fungi did show
evidence of increasing stability with increasing biodiversity (Setälä and McLean
2004). As did communities containing up to 72 species of bacteria (Bell et al. 2005).
However, soils contain far more species than generally used in such community
assembly experiments and effects of biodiversity are more evident in systems with
low diversity (Nielsen et al. 2011). Arbuscular mycorrhizal fungi and other plant
growth-promoting microorganisms have been used as inocula for biofertilisation and
phytostimulation or different soil types (Rana et al. 2012). In addition to using
indigenous inocula a growing number of inocula are being marketed which may
help to restore the functionality of remediated soil. The commercial inocula, however, typically include only a limited taxa richness, which is usually also not very
well defined (typically no molecular data on the taxa identity is available on the
product specification). Therefore, a range of diversity that has been present in
original soil before the remediation treatment is difficult to establish only with
using commercial inocula (Maček et al. 2016b). This has also been shown by the
study of Maček et al. (2016b) where both, commercial and indigenous inocula were
tested (see the next section for details). Nevertheless, the potential for the arbuscular
mycorrhizal fungi to establish in the remediated substrate has been shown also in this
soil type and long-term experiments are urgently needed in order to follow the
succession of the soil microbial and mycorrhizal fungal communities in such substrates (see Fig. 25.5 for the details on the mesocosm experiment with remediated
soil at Biotechnical Faculty, University of Ljubljana, Slovenia).
The use of commercial fungal inocula is still connected to a lot of uncertainties,
including persistence of inoculated organisms in the new environment, introduction
of invasive species, competitive exclusion etc. In a perspective paper Rodrigues and
Sanders (2015) address the role of community and population ecology in applying
arbuscular mycorrhizal fungi for improved food security. The authors argue that
despite the huge potential of the use of symbionts of plants for improving yields of
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Fig. 25.5 A mesocosm experiment set as a full factorial experiment with remediated (EDTA
washed) and original (toxic metal-contaminated) soil at the laboratory field of Department of
Agronomy, Biotechnical Faculty, University of Ljubljana, Slovenia. Contaminated soil from two
locations with different soil types has been selected for the experiment; acidic soil (pH < 6.1) from
Stossau, Arnoldstein (Austria) and calcareous soil (pH > 7.1) from Meža Valley (Slovenia). Factors
include soil type (acidic, calcareous), remediation treatment (original, remediated soil), plant (with
Lolium perenne L., no plants), inoculum (grassland rhizosphere soil with roots, no inoculum).
Temporal dynamics of the soil and plant root microbial community development (succession) and
function (archaea, bacteria and fungi), mycorrhiza development, soil chemical parameters and plant
responses have been investigated with sampling in regular intervals during the experiment, including the winter season. The experiment has been set as part of the activities within the Slovenian
Research Agency (ARRS) research project J4-7052 with I. Maček as the principal investigator
globally important crops, the application of arbuscular mycorrhizal fungi in agriculture is too simplistic and ignores basic ecological principals. Thus interdisciplinary
work with ecologists could significantly improve our understanding of the determinants of the survival, and ecological roles of the introduced microbes, and the effect
this has on plant production and soil health.
25.4.2 Diversity of Arbuscular Mycorrhizal Fungal
Communities in Remediated Soil
Establishing diverse communities of soil microorganisms, and importantly also
arbuscular mycorrhizal fungi are urgent for the restoration of biological activity of
the remediated soil. The first attempt to revitalise the EDTA-washed remediated soil
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with arbuscular mycorrhizal fungi was made in a pot experiment, where the newly
established diversity of the fungal community was evaluated using molecular tools,
at the time by using cloning and Sanger sequencing (Maček et al. 2016b).
The contaminated soil used in this experiment was collected from the upper
30 cm layer of a managed vegetable garden near an abandoned lead smelter in the
Meža Valley, Slovenia (Maček et al. 2016b). More than 300 years of active lead
mining and smelting was present in the area until 1990, leaving behind 6600 ha of
agricultural land polluted primarily with Pb, Zn, and Cd (Jelusic and Lestan 2014).
The site is home to more than 6000 inhabitants, with children being the most
endangered group regarding toxic metals impact on their health (Jez and Lestan
2015). Despite the adoption of the action programme for improving the quality of the
environment and reducing the health hazard for people in the Meža Valley in 2007,
the results of annual surveys of blood Pb concentration indicated that the number of
children with levels higher than 10 μg dL (toxicity threshold under Slovenian
legislation) did not drop over the first 6-year duration of the programme, thus
additional measures are immediately needed (Jez and Lestan 2015). After the
remediation of soil collected in Meža Valley using EDTA washing technique, the
initial metal concentrations of 1585 mg kg 1 Pb, 525 mg kg 1 Zn, and 8.8 mg kg 1
Cd were reduced to 313, 378, and 2.52 mg kg 1 for Pb, Zn, and Cd, respectively
(Jelusic and Lestan 2014). The effect of EDTA washing of the soil used also in the
study of Maček et al. (2016b), on soil properties, toxicity hazards, and plant biomass
and fitness has been described before (Jelusic and Lestan 2014; Jelusic et al. 2014).
Maček et al. (2016b) have quantified arbuscular mycorrhizal fungal colonisation
in plant roots as an indicator of mycorrhizal potential in toxic metal-contaminated
(original) vs. remediated soil, and after inoculation with commercial (Symbivit
Remed, Symbiom Ltd., Czech Republic2) and indigenous mycorrhizal inoculum
(rhizosphere soil and root particles of Plantago lanceolata L. roots sampled at the
original soil sampling location in Meža Valley, Slovenia). After soil remediation, the
mycorrhizal potential and capability of fungi to form arbuscules in roots of several
plant hosts (Plantago lanceolata L., Lolium perenne L., and Sorghum bicolor (L.)
Moench) were severely reduced to practically hardly detectable root colonisation
with fungal hyphae and no arbuscules present. This indicates that immediately after
remediation mycorrhizal potential of remediated soil is very low and that the
addition of inoculum into such substrate could accelerate the mycorrhiza establishment (Maček et al. 2016b). Further tests have shown that functional symbiosis could
be established in the remediated soil by the use of indigenous inoculum (rhizosphere
soil with root particles) as a good source of diverse fungal propagules for
revitalisation of remediated soil, which has in this experiment performed better
2
Commercial inoculum—Symbivit Remed, Symbiom Ltd., Czech Republic, that—according to the
producer’s specification—contains the following arbuscular mycorrhizal fungal isolates;
Rhizophagus irregularis, Funneliformis mosseae, Claroideoglomus claroideum, and Funneliformis
constrictus, isolated from temperate anthropogenic sites. Using molecular methods in our experiment, only Funneliformis mosseae could be amplified from the collected root particles that were an
integrative part of the Symbivit Remed inoculum (Maček et al. 2016b).
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483
(higher diversity of AM fungi in the final substrate), when compared to the commercial inoculum used (Maček et al. 2016b).
The evaluation of plant root colonisation however cannot give sufficient information on arbuscular mycorrhizal fungal diversity and community composition in
the plant roots. Thus, molecular methods based on the analyses of arbuscular
mycorrhizal fungal 18S rRNA SSU (small subunit ribosomal ribonucleic acid)
marker genes were used in order to explore whether the arbuscular mycorrhizal
fungal taxa present were distinct in toxic metal-polluted habitats in Meža Valley (the
metal-contaminated site where the original soil has been collected), and in inoculated
remediated soil (Maček et al. 2016b). In addition, arbuscular mycorrhizal fungal
diversity in plant roots growing in remediated and original soil has been analysed,
dependent on different inoculation methods (commercial vs. indigenous inoculum).
The experiment has confirmed functional mycorrhizal symbiosis with plants was
established either by commercial or indigenous inoculum addition. This was
followed in two time-windows, 3- and 5-months after experiment initiation (soil
remediation treatment), with progression in the development of mycorrhiza with
time, which has been confirmed also as a molecular signal (arbuscular mycorrhizal
fungal specific PCR (polymerase chain reaction) products of the 18S rRNA SSU
marker genes) 5-months after the initiation of the experiment. The results clearly
show that the use of the indigenous inoculum (grassland roots and rhizosphere soil)
resulted in higher arbuscular mycorrhizal fungal taxa richness in the roots of plants
growing in the remediated soil compared to the use of the commercial inoculum and
that the use of the indigenous inocula (rhizosphere soil) resembles the diversity of
the environmental (field) mycorrhizal communities to a greater extent. The only
taxon that has been detected in the commercial inoculum and later in the remediated
soil that has been inoculated by this inoculum type was Funneliformis mosseae. No
arbuscular mycorrhizal fungal specific PCR products were yielded from the roots of
Plantago lanceolata plants growing in non-inoculated remediated soil with a very
low level of fungal root colonisation intensity.
However, the question on how diverse arbuscular mycorrhizal fungal communities develop in remediated soil still stays as in the same study of Maček et al. (2016b)
only a limited number of arbuscular mycorrhizal taxa (OTUs) have been confirmed.
Most of the arbuscular mycorrhizal fungal sequences (45 sequences), both from the
indigenous inoculum samples and the community developed in the remediated soil
could be assigned to the genus Glomus, followed by the genera Rhizophagus, and
Funneliformis (both latter previously also known as Glomus, see Öpik et al. 2013 for
Glomeromycota taxa nomenclature including modifications) (Maček et al. 2016b). A
predominance of the taxa within the genus Glomus (old nomenclature before the
major modifications published in the years 2010 by Schüßler and Walker, and 2011
by Oehl et al., see also Öpik et al. 2013) has been reported from most of the studied
areas with severe toxic metal disturbance (e.g. Whitfield et al. 2004; Vallino et al.
2006; Zarei et al. 2008; Sonjak et al. 2009; Hassan et al. 2011), as well as other
disturbed sites like agricultural sites, phosphate-contaminated sites (Daniell et al.
2001; Renker et al. 2005), and sites with fungicide treatments (Helgason et al. 2007).
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This could however partially be conditioned also with the methodology used at the
time of these explorations into fungal communities.
Representatives of Funneliformis mosseae were the most frequently reported as
toxic metal-tolerant fungal taxa in several independent studies (Turnau et al. 2008;
Vallino et al. 2006; Zarei et al. 2008; Hassan et al. 2011), suggesting adaptation of
specific ecotypes of this species to toxic metal contamination (e.g. Turnau et al.
2008; Vallino et al. 2006, Zarei et al. 2008; Zarei et al. 2010; Hassan et al. 2011).
This group includes arbuscular mycorrhizal fungi with a ruderal strategy as reported
for Funneliformis mosseae, which is encountered as spores in natural soils and in
trap cultures but infrequently in natural plant roots (Öpik et al. 2014). Indeed,
Funneliformis mosseae has been reported to increase in abundance in many disturbed environments and competitive release has been suggested by Helgason et al.
(2007) as the mechanism behind the high abundance of Funneliformis mosseae after
using the fungicide benomyl to alter the community of arbuscular mycorrhizal fungi
in undisturbed monoliths of soil in a natural community. Sequences of Funneliformis
mosseae (VT67—virtual taxon, according to Öpik et al. 2013) represented also the
only arbuscular mycorrhizal fungal taxon that was detected using molecular markers
in the dry roots from the commercial inoculum (Symbivit Remed, Symbiom Ltd.,
Czech Republic) also used in the study with the remediated soil (Maček et al.
2016b).
Since in the study of Maček et al. (2016b) a low-throughput technique (cloning
and Sanger sequencing) was used it is possible that only the most abundant
arbuscular mycorrhizal fungal taxa have been reported. Therefore, more intensive
sampling could result in detecting additional taxa in the samples since the arbuscular
mycorrhizal fungal communities in this study were not sampled to saturation due to
methodological limitations. Nevertheless, the results of this study clearly show that
the most abundant taxa in the newly established communities were among the fungi
that are well known for their ruderal (opportunistic) strategy and are tolerant to
anthropogenic disturbance (e.g. Funneliformis mosseae).
25.5
Future Prospects
The study of Maček et al. (2016b) represented the first preliminary experiment with
the focus on arbuscular mycorrhizal fungal communities development in remediated
soil based on EDTA washing procedure. More work will be needed in the future to
follow the dynamics, stability, and succession of soil microbial communities in
remediated soil, along with the development of the soil functional diversity and
functional traits. An effort for further investigation of the arbuscular mycorrhizal
fungal community composition in the remediated soils on a larger scale (field
experiments) is necessary, including monitoring successional stages and seasonal
dynamics of the newly established communities, in order to thoroughly evaluate to
what extent they can resemble natural communities in healthy soils (Maček et al.
2016b). This is the goal of the project and experiment set with the remediated soil at
25
Remediation of Toxic Metal-Contaminated Soil and Its Revitalisation with. . .
485
University of Ljubljana, Biotechnical Faculty (Fig. 25.5) with the aim to follow
temporal dynamics of the soil and plant root microbial community throughout
several years. This goes along with evaluating the development (succession) of the
communities and functional traits of soil archaea, bacteria, and fungi, mycorrhiza
development, soil chemical parameters, and plant–soil responses. The remediated
substrate enables studies of succession of the microbial communities in the soil after
the treatment and depending on the revitalisation (inoculation) with a different range
of microbial inocula. The idea of this experiment (see Fig. 25.5 for details) is to study
the temporal dynamics, stability, and the extent of the recovery (resilience) of the
microbial communities in two remediated soil types (calcareous and acidic soil).
Moreover, studies of plant performance (plant–soil feedbacks) in the new substrate
will be included to target the most widely recognised function of soil, which is its
support of plant (food) production. The newly developed microbial community of a
known composition could also serve as a soil type specific inoculum that could be
used to inoculate larger amounts of remediated substrate for faster and more efficient
revitalisation.
25.6
Conclusion
Fertile soil is a valuable, limited resource, often contaminated with substances that
have negative impacts on life. Soil, heavily contaminated with toxic metals is
referred as a hazardous waste. Soil remediation is a solution, but the harsh procedures result in soil that has heavily reduced soil diversity. There still is little
knowledge of the establishment, succession, function, and dynamics of soil microbial communities in remediated soil. Moreover, the stability of soil microbial
community composition is another question getting increasing attention especially
in the light of global change. Therefore, in order to tackle the problem of soil toxic
metal contamination from different angles, interdisciplinary efforts should be taken
to combine the innovative soil remediation techniques with the integration of the
rapidly increasing knowledge on soil microbial ecology, driven by the latest development of molecular tools (e.g. next generation sequencing techniques). This will
give a better insight into the biological component of the remediated soil, including
soil microbial communities and populations. Application of indigenous inocula
(rhizosphere soil) has already been shown to result in a faster development of a
more diverse microbial community compared to the no inocula treatment (Maček
et al. 2016b), while commercial inocula have been shown to have only a limited
range of fungal diversity, often limited to only few or even a single generalist taxon.
Acknowledgements This work was supported by the Slovenian Research Agency (ARRS)
funding: basic research project J4-7052 and research programme P4-0085. All of the support
given is gratefully acknowledged by the author.
486
I. Maček
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