Journal of Plant Pathology (2010), 92 (4, Supplement ... - Sipav.org

Journal of Plant Pathology (2010), 92 (4, Supplement), S4.71-S4.105 Edizioni ETS Pisa, 2010 S4.71 

SEASONAL VARIATION IN ROOT INFECTION AND POP- 

ULATION LEVELS OF FUSARIUM spp. IN CITRUS NURS- 

ERIES IN EGYPT. Y.M. Ahmed 1 , H. El-Shimy 1 , A. Ippolito 2 , T. 

Yaseen 3 , A.M. D’Onghia 3 . 1 Plant Pathology Research Institute, 

Agriculture Research Center, Giza, Egypt. 2 Dipartimento di Protezione 

delle Piante e Microbiologia Applicata, Università degli Studi 

“Aldo Moro”, Via Amendola 165/A, 70126 Bari, Italy. 3 Centre International 

de Hautes Etudes Agronomiques Méditerranéennes, 

Mediterranean Agronomic Institute of Bari, Via Ceglie 9, 70010 

Valenzano (BA), Italy. E-mail: ippolito@agr.uniba.it 

Fusarium species are commonly associated with different citrus 

diseases, such as dry root rot, root rot, feeder root rot, wilt, 

twig dieback, and citrus decline. Fusaria are ubiquitous in citrus 

groves and nurseries, attacking feeder roots under stress conditions. 

This study aimed at monitoring the seasonal variation of 

Fusarium spp. in soil and feeders roots in Egyptian citrus nurseries 

with the purpose of detecting the most common species associated 

with diseases in the nursery plots. The study was conducted 

in two nurseries, located in different regions (Delta and 

Desert area). Soil and root samples were collected monthly from 

March to July from sour orange and Volkameriana lemon rootstocks. 

The inoculum density of Fusarium spp., expressed as 

colony forming unit (CFU) per gram, was estimated by soil dilution 

using semi selective media. The percentage of infected feeder 

root was assessed by culturing feeder root pieces on the same selective 

media. Fusarium isolates were grouped according to their 

morphological characteristics and classified by amplifying and sequencing 

a regions of the beta-tubulin gene (benA). In both nurseries 

Fusarium solani was the prevailing species, followed by F. 

oxysporum. The inoculm density of Fusarium and the percentage 

of root infection varied according to the rootstock, the environmental 

conditions and the nursery management, reaching the 

highest values during high temperature periods. 

PRELIMINARY SURVEY OF COMMON FUNGAL DIS- 

EASES OF MANGO IN SICILY. Y. Ahmed 1 , A. Ismail Mahmoud 

1 , T. Yaseen 2 , A.M. D’Onghia 2 , G. Cirvilleri 1 . 1 Dipartimento 

di Scienze e Tecnologie Fitosanitarie, Università degli Studi, Via 

S. Sofia 100, 95123 Catania, Italy. 2 Centre International de Hautes 

Etudes Agronomiques Méditerranéennes, Mediterranean Agronomic 

Institute of Bari, Via Ceglie 9, 70010 Valenzano (BA), Italy. 

E-mail: gcirvil@unict.it333 

Mango (Mangifera indica Linn.) is a promising crop in Sicily 

due to the favorable climatic and soil conditions. Mango cultivation 

was introduced a few years ago on a limited surface, mainly 

in the provinces of Palermo, Ragusa, Messina and Catania. Kensington 

was the main variety obtained by locally produced seeds, 

probably coming from Australia. In the near future, it is expected 

that commercial and backyard plantings of mango trees will increase, 

being a profitable crop. Being the phytosanitary state of 

this crop in Italy unknown, surveys were conducted in spring and 

in autumn in seven commercial groves located in different areas, 

to assess the occurrence of diseases and associated causal agents. 

Typical symptoms of die-back, gray leaf spot, anthracnose on 

twigs, branches, leaves and fruits, Phytophthora crown rot and 

root rot, and Armillaria root rot were observed. Samples were 

collected from symptomatic plant tissues and plated on different 

media. Pure fungal cultures were identified based on their morphological 

and microscopic features. Preliminary results indicated 

that foliar, fruit and soil-borne diseases were present in all the 

investigated areas. Pestalotiopsis mangiferae, Botrydiplodia spp., 

Colletotrichum spp., and Phytophthora nicotianae were the most 

prevalent pathogens isolated from leaves, branches, soil and 

roots. Other fungi were identified, including Pythium spp., Rhizoctonia 

solani, Fusarium spp. and Armillaria mellea. 

VARIABILITY OF SCLEROTIUM ROLFSII ISOLATES 

FROM ORNAMENTAL PLANTS AND TURFS IN ITALY. 

D. Aiello 1 , I. Castello 1 , V. Guarnaccia 1 , R. Caiazzo 2 , A. Carella 2 , 

E. Lahoz 2 , G. Polizzi 1 . 1 Dipartimento di Scienze e Tecnologie Fitosanitarie, 

Università degli Studi, Via S. Sofia 100, 95123 Catania, 

Italy. 2 CRA, Unità di Ricerca per le Colture Alternative al Tabacco, 

Via Pasquale Vitiello 106, 84018 Scafati (SA), Italy. E-mail: 

gpolizzi@unict.it 

Southern blight, caused by the soilborne fungus Sclerotium 

rolfsii is a serious disease of a wide variety of plants, including 

field, vegetable, fruit, ornamental crops and turfs. In recent years, 

southern blight was particularly damaging to ornamentals and 

turfs in eastern Sicily, where 37 isolates were recovered over a 2year 

period. Variability of these isolates was investigated based on 

morphology (mycelium growth rate and sclerotium formation), 

teleomorph formation on three different media, and mycelial 

compatibility. Fungal isolates varied considerably in growth rate 

at different temperatures, in the number of sclerotia per plate 

and in their dry and fresh weight. Only one isolate formed hymenia 

and basidia of Athelia rolfsii on potato dextrose agar 

(PDA) containing 2% activated charcoal. Five vegetative compatibility 

groups (VCGs) resulted from isolate pairings. In compatible 

reactions mycelia of the two isolates intermingled at the zone 

of interaction. Thirty-six out of 37 isolates showed compatibility 

with at least one isolates and were grouped into four groups, 

whereas one isolate was self-compatible and constituted another 

VCG. The pathogenic variability of five isolates belonging to different 

VCGs was assessed on Laurus nobilis seedlings. In addition, 

the variability in esterase and polygalacturonase electroforetic 

patterns was considered in relation to VCGs and the main 

characteristics of the isolates. Studies on the variability of S. rolfsii 

strains from a geographical region are important as they can 

help documenting their origin and the changes that might be occurring 

in the population. 

A PCR-BASED METHOD FOR THE IDENTIFICATION OF 

FUSARIUM FUJIKUROI AND F. PROLIFERATUM AND 

DETECTION IN RICE SEEDS. M.T. Amatulli 1 , D. Spadaro 1,2 , 

M.L. Gullino 1 , A. Garibaldi 1 . 1 Centro di Competenza per l’Innovazione 

in Campo Agro-Ambientale (AGROINNOVA), Università 

degli Studi di Torino, 10095 Grugliasco (TO), Italy. 2 Dipartimento 

di Valorizzazione e Protezione delle Risorse Agroforestali, Università 

degli Studi, Via Leonardo da Vinci 44, 10095 Grugliasco (TO), 

Italy. E-mail: mariateresa.amatulli@unito.it 

Northern Italy represents the largest production area (232,500 

ha in 2007) of rice in Europe. Several fungal diseases affect this 

crop. Among these, Fusarium spp. are important agents of plant 

and seed diseases. In the last decades, bakanae has emerged in 

Italian rice fields, becoming a serious problem for seed production 

and for seed companies. The causal agent of this disease is 

Fusarium fujikuroi a species of the Gibberella fujikuroi complex 

(GFC). F. fujikuroi is the most abundant, but other species can be 

isolated from rice, among which F. proliferatum, phylogenetically 

closely related to F. fujikuroi and morphologically indistinguishable 

from it. Multiple alignments of translation elongation factor 

gene sequences of various Fusarium spp., showed a deletion of 

six nucleotides in F. fujikuroi and two nucleotide polymorphisms 

in the same region in F. proliferatum. These sequence variants 

Edizioni ETS Pisa, 2010

S4.72 Journal of Plant Pathology (2010), 92 (4, Supplement), S4.71-S4.105 

were used to set up a PCR-based diagnostic protocol. The 

species-specific primer pairs FUJI1F/1R and PROLI1F/1R gave 

products of 179 and 188 bp, respectively. Primer specificity was 

confirmed by assaying DNA from different species in the GFC, 

as well as from 298 isolates present in our Fusarium spp. collection, 

found on rice plants and seeds. Primer sensitivity was tested 

on 10 pg of pure fungal DNA. Primers successfully detected fungal 

DNA from infected rice tissues and seeds. The developed 

PCR assay can be used for the rapid identification of these 

species and may represent a powerful tool for their detection in 

rice seeds. 

IDENTIFICATION AND CHARACTERIZATION OF 

BACILLUS SUBTILIS ET-1, A STRAIN WITH ANTIFUN- 

GAL ACTIVITY AGAINST FRUIT ROT PATHOGENS. A. 

Ambrico, M. Trupo, L. Lopez. Unità Tecnica Tecnologie Trisaia, 

Laboratorio Biotecnologie, Centro Ricerche ENEA Trisaia, ss 106 

Jonica km 419.5, 75026 Rotondella (MT), Italy. E-mail: 

alfredo.ambrico@enea.it 

A bacterial strain (ET-1) isolated from soil and used in these 

studies, was identified as Bacillus subtilis based on morphological 

and physiological tests, Biolog and the 16S rDNA sequence. This 

species is commonly regarded as a biological control agent. Botrytis 

cinerea and Penicillium digitatum are known to cause severe 

rotting of strawberries and citrus fruits during storage and shelf 

life. Inhibition of mycelial growth and conidial germination of the 

two fungal pathogens by a strain ET-1 cell-free supernatant was 

tested on agar substrate. Preliminary assays were also conducted 

on strawberry and lemon fruits to assess the ability of ET-1 bacterial 

suspension and ET-1 cell-free supernatant to inhibit the 

growth of fungal agent rots in post-harvest. In vitro, isolated ET-1 

significantly reduced mycelial growth of both fungal pathogens 

forming a clear-cut inhibition zone. Cell-free supernatant diluted 

1:32 and 1:128 caused over 95% inhibition on conidial germination 

of B. cinerea and P. digitatum, respectively. Promising results 

were obtained in vivo trials. The more efficient protection was 

observed on strawberry and lemon fruits treated with cell-free supernatant. 

Our results are in agreement with those of other studies 

and confirm the ability of strain ET-1 to secrete secondary 

metabolites with antifungal activity. Further investigation is needed 

to verify the effectiveness of ET-1 under real conditions of 

fruits storage and to identify the molecules involved in fungal inhibition. 

THE EXPRESSION OF THE GENE CODING FOR CERA- 

TO-PLATANIN IS MODULATED BY BIOTIC AND ABIOT- 

IC FACTORS. I. Baccelli 1 , R. Bernardi 2 , L. Carresi 1 , C. Comparini 

1 , L. Pazzagli 3 , A. Scala 1 . 1 Dipartimento di Biotecnologie 

Agrarie, Sezione di Protezione delle Piante, Università degli Studi, 

Via della Lastruccia 10, 50019 Sesto Fiorentino (FI), Italy. 2 Dipartimento 

di Biologia delle Piante Agrarie, Sezione di Genetica, Università 

degli Studi, Via Matteotti 1⁄B, 56124 Pisa, Italy. 3 Dipartimento 

di Scienze Biochimiche, Università degli Studi, Viale Morgagni 

50, 50134 Firenze, Italy. E-mail: ivan.baccelli@unifi.it 

Ceratocystis platani is the causal agent of canker stain, the 

most serious disease of plane trees. The fungus produces ceratoplatanin 

(CP), a protein of about 12.4 kDa acting as a PAMP in 

host and non-host plants. On plane leaves, CP elicits the transcription 

of defence-related genes earlier than C. platani. The 

amino acid sequence 1-119 of CP is a new protein domain, called 

“cerato-platanin domain”; thus, CP is the founding member of 

the cerato-platanin family (pfam PF07249). To date, a number of 

highly conserved proteins produced by Ascomycetes and Basidiomycetes 

proved to contain this domain. They have been reported 

to interact with plants and humans, but very little is known 

about the regulation of the genes coding for these proteins and 

about their primary role in the lifestyle of the producing fungi. 

With the present work we show that CP is released by the fungus 

during the interaction with the host plant, and the expression of 

the cp gene is highly modulated. This gene is expressed more rapidly 

when the fungus is inoculated on the plane leaves than when 

it is grown in axenic culture. Some other potential abiotic and biotic 

stressors have been investigated: temperature, H 2 O 2 , umbelliferone 

phytoalexin, matric water stress, light, growth on sawdust 

of susceptible and resistant plane or elm trees, and co-culture 

with Trichoderma atroviride P1 and T. harzianum T22. Gene 

expression was evaluated by qRT-PCR using TaqMan probes. 

The highest effect on the modulation of the cp gene was caused 

by temperature and matric water stress. 

EVOLUTION OF KIWIFRUIT BACTERIAL CANKER. 

G.M. Balestra, A. Rossetti, L. Ricci, M. Renzi, A. Quattrucci, 

M.C. Taratufolo, A. Mazzaglia. Dipartimento di Protezione delle 

Piante, Università degli Studi della Tuscia, Via S. Camillo de Lellis, 

01100 Viterbo, Italy. E-mail: balestra@unitus.it 

Italy is the first kiwifruit world exporter but this crop is economically 

important also for many other countries. Hayward (Actinidia 

deliciosa) is by far the most common green cultivar, whilst 

over the last ten years there has been a considerable increase of 

yellow cultivars (Actinidia chinensis), such as Hort 16 A-Zespri 

Gold and Jin Tao-Kiwi Gold. Among diseases affecting kiwifruit, 

the bacterial canker caused by Pseudomonas syringae pv. actinidiae 

(PSA) proved to be the most dangerous during the last years. 

PSA is able to infect quickly host plants causing severe damages 

on different plant organs and inducing typical symptoms such as 

abundant exudate production. PSA affects different Actinidia 

spp. and cultivars, survives within host tissues in winter, and its 

spreading during the vegetative season is favoured by heavy rains, 

late frosts and hail storms followed by mild temperatures. Pruning 

and harvest, as well as nutritional strategies, are crucial for 

PSA disease development. Different molecular approaches were 

used to assess the genetic relatedness among strains from several 

countries. Genetic fingerprints showed few but significant differences 

between Italian, Japanese and Korean bacterial populations, 

suggesting a separate origin for the disease. The implementation 

of effective control strategies by sustainable approaches 

such as natural compounds/antagonists, induced resistance and 

reduced amount of cupric salts, coupled with proper nutritional 

supports, is in progress. Recent findings are also reported on PSA 

biological cycle and worldwide spread. 

FUSARIUM GUTTIFORME, THE INCITANT OF PINEAP- 

PLE FUSARIOSIS, CAUSES MYCOTIC INFECTION TO 

HUMAN HOSTS. V. Balmas 1 , B. Scherm 1 , A. Marcello 1 , F. 

Spanu 1 , H. Harak 2 , K. O’Donnell 3 , T. Aoki 4 , Q. Migheli 1 . 1 Dipartimento 

di Protezione delle Piante, Unità di Ricerca Istituto 

Nazionale Biostrutture e Biosistemi, Università degli Studi, Via E. 

De Nicola 9, 07100 Sassari, Italy. 2 Ospedale di Sesto San Giovanni, 

Milano, Italy. 3 National Institute of Agrobiological Sciences, 

Genebank Unit, Tsukuba, Japan. 4 Microbial Genomics Research 

Unit, Agricultural Research Service, U.S. Department of Agriculture, 

Peoria, Illinois, USA. E-mail: balmas@uniss.it

Journal of Plant Pathology (2010), 92 (4, Supplement), S4.71-S4.105 S4.73 

During an extensive survey of clinically relevant Fusarium 

species (Migheli et al., 2010, Journal of Clinical Microbiology 48: 

1076-1084), fifty-eight fusaria isolated in Northern and Central 

Italy from 50 Italian patients between 2004 and 2007 were subjected 

to multilocus DNA sequence typing to characterize the 

spectrum of species and circulating sequence types associated 

with dermatological infections, especially onychomycoses and 

paronychia, and other fusarioses. Sequence typing revealed that 

isolates were nearly evenly divided among the Fusarium solani 

(FSSC, N =18), the F. oxysporum (FOSC, N = 20) and the Gibberella 

(Fusarium) fujikuroi (GFSC, N = 20) species complexes. 

Among the GFSC, a two-locus typing scheme was used to successfully 

identify 17 isolates as F. verticillioides, two as F. sacchari 

and one as F. guttiforme (the latter from paronychia on a 65-yearold 

female in good health). Since the only known host for F. guttiforme 

is pineapple (Ananas comosus), we wanted to determine 

whether the patient had come in contact with fresh pineapple 

fruits possibly harbouring this pathogen and to further characterize 

this isolate to see if it was pathogenic to pineapple. A followup 

interview revealed that the patient frequently consumed fresh 

pineapple after peeling it by hand. Results of a pathogenicity experiment 

revealed that the human pathogenic F. guttiforme isolate 

NRRL 53131 was able to induce tissue necrosis and to sporulate 

profusely after 14 days incubation at 25°C when inoculated on 

artificially wounded pineapple leaves. The same symptoms were 

induced by the ex-holotype positive control strain of F. guttiforme 

(NRRL 25295). 

FLUORESCENT AFLP ANALYSIS OF ALTERNARIA AL- 

TERNATA ISOLATES CAUSING BROWN SPOT OF CIT- 

RUS. P. Bella 1 , G. Ialacci 1 , M. Russo 2 , A. Catara 1,2 , V. Catara 1 . 

1 Dipartimento di Scienze e Tecnologie Fitosanitarie, Università 

degli Studi, Via Santa Sofia 100, 95123 Catania, Italy. 2 Parco Scientifico 

e Tecnologico della Sicilia, Zona Industriale, Via V. Lancia, 

95030 Catania, Italy. E-mail: patrizia.bella@unict.it 

Alternaria brown spot, caused by the tangerine pathotype of 

Alternaria alternata, seriously affects yield and fruit quality of citrus 

tangerines and their hybrids. After the first report in Italy in 

2000, the disease has become a real threat, especially for the more 

susceptible citrus cultivars, which require an adequate schedule 

of treatments. Since no information on the population structure 

of A. alternata in Italy is available, a collection of isolates already 

characterized by conidial morphology, pathogenicity tests and endoPG 

sequence analysis, was further compared by fluorescent 

amplified fragment length polymorphism analysis (fAFLP), in order 

to assess intra-population diversity. Pre-amp Primer Mix I 

(Invitrogen), containing adapter complementary AFLP 

primers for EcoRI and MseI sites, each with one selective nucleotide, 

was used in the pre-amplification reaction. To choose 

the most performing selective primer combination, selective amplification 

was preliminarily performed on few isolates using four 

different selective primer pairs, bearing at 3’ends two selective 

nucleotides. Three primer pairs were discarded since the number 

of picks they generated neither allowed a clear comparison of fingerprint 

patterns nor detected genetic variability. The primer 

combination, Ecosel2 (+GA)/Msesel(+CT), produced an adequate 

number of picks and was chosen for the analysis of the collection 

of A. alternata isolates. UPGMA-based cluster analysis 

from the similarity matrix obtained using Jaccard coefficient, revealed 

the presence of at least two sub-populations. Isolates obtained 

from different groves either clustered together or into two 

distinct clusters, regardless of the cultivar they came from. 

FIRST REPORT OF IMPATIENS NECROTIC SPOT VIRUS 

INFECTING ONCIDIUM IN ITALY. M.G. Bellardi 1 and C. 

Cavicchi 2 . 1 Dipartimento di Scienze e Tecnologie Agroambientali, 

Sezione di Patologia Vegetale, Università degli Studi, Viale Fanin 

42, 40127 Bologna, Italy. 2 Plesso Didattico G. Scarabelli, Università 

degli Studi di Bologna, Viale G. Ascari 15, 40026 Imola (BO), 

Italy. E-mail: mariagrazia.bellardi@unibo.it 

Oncidium is a large genus of orchids that includes over 600 

species. The quality of Ligurian Oncidium potted pants and cut 

flowers is highly appreciated in the Italian floral auction market, 

and the number of producers has increased in the Sanremo area 

in the last decade. During spring 2008, Impatiens necrotic spot 

virus (INSV) was found by DAS-ELISA in greenhouse-grown 

Oncidium orchids displaying leaf symptoms that ranged from 

necrotic concentric rings to necrotic lesions 1-2 cm in diameer. 

Subsequently, symptomatic leaves exhibited yellowing and necrosis 

along the veins but no flower symptoms. Oncidium plants 

were growing adjacent to Ranunculus hybrids infected by this 

tospovirus. To confirm the presence of INSV, total RNA was extracted 

from symptomatic Oncidium leaves and used for RT-PCR 

amplification of the nonstructural protein gene of INSV. The resulting 

primers directed the amplification of a PCR product of 

approximately 1300 nt. To verify that the amplified products 

were derived from INSV RNA, the PCR fragment was cloned 

and two independent clones were sequenced in both directions. 

Sequence analyses of the cloned PCR product showed 98.6% nucleotide 

sequence identity, and 98.2 and 99.2% amino acid identity 

and similarity, respectively with a previously published INSV- 

NSs sequence (Gene Bank accession No. NC_003624). Methods 

for reducing the risk of this disease include the use of healthy 

plant material, removal of weeds acting as hosts for the insect 

vector (Frankliniella occidentalis) or the use of insecticides to prevent 

virus transmission. This is thought to be the first report of 

INSV infecting Oncidium in Italy. 

STUDY OF TWO DIFFERENT HYDROPHOBINS IN 

GEOSMITHIA spp. P. Bettini 1 , L. Carresi 2 , C. Comparini 2 , G. 

Tomai 1 , R. Viganò 1 , L. Pazzagli 3 , A.L. Pepori 4 , A. Santini 4 , G. 

Cappugi 3 , F. Scala 5 , A. Scala 2 . 1 Dipartimento di Biologia 

Evoluzionistica “Leo Pardi”, Università degli Studi, Via Romana 

17, 50125 Firenze, Italy. 2 Dipartimento di Biotecnologie Agrarie, 

Sezione di Protezione delle Piante, Università degli Studi, Via della 

Lastruccia 10, 50019 Sesto Fiorentino (FI), Italy. 3 Dipartimento di 

Scienze Biochimiche, Università degli Studi, Firenze, Italy. 4 Istituto 

per la Protezione delle Piante del CNR. Via Madonna del Piano 10, 

50019 Sesto Fiorentino (FI), Italy. 5 Dipartimento di Arboricoltura, 

Botanica e Patologia Vegetale, Università degli Studi “Federico II”, 

Via Università 100, 80055 Portici (NA), Italy. E-mail: 

aniello.scala@unifi.it 

The genus Geosmithia includes several fungal species associated 

with phloem-feeding bark beetles. In previous studies, we 

showed that the gene encoding cerato-ulmin (cu) in Ophiostoma 

novo-ulmi was also present in a Geosmithia isolate obtained from 

an elm tree affected by Dutch Elm Disease (DED). To explain 

this result, we hypothesized a horizontal gene transfer (HGT). 

Thirty-six isolates of Geosmithia, collected from elm trees with 

DED symptoms, were used in this work to verify if other Geosmithiae 

contained the cu gene. Sequencing of the PCR products 

obtained with different primer pairs designed on the cu gene sequence 

revealed that fragments highly homologous to the cu gene 

were present in all the analyzed Geosmithia isolates. Culture filtrates 

of 16 Geosmithia isolates gave a positive response in ELISA 

assays using anti-CU antibodies; however, Western blotting and


mass spectrometry analyses showed that the MW of the protein 

was higher than that expected for CU. This protein was purified 

and characterized. Applying Edmann sequencing and Genome 

Walking techniques, we obtained a partial nucleotide sequence of 

the gene and an amino acid sequence of the protein, that were 

different from those known for CU. Our results show that isolates 

of Geosmithia spp. harbour two different hydrophobin 

genes: one, homologous to the cu gene from O. novo-ulmi, could 

have been acquired by HGT between the two fungal species, as 

they occupy the same habitat in elm trees. The other gene codes 

for a new hydrophobin, named Geo1, with a good homology level 

to the O. novo-ulmi CU protein. 

ACTIVITY OF THE MICROBIAL CONTROL AGENT 

BACILLUS SUBTILIS STRAIN QST 713 AGAINST BACTE- 

RIAL CANKER OF KIWIFRUIT. E. Biondi 1 , S. Mucini 1 , C. 

Lucchese 1 , E. Ladurner 2 , M. Benuzzi 2 , P. Minardi 3 , U. Mazzucchi 

1 . 1 Dipartimento di Scienze e Tecnologie Agroambientali, 


40, 40127 Bologna, Italy. 2 Intrachem Bio Italia, Servizio Tecnico, 

Ricerca e Sviluppo, Via Calcinaro 2085, 47023 Cesena, Italy. 3 Dipartimento 

di Morfofisiologia Veterinaria e Produzioni Animali, 

Università degli Studi, Via Tolara di Sopra 50, 40064 Ozzano dell’Emilia 

(BO), Italy. E-mail: umberto.mazzucchi@unibo.it 

Bacterial canker of kiwifruit, caused by Pseudomonas syringae 

pv. actinidiae (Psa), became relevant, especially in 2008 and 2009. 

Epidemics occurred in Lazio and Emilia Romagna (central and 

northern Italy) and the causal organisms were identified on the 

basis of phenotypic and genomic characteristics. In Italy, the control 

of bacterial canker relies mainly on agronomic prophylactic 

measures and on the use of copper-based products. In this study, 

we investigated the ability of Bacillus subtilis strain QST-713 (Serenade 

Max) to inhibit the growth of two different Psa strains in 

vitro, and its ability to survive and reduce the population of two 

rifampicin resistant pathogen strains on female flowers of Actinidia 

chinensis and A. deliciosa. The microbial control agent was 

able to survive on female flowers of both Actinidia species, reaching 

a population of ca. 10 5 CFU/flower 48 and 96 h after application. 

Moreover, the antagonist reduced the Psa population on A. 

chinensis flowers by more than one order of magnitude 48 h after 

its application. These preliminary results indicate that B. subtilis 

strain QST 713 may be a promising tool for the biological control 

of bacterial canker of kiwifruit, and provide insights into the interaction 

between the microbial control agent and Psa. 

ANTIFUNGAL ACTIVITY OF THREE NOVEL SAPONINS 

FROM ALLIUM CEPA. G. Bonanomi 1 , A. Gargiulo 1 , V. Antignani 

1 , V. Lanzotti 2 , F. Scala 1 . 1 Dipartimento di Arboricoltura, 

Botanica e Patologia Vegetale, Università degli Studi di 

Napoli“Federico II”, Via Università 100, 80055 Portici (NA), Italy. 

2 Dipartimento di Scienze degli Alimenti, Università degli Studi di 

Napoli “Federico II”, Via Università 100, 80055 Portici (NA), Italy. 

E-mail: giuliano.bonanomi@unina.it 

During their life cycle plants interact with a wide range of different 

microbial species, including pathogens. Thousands of diverse 

natural products are produced by plants and many of these 

are involved in plant defence. The phytochemical diversity of antimicrobial 

compounds include terpenoids, phenolics, phenylpropanoids, 

stilbens, alkaloids, glucosinolates, indole and 

saponins. In this study, the effects of three novel saponins 

(ACE15G3, ACE15E, ACE15D4) isolated from Allium cepa were 

tested on soil-borne pathogens (Fusarium oxysporum f. sp. lycopersici, 

Rhizoctonia solani and Sclerotium cepivorum), air-borne 

pathogens (Alternaria alternata, Aspergillus niger, Botrytis cinerea, 

Mucor sp., Phomopsis sp.) and two antagonistic fungi (Trichoderma 

atroviride and T. harzianum). Antifungal activity of all three 

saponins increases with their concentration and varied with the 

following rank: ACE15G3 > ACE15E ~ ACE15D4. F. oxysporum 

f. sp. lycopersici, S. cepivorum and R. solani were very little affected 

by saponins. Among the other fungi, B. cinerea and the two 

Trichoderma species were the most sensitive. We found a significant 

synergism in the antifungal activity of the three saponins 

against B. cinerea. Growth of this fungus was strongly inhibited 

when the three saponins were applied in combination. 

PLANT LITTER PHYTOTOXICITY AND SOIL-BORNE 

PATHOGENS CAN EXPLAIN TREE DIVERSITY GRADI- 

ENT AT GLOBAL SCALE. G. Bonanomi 1 , F. Giannino 2 , G. 

Incerti 4 , S. C. Dekker 3 , M. Rietkerk 3 , S. Mazzoleni 1 . 1 Dipartimento 

di Arboricoltura, Botanica e Patologia Vegetale, Università 

degli Studi di Napoli “Federico II”, Via Università 100, 80055 Portici 

(NA), Italy. 2 Dipartimento di Ingegneria Agraria e Agronomia 

del Territorio, Università degli Studi di Napoli “Federico II”, Via 

Università 100, 80055 Portici (NA), Italy. 3 Department of Environmental 

Sciences, Copernicus Institute, Utrecht University, PO Box 

80115, 3508 TC Utrecht, The Netherlands. 4 Dipartimento di Scienze 

della Vita, Università degli Studi, Via Giorgieri 10, 34127 Trieste, 

Italy. E-mail: giuliano.bonanomi@unina.it 

The diversity of plant species increases from the poles to the 

equator ranging from almost monospecific forests at high latitude, 

to intermediate species richness in temperate climates, to 

the hyper-diverse tropical forests. For plants in aquatic ecosystems, 

however, this does not occur. Current theories based on resources 

competition cannot explain such latitudinal patterns of 

plant diversity. Moreover, the co-occurrence of hyper-diverse 

stands in lowland terra firma forests and almost monospecific 

stands in mangroves and gallery riparian vegetation within the 

tropics remains enigmatic. Here we present a new mathematical 

model in which, besides the positive feedback of plant growth by 

nutrients release, litter decomposition and associated changes in 

soil microbial communities build up a species-specific negative 

feedback due to soil-borne pathogens and phytotoxicity released 

by the decaying plant litter. We validated the model by comparing 

it with extensive published data sets collected both across and 

within latitudinal or climatic zones. The model predicts correctly 

the number of tree species, their relative abundance as well as 

their biomass production in all environmental conditions providing 

a putative explanation also for the diversity variations observed 

within the tropics. The model advances in the direction of 

a unifying ecosystem theory and demonstrates a mechanistic link 

between the carbon cycle, the soil microbial communities and the 

tree diversity patterns. 

DISCOVERY OF NEW PHLOMIS SPECIES NATURALLY 

INFECTED WITH PHLOMIS MOTTLE VIRUS. D. Boscia 1 , 

P. Saldarelli 1 , A. De Stradis 1 , C. Vovlas 2 . 1 Istituto di Virologia 

Vegetale del CNR, UO Bari, Via Amendola 165/A, 70126 Bari, 

Italy. 2 Dipartimento di Protezione delle Piante e Microbiologia Applicata, 

Università degli Studi “Aldo Moro”, Via Amendola 165/A, 

70126 Bari, Italy. E-mail: d.boscia@ba.ivv.cnr.it 

Phlomis mottle virus (PhMV), a putative member of the family 

Flexiviridae, was isolated in 2008 from Phlomis fruticosa L.


(family Lamiaceae), a native Mediterranean plant widespread in 

the Greek Ionian islands and Epirus, on which it causes mottling 

and deformation of the leaves. More recently, similar symptoms 

were observed in five species of Phlomis, namely P. chrysophylla 

Boiss., P. italica L., P. purpurea L., P. viscosa Poir., and P. fruticosa 

growing in the Botanical Garden of the University of Bari. Leaf 

samples were collected, processed for observation in the electron 

microscope, and subjected to RT-PCR analysis using a set of the 

already described primers FloNAB-FloNABr. Immunoelectron 

microscopy (IEM) assays were also done using a polyclonal antiserum 

raised in rabbits immunized with purified PhMV particles. 

Electron microscope observation of leaf dips from all Phlomis 

species revealed the consistent presence of filamentous flexuous 

particles ca. 850 nm long, resembling virions of PhMV, which 

were decorated by the antiserum to PhMV. RT-PCR assays amplified 

a single product of the expected size of 465 bp from all tested 

species. The results of this investigation widen the natural host 

range and geographical distribution of PhMV. 

PRELIMINARY RESULTS ON DISTRIBUTION AND DI- 

VERSITY OF GRAPEVINE YELLOWS IN TUSCANY. H. 

Bouyahia 1 , D. Rizzo 2 , S. Paltrinieri 3 , M. Della Bartola 1 , P. Braccini 

2 , C. Milano 4 , A. Materazzi 1 , A. Bertaccini 3 . 1 Dipartimento di 

Coltivazione e Difesa delle Specie Legnose “G. Scaramuzzi”, 

Sezione di Patologia Vegetale, Università degli Studi, Via del 

Borghetto 80, 56124, Pisa, Italy. 2 Agenzia Regionale per lo Sviluppo 

e l’Innovazione nel Settore Agricolo-Forestale (ARSIA), Laboratorio 

di Diagnostica Fitopatologica, Via dei Fiori 8, 51012 Pescia 

(PT), Italy. 3 Dipartimento di Scienze e Tecnologie Agroambientali, 


42, 40127 Bologna, Italy. 4 Dipartimento Provinciale ARPAT, Unità 

Operativa Agroecosistemi e Alimenti, Via Ponte alle Mosse 211, 

50144 Firenze, Italy. E-mail: hbouyahia@agr.unipi.it 

Grapevine yellows (GY) are an important threat to grapevine 

industry in Europe. In Italy, GY are mostly represented by 

Flavescence dorée (FD) and Bois noir (BN) associated with infection 

by 16SrV and 16SrXII phytoplasma ribosomal groups, respectively. 

In the frame of a regional strategy aimed at monitoring 

GY presence, an extensive survey was carried out covering the 

ten provinces of Tuscany (central Italy), and collecting 585 leaf 

samples in commercial vineyards inspected in summer 2009. 

Preference was given to samples showing symptoms referable to 

GY diseases. To allow tracking of FD-positive samples for uprooting, 

all vines were marked and localised by “Global Positioning 

System-GPS”. Nested-PCR and Real-time PCR confirmed 

that BN is largely the most important GY in Tuscany with an 

ubiquitous distribution. In fact, it was present in almost all surveyed 

vineyard with an overall incidence of 70% (418 out of 585 

samples tested). Alarming presence of FD was recorded in 23 

samples distributed in five provinces, i.e. Arezzo, Massa-Carrara, 

Florence, Siena and Lucca. Molecular characterization performed 

with PCR/RFLP analysis on 16S ribosomal and on SecY 

genes confirmed the presence of either FD-C and FD-D subgroups. 

FD-D is reported for the first time in Tuscany and is 

identical to the strain described earlier in France and in Veneto 

(northern Italy). Further investigations on insect vectors, natural 

reservoirs and molecular characterization of grapevine-infecting 

phytoplasmas are needed to monitor and understand the epidemiology 

GY diseases in Tuscany. 

EXTENSIVE SURVEY OF VIRUSES INFECTING AN 

OLIVE VARIETAL COLLECTION IN TUSCANY. H. 

Bouyahia 1 , D. Rizzo 2 , M. Della Bartola 1 and A. Materazzi 1 1 Dipartimento 

di Coltivazione e Difesa delle Specie Legnose “G. Scaramuzzi”, 

Sezione di Patologia vegetale, Università degli Studi, Via 

del Borghetto 80, 56124 Pisa, Italy. 2 Agenzia Regionale per lo 

Sviluppo e l’Innovazione nel Settore Agricolo-Forestale (ARSIA), 

Laboratorio di Diagnostica Fitopatologica, Via dei Fiori 8, 51012 

Pescia (PT), Italy. E-mail: hbouyahia@agr.unipi.it 

The occurrence was investigsted of olive-infecting viruses in 

the regional varietal collection at the experimental station “Santa 

Paolina” (CNR-IVALSA, Follonica-Grosseto). Starting from 

2004, a total of 245 mother plants belonging to 26 different Tuscan 

olive varieties were sampled an tested for the presence of 

nine olive viruses. Largely grown varieties like Leccino, Frantoio 

and Moraiolo and the less known Correggiolo, Grappolo, Leccio 

del Corno, etc. were included. Hardwood cuttings taken from 

different part of the canopy were sampled twice a year (spring 

and autumn). Cortical scrapings were powdered in liquid nitrogen 

and total RNA was extracted. During the first years, cDNA 

synthesis followed by PCR was employed and successively replaced 

by one-Step RT-PCR for the detection of Arabis mosaic 

virus (ArMV), Cherry leafroll virus (CLRV) Cucumber mosaic 

virus (CMV), Olive latent virus 1 (OLV-1), Olive latent virus 2 

(OLV-2), Olive ringspot virus (OLRSV), Olive leaf yellowing-associated 

virus (OLYaV), Strawberry latent ringspot virus (SLRSV) 

and Tobacco necrosis virus (TNV). Seventeen of 245 samples 

(6.9%) were infected. Of the nine virus tested for only ArMV, 

CLRV, OLYaV and SLRSV were detected and no cases of mixed 

infections were found. Specifically, six mother plants of cvs Leccino, 

Frantoio and San Fancesco were infected with OLYaV. Ar- 

MV was found in cvs Correggiolo, Olivastra and Ornellaia and 

four mother plants of cvs Moraiolo and Maurino were positive to 

CLRV. Two positive samples to SLRV were detected in cvs Maurino 

and Grappolo. As previously reported, we confirmed a satisfactory 

sanitary status of olive germplasm in Tuscany. 

Work partially funded by “Progetto Interregionale-Qualificazione 

del vivaismo olivicolo – OLVIVA”. 

INCIDENCE AND DISTRIBUTION OF BOIS NOIR IN 

YOUNG VINEYARDS IN TUSCANY. P. Braccini 1 , D. Rizzo 1 , 

T. Cinelli 2 , G. Marchi 2 . 1 Agenzia Regionale per lo Sviluppo e l’Innovazione 

nel Settore Agricolo-Forestale (ARSIA), Via Pietrapiana 

30, 50121 Firenze, Italy. 2 Dipartimento di Biotecnologie Agrarie, 

Sezione di Protezione delle Piante, Università degli Studi, Piazzale 

delle Cascine 28, 50144 Firenze, Italy. E-mail: guido.marchi@ 

unifi.it 

The spread of bois noir was monitored from 2005 to 2009 in 

seven vineyards of cv. Sangiovese established in 2001 in Tuscany 

(central Italy). In each season the number and position of the 

vines showing typical symptoms (leaf discolouration, downward 

rolling of leaves, incomplete lignification of canes, shrivelled 

bunches) were recorded. Only the presence of 16SrXII-A phytoplasmas 

was ascertained by PCR analysis on bulk samples of 

symptomatic leaves. With the purpose of determining the spatial 

pattern of the disease (random vs. aggregated or clustered), for 

each year of assessment, field data on annual incidence were analyzed 

at three spatial levels (hierarchies) including: (i) adjacent 

vines within the row and across the rows (ordinary runs analysis), 

(ii) within vines grouped into sampling units (distribution analysis) 

and (iii) among groups of plants at some distance from one 

another (SADIE-Spatial Analysis by Distance Indices). As a 

whole, the pattern of temporal disease incidence showed a similar 

trend in six of the seven study sites: annual incidence rate progressively 

decreased from year to year after the first or second


season of survey. Among the other epidemiological phenomena 

observed the most important were: (i) a sharp decrease in the frequency 

of new disease records in previously unaffected 

grapevines; (ii) a random distribution of disease incidence for the 

majority of the plots and assessment periods at all spatial levels of 

analysis, suggesting the lack of disease spread between adjacent 

vines. Studies designed to characterize the role of insect vectors 

in disease spread are in progress. 

MYCORRHIZAL SYMBIOSES AND PLANT PATHOLOGY. 

S. Burruano 1 , P. Corda 2 , A. Franceschini 2 , G. Innocenti 5 , M. Iotti 

5 , E. Lancellotti 2 , L. Montecchio 3 , S. Mutto Accordi 3 , F. Piattoni 

5 , L. Scattolin 3 , L. Torta 1 , A. Vannini 4 , A.M. Vettraino 4 , A. 

Zambonelli 5 . 1 Dipartimento di Scienze Entomologiche, Fitopatologiche, 

Microbiologiche, Agrarie e Zootecniche, Università degli 

Studi, Viale delle Scienze 2, 90128 Palermo, Italy. 2 Dipartimento di 

Protezione delle Piante, Università degli Studi, Via De Nicola, 

07100 Sassari, Italy. 3 Dipartimento del Territorio e dei Sistemi 

Agroforestali, Università degli Studi, Viale dell’Università, 35020 

Legnaro, Italy. 4 Dipartimento di Protezione delle Piante, Università 

degli Studi della Tuscia, Via S. Camillo de Lellis, 01100 Viterbo, 

Italy. 5 Dipartimento di Protezione e Valorizzazione Agroalimentare, 

Università degli Studi, Via Fanin, 40127 Bologna, Italy. 

E-mail montecchio@unipd.it 

The firs detailed morphological description of a mycorrhiza 

was by Giuseppe Gibelli in 1883, in a paper on chestnut ink disease. 

Since then, much of the published literature reported the 

importance of mutualistic mycorrhizal symbioses for the improvement 

of the vegetative and health status of plants, mainly by 

means of an increase in water and mineral nutrition, and by protection 

from fungal parasites. Rare examples of parasitic mycorrhizal 

symbioses are also known. In natural ecosystems, almost all 

terrestrial plants are associated with a wide community of mycorrhizal 

fungal species, whose composition is the result of quick dynamic 

interactions involving vegetative, nutritional, physiological 

and pathological features. Since a significant correlation between 

plant health and mycorrhizal community composition is well established, 

a synthetic evaluation of the plant or plantation fitness, 

generally difficult to assess, can be deduced from the mycorrhizal 

status. An in-depth examination of the role of mycorrhizae, the 

majority of which are still undescribed, and of the dynamics regulating 

their presence and frequency in any given ecosystem, are 

therefore of main importance in plants health management. 

Thanks to the availability of modern microscopic, enzymatic, molecular 

and biostatistics methods, the Italian research on this topic 

is in constant growth at the international level, also in applyed 

sectors, i.e. production of artificially mycorrhized plants to be 

employed in unfavourable sites, or production of high-priced 

sporocarps. 

HIGH SUSCEPTIBILITY OF THE TRIPLOID HYBRID 

LEMOX TO MAL SECCO DISEASE CAUSED BY PHOMA 

TRACHEIPHILA. S.O. Cacciola 1 , A. De Patrizio 2 , F. Raudino 3 , 

A. Pane 3 , V. Lo Giudice 4 , G. Magnano di San Lio 5 . 1 Dipartimento 

di Chimica Biologica, Chimica Medica e Biologia Molecolare, 

Università degli Studi, Viale Andrea Doria 6, 95125 Catania, Italy. 

2 Dipartimento di Scienze Entomologiche, Fitopatologiche, Microbiologiche, 

Agrarie e Zootecniche, Università degli Studi, Viale delle 

Scienze 2, 90128 Palermo, Italy. 3 Dipartimento di Scienze e Tecnologie 

Fitosanitarie, Università degli Studi, Via S. Sofia 100, 

95123 Catania, Italy. 4 Via Calvario 45, 95030 Mascalucia (CT), 

Italy. 5 Dipartimento di Gestione dei Sistemi Agrari e Forestali, 

Università Mediterranea degli Studi, Località Feo di Vito, 89122 

Reggio Calabria, Italy. E-mail: olga.cacciola@unict.it 

‘Lemox’, a triploid obtained by crossing the tetraploid lemon 

‘Doppio Lentini’ and the spontaneous lemon hybrid ‘Femminello’ 

x ‘Pera del Commendatore’, is a seedless and early bearing 

new lemon cultivar, with a good fruit size and high yields. In this 

study, the susceptibility of ‘Lemox’ to mal secco disease was tested 

in comparison with commercial lemon cultivars, including nucellar 

and old clones of ‘Monachello’, ‘Femminello’, ‘Femminello 

Zagara bianca’, ‘Cerza VCR’, ‘Lunario’, ‘Femminello Siracusano 

2Kr’, as well as three triploid hybrids of ‘Femminello’ x allotetraploid 

somatic hybrids of ‘Valencia’ sweet orange x ‘Femminello’(VF), 

‘Milam’ lemon x ‘Femminello’(MF), and Key lime x ‘Valencia’sweet 

orange (KlV), respectively. In autumn 2008, 1-yearold 

potted trees on sour orange rootstock were stem-inoculated 

with a conidial suspension (10 6 conidia/ml) of a virulent isolate of 

Phoma tracheiphila. In spring 2009, symptom severity was rated 

visually and the extent of xylem colonization by the fungus was 

assessed by both isolation on potato-dextrose-agar and a PCRbased 

assay with specific primers (PtFOR2 and PtREV2). 

‘Lemox’ and ‘Femminello Siracusano 2Kr’ proved to be the most 

susceptible cultivars, followed by ‘Lunario’ and ‘Femminello’, 

wheres both nucellar and old clones of ‘Monachello’ were tolerant. 

‘Cerza VCR’ and ‘Femminello Zagara bianca’ were moderately 

tolerant. The MF hybrid proved to be very susceptible 

whereas the VF and KLV hybrids were moderately tolerant. 

However, the tolerance to mal secco of these two hybrids was not 

comparable to that of ‘Monachello’. The test was repeated in 

2009-2010, yielding similar results. 

EFFECT OF DOWNY AND POWDERY MILDEWS ON 

GRAPEVINE BERRY JUICE COMPOSITION. T. Caffi, S.E. 

Legler, V. Rossi. Istituto di Entomologia e Patologia Vegetale, Università 

Cattolica del Sacro Cuore, Via E. Parmense 84, 29122 Piacenza, 

Italy. E-mail: tito.caffi@unicatt.it 

Plasmopara viticola and Erisyphe necator are the causal agents 

of downy and powdery mildew of grapevine, respectively. They 

can cause severe yield losses but their effect on the quality components 

of bunches is little investigated. Clusters of different redand 

white-berried cultivars of Vitis vinifera were hand-harvested 

from untreated plots in commercial vineyards of Emilia-Romagna, 

Piedmont, and Lombardy. Individual berries were removed 

from clusters and classified as follows: (i) healthy from 

healthy clusters; (ii) healthy from affected clusters; (iii) with a single 

downy mildew lesion, or powdery mildew colony; (iv) with 

multiple lesions or colonies; (v) totally affected or cracked. Subsamples 

of the berries belonging to the same category were 

crushed, de-stemmed, and pressed. Juice yields were determined, 

and chemical and physical measurements were performed on the 

juice (i.e., °Brix, pH, treatable acidity, malic and tartaric acids, 

and polyphenols). Both pathogens negatively affected juice yield 

and average weight of berries, and modified quality components 

of the juice. For instance, powdery mildew increased °Brix from 

18.3 to 23.3, while downy mildew increased treatable acidity by 

77% in average. Equations were developed for estimating the impact 

of downy and powdery mildews on quality of bunches. 

EPIDEMIC DEVELOPMENT OF RHIZOCTONIA SOLANI 

ON BRASSICA OLERACEA var. ACEPHALA, AND CHARAC- 

TERIZATION OF ISOLATES. R. Caiazzo, A. Carella, R. Nicoletti, 

F. Raimo, F.A. Porrone, E. Lahoz. CRA, Unità CAT, Via P.


Vitiello 108, 84018 Scafati (SA), Italy. E-mail: rosa.caiazzo@ 

entecra.it 

Brassica oleracea L. var. acephala known as ‘torzella’ was in the 

past largely cultivated in southern Italy, then abandoned, and 

revaluated in recent years. In a farm in Scafati, where torzella was 

cropped for the first time, collar rot symptoms were observed on 

about 20% of the plants. Isolates of Rhizoctonia solani Kühn, 

showing the typical hyphal branching and multinucleate cells, 

were recovered from affected tissues. Determination of anastomosis 

groups was carried out by pairing isolates with tester strains on 

2% water agar (WA) in Petri plates. Hyphal anastomosis was only 

observed with tester isolates of AG-4, producing both C2 and C3 

reactions. Moreover, typical AG-4 isozyme patterns were obtained 

after polygalacturonase gel electrophoresis. The clonal condition 

of the isolates was also assessed by examining tuft formation in 

pairings on PDA supplemented with 0.5% activated charcoal. Finally, 

a biomolecular analysis was carried out by means of RFLPs 

of rDNA-ITS by using HincII and HpaI as restriction enzymes to 

assign torzella isolates to one of the three homogeneous groups 

(HG-I, -II and -III) so far characterized within R. solani AG-4. 

Pathogenicity test confirmed the ability of R. solani AG-4 isolates 

to induce collar rot. These isolates were recovered from symptomatic 

tissues, thus fulfilling Koch’s postulates. 

TRANS-RESVERATROL AND LEAF SYMPTOM EXPRES- 

SION IN ESCA OF GRAPEVINE. F. Calzarano 1 , V. D’Agostino 

1 , F. Osti 2 , S. Di Marco 2 . 1 Dipartimento di Scienze degli Alimenti, 

Università degli Studi, Via C.R. Lerici 1, 64023 Mosciano 

Sant’Angelo (TE), Italy. 2 Istituto di Biometeorologia del CNR, Via 

P. Gobetti 101, 40129 Bologna, Italy. E-mail: fcalzarano@unite.it 

Esca of grapevine is a serious disease some aspects of which, 

including the nature of foliar symptoms, are still poorly understood. 

Leaves collected at different phenological growth stages 

from symptomatic grapevines cv. Trebbiano d’Abruzzo showed 

the presence of higher concentrations of trans-resveratrol, the 

major stilbene produced in grapevine, compared to leaves from 

healthy plants. Increased levels of trans-resveratrol are supposed 

to be associated with the host-plant defence response, but this 

condition raises questions on the possible role of the stilbene in 

the foliar symptom development. Symptomatic leaves collected in 

2009 were divided into 4 classes estimating symptom severity as 

the percentage of chlorosis and necrosis on the total leaf area. For 

each class, leaves were collected at pre-bunch closure, post-veraison 

and harvest, and the level of trans-resveratrol was determined. 

The results showed higher concentrations of trans-resveratrol 

in symptomatic leaves collected at pre-bunch closure compared 

to what observed at the other growth stages. These results 

are in agreement with our previous findings, i.e. the stronger response 

of the leaves might be correlated with leaf functioning at 

the different phenological growth stages. Moreover, the increase 

of trans-resveratrol concentration with increased severity of leaf 

symptoms seems to exclude an involvement of the stilbene in 

symptom development. Further investigations on cut leaves inoculated 

with culture filtrates of the pathogen and/or trans-resveratrol 

may lead to a better understanding of the relationship between 

the stilbene and foliar symptom expression. 

THREE BASIDIOMYCETES CONTAMINATING COM- 

POST FOR CULTIVATION OF PLEUROTUS ERYNGII. I. 

Camele, L. Altieri, G.L. Rana. Dipartimento di Biologia, Difesa e 

Biotecnologie Agro-Forestali, Università degli Studi della Basilica- 

ta, Via Ateneo Lucano 10, 85100, Potenza, Italy. E-mail: 

ippolito.camele@unibas.it 

The basidiomycetes Oxyporus latemarginatus, Schizophyllum 

commune and Bjerkandera adusta were found contaminating substrate 

commonly used to grow Pleurotus eryngii in southern Italy 

in 2009. More specifically, basidiomes of O. latemarginatus and S. 

commune appeared on the compost surface at beginning of a cultivation 

in Sardinia and in an incubation tunnel of ITALMIKO 

agricultural farm in Basilicata, respectively. B. adusta formed 

sporophores after the first production flush in a P. eryngii autumnal 

culture in Apulia. Identification of the three contaminants 

was at first achieved on the basis of their macro- and microscopic 

features. Molecular analysis, done by PCR and nucleotide sequencing, 

confirmed the identity of the first and second contaminant 

as O. latemarginatus and B. adusta. DNA was extracted from 

pure cultures of both contaminants and amplified with primers 

ITS4/ITS5. Nucleotide sequences of O. latemarginatus (isolates 

n. 947) and B. adusta (isolate n. 1147) were 606 and 609 bp in 

size and showed a 99% similarity with GenBank sequences of the 

two fungi (accession numbers AF 232721 and AJ 006672). Two 

nucleotide sequences of the above isolates of O. latemarginatus 

and B. adusta were deposited at the European Molecular Biology 

Laboratory (UK) with access codes FN 252852 and FN995241, 

respectively. This is the first report of O. latemarginatus and S. 

commune as contaminants and competitors of P. eryngii in exploiting 

cultivation compost. By contrast, B. adusta was already 

known as contaminant of the same substrate. 

EFFECT OF ORGANIC AMENDMENTS AND MICRO- 

BIAL DIVERSITY ON SOIL FUNGISTASIS. M. Capodilupo, 

G. Bonanomi, M. Ceniccola, V. Antignani, F. Scala. Dipartimento 

di Arboricoltura, Botanica e Patologia Vegetale, Università degli 

Studi di Napoli “Federico II”, Via Università 100, 80055 Portici 

(NA), Italy. E-mail: felice.scala@unina.it 

Soil fungistasis is defined as the inhibition of fungal propagule 

germination under favourable conditions. In this study, we analyzed 

the effect on fungistasis of four different carbon sources 

(glucose, starch, PDB and Medicago sativa hay) at different times 

of decomposition (0, 2, 4, 7, 28 and 46 days) applied at three concentration 

levels (3%, 0.3% and 0.03%). Fungistasis was assessed 

by germination tests carried out with four fungi: Aspergillus 

flavus, Botrytis cinerea, Mucor sp. and Trichoderma 

harzianum. Microcosms were characterized for pH, electrical 

conductivity and fluoresceine diacetate hydrolytic activity (FDA). 

We also tested the influence of microbial diversity on fungistasis. 

Microbial consortia with three levels of diversity (1, 3 and 9 

species) were assembled from a pool of known microorganisms 

selected among different functional groups. In this case, fungistasis 

was assessed by using Rhizoctonia solani as a target fungus. We 

found that fungistasis was rapidly lost after the application of all 

organic amendments but it was also restored, usually within 7-28 

days, according to the type and the amount of amendment. Mucor 

sp. was the most sensitive fungus while A. flavus was the less 

affected. Fungistasis towards R. solani was enhanced and much 

less variable in the presence of microcosms of increasing microbial 

diversity. We conclude that soil fungistasis is strictly dependent 

on decomposition of organic matter and diversity of the microbial 

communities. 

OCCURRENCE OF LECANICILLIUM MUSCARIUM AS 

AN ANTAGONIST OF SOIL-BORNE FUNGAL


PATHOGENS OF PERENNIAL WALLROCKET. A. Carella 

and R. Nicoletti. CRA, Unità di Ricerca per le Colture Alternative 

al Tabacco, Via P. Vitiello 108, 84018 Scafati (SA), Italy. E-mail: 

rosario.nicoletti@entecra.it 

The role of fungal antagonists in plant protection may be 

quite relevant in short-cycle crops where the use of fungicides is 

restricted. During an investigation on crown and root rot of 

perennial wallrocket (Diplotaxis tenuifolia) in the Piana del Sele 

area, the occurrence of fungal antagonists was observed in the 

isolation plates of the causal agents, Rhizoctonia solani and Sclerotinia 

sclerotiorum. Besides some well-known mycoparasites, a 

fungus producing Verticillium-like conidiophores was recovered 

from subcultures of both pathogens which was identified as 

Lecanicillium muscarium (Petch) Zare et Gams. While its antagonistic 

ability against several rusts and powdery mildews is already 

documented, to our knowledge this is the first report in relation 

to the above-mentioned soil-borne pathogens. Their mycelial mat 

was slowly overgrown by L. muscarium isolates in PDA cultures, 

and no colonies originated as mycelial plugs were transferred into 

new plates. S. sclerotiorum sclerotia harvested as soon as they had 

developed were colonized by the mycoparasite, as they assumed a 

soft consistency and failed to germinate when placed on fresh 

medium a month later. Mycoparasitic interactions in dual cultures 

were not obvious since coiling or growth inside hyphae of 

the suscept did not occur. However, a depressed growth of the 

hyphae was indicative of the likely establishment of a trophic relationship, 

as we have previously ascertained electron microscopically 

for other R. solani antagonists. Mycoparasitic aptitude by 

isolates of L. muscarium was also supported by the production of 

chitinases and glucanases in liquid cultures. 

DIFFERENTIAL COLONIZATION OF RESISTANT AND 

SUSCEPTIBLE MELON GENOTYPES BY FUSARIUM 

OXYSPORUM f. sp. MELONIS. V. Catalano 1 , A. Haegi 1,2 , L 

Luongo 1 , N. Ficcadenti 3 , A. Belisario 1 . 1 CRA, Centro di Ricerca 

per la Patologia Vegetale (CRA-PAV), Via C.G. Bertero 22, 00156 

Roma, Italy. 2 CRA, Unità di Ricerca per il Vivaismo e la Gestione 

del Verde Ambientale ed Ornamentale (CRA-VIV), Via dei Fiori 8, 

51012 Pescia (PT), Italy. 3 CRA, Unità di Ricerca per l’Orticoltura 

(CRA-ORA), Via Salaria 1, 63030 Monsampolo del Tronto (AP), 

Italy. E-mail: alessandra.belisario@entecra.it 

Fusarium wilt of melon, caused by the soil-borne fungus 

Fusarium oxysporum f. sp. melonis (FOM), is the most important 

and least controllable disease. The most effective control is the 

use of resistant host genotypes. Investigations were conducted to 

assess the qualitative and quantitative traits of FOM colonization 

in the compatible and incompatible interactions. The absence of 

symptoms on susceptible scions was considered of particular interest. 

To this purpose, FOM race 1 (ISPaVe1070) and race 1,2 

(ISPaVe1018) were used to inoculate grafted melon plants. The 

resistant DH line NAD-1 and the susceptible Charentais-T were 

used in all possible combinations. Pathogen colonization of the 

stem at different heights and at 2, 4, 14 and 18 days post inoculation 

(dpi) was evaluated. Assessments were performed both by 

direct isolations from stem fragments and by PCR with a selective 

primer pair. The results showed that in plants with resistant rootstock 

no disease symptoms appeared at 18 dpi, and the two fungal 

strains were reisolated mainly from the rootstocks and rarely 

from the susceptible scions. Conversely, in plants with susceptible 

rootstock disease symptoms became obvious at 7 dpi and the 

plants died at 18 dpi. The two fungal strains were always reisolated 

form the whole stem. PCR confirmed the outcome of direct 

reisolations and showed to be more sensitive. Plants with resist- 

ant rootstock inoculated with either FOM race 1 or race 1,2 remained 

symptomless until the end of the experiment, notwithstanding 

the presence.of the fungus. 

CHARACTERIZATION OF A NEW ILARVIRUS SPECIES 

FROM VIOLA TRICOLOR. M. Ciuffo, R. Lenzi, V. Masenga, 

M. Turina. Istituto di Virologia Vegetale del CNR, Strada delle 

Cacce 73, 10135 Torino, Italy. E-mail: m.turina@ivv.cnr.it 

In 2009, a number of samples of Viola tricolor from Lombardy 

(northern Italy) showing white mosaic and deformation of 

younger leaves were mechanically inoculated on a range of host 

plants. Electron microscopy observation failed to detect virus 

particles in the original sample, but inoculated Nicotiana benthamiana 

plants showed leaf deformations that could be easily 

mechanically transmitted. Numerous attempts to purify the virus 

using standard procedures failed. We therefore proceeded to isolate 

dsRNA from infected N. benthamiana. A random primer cD- 

NA library from dsRNA yielded clones specific to infected 

plants. Sequence analysis showed some similarity with Prune 

dwarf virus (Ilarvirus, Bromoviridae) RNA-3 coding region. From 

sequence information from the initial clone, specific oligonucleotides 

were designed to extend cloning and sequencing to other 

RNA-3. Region. The full length coding sequence is now available 

of the putative movement protein (MP) and coat protein 

(CP). Phylogenetic analysis of a number of Ilarvirus sequences 

suggests that the virus under stidy is a new Ilarvirus species, for 

which the name Viola white mosaic virus (VWMV) is proposed. 

Specific oligonucleotides were used in RT-PCR for diagnostic 

purposes in a number of viola samples. 

WIDESPREAD OCCURRENCE OF RANUNCULUS LA- 

TENT VIRUS (MACLURAVIRUS, POTYVIRIDAE) IN ARTI- 

CHOKE CROPS IN ITALY. M. Ciuffo 1 , R. Lenzi 1 , M. Testa 2 , 

M. Turina 1 . 1 Istituto di Virologia Vegetale del CNR, Strada delle 

Cacce 73, 10135 Torino, Italy. 2 Dipartimento per la Ricerca nelle 

Produzioni Vegetali, AGRIS Sardegna, Cagliari, Italy. E-mail: m.turina@ivv.cnr.it 

In 2004 four new virus species belonging to the family Potyviridae, 

were found in Ranunculus asiaticus in Liguria (Northern 

Italy). One of these, the macluravirus Ranunculus latent virus 

(RaLV) was detected by DAS ELISA in symptomatic and symptomless 

artichoke plants. Several other samples from Liguria, Sardinia 

and Latium were tested in the following years and most of 

them were positive for RaLV. The presence of this virus in the 

samples was also confirmed by specific western blot analysis. A 

fragment of the sequence corresponding to the 3’ proximal region 

of the genome of about 500 bp was amplified cloned and sequenced 

using RaLV specific primers. Comparison of the artichoke 

isolates from Liguria, Sardinia and Latium, with the ranunculus 

isolates of RaLV showed 98% identity at the nucleotide level. 

The sequence of the artichoke isolate was than extended upstream 

to cover a part of the NIa coding sequence. The NIa fragment 

from RaLV showed 80% identity with Artichoke latent virus 

(ArLV), a virus known in artichoke since the early sixties. Due to 

the similarity with ArLV we tried to detect RaLV infected samples 

with an ArLV commercial probe, but no reaction was obtained. 

Based on our results RaLV is distinct from ArLV; its presence in 

artichoke germplasm is cause of concern, particularly because it 

seems to exacerbate symptoms expression of other viruses when 

present in mixed infections.


ANTIFUNGAL ACTIVITY OF ALCOHOLIC EXTRACT 

FROM SPARTIUM JUNCEUM : PRELIMINARY SCREEN- 

ING STUDIES. F. Clematis, C. Pasini, P. Curir. CRA, Unità di 

Ricerca per la Floricoltura e le Specie Ornamentali (CRA-FSO), 

Corso Inglesi 08, 18038 Sanremo (IM), Italy. E-mail: f.clematis@ 

istflori.it 

The antifungal properties of Spartium junceum, a perennial 

shrubs native to the Mediterranean area and widespread throughout 

Italy, were tested against eight selected fungal pathogens. 

Plant material (stem and leaves) were extracted with ethanol (1:1) 

in a soxhlet apparatus under refluxing conditions for 3 h. The efficacy 

of plant extracts was tested in vitro by poisoned food technique 

at different concentrations (2, 1.6, and 1%). The best inhibition 

values ranged between 21.3 and 46%. The highest concentration 

of plant extract (2%) showed significant reduction on the 

growth of Fusarium oxysporum isolated from Danae racemosa and 

F. oxysporum f. sp. dianthi, while the lowest concentration (1%) 

was already effective in suppressing Alternaria dianthi and Botrytis 

cinerea. A. dianthi was inhibited by 46.0% when 2% of plant 

extract was applied, B. cinerea and both F. oxysporum strains 

were inhibited by 45.3 and 37.3%, respectively. This preliminary 

screening showed significant inhibition activity of S. junceum extracts 

already at low concentrations. Preliminary TLC and spectrophotometer 

analysis showed the presence of phenolic compounds 

in the extracts using UV absorbance at 275 nm. Investigations 

are in progress to study the phytochemical characteristics 

of plant extract by HPLC and TLC. 

AN APPROACH TO GRAPEVINE SANITARY SELEC- 

TION IN SARDINIA: FIELD STUDY AND SEROLOGICAL 

DIAGNOSIS. L. Cogotzi, V.A. Prota, R. Garau. Dipartimento di 

Protezione delle Piante, Sezione di Patologia Vegetale, Università 

degli Studi, Via Enrico De Nicola 1, 07100 Sassari, Italy. E-mail: 

lcogotzi@uniss.it 

Strategies to control viruses are essentially preventive and 

based on sanitary selection programs. Within this project, the creation 

of a germplasm collection will aim at progressive sanitary 

selection. Field studies, carried out over two years (2008-2009), 

focused on eleven local grapevine varieties: Vermentino, Cannonau, 

Monica, Bovale, Malvasia, Carignano, Nuragus, Nebbiolo, 

Moscato, Caricajola, and Nasco. Selected vineyards were inspected 

in spring, summer and autumn. Symptoms of virus and 

phytoplasma infections were common in these vineyards. Almost 

5000 essentially symptomless vines were selected in the course of 

numerous field surveys carried out throughout the entire region 

and were tested serologically (ELISA). We focused on the presence 

of two types of viruses associated with leafroll disease 

(GLRaV-2, GLRaV-3) that cause severe losses, reduce yield and 

impact negatively fruit quality. Grapevine fanleaf virus (GFLV) 

and Grapevine virus A (GVA), an agent associated with Kober 

stem grooving were also looked for on a lower number of plants. 

Results showed that, in Sardinia, infection rates of GLRaV-2, 

GLRaV-3, GFLV and GVA range between 3 and 70%, 5 and 

80%, 0 and 40% and 5 and 60%, respectively. ‘Carignano’ from 

the Sulcis area (south-west Sardinia) had the highest percentage 

of virus-free plants while ‘Nuragus’ and ‘Malvasia, both from the 

south of Sardinia (Parteolla area) had the lowest. Selected plants 

will undergo further sanitary selection. 

Work funded by the Con.Vi.Sar. consortium of Sardinia 

PLANT VIRUSES AS POSSIBLE PLATFORMS FOR MOLE- 

CULAR FARMING. V. Condelli, A. Vitti, M. Nuzzaci, M.T. 

Lanorte, P. Piazzolla. Dipartimento di Biologia Difesa e Biotecnologie 

Agro-Forestali, Università degli Studi della Basilicata, Campus 

di Macchia Romana, Via dell’Ateneo Lucano 10, 85100 Potenza, 

Italy. E-mail: valentina.condelli@unibas.it 

Plant viruses are emerging as an attractive system for the expression 

of foreign epitopes used as immunogens for the development 

of innovative vaccination strategies. In such a way, plant 

viruses carrying on their coat protein (CP) peptides of medical 

interest can be considered, in association with their hosts, right 

partners of biological systems devoted to pursue the goal of functioning 

as medical molecular farming. Cucumber mosaic virus 

(CMV) is one of the plant viruses used as a carrier of foreign epitopes 

because of its wide host range, which comprises edible 

plants (celery, lettuce, cucumber, tomato, carrot, pepper, banana, 

ecc). The results of immunological experiments, obtained by using 

CMV as a presentation system for the Hepatitis C virus- and 

Alzheimer’s disease-derived epitopes, in association with the 

proven stability of CMV in gastrointestinal environment, seem to 

open a new prospect for the development of effective vaccine 

candidates. These data could be considered of paramount importance 

to health and medicine, thus suggesting to extend this strategy 

to other human and animal diseases, such as Influenza. Alternatively, 

another plant virus, Potato virus A (PVA) was selected as 

a carrier because the filamentous shape of its particles offers no 

packaging limitation for rather large genome insertion. A molecular 

modeling approach has been used to identify the possible insertion 

points of foreign determinants, in order to get the best 

conditions of stability and infectivity of chimeric virus particles. 

MYCOTOXIGENIC FUSARIUM SPECIES AND MYCO- 

TOXINS IN WHEAT GRAIN IN 2008 IN UMBRIA. L. Covarelli, 

G. Beccari, M. Tinelli, G. Santoponte. Dipartimento di 

Scienze Agrarie e Ambientali, Università degli Studi, Borgo XX 

Giugno 74, 06121 Perugia, Italy. E-mail: lorenzo.covarelli@ 

unipg.it 

In 2008, a study was conducted on 53 durum wheat (DW) 

and 36 soft wheat (SW) grain samples harvested in Umbria (central 

Italy). After visual observation of Fusarium damaged kernels, 

samples were subjected to fungal isolation on PDA, DNA extraction 

for Fusarium detection and identification by PCR, mycotoxin 

extraction for deoxynivalenol (DON) and T-2 toxin determination.. 

DW and SW samples had 34% and 19% Fusarium-damaged 

kernels while average the incidence of Fusarium infections 

was 40% and 33%, respectively, in the two wheat species. PCR 

assays showed that the most frequent species were F. graminearum, 

F. avenaceum and F. culmorum. F. equiseti was also detected 

by PCR but was not isolated on PDA while F. poae was mainly 

detected by traditional isolation rather than by PCR. DON levels 

exceeded the EU legal limits in 9% of DW and 8% of SW samples, 

reaching contamination levels of 53 mg kg -1 in DW (average 

2 mg kg -1 ) and 10 mg kg -1 in SW (average 0.7 mg kg -1 ). T-2 toxin 

was constantly detected, with peaks of 187 µg kg -1 in DW (average 

62 µg kg -1 ) and 78 µg kg -1 in SW (average 40 µg kg -1 ). Our results 

indicate that, even if previous studies carried out in Umbria 

showed a low mycotoxins risk, in years like 2008 that was characterized 

by climatic conditions extremely favourable to Fusarium 

head blight, wheat contamination may represent an important 

problem also in the examined area.


DETECTION OF FUSARIUM spp. IN ASPARAGUS TIS- 

SUES AND SOIL BY POLYMERASE CHAIN REACTION. 

L. Cozzolino, G. Popolo, A. Zoina. Dipartimento di Arboricoltura, 

Botanica e Patologia Vegetale, Università degli Studi di 

Napoli “Federico II”, Via Università 100, 80055 Portici (NA), Italy. 

E-mail: lucia.cozzolino@unina.it 

Fusarium oxysporum f. sp. asparagi and Fusarium proliferatum 

are very dangerous pathogens of asparagus (Asparagus officinalis) 

associated with crown and root rot. Both fungal species can produce 

mycotoxins, thus their early detection is of great importance 

for prevention of pathogenic and toxigenic risks arising from infections. 

One of the most affected places of Campania (southern 

Italy) is the asparagus cropping area around Aversa, where in 

2005 and 2006 repeated surveys were carried out in different 

farms where many samples consisting of roots, rhizomes and soil 

were collected. A molecular diagnostic method was developed 

using a PCR assay based on partial calmodulin gene sequence using 

primers CLOX1/CLOX2, specific for F .oxysporum f. sp. asparagi 

and primers CLPRO1/CLPRO2, specific for F. proliferatum. 

PCR assays were performed on DNA extracted from plant 

material and infested soil and confirmed the occurrence of F. 

oxysporum f. sp. asparagi only. The sensitivity threshold of the detection 

protocol from soil was about 10 4 propagules/g of soil. 

Only F. oxysporum f. sp. asparagi was found as the agent of root 

rotting. Since this pathogen is specific to asparagus, and is unable 

to attack other crops, cultural practices such as the use of resistant 

cultivars and crop rotation can be very helpful in controlling 

the disease. 

TURNIP MOSAIC VIRUS INFECTING ERUCA SATIVA IN 

SICILY. S. Davino 1 , M. Davino 2 , L. Cavicchi 3 , M.G. Bellardi 4 . 

1 Dipartimento di Scienze Entomologiche, Fitopatologiche, Microbiologiche, 

Agrarie e Zootecniche, Sezione di Patologia Vegetale e Microbiologia 

Agraria, Università degli Studi, Viale delle Scienze, 

90128 Palermo, Italy. 2 Dipartimento di Scienze e Tecnologie Fitosanitarie, 

Università degli Studi, Via Santa Sofia 100, 95123 

Catania, Italy. 3 Plesso Didattico G. Scarabelli, Università degli Studi 

di Bologna, Viale G. Ascari 15, 40026 Imola (BO), Italy. 4 Dipartimento 

di Protezione e Valorizzazione Agroalimentare, Università 

“Alma Mater Studiorum”, Viale Fanin 42, 40127 Bologna, Italy. Email: 

davino@unipa.it 

In the Spring 2010, plants of Eruca sativa (family Brassicaceae), 

also known as rocket, grown in a private garden of Sicily 

(southern Italy) were observed, showing a severe virus-like disease 

consisting of mosaic, interveinal yellowing and/or dark 

greening areas on crinkled leaves and stunting. Preliminary electron 

microscopy observations of leaf-dips revealed the presence 

of filamentous particles 700-750 nm long. Considering that in 

1959, this species has been reported in Italy as a natural host of 

Turnip mosaic virus (TuMV), symptomatic leaf samples were tested 

serologically to verify the presence of this potyvirus. Both 

ISEM and PAS-ELISA analyses were positive for TuMV. Mechanical 

inoculations carried out using rocket leaf sap allowed 

transmission of the virus to Chenopodium murale (local symptoms) 

and C. quinoa. (necrotic spots and systemic veinal flecks). 

None of the other species belonging to family Brassicaceae were 

infected. To further characterize the virus at the molecular level, 

RT-PCR of CP gene of the isolate under study was amplified with 

the primers TuMV 3pr: 5’-CTAGCATACAACTTCATAAC-3’ 

and TuMV 5pr: 5’-TGTGTTATCACCAGGCAG-3’. The amplified 

product was cloned and sequenced and the sequence obtained 

was compared with that of other isolates retrieved from 

GenBank. Results indicated that the homology level of CP genes 

is probably related to the major differences in host specificity, raher 

than to geographic distribution. Notwhistanding the widespread 

presence of TuMV Italy, this virus does not appear to be 

associated with serious disease outbreaks. 

CITRUS TRISTEZA VIRUS: TEN YEARS OF INVESTIGA- 

TION IN SICILY. S. Davino 1 , M. Guardo 2 , A. Caruso 2 , M. 

Davino 3 . 1 Dipartimento di Scienze Entomologiche, Fitopatologiche, 

Microbiologiche, Agrarie e Zootecniche, Sezione di Patologia 

Vegetale e Microbiologia Agraria, Università degli Studi, Viale 

delle Scienze, 90128 Palermo, Italy. 2 CRA, Istituto Sperimentale 

per l’Agrumicoltura, Corso Savoia 190, 95024 Acireale (CT), Italy. 

3 Dipartimento di Scienze e Tecnologie Fitosanitarie, Sezione di Patologia 

Vegetale, Università degli Studi, Via Santa Sofia 100, 95123 

Catania, Italy. E-mail: davino@unipa.it 

Citrus tristeza virus (CTV) is the causal agent of a severe citrus 

disease. In the past, citrus budsticks imported illegally from 

abroad were infected by CTV. In 2000, in the course of a survey 

for selecting superior old citrus lines in the area of Cassibile 

(Syracuse) trees of Fortune, Nova, Satsuma mandarins and Marsh 

grapefruit grafted on sour orange rootstock showed stunting, decline, 

dieback small-size fruits and pin-holing on the bark below 

the bud union line. The samples collected and analyzed by DAS- 

ELISA and DTBIA for CTV infection were positive. Total RNA 

was extracted from 50 plants and tested by RT-PCR using 

primers specific for the genes encoding protein p20 and p23. In 

all cases, DNA fragments of the expected size were amplified. 

About two years later, thousands of CTV infections were ascertained 

in old cv. Tarocco sweet orange grafted on sour orange in 

the area of Catania. To study the genetic variation of CTV populations, 

150 samples from each sampled area (Catania-Baé, Cassibile 

and Catania-Motta S. Anastasia) were examined by SSCP 

and nucleotide sequence analysis of protein p20. All isolates from 

the same area showed the same SSCP pattern, whereas for each 

area a different SSCP pattern was obtained. The isolates from 

Cassibile and Motta S. Anastasia had a nucleotide identity higher 

than 99% with a mild CTV isolate from Spain (T385) and about 

92% with the severe isolate from Israel (CTV-VT), whereas the 

isolate from Catania-Baé was similar (> 99%) to severe isolates 

from California (SY568) and Japan (NUagA). From 2000 to 2009 

over 60,000 samples were collected in Sicily where propagating 

material was introduced or declining trees were present. The results 

showed that different varieties are affected by CTV. 

SPREAD OF PEPINO MOSAIC VIRUS TO DIFFERENT 

TOMATO GENOTYPE CROPS BY BUMBLE BEES. S. Davino 

1 , G. Iacono 2 , M. Davino 2 . 1 Dipartimento di Scienze Entomologiche, 

Fitopatologiche, Microbiologiche, Agrarie e Zootecniche, 

Sezione di Patologia Vegetale e Microbiologia Agraria, Università 

degli Studi, Viale delle Scienze, 90128 Palermo, Italy. 2 Dipartimento 

di Scienze e Tecnologie Fitosanitarie, Sezione di Patologia Vegetale, 

Università degli Studi, Via Santa Sofia 100, 95123 Catania, 

Italy. E-mail: davino@unipa.it 

Pepino mosaic virus (PepMV, genus Potexvirus family Flexviridae), 

first reported form pepino (Solanum muricatum Ait.) in 

Perù, was shortly after discovered in tomato (Solanum lycopersicon) 

In 1999, PepMV was widespread in tomato throughout Europe 

including Italy (Sardinia and Sicily). PepMV is highly infectious, 

being readily transmitted to healthy plants during routine 

cultivation procedures and by plant-to-plant contact. PepMV is 

also transmitted via contaminated seeds, which could serve as the


primary inoculum for transmission mechanical means or by Bumble 

bees. How PepMV infection initiates in new areas is now understood, 

but tomato seed has been suspected as one of the key 

components of worldwide epidemics. The objective of this study 

was to investigate the role of PepMV transmission by Bumble 

bees. Observations were carried out in different farms in Ragusa 

province. The tomato genotypes were Genio and Shereen (severe 

symptoms on the fruits), Piccadilly (mild symptoms) and Belize 

(symptomless) In some farms 70 Bumble bees were used per 

1,000 mq (thesis A), while in others 140 or more (thesis B). At 

the beginning of the observations 10% of plants were infected. 

By the end of March, 30% of plants were infected in the farms A, 

while in B the infection rate was between 60 and 90%. The cultivation 

procedures and transplant dates were practically identical 

in all farms. On the basis of our observations we can affirm that a 

high number of Bumble bees significantly contributed to the 

spread of PepMV. 

INHIBITORY EFFECT OF FURFURALS CONTAINED IN 

THE STEAM-EXPLODED BIOMASS OF MISCANTHUS 

SINENSIS AGAINST SOIL-BORNE PLANT PATHOGENS. 

U. De Corato 1 , N. Sharma 2 , O. Maccioni 2 , F. Zimbardi 3 . 1 Agenzia 

Nazionale per le Nuove Tecnologie, l’Energia e lo Sviluppo Economico 

Sostenibile (ENEA), Unità Tecnica Efficienza Energetica, 

Servizio Agricoltura, Centro di Consulenza Energetica Integrata di 

Bari, Via Roberto Da Bari 119, 70122 Bari, Italy. 2 Laboratorio di 

Biotecnologie dell’ENEA, Centro Ricerche Trisaia, S.S. 106 Jonica 

Km. 419.500, 75026 Rotondella (MT), Italy. 3 Laboratorio di Biomasse 

e Solare Termico dell’ENEA, Centro Ricerche Trisaia, S.S. 

106 Jonica Km. 419.500, 75026 Rotondella (MT), Italy. E-mail: 

ugo.decorato@enea.it 

The Steam-Exploded Biomass (SEB) of Miscanthus sinensis, a 

herbaceous perennial species growing up to 3-4 m in height, with 

an annual yield of 20-25 tons/ha, is a good renewable energy resource. 

SEB could also be useful in crop protection, as an alternative 

to the use of compost in the greenhouse against soil-borne 

plant pathogens. Detailed studies on disease suppressiveness of 

SEB yielded positive results against three pathosystems. In this 

work, we studied the inhibitory effect of furfurals contained in 

the SEB against Phytophthora nicotianae, Pythium ultimum, 

Fusarium oxysporum f. sp. lactucae, Fusarium oxysporum f. sp. 

melonis and Rhizoctonia solani. Analysis of microbial growth inhibitors 

was carried out by HPLC, and inhibition of furfurals was 

tested in vitro. Furfural, 5–HMF, lignosulfonates, acetic and 

formic acid were detected at a concentration of 2.93, 0.28, 4.12, 

10.07, 1.88 g/l, respectively. P. ultimum, P. nicotianae and R. 

solani were inhibited by the addition to PDA of 3.2 g/l furfural 

and 0.48 g/l 5–HMF. However, both furfurals were not efficient 

inhibitors of F. oxysporum f. sp. lactucae and melonis at the same 

concentrations. An increase of furfural and 5–HMF concentration 

in the growing medium correlated positively with inhibition 

of P. ultimum, P. nicotianae and R. solani, but not with the growth 

of F. oxysporum. Disease suppressiveness of SEB could be related 

to its content of furfurals, organic acids and lignosulfonates produced 

during processing of fresh biomass in a pilot plant of 

Steam-Explosion. 

THE INFLUENCE OF YEAST ORIGIN AND IDENTITY 

ON MODES OF BIODEGRADATION OF PATULIN BY 

BASIDIOMYCETOUS PINK YEASTS. F. De Curtis 1 , R. 

Quici 1 , G. Ianiri 1 , L. Palmgren 2 , V. De Cicco 1 , R. Castoria 1 , 

S.A.I. Wright 1,2 . Dipartimento di Scienze Animali, Vegetali e del- 

l’Ambiente, Università del Molise, Via F. De Sanctis, 86100 Campobasso, 

Italy. 2 Department of Plant and Environmental Sciences, 

University of Gothenburg, Göteborg, Sweden. E-mail: decurtis 

@unimol.it 

Pink yeasts belonging to the genera Sporobolomyces/Sporodiobolus, 

Rhodosporidium/Rhodotorula, Cystofilobasidium/Cryptococcus 

and Erythrobasidium are natural inhabitants of many agricultural 

and wild plant species. Their natural role in the microbial 

ecology of the phylloplane is not sufficiently understood. 

When isolated and purified, many are able to suppress plant diseases 

and some can biodegrade patulin. Pink yeasts were isolated 

from the phyllosphere of agricultural and wild plants in various 

climatic zones, islands, coastal, hilly and mountainous regions of 

south-central Italy. Yeasts were purified and characterized by 

morphological and molecular methods. Subsequently, they were 

tested for the ability to degrade patulin, a mycotoxin produced 

by Penicillium expansum, the causal agent of blue mould. The 

highest degree of yeast biodiversity was recorded in the Tremiti 

islands, because more species of plants were sampled and because 

of the absence of intense agricultural activity. Most of the 

170 pink yeasts isolated belonged to the genus Rhodosporidium. 

More yeasts were isolated from apple and fig trees than from 

olive in three comparable locations. Fig trees had a more diverse 

epiphytic microflora than did apple and olive. Interestingly, apples 

appear to host predominantly two different types of pink 

yeasts, as shown by analysis of morphological traits, RFLP (restriction 

fragment length polymorphism) and sequencing of the 

ITS (internal transcribed spacer) regions. We have evidence suggesting 

that members of specific phylogenetic groups are capable 

of degrading patulin. No isolates from wild plants were able to 

biodegrade patulin. 

CHARACTERIZATION OF RESISTANCE TO QOI FUNGI- 

CIDES IN BOTRYOTINIA FUCKELIANA (BOTRYTIS 

CINEREA). R.M. De Miccolis Angelini, C. Rotolo, S. Pollastro, 

A. Santomauro, M. Masiello, F. Faretra. Dipartimento di Protezione 

delle Piante e Microbiologia Applicata, Università degli 

Studi “Aldo Moro”, Via Amendola 165/A, 70126 Bari, Italy. 

E-mail: faretra@agr.uniba.it 

Botryotinia fuckeliana (Botrytis cinerea), the grey mould fungus, 

is well known for its adaptability to adverse conditions that 

frequently lead to acquired resistance to fungicides. QoIs include 

fungicides effective against a broad spectrum of fungi. They act 

as mitochondrial respiration inhibitors at the cytochrome bc 1 - 

complex level and are considered at high risk of acquired resistance. 

The response of B. fuckeliana field isolates, collected prevalently 

from grapevine in Italy, France, Germany and Japan, to 

several QoI fungicides was evaluated by colony-growth and conidial-germination 

assays. Media were amended with various concentrations 

of QoIs along with salicylhydroxamic acid, a specific 

inhibitor of alternative oxidase. Tested fungicides showed different 

effectiveness against B. fuckeliana. Positive cross resistance 

between QoI fungicides belonging to different chemical groups 

was observed. One-hundred-thirty-four isolates were characterized, 

35 of which showed a high level of resistance (RF ≥ 200). 

All isolates carried the G143A mutation in the mithocondrial cytochrome 

b (cytb) gene, as shown by CAPS (Cleaved Amplified 

Polymorphic Sequences) analysis with the AluI enzyme. Classical 

genetic analysis confirmed that QoI-resistance is maternally inherited 

by ascospore progeny of sexual crosses. The polymorphic 

structure of the cytb gene of B. fuckeliana was investigated in 52 

sensitive and 47 resistant isolates. A 1.197-bp intron (group I) 

was detected, flanking downstream codon 143 of the gene exclu-


sively in 15% of QoI-sensitive isolates. These findings corroborate 

the hypothesis that the presence of the intron can prevent 

the occurrence of the G143A mutation and QoI resistance as it 

occurs in other phytopathogenic fungi. 

LABORATORY ASSAYS ON NON-ENZYMATIC PROCESS- 

ES ASSOCIATED WITH IRON AND ESCA TRACHEOMI- 

COTIC FUNGI. S. Di Marco and F. Osti. Istituto di Biometeorologia 

del CNR, Via P. Gobetti 101, 40129 Bologna, Italy. E-mail: 

s.dimarco@ibimet.cnr.it 

The tracheomycotic fungi Phaeomoniella chlamydospora and 

Phaeoacremonium aleophilum are considered the main pathogens 

causing esca of grapevine, a widespread wood disease that affects 

vine yield and longevity. These pathogens can produce toxins and 

extracellular enzymes in a complex infection process not yet fully 

understood. The possibility that fungi employ also non-enzymatic 

means in such process was investigated. Biological tests were carried 

out in the laboratory to examine the role of iron in promoting 

non-enzymatic processes with the production of radicals. P. 

chlamydospora and P. aleophilum produced siderophores, and reduced 

ferric iron. Although both fungi lack specific enzymatic activity, 

they were able to degrade crystalline cellulose, but only in 

the presence of iron. It was therefore hypothesized that the activation 

of iron-dependent cellulose-degrading mechanisms that 

supplement the degrading activity of the enzymes, may have taken 

place with the production of hydroxyl radicals. This hypothesis 

was confirmed by a specific spectrophotometric test as capacity 

of the radicals to break down 2-deoxi-D-ribose. The present 

study has shown that P. chlamydospora and P. aleophilum can activate 

iron-dependent non-enzymatic mechanisms, amplifying their 

pathogenic capacity. These findings, if confirmed by further 

study, may provide a new way to explain the nature of 

grapevine/pathogen relationship in term of wood degradation 

and/or foliar symptom formation, leading to a better understanding 

of the esca complex infection process. 

SEVERE ATTACK OF OLIVE KNOT IN THE PROVINCE 

OF PISTOIA. S. Di Napoli 1 , D. Rizzo 2 , A. Voirgar 1 . 1 Istituto 

Tecnico Agrario Statale “D. Anzilotti”, Viale Ricciano 5, 51017 Pescia 

(PT), Italy. 2 Agenzia Regionale per lo Sviluppo e l’Innovazione 

nel Settore Agricolo-Forestale (ARSIA), Laboratorio di Diagnostica 

Fitopatologica, Via dei Fiori 8, 51012 Pescia (PT), Italy. E-mail: 

domenico.rizzo@arsia.toscana.it 

In late spring-early summer 2010, many cases of severely damaged 

olive trees were reported to occur in Valdinievole, an extended 

area in the south-western part of the province of Pistoia 

(Tuscany). Samples collected by local extension services and olive 

growers showed symptoms consistent with those induced by olive 

knot. In fact, trees were stunted and had desiccated leaves, twigs 

and terminal branches. Galls were present on the entire canopy 

including twigs, young branches, trunk and leaves. Most severe 

symptoms were observed in cv. Frantoio, known to be particularly 

susceptible to olive knot. To verify the occurrence of 

Pseudomonas savastanoi pv. savastanoi, DNA was extracted from 

symptomatic organs and PCR was performed. As expected, the 

presence of the bacterium was ascertained in all tested samples. 

To understand the cause of such outbreak, data recorded from 

different meteorological stations in Pistoia were collected and 

analysed. It was found that that numerous cold waves occurred 

during winter 2010 and unusual low temperatures were recorded. 

In addition, the following spring was characterized by frequent 

and intense rainfalls. Thus, we conclude that the wide distribu- 

tion and severity of the disease observed in 2010 can be credited 

in part to lesions induced by agricultural practices and to exceptional 

environmental conditions which favoured the infection by 

P. savastanoi. 

CANKER AND DIEBACK OF SYCAMORE MAPLE 

CAUSED BY EUTYPA FLAVOVIRENS IN ITALY. R. 

Faedda 1 , A. Pane 1 , A. Sidoti 2 , G. Granata 1 . 1 Dipartimento di 

Scienze e Tecnologie Fitosanitarie, Università degli Studi, Via S. 

Sofia 100, 95123 Catania, Italy. 2 Regione Siciliana, Azienda Regionale 

Foreste Demaniali, UOB n.3, Via della Libertà 97, 90143 

Palermo, Italy. E-mail: granatag@unict.it 

Sycamore maple (Acer pseudoplatanus, family Sapindaceae) is 

a deciduous tree native to Europe and southwestern Asia. In Italy 

it grows from the Alps to Sicily. Since spring 2007, cankers and 

dieback of entire branches of sycamore trees was observed in 

eastern Sicily. Cankers girdled entire branches and trunks. Yellowing 

and death of foliage above the cankers was also observed. 

Numerous dark pycnidia occurred on the surface of the necrotic 

bark of cankers. Colonies of Cytosporina flavovirens (Sacc.) 

Grove [anamorph of Eutypa flavovirens (Pers.) Tul. & C. Tul 

(syn. Diatrype flavovirens)] were consistently obtained from 

symptomatic tissues on oatmeal agar (OA) , producing spindly 

and aseptate conidia (17-23 x 1.5-1.8 µm). The fungus was identified 

on the basis of morphological and cultural characteristics in 

comparison with reference isolates supplied by the Centraalbureau 

voor Schimmelcultures (Utrecht, The Netherlands). Ribosomal 

DNA (rDNA) sequences of the internal transcribed spacer 

region (ITS1-5.8S-ITS2) from sycamore isolates were amplified 

and sequenced. Consensus sequences showed 98% similarity 

with E. flavovirens isolates deposited in GenBank (accession Nos. 

AJ302457, AJ302430 and AJ302429). Pathogenicity tests were 

performed on ten 3-year-old seedlings of sycamore maple. A 

patch of bark was removed from the stems and replaced with a 5mm-diameter 

OA plugs colonized by the fungus. Ten control 

plants were inoculated with sterile OA plugs. All trees were 

grown outdoors. After ten months, cankers and canopy wilting 

were observed on inoculated sycamores, whereas control plants 

remained symptomless. E. flavovirens was reisolated from symptomatic 

tissues. Several species of Eutypa, Diatrype, Eutypella and 

Valsa are known to occur on A. pseudoplatanus. To our knowledge, 

this is the first report of E. flavovirens on sycamore maple 

in Italy. 

PROTEOMIC STUDY OF THE FOUR-WAY INTERACTION 

BETWEEN TRICHODERMA ATROVIRIDE, PSEUDO- 

MONAS FLUORESCENS, RHIZOCTONIA SOLANI AND 

TOMATO. M. Faraji 1 , M. Ruocco 2 , S.L. Woo 3 , M. Ahmadzadeh 1 , 

K. Behbodi 1 , A.M. El-Tabey Eid 3 , M. Lorito 3 . 1 Department of 

Plant Protection, Section of Agricultural and Natural Resources, University 

of Tehran, Karaj, Iran. 2 Istituto per la Protezione delle Piante 

del CNR, Via Università 130, 80055 Portici (NA), Italy. 3 Dipartimento 

di Arboricoltura, Botanica e Patologia Vegetale, Università 

degli Studi di Napoli “Federico II”, Via Università 100, 80055 Portici 

(NA), Italy. E-mail: matteo.lorito@unina.it 

The use of biocontrol agents (BCAs) in synergistic combinations 

is a promising approach for the control of plant diseases 

and should be a priority in agricultural research. The efficacy, in 

terms of plant growth promotion and pathogen protection, provided 

by some elite bacterial (Pseudomonas fluorescen) and fungal 

(Trichoderma spp.) BCA strains has been widely demonstrated. 

The three-way interaction between plant, pathogen and antago-


nist, e.g. Trichoderma or P. fluorescens, has been previously investigated 

by using functional genomics and proteomic techniques. 

In this study we propose testing the contemporary presence of 

both BCAs in a very complex four-player system. The main strategy 

of this research was to use a proteomic approach in order to 

determine which molecular factors are activated in the interactions, 

identify those compounds that have an important affect on 

the activity of either or both BCAs during their concurrent interaction 

with tomato roots and/or pathogen, and the relative plant 

molecular responses to the different microorganisms. A method 

has been developed and optimized to obtain 2-D gel maps of separately 

collected proteomes from each partner, as well as all the 

possible combinations of the components. Several plant and microbial 

differentially accumulated proteins, selected from the 

four interacting proteomes, have been isolated and are being 

identified and characterized. 

FIRST OBSERVATIONS ON THE FUNGAL ENDOPHYT- 

IC COMMUNITY OF OLEA EUROPAEA IN SICILY. V. Ferraro, 

S. Lo Piccolo, G. Conigliaro, V. Mondello, L. Torta, S. Burruano. 

Dipartimento di Scienze Entomologiche, Fitopatologiche, 

Microbiologiche, Agrarie e Zootecniche, Università degli Studi, 

Viale delle Scienze 2, 90128 Palermo, Italy. E-mail: santella@unipa.it 

During an etiological and epidemiological study on a new decline 

(foliar chlorosis and withering twigs) of Olea europaea in 

Sicily, the composition of the endophytic community of symptomless 

and symptomatic olive tree organs was investigated. Two 

Sicilian olive-groves, located in a hill and in a plain, respectively, 

comparable for plant age, cultivar and agricultural management, 

were taken into consideration from spring 2007 to autumn 2008. 

Samples from healthy and diseased leaves and twigs were collected 

to isolate fungal endophytes. The fungal endophytic community 

varied quantitatively and qualitatively in relation to (i) type 

and health condition of the sampled organ; (ii) site of collection 

and (iii) survey period. Fungi belonging to the genera Alternaria, 

Aureobasidium, Camarosporium, Cladosporium, Diplodia, Phoma, 

Pleospora Septoria, Stemphylium, and several Mycelia sterilia were 

detected in almost all samples. The overall colonization rates 

(CR) were always higher in leaves than twigs, in both localities. 

Moreover, CR showed variability also in relation to the health 

condition of the organs. In fact, both in the hill and the plain, the 

endophytic colonization was higher in symptomatic organs than 

in the healthy ones. Although many taxa were detected, the results 

of this investigation suggest that the relatively stable endophytic 

fungal community residing in O. europaea is characterized 

by a constant composition of common taxa. Studies to achieve 

fungal identification and to evaluate the role of fungal endophytes 

in O. europaea are in progress. 

CHARACTERIZATION OF PSEUDOMONAS spp. STRAINS, 

CAUSAL AGENTS OF CANKERS AND WILT OF STONE 

FRUITS IN SARDINIA. M. Fiori, V. Ligios, A. Schiaffino. Dipartimento 

di Protezione delle Piante, Università degli Studi, Via E. De 

Nicola, 07100 Sassari, Italy. E-mail: fiorim@uniss.it 

A collection of 94 bacterial strains was obtained from apricot, 

nectarine, peach and plum trees showing cankers on twigs, stems 

and branches, gum exudates, bud necrosis, wilting of shoots, and 

spots on the leaves. Thirteen strains, selected on the basis of morphology 

on NSA and hypersensitivity reaction on tobacco leaves, 

were able to infect cherry plum GF31, watermelon and vegetable 

marrow plantlets; two strains infected pepper and tomato and 

five lilac and lemon fruits. On the basis of the results obtained by 

Gram reaction, catalase, poly-hydroxybutyrate, fluorescence on 

KB and LOPAT tests, five strains were identified as Pseudomonas 

syringae (Ps), three as P. viridiflava (Pv), four as P. fluorescens. 

One isolate was not identified. Biolog and serological agglutination 

tests partially confirmed the above reported results, allowing 

the assignment of three strains of Pseudomonas syringae to the patovar 

syringae (Pss). Ater fatty acid analysis on five isolates, one 

was ascribed to Ps (S.I., 0.967), one to Pv (S.I., 0.979), one to P. 

putida (S.I., 0.847), while two were not identified. The analysis of 

the thirteen strains with BOX-PCR produced 5 reaction profiles. 

One of them was characteristic of the type strain Pss CFBP 700. 

SyrB gene amplification of the three Pss strains produced the specific 

band of 752 bp like the type strain Pss CFBP 700, confirming 

their identification. In conclusion, while Pss is already present 

in Italy in all plant species analyzed, Pv was not yet reported in 

Europe on apricots and nectarine. 

EFFECT OF WATER ACTIVITY AND FUNGICIDES ON 

COMPETING ABILITIES OF COMMON MAIZE FUNGI. 

S. Formenti 1 , N. Magan 2 , A. Pietri 3 , P. Battilani 1 . 1 Institute of 

Entomology and Plant Pathology, Università Cattolica del Sacro 

Cuore, Via Emilia Parmense 84, 29122 Piacenza, Italy. 2 Cranfield 

Health, Vincent Building, Cranfield University, Bedford MK43 

0AL, UK. 3 Institute of Science of Food and Nutrition, Università 

Cattolica del Sacro Cuore, Via Emilia Parmense 84, 29122 Piacenza, 

Italy. E-mail: paola.battilani@unicatt.it 

Maize is colonised by a mixture of spoilage fungi in pre- and 

post-harvest. The occurrence of certain dominant fungal species 

depends on several abiotic and biotic factors that determine their 

prevalence in the maize grain ecosystem. In order to understand 

when Fusarium spp. are able to dominate the maize ecosystem, it 

is necessary to clarify the complex interactions which occur between 

abiotic and biotic factors and their impact on the growth 

and between fungal interactions. The aims of this study were: (i) 

to determine the competitiveness of F. verticillioides against a 

range of fungi commonly growing on maize (F. proliferatum, A. 

flavus, A. ochraceus, A. niger and P. verrucosum), on artificial media 

under different a w levels (0.99; 0.98; 0.95); (ii) to establish 

how the presence of sub-optimal concentrations (50% effective 

dose) of commercial fungicides (tebuconazole, prothioconazole 

and prochloraz) affects interspecific interactions. The results 

showed how F. verticillioides was able to compete effectively in 

dual culture with other fungal species commonly isolated from 

maize, although the dominance against some species was modified 

by a w and the presence of fungicides. The dominance of F. 

verticillioides without the presence of sub-optimal active ingredients 

could be mainly due to its ability to grow rapidly and invasively. 

A. flavus was the most competitive species at sub-optimal 

levels of active ingredients tested, which is not surprising because 

the tested products are considered as effective mainly towards 

Fusaria. However, no important variation in the inter and intraspecific 

interaction of common fungi in maize was noticed by the 

addition of fungicides. 

ABILITY OF SOME SOIL FUNGI TO DECREASE THE AC- 

TIVE PRINCIPLE CONCENTRATION OF BIOFUMIGA- 

TION BY BRASSICA CARINATA SEED MEAL. S. Galletti 1 , 

L. Ugolini 1 , P.L. Burzi 1 , S. Cianchetta 1 , R. Roberti 2 , C. Cerato 1 . 

1 CRA, Centro di Ricerca per le Colture Industriali, Via di Corticella 

133, 40128 Bologna, Italy. 2 Dipartimento di Protezione e Valo-


rizzazione Agro-Alimentare, Università degli Studi, Viale Fanin 42, 

40127 Bologna, Italy. E-mail: stefania.galletti@entecra.it 

Biofumigation is a low-impact method alternative to chemical 

disinfestation of soil, based on the glucosinolate-myrosinase defensive 

system of Brassicaceae plants, ploughed into the soil as 

green manure or seed meal. The enzymatic degradation of glucosinolates 

by myrosinase occurring in the soil delivers volatile 

isothiocyanates, which are very effective against several soil 

pathogens. The combination of this technique with a biological 

control agent like the filamentous fungus Trichoderma, selected 

for tolerance to allyl-isothiocyanates in previous trials, gave interesting 

synergic effects in controlling sugar beet damping-off by 

Pythium ultimum. On the other hand, it was found that a non 

pathogenic isolate of Aspergillus flavus was able to actively reduce 

the concentration of allyl-isothiocyanate in soil during biofumigation, 

thus hampering its effectiveness. A screening was then carried 

out in vitro among different non-pathogenic fungal isolates 

in order to investigate their interaction with allyl-isothiocyanate. 

The results showed differential responses: in particular some isolates 

of Trichoderma were able to decrease allyl-isothiocyanate 

concentration similarly to A. flavus, while the same Trichoderma 

isolate used in the biofumigation experiment against P. ultimum, 

was not. These findings suggest that the soil fungal community 

could be one of the causes of the reduced isothiocyanate concentration 

often found in biofumigated soil with respect to expectations. 

Therefore, the use of Trichoderma isolates, selected for 

their antagonistic behaviour against the pathogens, if employed in 

combination with biofumigation, should be carefully evaluated 

not only for their tolerance to isothiocyanates, but also for their 

ability to interact with these molecules. 

COMPARATIVE PROFILING OF SMALL RNA POPULA- 

TIONS IN VIRUS-INFECTED AND HEALTHY 

GRAPEVINE PLANTS. A. Giampetruzzi 1 , A. Gisel 3 , P. La 

Notte 2 , C. Pirolo 1 , P. Saldarelli 2 . 1 Dipartimento di Protezione 

delle Piante e Microbiologia Applicata, Università degli Studi “Aldo 

Moro”, Via Amendola 165/A, 70126 Bari, Italy. 2 Istituto di Virologia 

Vegetale del CNR, UO Bari, Via Amendola 165/A, 70126 Bari, 

Italy. 3 Istituto di Tecnologie Biomediche del CNR, Via Amendola 

122/O, 70126 Bari, Italy. E-mail: p.saldarelli@ba.ivv.cnr.it 

RNA silencing in plants is a process operating through a multitude 

of small RNAs (sRNAs) 18-24 nt in lenght, that maintain 

genome integrity, control plant development, and respond to abiotic 

and biotic stresses. The large majority of sRNAs derives from 

the host genome and mediates gene regulation through targetgene 

cleavage, translational inhibition or DNA methylation. 

However, in virus-infected plants some sRNAs originate from 

replicating viral genomes in response to infections. Grapevine 

fanleaf virus (GFLV), one of the most important grapevine viruses, 

causes fanleaf degeneration and is responsible for severe yield 

losses. The sRNAs fractions from leaves of a healthy (S5) and a 

fanleaf-infected vine (S3) with the same genotype, were sequenced 

by Illumina Genome Analyzer II. The 21-24 nucleotide 

size classes dominated both libraries, but while healthy S5 tissues 

showed a prevalent 24 nt over the 21 nt population, the opposite 

occurred in infected S3 tissues. BLASTN search, together with a 

de novo assembly approach, confirmed the presence of actively 

replicating GFLV in the S3 vine, and a small number of reads also 

from Grapevine rupestris stem pitting-associated virus 

(GRSPaV), Hop stunt viroid (HSVd) and Grapevine yellow speckle 

viroid 1 (GYSVd1). The same analysis on the healthy vine (S5), 

confirmed its good sanitary status, as it only showed contig homologies 

to grapevine genes. Characterization of known 

grapevine microRNAs (miRNAs) done on both libraries, showed 

clearly different profiles. In particular, miRNA166, which is involved 

in morphogenesis in A. thaliana and aspen, is among the 

overexpressed miRNAs in the infected vine. 

PRESENCE AND ABUNDANCE OF ROOT ROT, BUTT 

ROT AND STEM ROT FUNGI IN PROTECTION 

FORESTS OF THE WESTERN ALPS. L. Giordano, G. Nicolotti, 

P. Gonthier. Dipartimento di Valorizzazione e Protezione 

delle Risorse Agroforestali, Università degli Studi di Torino, Via 

Leonardo da Vinci 44, 10095 Grugliasco (TO), Italy. E-mail: paolo. 

gonthier@unito.it 

Protection forests have an important role in reducing the effects 

of natural hazards, such as avalanches, rock falls and debris 

flow. An adequate understanding of the disturbances that these 

forests may undergo is a prerequisite for the successful maintenance 

of the protection function. Wood decay fungi may affect 

the stability of trees and thus they may behave as natural disturbances. 

In this work we investigated the presence of root rot, butt 

rot and stem rot fungi in some protection forests of Piedmont 

and Aosta Valley (western Italian Alps). Tree species composition 

was variable depending on sites, and included both conifers and 

broadleaves. Fungi were identified through traditional techniques 

or DNA sequencing. The most frequent fungi were Fomitopsis 

pinicola, Heterobasidion annosum sensu lato and Stereum sanguinolentum. 

In a Norway spruce-dominated subalpine stand, approximately 

150 trees were sampled at the root collar by drilling 

them with a 4 mm diameter, 43 cm long bit. Fungal DNA was extracted 

from wood and analyzed by using universal and taxonspecific 

primers. A high percentage (56%) of the trees was infected 

by root and butt rot fungi. H. annosum sensu lato accounted 

for 70% of infected trees, while Armillaria mellea sensu lato for 

8%. Remaining trees were infected either by both fungi or by unknown 

fungi. These results suggest that H. annosum sensu lato is 

by far the most frequent root and butt rot agent in the site. 

ULTRASTRUCTURE OF THE ALDER RUST MELAMP- 

SORIDIUM HIRATSUKANUM ON GREY ALDER (ALNUS 

INCANA). B. Ginetti 1 , G. Maresi 2 , S. Moricca 1 . 1 Dipartimento 

di Biotecnologie Agrarie, Sezione di Protezione delle Piante, Piazzale 

delle Cascine 28, 50144 Firenze, Italy. 2 FEM-IASMA-Centro 

Trasferimento Tecnologico, Via E. Mach, 38010 San Michele 

all’Adige (TN), Italy. E-mail: beatrice.ginetti@unifi.it 

The alder rust pathogen Melampsoridium hiratsukanum S. Ito 

ex Hirats. is currently spreading epidemically across several European 

countries. Outbreaks were reported in the past 15 years from 

the northernmost countries (Finland, Norway, Latvia) down to 

Hungary, the Balcan Peninsula and Turkey. It has been conjectured 

that this heteroecious rust, considered to be native to eastern 

Asia, may have been introduced into Baltic states with contaminated 

commercial material (seedlings) transported through 

the Baltic sea. The fungus is closely related to Melampsoridium betulinum 

and M. alni, two rusts infecting also hosts in the family 

Betulaceae (Alnus spp. and Betula spp). However, based on host 

specificity (M. hiratsukanum does not infect Betula sp.) and micromorphometric 

data (spore dimensions, spore ornamentation and 

length of ostiolar cells) M. hiratsukanum was kept separate from 

the above congeneric rusts. In spite of the many reports on the 

distinguishing features of M. hiratsukanum, a clear identification 

of this rust at the ultrastructural level was never achieved. In the 

present work, the ultrastructure of M. hiratsukanum on grey alder


(the fungus was never observed in Europe on the alternate host 

Larix sp.) was investigated under the scanning electron microscope 

(SEM). The dimension and morphology of urediniospores, 

the distribution of spines over the spore surface, and the other 

distinguishing features of this rust were examined in detail. 

PRESENCE AND ABUNDANCE OF ROOT ROT, BUTT 

ROT AND STEM ROT FUNGI IN PROTECTION 

FORESTS OF THE WESTERN ALPS. L. Giordano, G. Nicolotti, 

P. Gonthier. Dipartimento di Valorizzazione e Protezione 

delle Risorse Agroforestali, Università degli Studi, Via Leonardo da 

Vinci 44, 10095 Grugliasco (TO), Italy. E-mail: paolo.gonthier@ 

unito.it 

Protection forests have an important role in reducing the effects 

of natural hazards, such as avalanches, rock falls and debris 

flow. An adequate understanding of the disturbances that these 

forests may undergo is a prerequisite for the successful maintenance 

of the protection function. Wood decay fungi may affect 

the stability of trees and thus they may behave as natural disturbances. 

In this work we investigated the presence of root rot, butt 

rot and stem rot fungi in some protection forests of Piedmont 

and Aosta Valley (western Italian Alps). Tree species composition 

was variable depending on sites, and included both conifers and 

broadleaves. Fungi were identified through traditional techniques 

or DNA sequencing. The most frequent fungi were Fomitopsis 

pinicola, Heterobasidion annosum sensu lato and Stereum sanguinolentum. 

In a Norway spruce-dominated subalpine stand, approximately 

150 trees were sampled at the root collar by drilling 

them with a 4 mm diameter, 43 cm long bit. Fungal DNA was extracted 

from wood and analyzed by using universal and taxonspecific 

primers. The majority of the trees (56%) was infected by 

root and butt rot fungi. H. annosum sensu lato accounted for the 

70% of the infected trees, while Armillaria mellea sensu lato for 

8%. Remaining trees were infected either by both fungi or by unknown 

fungi. These results suggest that H. annosum sensu lato is 

by far the most frequent root and butt rot agent in the site. 

ALTERNATIVE HOSTS OF CASSAVA MOSAIC BEGO- 

MOVIRUSES IN TANZANIA. D. Guastella 1 , C. Busungu 2 , J. 

P. Legg 2 and M. Tessitori 1 . 1 Dipartimento di Scienze Fitosanitarie, 

Sezione Patologia Vegetale, Università di Catania, Via S. Sofia 100, 

95123 Catania, Italy. 2 IITA-Tanzania, P.O. Box 34441, Dar es 

Salaam, Tanzania. E-mail: mtessitori@unict.it 

Cassava mosaic disease (CMD), the most important disease of 

this staple food crop in Africa, is caused by eight cassava mosaic 

begomoviruses (CMBs), seven of which occur in Africa. All viruses 

involved in the disease are efficiently transmitted in the field 

by Bemisia tabaci. Cassava was introduced to Africa in the 16 th 

century, so it is recognized that this crop must have become infected 

by CMBs originating from wild African plant species. To 

date, however, little is known about the wild hosts of CMBs. This 

is an important shortcoming, since wild hosts may potentially 

play a significant role in the epidemiology of CMD. During a 

May 2010 survey that covered about 50,000 km 2 of north-western 

Tanzania, more than 30 samples were collected from different 

species of weeds or cultivated plants showing mosaic symptoms. 

PCR using universal primers (UniF and UniR) confirmed the association 

of wild cassava (Manihot glaziovii) with CMBs in Tanzania. 

Moreover, new hosts of these viruses were identified, i.e. 

Combretum confertum, Arachis hypogaea, Hibiscus cannabinus, 

Leucaena leucocephala and Conyza sumatrensis. Subsequent PCR 

by specific primers for both African cassava mosaic virus (ACMV) 

and East African cassava mosaic virus (EACMV) allowed the identification 

of specific virus infections. L. leucocephala and C. sumatrensis 

were infected by both ACMV and EACMV. Sequencing 

studies are currently underway to further characterize these novel 

CMB infections of non-cassava hosts. 

BOTRYOSPHAERIA SPECIES ASSOCIATED WITH ESCA- 

DISEASED GRAPEVINE IN SOUTHERN ITALY AND 

LEBANON. W. Habib, S. Pollastro, F. Faretra. Dipartimento di 

Protezione delle Piante e Microbiologia Applicata, Università degli 

Studi “Aldo Moro”, Via Amendola 165/A, 70126 Bari, Italy. Email: 

faretra@agr.uniba.it. 

Botryosphaeriaceae are fungi frequently isolated from vines 

showing decline or dieback symptoms in several countries. Wood 

symptoms such as cankers, sectorial V-shaped necroses in wood, 

bud mortality, shoot dieback and graft union failure have been 

associated with different species of Botryosphaeriaceae, and different 

disease names have been given to certain types of symptoms. 

In particular, in Mediterranean countries, black dead arm 

(BDA) was retained an important vine-decline disease in addition 

to Eutypa dieback and esca. In the present work, morpho-taxonomic 

characterization and sequence analysis of rDNA internal 

transcribed spacer regions (ITS) were used to identify 

Botryosphaeriaceae isolates obtained from esca-affected vines in 

Apulia (southern Italy) and Lebanon. According to the morphotaxonomic 

approach, three main groups of isolates were recognized: 

(i) characterized by pigmented, thick-walled conidia, including 

the genera Diplodia, Lasiodiplodia, and Dothiorella; (ii) 

characterized by hyaline, thin-walled conidia, comprising the 

genera Fusicoccum and Neofusicoccum; (iii) isolates which did not 

differentiate conidia in culture. Molecular identification of 

Botryosphaeriaceae species was based on the comparison of ITS 

sequences with those available in GenBank. In agreement with 

the results obtained in other countries, 55 tested isolates were 

identified as Diplodia seriata (32.7%), Neofusicoccum parvum 

(30.9%), Fusicoccum aesculi (12.7%), Neofusicoccum australe 

(9.1%), Neofusicoccum luteum (5.5%), and Neofusicoccum vitifusiforme 

(3.6%). The species Dothiorella viticola, Diplodia mutila, 

and Lasiodiplodia theobromae were represented by one isolate 

each. Further studies on the distribution and pathogenicity of 

Botryosphaeriaceae fungi are therefore essential to clarify their 

possible role in grapevine decline. 

RAPD ANALYSIS IN BOTRYOSPHAERIACEAE FUNGI. 

W. Habib, S. Pollastro, F. Faretra. Dipartimento di Protezione 


Moro”, Via Amendola 165/A, 70126 Bari, Italy. E-mail: faretra@agr.uniba.it. 

For a long time, taxonomic studies on Botryosphaeriaceae 

fungi were based on morphological characters which exhibit high 

variability. Host specificity cannot be of help as a taxonomic criterion 

since several host plants can be colonized by a single fungal 

species and different species can occur on the same host 

plant. In the present study, 55 isolates of Botryosphaeriaceae fungi 

(Diplodia mutila, Diplodia seriata, Dothiorella viticola, Fusicoccum 

aesculi, Lasiodiplodia theobromae, Neofusicoccum australe, 

Neofusicoccum luteum, Neofusicoccum parvum, Neofusicoccum vitifusiforme) 

were collected from standing vines showing symptoms 

of cankers, dieback and esca in commercial vineyards and 

from rootstocks in nurseries of southern Italy and Lebanon.


These isolates were identified by morphological and molecular 

methods. Fungal DNA was subjected to RAPD (Random Amplified 

Polymorphic DNA) analysis using 20 decamer primers. A total 

of 248 markers (from 209 to 1,800 bp in size) were obtained. 

The number of RAPD markers generated per primer varied between 

15 and 28. Similarity matrix using Dice coefficient for pairwise 

comparisons gave a dendrogram that grouped the isolates in 

clusters corresponding to the anamorph. A broad inter-specific 

variability resulted, each species being well distinguished from 

the most closely related ones. RAPD analysis confirmed to be a 

reliable technique for investigating species differentiation and genetic 

diversity. The technique can be of help in the identification 

of some Botryosphaeriaceae species, being cheaper and faster 

than the commonly used methods based on sequencing of ITS region, 

often integrated with partial sequencing of EF1-α or βtubulin 

genes. 

DEVELOPMENT OF A MACROARRAY SYSTEM FOR 

THE DIAGNOSIS OF ROOT ROT AND TRACHEOMYCO- 

SIS OF ORNAMENTAL PLANTS. A. Haegi 1,2 , M. Paoli 3 , D. 

Rizzo 3 , L. Riccioni 2 , A. Grassotti 1 . 1 CRA, Unità di Ricerca per il 

Vivaismo e la Gestione del Verde Ambientale ed Ornamentale 

(CRA-VIV), Via dei Fiori 8, 51012 Pescia (PT), Italy. 2 CRA, Centro 

di Ricerca per la Patologia Vegetale (CRA-PAV), Via C.G. Bertero 

22, 00156 Roma, Italy. 3 ARSIA, Via dei Fiori 8, 51012 Pescia 

(PT), Italy. E-mail: anita.haegi@entecra.it 

The industry of ornamental plants is characterized by continuous 

introduction of new crops and new production technologies 

that creates new opportunities for pathogens to exploit. In this 

framework an early and precise identification of pathogens is critical 

for determining defence strategy. In this work we addressed 

root rot and tracheomycosis diseases of perennial ornamental 

plants. Since phytosanitary surveys show an epidemiological complex 

situation, a macroarray device was chosen because it can detect 

multiple pathogens in a single assay, is sensitive, rapid and 

does not require a sophisticated equipment. Surveys were conducted 

in some Tuscan farms growing ornamentals to assess the 

main phytosanitary problems. Samples were collected and the 

fungi isolated from infected tissues were identified by morphology 

and molecular tools. On the same samples, a DNA extraction 

protocol from infected plant material have been set up, and the 

resulting DNA samples were amplified with ITS5/ITS4 primers 

for fungi and ITS6/ITS4 primers for oomycetes. The technique 

for macroarray procedure was set up using direct labelling (Gene 

Images AlkPhos, Amersham). Briefly, oligonucleotide detectors 

were immobilized on a nylon membrane and hybridized with the 

sample DNA, previously amplified and labelled. For each 

pathogen to be tested oligonucleotide detectors were looked for 

in the literature and validated or designed ex novo. Oligonucleotide 

detectors for Fusarium oxysporum, Phytophthora spp. 

and Pythium spp. were found in the literature and are now under 

validation. Four oligo detectors for Phytophthora cactorum have 

also been designed. 

IN VITRO STUDY OF FUM GENES EXPRESSION AND 

FUMONISINS PRODUCTION IN FUSARIUM VERTICIL- 

LIOIDES UNDER DIFFERENT ECOLOGICAL CONDI- 

TIONS. I. Lazzaro 1 , A. Susca 2 , G. Mulè 2 , A. Ritieni 3 , P. Battilani 

1 . 1 Istituto di Entomologia e Patologia Vegetale, Università Cattolica 

del Sacro Cuore, Via Emilia Parmense 84, 29100 Piacenza, 

Italy. 2 Istituto di Scienze delle Produzioni Alimentari del CNR, Via 

Amendola 122/O, 70126 Bari, Italy. 3 Istituto di Scienze degli Ali- 

menti, Università degli Studi “Federico II”, 80055 Napoli, Italy. Email: 

paola.battilani@unicatt.it 

F. verticillioides, a fungus associated with maize ears all over 

the world, induces Fusarium pink ear rot and produces fumonisins 

(FBs), mycotoxins found in maize kernels and their derivatives. 

FBs are toxic to humans, FB 1 in particular, which is classified 

as possibly carcinogenic. Fumonisins biosynthetic pathway is 

regulated by several genes belonging to the FUM cluster whose 

behaviour in different ecological conditions is poorly studied. 

The aim of this work was to investigate the behaviour of two F. 

verticillioides strains grown in vitro both on FB-inducing (Malt 

extract agar, MEA) and FB-inhibiting (Czapeck yeast agar, CYA) 

media. Fungal growth, FBs production and gene expression were 

studied at different temperature (T; 20-30) and water activity (a w; 

0.90-0.99) regimes, in liquid cultures incubated for 7-21 days. 

The expression of two genes, FUM21 and FUM2, was considered; 

the relative quantification of gene expression was performed 

by Real time PCR. Results showed that, with a w fixed at 

0.99, maximum FUM21 and FUM2 expression was at 30°C after 

14 days of incubation, while FBs production increased from 7 to 

21 days. At fixed T (25°C), fungal growth at 0.90 a w was not observed 

after 21 days of incubation. Gene expression increased 

with incubation time both at 0.95 and 0.99 a w ; FB production increased 

till 21 and 14 days respectively at 0.95 and 0.99 a w . In all 

conditions studied the expression of the two genes considered 

followed a very similar trend, but FUM2 was much more expressed 

than FUM21 and more influenced by a w conditions. 

TRICHODERMA VIRIDE AND PHOMOPSIS sp. ASSOCI- 

ATED WITH A DECLINE OF PINUS NIGRA PLANTLETS 

IN A REFORESTATION AREA OF CENTRAL ITALY. M.G. 

Li Destri Nicosia, S. Mosca, G.E. Agosteo, R. Mercurio, L. 

Schena. Dipartimento di Gestione dei Sistemi Agrari e Forestali, 

Università degli Studi Mediterranea, Località Feo di Vito, 89122 

Reggio Calabria, Italy. E-mail: lschena@unirc.it 

In winter 2009, a decline was observed on 4-year-old plantlets 

of Pinus nigra in a reforestation area of Abruzzo National Park 

(central Italy). More than 70% of the plantlets died during the 

first year after transplanting while survivors were stunted and 

showed leaf chlorosis as well as root and crown rot. Four fungal 

morphotypes, grouped according to the morphology of colonies 

and reproductive structures, were isolated from the necrotic subcortical 

tissues of the basal stem of symptomatic plantlets. ITS regions 

of representative isolates of each morphotype were examined 

by BLAST analysis and compared with available sequences 

in GenBank. Three isolates were identified as Phomopsis sp., Trichoderma 

viride and T. harzianum, respectively, based on a 99- 

100% identity with deposited sequences. Conversely, a morphotype 

that did not produce conidia was not identified since, surprisingly, 

its ITS sequence matched sequences of multiple taxonomic 

groups. Pathogenicity tests were conducted on both cuttings 

and potted 4-year-old plantlets of P. nigra by inserting a 

plug of PDA with actively growing mycelium under the bark. T. 

viride and Phomopsis sp. caused necrosis of subcortical tissues 

around the inoculation site. Lesions caused by T. viride were significantly 

more extended than those caused by Phomopsis sp. 

Both fungi were reisolated from symptomatic tissues. No symptoms 

were observed on cuttings and plantlets inoculated with 

sterile agar, T. harzianum or the unidentified fungus. Both Phomopsis 

sp. and T. viride were reported previously as tree 

pathogens. However their actual role in the decline of P. nigra 

should be further investigated.


EMERGING BOTRYOSPHAERIACEAE IN FOREST 

ECOSYSTEMS IN SARDINIA. B.T. Linaldeddu, B. Scanu, A. 

Schiaffino, A. Franceschini. Dipartimento di Protezione delle 

Piante, Sezione di Patologia Vegetale, Università degli Studi, Via E. 

De Nicola 9, 07100 Sassari, Italy. E-mail: ben@uniss.it 

Fungal species belonging to the family Botryosphaeriaceae are 

well known as endophytes and pathogens of woody plants worldwide. 

Beginning in 2008, a field survey was conducted to study 

Botryosphaeriaceae that occur on declining trees and shrubs of 

Mediterranean maquis, in various Sardinian ecosystems. Fungal 

isolates from symptomatic plants were identified on the basis of 

morphological features, analysis of nucleotide sequences of the 

internal transcribed spacer region (ITS1-5.8S-ITS2) of rDNA 

and partial sequence of the translation elongation factor 1-_ gene. 

Seven species were constantly isolated from diseased plants: 

Diplodia corticola (from holm oak and kermes oak), Diplodia seriata 

(field elm), Diplodia sp. (narrow-leaved ash), Diplodia scrobiculata 

(strawberry tree), Neofusicoccum vitifusiforme (narrowleaved 

ash) and two new species belonging to the genus Dothiorella 

(common hazel). Pathogenicity of all fungal species was 

verified by stem inoculation of seedlings of the same host from 

which they were isolated, under controlled laboratory conditions. 

All fungal species, except for a Dothiorella taxon, proved to be 

pathogenic to their host. The results obtained emphasize that 

many Botryosphaeriaceae may represent a serious threat for 

Mediterranean trees and shrubs. Our findings have contributed 

to improve the knowledge of N. vitifusiforme and D. scrobiculata 

by expanding their host range that includes now narrow-leaved 

ash and strawberry tree, respectively. 

CERATO-PLATANIN AND CERATO-POPULIN INDUCE 

DIFFERENTIAL RESISTANCE RESPONSES IN PLANE 

LEAVES. L. Lombardi 1 , I. Baccelli 3 , R. Bernardi 2 , G. Cappugi 4 , 

L. Pazzagli 4 , P. Picciarelli 2 , A. Scala 4 . 1 Dipartimento di Biologia, 

Università, Via Luca Ghini 5, 56126 Pisa, Italy. 2 Dipartimento di 

Biotecnologie Agrarie, Sezione di Protezione delle Piante, Laboratorio 

di Patologia Vegetale Molecolare, Università degli Studi, Via 

della Lastruccia 10, 50019 Sesto Fiorentino (FI), Italy. 3 Dipartimento 

di Biologia delle Piante Agrarie, Università, Via della Piagge 

23, 56124 Pisa, Italy. 4 Dipartimento di Scienze Biochimiche, Università 

degli Studi, Viale Morgagni 50, 50134 Firenze, Italy. E-mail: 

aniello.scala@unifi.it 

Cerato-platanin (CP) and cerato-populin (Pop1) are small 

proteins produced by the phytopathogenic fungi Ceratocystis platani 

and C. populicola, respectively. CP and Pop1 behave as 

PAMPs, since they elicit typical defense responses in various host 

and non-host plants. CP and Pop1 are well-structured α/β proteins 

with an identity of about 63% and the conservative substitution 

of approximately 12% of the amino acids. Moreover, the 

analysis by circular dichroism shows differences in the secondary 

structure between the two proteins. The present work aimed at 

ascertaining whether these structural differences are reflected in 

differences in their eliciting activity in plane leaves, which represents 

a study model on which we have been working for a long 

time and know deeply. We examined the ability of CP and Pop1 

to induce in plane leaves: (i) production of hydrogen peroxide 

and nitric oxide by using the specific probe 2’-7’-dichlorodihydrofluorescein 

diacetate and the fluorescent dye 4,5 diaminofluorescein 

diacetate, respectively; (ii) programmed cell death by in 

situ detection of DNA fragmentation (TUNEL assay); (iii) the expression 

of the genes PR5 (thaumatin), LTP (lipid transfer protein) 

e APX (ascorbate peroxidase). The inhibition of fungal 

growth on plane leaves treated with CP and Pop1 was also as- 

sessed. Results showed marked differences in the eliciting capacity 

of the two proteins. In particular the plane resistance responses 

are activated earlier by CP than by Pop1. These results are the 

basis for identifying the protein region(s) involved in the PAMP 

activity. 

MOLECURAL DETECTION OF BISCOGNIAUXIA NUM- 

MULARIA IN SYMPTOMLESS BEECH TREES IN THE 

APENNINE MOUNTAINS. N. Luchi 1 , B. Ceccarelli 2 , A.M. 

Vettraino 2 , A. Vannini 2 , P. Capretti 1 . 1 Dipartimento di Biotecnologie 

Agrarie, Sezione di Protezione delle Piante, Università degli 

Studi, Piazzale delle Cascine 28, 50144, Firenze, Italy. 2 Dipartimento 

di Protezione delle Piante, Università della Tuscia, Via S. 

Camillo De Lellis, 01100 Viterbo, Italy. E-mail: nicola.luchi@ 

unifi.it 

Spreading of weak fungal parasites on forest trees growing at 

the border of their natural range area is one of the expected consequences 

of global climate change. Studies on Fagus sylvatica 

show that the spread of populations towards the southern limit of 

the species distribution are limited strongly by drought. Biscogniauxia 

nummularia is an endophyte fungus of European beech, 

generally living in symptomless trees, but also able to cause stripcankers 

and wood decay on individuals stressed by drought. In 

the present work the latent phase of the fungus in symptomless 

host tissues was studied taking samples from apparently healthy 

beech trees growing in two different Fagus type associations: (i) 

Luzulo-Fagion Wood, sub continental climate (Gavinana, Pistoia); 

(ii) Acremonio-Fagion wood, Mediterranean climate with 

dry summers (Monti Cimini, Viterbo). Samples consisiting of 1to 

2-year-old twigs were collected in different seasons and used 

both for isolation of the fungus and DNA extractions following 

the referenced method described by the authors (Lett. Appl. Microbiol. 

43: 33-38. 2006) . Real time PCR afforded higher fungal 

detection rates than isolation methods. The occurrence of B. 

nummularia as endophyte showed seasonal fluctuation in area (i) 

(Gavinana) and was scarce in autumn and winter (5-20%) with 

respect to summer (40-100%), whereas it was constant in area (ii) 

(Monti Cimini), where detection of fungal DNA ranged from 80- 

100% of the samples throughout the year. Higher level of B. 

nummularia detection in area (ii), much more prone to drought 

stress, confirms the role of this fungus as bioindicator of water 

deficit. 

HETEROBASIDION ABIETINUM POPULATION GENET- 

IC STRUCTURE IN RESPECT TO THE EUROPEAN 

ABIES POPULATION. N. Luchi 1 , D. Paffetti 3 , K. Korhonen 2 , 

J. Hantula 2 , M.E. Sanchez 4 , T.D. Lehtijärvi 5 , A. Lehtijärvi 5 , P. 

Capretti 1 . 1 Dipartimento di Biotecnologie Agrarie, Sezione di Protezione 

delle Piante, Università degli Studi, Piazzale delle Cascine 

28, 50144 Firenze, Italy. 2 METLA, FI-01301 Vantaa, Finland. 

3 DISTAF, Università degli Studi, Via S. Bonaventura 13, 50145 

Firenze, Italy. 4 Departamento de Agronomìa, Patologìa Agroforestal, 

Universidad de Córdoba, Spain. 5 Süleyman Demirel University, 

Faculty of Forestry, Isparta, Turkey. E-mail: nicola.luchi@ 

unifi.it 

Heterobasidion abietinum is one of fungal pathogens colonizing 

almost all Abies species occurring in Europe. However consequences 

of its attacks may be different according to environmental 

characters. During the last glacial age in Europe both fungal 

and host populations, including Abies species survived in different 

refuges in mountain areas or in the southern Mediterranean


peninsulas. Later, northbound migrations enabled those populations 

to recolonize their present natural habitats. To investigate 

the genetic divergence of H. abietinum populations, a collection 

of isolates from central to southern Europe (Spain, Italy, Greece 

and Turkey), was analysed using minisatellites (DAMD-M13) and 

microsatellites (RAMS). Genetic variation within and among 

groups of populations was compared and a dendrogram was constructed 

with the Neighbor-Joining method. Clusters generally 

showed that isolates grouped according to their geographical origin. 

However those from southern Spain (from Abies pinsapo) 

were clearly differentiated from the others. More than 20 main 

haplotypes of H. abietinum were observed. Their number was 

higher in the central part of the area considered in this study and 

lower in the peripheral regions. In the western part of the distribution 

area (Andalucìa, Spain) their number was very scarce. 

This study confirms the hypothesized relationship between variability 

of H. abietinum population and migration history of Abies 

species in Europe. The occurrence of several haplotypes in the 

eastern part of the distribution area could be a consequence of 

historical trade routes; in this case man activities may have helped 

fungal spreading. 

INFLUENCES OF MULCHES ON SOLARIZED SOIL- 

BORNE MICROBIAL COMMUNITY. A. Luvisi 1 , A. Panattoni 

1 , A. Colosimo 1 , F. Filippi 2 , G. Magnani 2 , E. Triolo 1 . 1 Dipartimento 


Università degli Studi, Via del Borghetto 80, 56124 Pisa, 

Italy. 2 Dipartimento di Biologia delle Piante Agrarie, Università 

degli Studi, Viale delle Piagge 23, 56124 Pisa, Italy. E-mail: aluvisi 

@agr.unipi.it 

To investigate the effectiveness of plastic mulches for soil solarization, 

transparent polyethylene (PE), Evalux (EL) and HT 

Supersol (HT) (Agriplast, RG, Italy) were used. The experiment 

was carried out in sandy soil in 2008 and 2009, from 20 July to 20 

August, near Pisa (Italy). Artificial soil infestation with Sclerotinia 

minor Jagger was carried out and untreated plots were used as 

control. Temperatures in the soil profile were monitored at 3-5 

cm depth. Seedlings of Lactuca sativa cv. Justine were planted after 

solarization. Sclerotinia-induced lettuce drop was evaluated 

by counting the number of healthy and diseased plants in the 

plots. Soil samples were collected after solarization at a depth of 

3-5 cm to evaluate total fungi (F), Trichoderma spp. (T) and actinomycetes 

(A) as CFU/g of soil, using PDA, P190 and water-agar 

medium, respectively. Microbial community analysis using Biolog 

EcoPlates was performed to estimate total activity, Shannon- 

Weaver index and Evenness. Different temperature profiles were 

registered and PE, EL and HT increased maximum soil temperature 

by 4.3, 6.7 and 7.2°C respectively. Lettuce drop was reduced 

by more than 90% compared to control, with no significant differences 

between treatments. In HT-treated soil, levels of F, T 

and A were more than 31.2, 2.5 and 39.0% higher than in PE- or 

EL-treated soils. Biolog parameters confirmed the milder effects 

of HT film on non pathogenic microbial population. Improved 

plastic mulches can control soil-borne pathogens efficiently, limiting 

shifts in the soil microflora. 

SANITARY SELECTION OF GRAPEVINE SUPPORTED 

BY RFID SYSTEM. A. Luvisi 1 , A. Panattoni 1 , A. Colosimo 1 , M. 

Pagano 2 , R. Bandinelli 3 , E. Rinaldelli 2 , E. Triolo 1 . 1 Dipartimento 

di Coltivazione e Difesa delle Specie Legnose “G. Scaramuzzi”, Università 

degli Studi, Via del Borghetto 80, 56124 Pisa, Italy. 2 Dipartimento 

di Scienze delle Produzioni Vegetali, del Suolo dell’Am- 

biente Agroforestale, Università degli Studi di Firenze, Viale delle 

Idee 30, 50019 Sesto Fiorentino (FI), Italy. 3 Associazione Toscana 

Costitutori Viticoli TOS.CO.VIT., Via Vecchia di Marina 6, 56010 

San Piero a Grado (PI), Italy. E-mail: aluvisi@agr.unipi.it 

Radio-frequency identification (RFID) technology was suggested 

for tracking of numerous and diverse materials in plant 

pathology trials. Keeping track of the sample identification number 

and of the precise location where samples were collected represents 

an essential step. Phenotypic, sanitary and genomic data 

can also be added to RFID tags associated to the assayed plants. 

Grapevine selection is based on sub-sequential steps in which 

many grapevines are monitored in vineyard(s) for years, as well as 

their relative propagated grapevines used for indexing, comparatives 

studies or conservation in screenhouses. These steps can be 

supported by RFID technology. Traditional labels may undergo 

discoloration, degradation, loss or removal: these issues represent 

critical points in plant selection, considering the long periods in 

which plants have to be monitored. The association of RFID tag 

to plant reduce the occurrence of errors or losses, in particular if 

microchips are inserted in the grapevine, making impossible the 

removal or errors in data-to-plant association. The tagging trial, 

started in 2009, regarded accessions of Vitis vinifera cv Sangiovese 

selected in Montalcino (Siena, Italy) for clonal selection 

purpose, propagated materials stored in screenhouse, grafted cuttings 

used for indexing with biological indicators. RFID wristband 

was used for tagging non-grafted plants, whereas microchips 

were implanted in the grafted ones. A database was created 

to monitor the tagged plants during the whole sanitary selection 

procedure, as well as for storing and updating data pertaining 

to each plant and relative propagated material. 

DETECTION OF PHYTOPHTHORA CAMBIVORA IN 

SOIL PARTICLES BY REAL TIME QUANTITATIVE PCR 

ASSAY. V. Mancini, N. Luchi, P. Capretti. Dipartimento di 

Biotecnologie Agrarie, Sezione di Protezione delle Piante, Università 

degli Studi, Piazzale delle Cascine 28, 50144 Firenze, Italy. 

E-mail: vale.mancini@yahoo.it 

Phytophthora cambivora is one of the most harmful pathogens 

causing root rot, collar rot and or stem canker of Castanea sativa 

and a well known agent of “ink disease”. According to the data 

collected since 2002 by the regional monitoring service (META 

http://meta.arsia.toscana.it/) disease centres are increasing in 

Tuscany (central Italy), where a number of new foci are detected 

annually in coppice forests and orchards. As disease centres are 

mainly found near streams and along roads and trails it could be 

useful to setup a protocol to detect the source of infection. Aim 

of this work was the optimization of a real time PCR assay, by using 

SYBR Green chemistry, to detect and quantify the occurrence 

of P. cambivora from potential sources of infection as plant tissues 

and soil particles. To this purpose DNA was extracted from 

chestnut tissue according to the method described by the authors 

(Lett. Appl. Microbiol. 41: 61-68, 2005) Later on, soil samples 

were artificially infected with P. cambivora mycelium. Real time 

PCR was developed using genus- and specie-specific primers. 

Notwhitstanding some failures, probably due to PCR inhibitors, 

real-time PCR proved to be an efficient method for detecting and 

quantifying P. cambivora from both host tissues and soil. The 

presence of P. cambivora DNA in soil samples, confirmed the hypothesis 

that the soil can be a source of infection.


SHOOT AND TIP BLIGHT BY DIPLODIA PINEA AND D. 

SCROBICULATA DETECTED BY MOLECULAR METH- 

ODS ON PSEUDOTSUGA MENZIESII IN CENTRAL 

ITALY. V. Mancini, N. Luchi, P. Capretti. Dipartimento di Biotecnologie 

Agrarie, Sezione di Protezione delle Piante, Università 

degli Studi, Piazzale delle Cascine 28, 50144 Firenze, Italy. E-mail: 

vale.mancini@yahoo.it 

The occurrence of Diplodia pinea as the causal agent of tip 

and shoot blight has often been reported from Pinus spp. and 

other conifers. In Italy, D. pinea is quite frequent on pines but also 

D. scrobiculata has occasionally been found on different hosts 

species in Sardinia and other localities of southern Italy. Recently 

in Tuscany (central Italy) during the annual regional monitoring 

survey (META http://meta.arsia.toscana.it/ ) symptoms of crown 

disease were observed in Douglas fir (Pseudotsuga menziesii) 

stands. The disease was especially frequent in the lower part of 

the crowns of 20- to 40-year-old trees. Terminal shoots and small 

branches were completely defoliated, the bark was necrotic and 

showed sporadic black picnidia. In the last few years a fungus 

was isolated from branches and shoots. Single spore colonies 

were obtained, grown in pure culture and DNA was extracted. 

Diplodia was identified on the basis of both morphological (fungal 

structures and colonies) and molecular methods. DNA amplification 

assays using specific primers available in the literature 

confirmed the identification of the fungal species as D. pinea in 

most of the samples but revealed also the occurrence of D. scrobiculata 

in some of the Douglas fir trees. 

DIPLODIA PINEA DETECTION ON LEPTOGLOSSUS OC- 

CIDENTALIS (INSECT VECTOR) BY REAL TIME-PCR. V. 

Mancini, N. Luchi, M. Feducci, P. Capretti. Dipartimento di 

Biotecnologie Agrarie, Sezione di Protezione delle Piante, Università 

degli Studi, Piazzale delle Cascine 28, 50144 Firenze, Italy. 

E-mail: vale.mancini@yahoo.it 

Leptoglossus occidentalis (Hemiptera: Coreidae), an insect native 

to North America, is present in Italy since 1999. It now occurs 

mainly in Pinus pinea stands where it is thought to cause extensive 

damage to seed production. Considering that this insect 

has the same habitat (cones of conifers) of Diplodia pinea, a fungus 

responsible for damaging pine cones, it is possible that it may 

be involved in spreading fungal conidia. Real time PCR was used 

for detecting and quantify fungal DNA on both cones and L. occidentalis 

bodies. Materials for PCR assays were symptomless 1year-old 

cones collected in S. Rossore (Pisa), individuals of L. occidentalis 

sampled in a conifer forest (Vallombrosa, Florence) and 

insectes reared in the laboratory. Some of the latter were washed 

with a conidial suspension of D. pinea, others were placed in a 

cage and allowed to walk on pine cones infected by D. pinea and 

still others, coming directly from breeding colonies, served as 

negative control. DNA was extracted from all samples (cones, insects 

and insects’ washing waters) and PCR was carried out using 

fungus-specific primers. D. pinea was detected on all cones 

analysed and, frequently, on L. occidentalis bodies This is taken as 

an indication that L. occidentalis may have a role as possible vector 

of D. pinea. 

INTERACTION OF CERATO-PLATANIN AND CERATO- 

POPULIN WITH INANIMATE AND PLANE LEAF SUR- 

FACES: A STRUCTURAL STUDY. F. Martellini 1 , L. Pazzagli 1 , 

L. Carresi 2 , F. Sbrana 3 , B. Tiribilli 4 , B. Pantera 1 , G. Cappugi 1 , F. 

Faoro 5 , A. Scala 2 . 1 Dipartimento di Scienze Biochimiche, Univer- 

sità degli Studi, Viale Morgagni 50, 50134 Firenze, Italy. 2 Dipartimento 

di Biotecnologie Agrarie, Sezione di Protezione delle Piante, 

Laboratorio di Patologia Vegetale Molecolare, Università degli Studi, 

Via della Lastruccia 10, 50019 Sesto Fiorentino (FI), Italy. 3 Dipartimento 

di Ingegneria Biofisica ed Elettronica, Università degli 

Studi, Via Opera Pia 11A, 16145 Genova, Italy. 4 Istituto dei Sistemi 

Complessi del CNR, Via Madonna del Piano 10, 50019 Sesto 

Fiorentino (FI), Italy. 5 Dipartimento di Produzione Vegetale, Università 

degli Studi, Via Celoria 2, 20133 Milano, Italy. E-mail: 

federica.martellini@unifi.it 

Cerato-platanin (CP) and cerato-populin (Pop1) are proteins 

abundantly secreted by and localized in the cell wall of Ceratocystis 

platani and C. populicola, respectively. Both are assumed to 

play a role in plant interaction, since they induce accumulation of 

H 2 O 2 and NO, programmed plant cell death, overexpression of 

defence genes, phytoalexin synthesis and restriction of conidia 

growth. Thus, CP and Pop1 appear to act as PAMPs able to activate 

effective primary defence systems. CP and Pop1 are members 

of the “cerato-platanin family” containing proteins involved 

in many microbe-host interactions acting as phytotoxins, elicitors 

of defence responses or human allergens. Cellular localization in 

fungi suggests a role of these proteins in interaction with host 

surfaces. To investigate the mechanism of interaction, in vitro and 

in vivo experiments have been performed. CP and Pop1 strongly 

interacted with Teflon, a colloidal suspension used to mimic hydrophobic 

surfaces. During reaction, these proteins lost their native 

structure and adopted an unfolding conformation with a 

small percentage of α-helix. Moreover, CP and Pop1 were adsorbed 

on hydrophobic surfaces (silanized mica, gold sheets and 

graphite) and appeared as supra-molecular aggregates, which resemble 

the ordered assemblages that the proteins form in vitro, 

and are able to enhance host-defences. In vivo, the proteins 

seemed to interact with the hydrophobic cuticle of the plane 

leaves and did not penetrate the cell wall and the membrane. The 

results suggest that CP and Pop1 interact with hydrophobic components 

of the host before inducing defence events. 

ANTIFUNGAL ACTIVITY OF TERPENES IDENTIFIED 

IN THE LEAVES OF ROSMARINUS OFFICINALIS. V. Martini 

1,2 , C. Comparini 2 , P. Capretti 2 , M. Michelozzi 1 , A. Scala 2 . 

1 Dipartimento di Biotecnologie Agrarie, Sezione di Protezione delle 

Piante, Università degli Studi, Piazzale delle Cascine 28, 50019 Sesto 

Fiorentino (FI), Italy. 2 Istituto di Genetica Vegetale del CNR, Via 

Madonna del Piano, 50019 Sesto Fiorentino (FI), Italy. E-mail: 

martini@imgpf.fi.cnr.it 

Plants of Rosmarinus officinalis are widely distributed in Europe, 

Asia and Africa. Mediterranean is the area where spontaneous 

plants are commonly found. This plant is known for its use 

in cookery, and the increasing interest for its pharmaceutical 

properties. Two groups of compounds are mainly responsible of 

the biological activities of rosemary: the volatile fraction and the 

phenolic constituents. Alternaria leaf spot of rosemary has been 

reported in various Italian regions, as the cause of black spots on 

leaves and stems followed by defoliation. The aim of the present 

work was to investigate the antifungal activity of different rosemary 

monoterpenes against a strain of Alternaria alternata (Fr.) 

Keissl. recently isolated from an Alternaria-diseased rosemary 

plant. Their antifungal activity was evaluated as inhibition of the 

mycelial growth using “special vials” containing potato dextrose 

broth and increasing concentrations (0.025, 0.1, 0.4, 1.6, 6.4, 10.0 

and 100 mM) of 1,8-cineole, (+)-alpha-pinene, (-)-alpha-pinene, 

(+)-limonene, (-)-limonene, (-)-beta-pinene, (+)-beta-pinene, 

myrcene and linalool. The minimum inhibitory concentration


(MIC) of these terpenes ranged from 0.025 to 1.6 mM. The lowest 

values of MIC were observed for myrcene and (-)-alphapinene, 

whereas the least toxic compounds were (+)-beta-pinene, 

1.8-cineole and linalool. These results are discussed in relation to 

the variability in constitutive terpene composition detected in 

four different Italian provenances (one Sardinian and three from 

Tuscany: Alberese, Isle of Elba and Isle of Giglio). 

THE ROLE OF VOLATILE COMPOUNDS IN THE CHEMI- 

CAL DEFENSE SYSTEM OF PICEA SITCHENSIS AGAINST 

HETEROBASIDION ANNOSUM ATTACK. V. Martini 1 , S. 

Woodward 2 , G. Deflorio 1 , P. Capretti 3 , M. Michelozzi 1 . 1 Istituto 

di Genetica Vegetale del CNR, Via Madonna del Piano 10, 50019 

Sesto Fiorentino (FI), Italy. 2 University of Aberdeen, School of Biological 

Sciences, Department of Plant and Soil Science, Cruickshank 

Building, St. Machar Drive, Aberdeen AB24 3UU, Scotland, UK. 

3 Dipartimento di Biotecnologie Agrarie, Sezione di Difesa delle 

Piante, Università degli Studi, Piazzale delle Cascine 28, 50144 

Firenze, Italy. E-mail: marco.michelozzi@igv.cnr.it 

Picea sitchensis (Bong.) is very susceptible to Heterobasidion 

annosum (Fr.) Bref., the agent of root and butt-rot of conifers 

that usually enters the host through wounds or stumps, causing 

wood decay, with significant economic losses when monoculture 

plantations are attacked. In some cases, host tree populations 

may remain free of infection but the basis for this apparent resistance 

to the pathogen is unknown. Plants produce a vast array 

of secondary metabolites such as terpenoids and phenolics, to defend 

themselves against their natural enemies. The aim of this 

study was to examine the variation in the chemical responses to 

H. annosum attack in P. sitchensis. Terpene composition was analyzed 

in cortical tissue samples of four Sitka spruce clones growing 

at the Scootmore site (Ref: NJ172392; Moray, Scotland, UK). 

The Sitka spruce clones included the two from among 30 tested 

in a previous screening trial forming the shortest stem bark lesions 

(Clones 20198 and 20206) and the two forming the longest 

stem bark lesions (Clones 20179 and 20204) following inoculation 

with H. annosum. The results showed significant variation in 

the constitutive terpene profiles between the different clones. 

Proportions of some volatile terpenes varied in the secondary 

resin produced in bark tissues surrounding the lesions. Total absolute 

quantities of monoterpenes were significantly higher in the 

secondary resin than in the primary resin, in both resistant and 

susceptible clones. This effect was more evident in tissues following 

inoculation, compared with the pseudo-inoculated samples. 

These findings are discussed in the context of ecological interactions 

and show that terpenoid metabolism may provide useful 

biochemical markers for resistance to H. annosum in selection 

and breeding programmes. 

FURTHER DATA ON THE DISTRIBUTION OF MAIN 

GRAPEVINE VIRUSES IN TUSCANY. A. Materazzi 1 , D. Rizzo 

2 , H. Bouyahia 1 , P. Braccini 2 , M. Della Bartola 1 . 1 Dipartimento 


Sezione di Patologia Vegetale, Via del Borghetto 80, 56124 Pisa, 

Italy. 2 Agenzia Regionale per lo Sviluppo e l’Innovazione nel Settore 

Agricolo-Forestale (ARSIA), Laboratorio di Diagnostica 

Fitopatologica, Via dei Fiori 8, 51012 Pescia (PT), Italy. E-mail: 

amatazzi@agr.unipi.it 

From 2005 to 2009, 451 accessions of V. vinifera belonging to 

major and minor varieties, were collected from the most important 

and the emerging grape-growing areas of Tuscany and sub- 

jected to virological tests. The sanitary status was evaluated by 

symptoms observation in the field and subsequently by laboratory 

analysis (ELISA and/or RT-PCR). The presence of the following 

viruses was investigated: Arabis mosaic virus (ArMV), 

Grapevine fanlealf virus (GFLV), Grapevine leafroll-associated 

virus 1(GLRaV-1), 2 (GLRaV-2), and 3 (GLRaV-3), Grapevine 

virus A (GVA) and B (GVB), and Grapevine fleck virus (GFkV). 

Results showed that 308 (68,3%) vines were infected with at least 

one virus. In detail, 166 plants had single infections and 142 had 

mixed infections. The most widely spread virus was GLRaV-3, 

detected in 164 of 308 vines (53.2%). GFkV infections concerned 

128 (41.6%) vines, while GLRaV-1 and GVA were found 

in 88 (28.6%) vines. GFLV, singly or in association with other 

viruses, was detected in 46 accessions (14.9%). Analysis confirmed 

the limited distribution in Tuscan vineyards of GLRaV-2 

and GVB, that were found in 6 and 4 vines, respectively. Except 

for the viticultural areas of Garfagnana and Media Valle del Serchio, 

where an unusually high incidence of ArMV was recently 

reported, the occurrence of this virus was confirmed to be very 

low. In fact, ArMV was detected in only 2 accessions, always in 

association with GFLV. 

ARE POINT MUTATIONS IN THE CYP51 GENE ASSOCI- 

ATED WITH RESISTANCE TO DMI FUNGICIDES IN 

ERYSIPHE NECATOR? M. Miazzi 1 , H. Hajjeh 2 , F. Faretra 1 . 1 Dipartimento 

di Protezione delle Piante e Microbiologia Applicata, 

Università degli Studi “Aldo Moro”, Via Amendola 165/A, 70126 

Bari, Italy. 2 PTU-K Palestinian Technical University Kadoori, P.O. 

Box 7, Tulkarm, Palestine. E-mail: m.miazzi@agr.uniba.it 

Sterol 14±-demethylation inhibitors (DMIs) fungicides are still 

important in grapevine protection against powdery mildew, although 

acquired resistance has been reported since 1989. Previous 

studies showed that the point mutation A495T in the CYP51 

gene, coding for a cytochrome P450, is responsible for a high level 

of resistance in E. necator. An allele-specific PCR assay with 

primers MUT1 (5’-AATTTGGACAATCAA-3’) and U14DM 

(5’ATGTACATTGCTGACATTTTGTCGG-3’) was performed 

on 50 fungal isolates sampled in 7 vineyards. Only four isolates 

(X109, X112, X113 and X115) carried the point mutation yielding 

a DNA band of the expected size. The response of E. necator isolates 

to tebuconazole was evaluated through an in vitro bioassay 

on grapevine leaf disks. Wild-type sensitive isolates showed EC 50 

≤0.1-3 µg ml -1 and MIC = 1-6 µg ml -1 , while mutants showed 

EC 50 = 6-10 µg ml -1 and MIC = 10 µg ml -1 . Isolate X109, although 

carrying the point mutation, was normally sensitive (EC 50 = 0.3 µg 

ml -1 and MIC = 3 µg ml -1 ). In these four isolates, the whole 

CYP51 gene was amplified with the primer pairs C14 (5’-TAAG- 

GTAGTATTGAGGCGGG-3’) and C14R (5’-TTCTAACCC- 

TAACACCTGCC-3’) and sequenced. The alignment with the nucleotide 

sequence available in GenBank (accession No. U83840) 

confirmed the presence of the point mutation A495T, but additional 

point mutations were detected. Results suggest that the 

A495T mutation is not strictly associated with resistance to DMIs 

in E. necator. Different mutations in the same gene or in other 

genes may be significant, but remain to be clarified. 

RESISTANCE OF PODOSPHAERA XANTHII TO QoI 

FUNGICIDES IN APULIA. M. Miazzi, C. La Guardia, F. Faretra. 

Dipartimento di Protezione delle Piante e Microbiologia Applicata, 


70126 Bari, Italy. E-mail: faretra@agr.uniba.it


Powdery mildew, caused mostly by Podosphaera xanthii, is an 

important disease of cucurbits in the Mediterranean basin. The 

fungicides known as QoI are largely used to control the disease, 

but resistance has been observed in many phytopathogenic fungi, 

including P. xanthii. Here we present the results of a monitoring 

of resistance to trifloxystrobin in P. xanthii populations in Apulia. 

Sixty-four isolates of P. xanthii were sampled from 32 cucurbit 

fields in 2002-2007, and assayed at seven trifloxystrobin (Flint®) 

concentrations. Alternative respiration was inhibited by adding 1 

mg ml -1 of salicylhydroxamic acid to fungicide suspensions. Portions 

of zucchini cotyledons (cv. Diamant 1) were dipped for 1 

min in the fungicide suspension, placed on Blaich medium in 

Petri dishes, inoculated at a single point with about 20 conidia, 

and maintained at 25°C under a 12 h photoperiod. After 10 days, 

the percentage of infected surface was estimated using an empirical 

scale based on six infection classes, and EC 50 and MIC were 

assessed. Results pointed out a high variability in the response to 

trifloxystrobin, with EC 50 ranging from 375 µg ml -1 , and 

MIC from 10 to >375 µg ml -1 . About half of isolates had EC 50 

>375 µg ml -1 trifloxystrobin, a concentration three times greater 

than the normal field rate (125 µg ml -1 ), with no differences attributable 

to the host plants or the geographical origin. In conclusion, 

resistance to QoIs is very common in Apulia, and this 

should induce growers to implement more stringent anti-resistance 

strategies. 

FIRST REPORT OF LEAF SPOT CAUSED BY STEMPHYLI- 

UM HERBARUM ON BORAGO OFFICINALIS. L.C. Moretti, 

M. Quaglia, M. Orfei, C. Cappelli. Dipartimento di Scienze Agrarie 

e Ambientali, Università degli Studi, Borgo XX Giugno 74, 06121 

Perugia, Italy. E-mail: chiaraluce.moretti@unipg.it 

Borage (Borago officinalis L.) is an ornamental plant, widely 

present in nature in different areas of Italy. During spring 2009, 

in private gardens and on the border of Trasimeno lake (Central 

Italy), leaf spots on cultivated and wild borage plants were observed. 

Initial symptoms were recorded on basal leaves as small 

brown circular spots (2 mm diameter), greyish-white in the middle 

and surrounded by a reddish halo. The symptoms developed 

on the upper leaves and later severe yellowing and necrosis of the 

leaf tissue were observed. Due to unfavourable climate conditions 

(hot temperature and low humidity), new foliages did not 

show symptoms and the disease disappeared but the plants, especially 

those present in the gardens, seemed less vigorous. A fungus, 

morphologically identified as Stemphylium herbarum Simmonds 

[anamorph of Pleospora herbarum (Fr.) Rabenh.], was 

consistently isolated. To assess its pathogenicity to borage plants, 

two fungal isolates were used. Plants were sprayed with conidial 

suspensions (5x10 5 conidia ml -1 ) from 10-day-old agar cultures. 

After 7-8 days, the plants inoculated with both fungal isolates 

showed small spots, identical to those observed under natural 

conditions, whereas no symptoms were observed on control 

plants sprayed with water. S. herbarum was consistently reisolated 

from inoculated borage leaves. The fungus is known to cause leaf 

spots on several herbaceous hosts, including lettuce and onion. 

However, to our knowledge, this is the first report of a leaf spot 

disease of borage caused by S. herbarum. 

MULTIPLE BOTRYOSPHAERIACEAE INFECTION IN 

FOREST TREES: SYNERGISTIC OR ANTAGONISTIC IN- 

TERACTION? S. Moricca 1 , A. Uccello 1 , E. Turco 2 , B. Ginetti 1 , 

A. Ragazzi 1 . 1 Dipartimento di Biotecnologie Agrarie, Sezione di 

Protezione delle Piante, Piazzale delle Cascine 28, 50144 Firenze, 

Italy. 2 Istituto per la Protezione delle Piante del CNR, Via Madonna 

del Piano, 50019 Sesto Fiorentino (FI), Italy. E-mail: andrea.uccello@unifi.it 

Botryosphaeriaceae an Ascomycetes family well known as endophytes 

of woody hosts under temperate and subtropical climates. 

It includes several species that may turn to a pathogenic 

habitus when hosts undergo physiological stress. Previous pathogenicity 

studies have focused their attention on the interactions 

between a single fungal species and their hosts. However, the 

type of interaction established when two or more botryosphaeriaceous 

fungi colonize the same host tissues is till unknown. Isolates 

of Botryosphaeria dothidea, Diplodia seriata and Neofusicoccum 

parvum were tested on two-year-old seedlings of Acer 

campestre, Carpinus betulus, Fraxinus excelsa and Quercus cerris 

to verify disease severity and to evaluate the effect of single and 

combined infections. N. parvum proved the most virulent of the 

three species, causing lesions significantly larger than those 

caused by the other species. Seedlings inoculated with the mixture 

of isolates showed smaller lesions than those caused by N. 

parvum alone. These preliminary results allow us to hypothesize 

that a sort of antagonistic interaction may occur between 

Botryosphaeriaceae in host tissues. 

STUDIES ON THE AETIOLOGY OF THE DISEASE 

KNOWN AS OCHRACEOUS LEAF SPOTS OF APPLE IN 

FRIULI VENEZIA GIULIA. S. Moruzzi, M. Martini, R. 

Musetti, P. Ermacora, S. Borselli and R. Osler. Dipartimento di 

Biologia e Protezione delle Piante, Università degli Studi, Via delle 

Scienze 208, 33100 Udine, Italy. E-mail: serena.moruzzi@uniud.it 

An apple disease known as ochraceous leaf spots is known 

since 1950, but its aetiology is still unclear. The disease is common, 

especially in orchards where apples are grown under organic 

regime. In Friuli Venezia Giulia, this disease is present in different 

areas, in orchards of commercial and autochthonous apple 

varieties. Symptomatic leaves were sampled in three farms to 

study disease aetiology. In total 825 fungal colonies were obtained, 

grouped according to morphological features, and characterized 

by molecular tools, by analysis of rDNA ITS region. Isolations 

yielded fungi belonging to Phoma spp. (about 37% subdivided 

in: P. macrostoma, 16.9%; P. glomerata, 4%; P. epicoccina, 

15%; P. exigua, 2.5%), Alternaria spp. (about 35%), and other 

minor genera. In pathogenicity tests, leaves inoculated with all 

four Phoma species developed classical necrotic lesions, while Alternaria 

spp. developed some atypical necrosis, suggesting Phoma 

spp. as the most probable causal agent of leaf spots. Portions of 

symptomless leaves were also investigated: Phoma spp. accounted 

for 23% of the obtained colonies and Alternaria spp. for 58%, 

indicating that the latter fungal genus might behave mainly as a 

plant endophyte. In plants with leaf symptoms, part of the xylem 

appeared to be damaged under the electron microscope. Therefore, 

further investigations were conducted using different techniques, 

showing that P. macrostoma colonized also young branches. 

P. macrostoma isolates from leaves and young branches were 

characterized molecularly based on the ITS and ″-tubulin gene 

sequences. No molecular differences were found in these genomic 

regions among fungal isolates. 

FUNGAL ENDOPHYTES IN NEEDLES AND BRANCHES 

OF PICEA ABIES FROM THE PANEVEGGIO FOREST. R. 

Musetti 1 , R. Polizzotto 1 , F. De Luca 2 , S. Grisan 1 , R. Osler 1 . 1 Dipartimento 

di Biologia e Protezione delle Piante, Università degli

S4.92 Journal of Plant Pathology (2010), 92 (4, Supplement), S4.71-S4.105 

Studi, Via delle Scienze 208, 33100 Udine, Italy. 2 Ente Parco Paneveggio-Pale 

di San Martino, Via Castelpietra 2, 38054 Tonadico 

(TN), Italy. E-mail: rita.musetti@uniud.it 

Norway spruce (Picea abies) is a dominant tree species in large 

areas of Europe and northern Asia. The fungal endophytes of Norway 

spruce needles were described in several papers and reported 

from symptomless spruce branches, stem and bark. However, the 

whole assemblage of mycota associated with Norway spruce is still 

largely unknown. The roles of endophytic fungi remain to be clarified. 

Carroll et al. (Sydowia 29: 87-103, 1977) proposed that some 

endophytic fungi living in coniferous needles could have a mutualistic 

relationship with the host tree, through enhanced resistance to 

herbivores and plant pathogens. Our aim was to determine the 

fungal population of healthy looking Norway spruce trees growing 

in the Paneveggio forest (north-east Italy) to increase the knowledge 

on the contribution of fungi to the microbial diversity of 

spruce forests. A preliminary screening was carried out using needle 

and branch segments (30+30) obtained from 10 plants that 

were sterilized by immersion in 3% sodium hypochlorite, rinsed in 

sterile water and incubated in PDA medium at 23°C under an alternating 

light/dark cycle consisting of 8 h of cool-white fluorescent 

daylight and 16 h darkness. Colonies were subcultured in 

PDA until pure cultures were obtained. On the whole, 38 fungal 

endophytes were recovered, belonging to 13 genera, the most representative 

being Alternaria, Aureobasidium and Aspergillus. The 

characterization of the fungal community associated with host 

plants not only helps us to estimate the diversity of fungi but provides 

raw material for further investigations on the interactive 

mechanisms among the two partners. 

PHOTOSYNTHETIC RESPONSE OF EUCALIPTUS TO 

BORON TOXICITY. C. Nali 1 , A. Francini 1 , E. Pellegrini 1 , S. 

Loppi 2 , G. Lorenzini 1 . 1 Dipartimento di Coltivazione e Difesa 

delle Specie Legnose “G. Scaramuzzi”, Università degli Studi, Via 

del Borghetto 80, 56124 Pisa, Italy. 2 Dipartimento di Scienze Ambientali 

“G. Sarfatti”, Università degli Studi, Via P.A. Mattioli 4, 

53100 Siena, Italy. E-mail: cristina.nali@agr.unipi.it 

Boron (B) toxicity is an important disorder that causes negative 

physiological effects such as decrease in leaf chlorophyll content, 

inhibition of photosynthesis and increased membrane leakiness. 

To gain an insight into the role of photosynthetic mechanisms 

in response to B toxicity, physiological parameters were analyzed 

in Eucaliptus globulus plants treated with 0.1 (control), 1 

and 10 mg l -1 (excess) H 3 BO 3 in nutrient solution for 12 weeks. 

After 42 days, plants grown with excess B developed leaf symptoms 

in the form of marginal necrosis. At the end of treatment, 

CO 2 assimilation and stomatal conductance decreased (-71% and 

-30%, respectively, compared to control) when plants were supplied 

with 10 mg l -1 H 3 BO 3 . Growth reduction (-30%) and increase 

of B concentration in the roots as a consequence of the 

treatment were also observed. Results indicate that B excess leads 

to: (i) visible injury in mature leaves; (ii) reduced root growth; 

(iii) increase in B concentration in all parts of the plant in leaves 

> stems > roots in the order; (iv) strong decrease in photosynthetic 

activity, because of structural damage of membranes. Under 

these circumstances, E. globulus should be regarded as sensitive 

to B toxicity. 

IDENTIFICATION, EXPRESSION AND CHARACTERIZA- 

TION OF NEW GRAPE DEFENSINS. V. Nanni 1 , L. Giacomelli 

2 , R. Hughes 3 , M. Banfield 3 , C. Moser 2 , M. Zanetti 4 , M. 

Dalla Serra 4 , P. Bertolini 1 , E. Baraldi 1 . 1 Dipartimento di Protezione 

e Valorizzazione Agroalimentare, Laboratorio di Biotecnologie, 

Università degli Studi, Viale Fanin 46, 40127 Bologna, 

Italy. 2 Centro Ricerca e Innovazione, Fondazione Edmund Mach, 

Via Mach 1, San Michele all’Adige (TN), Italy. 3 Department of Biological 

Chemistry, John Innes Centre, Norwich Research Park, Norwich, 

NR4 7UH, UK. 4 Istituto di Biofisica, Fondazione Bruno 

Kessler, Via Sommarive 18, 38123 Povo (TN), Italy. E-mail: elena.baraldi@unibo.it 

Plant defensins are small, highly basic, cysteine-rich peptides 

structurally related to defensins of other organisms. By scanning 

the Vitis vinifera Pinot noir genome using a combination of 

HMM and BLAST searches, different defensin-like sequences 

(DEFLs) were identified, and transcript accumulation was analyzed 

in different tissues and in fruits infected by Botrytis cinerea. 

We selected three putative genes coding for putative defensins, 

which have different expression pattern and cysteine arrangement. 

The corresponding proteins were expressed in E. coli and 

purified to homogeneity. The recombinant proteins exhibited different 

activity against B. cinerea, showing a possible correlation 

between gene expression and antifungal activity. Using a simple 

prokaryotic expression system coupled to in vitro testing, we 

have set up an useful system which will allow the functional characterization 

of all members of the grape defensin family. 

PATULIN ACCUMULATION BY PENICILLIUM EXPAN- 

SUM ISOLATES IN DIFFERENT FRUITS. F. Neri, I. Donati, 

F. Veronesi, D. Mazzoni and M. Mari. Dipartimento di Protezione 

e Valorizzazione Agroalimentare, Centro per la Protezione e 

Conservazione dei Prodotti Ortofrutticoli “G.C. Pratella”, Università 

degli Studi, Via Gandolfi 19, 40057 Cadriano (BO), Italy. Email: 

fiorella.neri@unibo.it 

To determine the accumulation of patulin in different fruits, 

four isolates of Penicillium expansum were cultured on fruit 

puree agar media (PAMs) and inoculated in wounded fruits of 

common (pear and apple) and less common (apricot, peach, 

strawberry and kiwifruit) hosts. The concentration of patulin accumulated 

in mycelia grown on fruit PAMs was higher than that 

detected in infected fruit tissues. Three P. expansum isolates accumulated 

patulin when grown on all fruit PAMs, while one isolate 

produced patulin only on apricot PAM. Apple PAM substrates 

were the most suitable for in vitro patulin accumulation (maximum 

concentration 173.1 and 74.1 µg/ml in ‘Pink Lady and 

‘Golden Delicious’ PAMs, respectively). However, infected tissue 

of cv. Golden Delicious showed lower average accumulation of 

patulin (1.7 µg/ml) compared with cv. Pink Lady (19.1 µg/ml), 

and no significant differences in patulin concentrations were 

found among ‘Golden Delicious’ apples and tested cultivars of 

pear, kiwifruit and strawberry. Peaches were highly susceptible to 

patulin accumulation, showing average concentrations of 27.4 

and 18.6 µg/ml in vitro and in vivo, respectively. Apricots were 

also consistently positive to patulin accumulation, both in vitro 

and in vivo. Our study showed the potential of some uncommon 

hosts of P. expansum to support patulin production, indicating 

that a steady monitoring of patulin contamination should be carried 

out in fruit substrates other than apples and pears. 

EFFECT OF DEOXYNIVALENOL-PRODUCING FUSARI- 

UM GRAMINEARUM STRAINS ON SEEDS OF TOLER- 

ANT AND SUSCEPTIBLE TRITICUM AESTIVUM VARI- 

ETIES. C. Nobili 1 , A. Ricelli 2 , M. Reverberi 3 , V. Scala 4 , G. Au-


reli 4 , S. D’Angeli, M.M. Altamura, A.A. Fabbri 3 , C. Fanelli 3 . 

1 UT-AGRINN, ENEA, Via Anguillarese 301, 00123 S.M. di Galeria 

(RM), Italy. 2 Istituto di Chimica Biomolecolare del CNR, Piazzale 

Aldo Moro 5, 00185 Roma, Italy. 3 Dipartimento di Biologia 

Vegetale, Università degli Studi “La Sapienza”, Largo Cristina di 

Svezia 24, 00165 Roma, Italy. 4 CRA, Unità di Ricerca della Valorizzazione 

Qualitativa dei Cereali (CRA-QCE), Via Cassia 166, 

00191 Roma, Italy. E-mail: chiara.nobili@enea.it 

To shorten the screening of wheat varieties tolerant to Fusarium 

head blight (FHB) and to deoxynivalenol (DON) synthesis 

the development of rapid and reliable assays is required. In this 

work, active but ungerminated seeds of two Triticum aestivum varieties, 

Blasco and Sagittario, respectively tolerant and susceptible 

to FHB, were inoculated with two F. graminearum strains (Fg126 

and Fg8308), having a different toxigenic profile. Wheat seeds reacted 

to F. graminearum infection by early production of reactive 

oxygen species (ROS) and activating antioxidant enzymes. Whilst 

cv. Blasco showed an important antioxidant reaction which apparently 

lead to a marked decrease in ROS content, cv. Sagittario 

partly missed this counteraction. Compared to cv. Blasco, cv. 

Sagittario also produced more 9-hydroxyoctadecenoic acid (9- 

HODE), considered a susceptibility factor toward mycotoxigenic 

fungi. Moreover, some genes related to fungal aggressiveness 

were up-regulated in Fg126 when grown on susceptible wheat 

seeds and some plant defence genes advanced their expression 

into cv. Blasco as compared with cv. Sagittario. Finally, it turned 

out that DON production may trigger apoptosis, occurring very 

quickly after fungal inoculation and with the typical formation of 

pre-apoptotic vesicles into aleuronic cells. As a matter of fact, 

wheat seeds, irrespective of the variety, are able to partly (5-10%) 

convert DON in its less toxic glucosylated form (3-Glu-DON). 

Each of these parameters might be considered as a wheat tolerance 

marker and used for diagnostic purposes or, through a forward 

genetic approach, for selecting wheat varieties hampering 

DON biosynthesis. 

ALARMING SPREAD OF PLUM POX VIRUS STRAIN M 

IN SOME AREAS OF SOUTHERN ITALY. F. Palmisano 1 , M. 

Calderaro 2 , D. Boscia 1 . 1 Istituto di Virologia Vegetale del CNR, 

UOS Bari, Via Amendola 165/A, 70126 Bari, Italy. 2 Dipartimento 

di Protezione delle Piante e Microbiologia Applicata, Università 

degli Studi “Aldo Moro”, Via Amendola 165/A, 70126 Bari, Italy. 

E-mail: d.boscia@ba.ivv.cnr.it 

Plum pox virus (PPV), the causal agent of Sharka, the most 

harmful virus disease of stone fruits, is characterised by a great 

variability represented by seven types or strains, among which 

“Marcus” (PPV-M) is considered the most dangerous, particularly 

for the peach industry. Sharka appeared in the south-eastern 

part of Italy (Basilicata and Apulia) in 1987 with few outbreaks of 

strain “Dideron” (PPV-D), and was maintained under control for 

long time, thanks to the timely eradication of all infection foci. 

The situation turned to worse in 2007, when PPV-M appeared 

for the first time in Basilicata, followed two years later by a couple 

of outbreaks of the same strain in northern Apulia. Although 

eradication actions were intensified by the local Plant Protection 

Services, during spring 2010 new outbreaks popped up in both 

regions. Object of this study was the characterization of PPV 

samples collected in different places, for identifying the viral 

strains involved and studying the level of variability among them. 

Isolates were first analysed by ELISA with strain-specific monoclonal 

antibodies. Serological characterization was followed by 

molecular characterization based on: (i) RT-PCR; (ii) sequencing 

of amplicons; (iii) multiple sequence alignments; (iv) phylogenetic 

analysis. Results showed that the majority of the outbreaks were 

caused by PPV-M, thus confirming the high rate of natural transmission 

of this strain and the need for enforcing eradication and, 

even more, for preventing the introduction in the region of infected 

nursery productions from elsewhere. 

SUPPRESSIVE EFFECT OF AERATED COMPOST TEAS 

PRODUCED IN WATER AND IN WHEY ON PLANT FUN- 

GAL PATHOGENS. C. Pane 1 , G. Celano 2 , D. Villecco 1 , M. Zaccardelli 

1 . 1 CRA, Centro di Ricerca per l’Orticoltura di Pontecagnano, 

S.S. 18 n. 204, 84091 Battipaglia (SA), Italy. 2 Dipartimento 

Scienze dei Sistemi Colturali, Forestali e dell’Ambiente, 

Università degli Studi della Basilicata, Via Nazario Sauro 85, 85100 

Potenza, Italy. E-mail: catello.pane@entecra.it 

Compost teas are fermented extracts of composted materials 

used for their ability to decrease plant diseases. A compost extractor 

in liquid phase, with a forced air-blowing system, assembled 

using farmer facilities, was used to produce “on farm” aerated 

compost teas (ACTs) from five types of compost, in a 14-day 

fermentation cycle. Solid feedstocks, representing one biowaste 

compost and four composted tomato residues, were separately 

extracted in water (waACTs) and whey (whACTs). The ten teas 

were tested for their ability to inhibit the in vitro growth of several 

soil-borne (Fusarium solani, Verticillium dahliae, F. oxysporum 

f. sp. lycopersici, Rhizoctonia solani, Sclerotinia minor, 

Pyrenochaeta lycopersici and Sclerotium rolfsii) and air-borne (Alternaria 

solani, A. radicina, A. dauci, Botrytis cinerea, and Colletrichum 

lindemuthianum) pathogens. Moreover, applications of 

ACTs were also used in greenhouse trials to assess their suppressive 

effect on gray mold (B. cinerea) on tomato plants. All ACTs 

significantly inhibited the mycelial growth of A. dauci (26-48%), 

A. radicina (27-66%), B. cinerea (13-30%), F. solani (34-24%), A. 

solani (45-17%), P. lycopersici (50-13%) and C. lindemuthianum 

(31-47%). The other pathogens were affected weakly. In the in 

planta assays, waACT consistently provided the highest suppression 

of gray mold, with >78% reduction, compared to the 50% 

reduction of lesion size due to waACTs applications. Generally, 

waACTs were evaluated as more effective than whACTs. Future 

prospectives consist in testing the best ACTs as potential alternatives 

to the use of synthetic chemical fungicides for disease control 

in the open field. 

RESPONSE OF MICROBIAL COMMUNITIES TO COM- 

POST AMENDMENT OF SOIL AND EFFECT ON DIS- 

EASE SUPPRESSIVENESS. C. Pane, D. Villecco, M. Zaccardelli. 

CRA, Centro di Ricerca per l’Orticoltura di Pontecagnano, 

S.S. 18 n. 204, 84091 Battipaglia (SA), Italy. E-mail: catello.pane@ 

entecra.it 

Compost can be used to improve organic matter in cultivated 

soils, so as to reduce the use of mineral fertilizers, stimulate soil 

microbial activities, and improve suppressiveness of soil-borne 

pathogens. In this study, the response of the soil-borne microflora 

to soil waste compost amendment was evaluated at functional 

biodiversity (Biolog CLPPs) level and at global (CO 2 release, 

beta-glucosidase, FDA hydrolysis) level, in a short post-amendment 

period. Rhizoctonia damping-off suppressiveness was also 

measured in a laboratory experiments on the host Lepidium 

sativum. Soil chemical parameters such as electrical conductivity 

(EC) and pH were monitored at the same time. Other than compost-amended 

(CA) soils, mineral fertilized (MF) and non-treated 

(NT) soils were used as control. The addition of compost in-


creased all microbial activities, suppressivity levels and EC for the 

whole experimental period, whereas pH did not changed. Conversely, 

Biolog CLPPs were enhanced in compost-amended 

soil at the beginning, but decreased with the time and disappeared 

at the end of the incubation period. FDA hydrolysis rate 

was strongly positively correlated to EC, soil beta-glucosidase activity 

and Biolog CLPPs. Results indicate that compost amendment 

affected microbial activities, both at the global and functional 

level, as a consequence of supplied carbon source. CO 2 release 

and soil beta-glucosidase activity were strongly auto-correlated 

and negatively related to the percentage of Rhizoctonia diseased 

plants, suggesting a mechanism of general suppression, 

where antagonistic microbial populations are stimulated by compost 

addition. 

A PROTEOMIC APPROACH TO THE STUDY OF THE 

ROLE OF CERATO-PLATANIN IN CERATOCYSTIS PLA- 

TANI AND IN THE CANKER STAIN DISEASE. B. Pantera 1 , 

L. Pazzagli 1 , L. Carresi 2 , C. Comparini 2 , F. Martellini 1 , M. Capuana 

3 , G. Cappugi 1 , I. Baccelli 2 , R. Bernardi 4 , A. Scala 2 . 1 Dipartimento 

di Scienze Biochimiche, Università degli Studi, Viale Morgagni 

50, 50134 Firenze, Italy. 2 Dipartimento di Biotecnologie 

Agrarie, Sezione di Protezione delle Piante, Laboratorio di Patologia 

Vegetale Molecolare, Università degli Studi, Via della Lastruccia 

10, 50019 Sesto Fiorentino (FI), Italy. 3 Istituto di Genetica Vegetale 

del CNR, Via Madonna del Piano 10, 50019 Sesto Fiorentino 

(FI), Italy. 4 Dipartimento di Biologia delle Piante Agrarie, Università 

degli Studi, Via della Piagge 23, 56124 Pisa, Italy. E-mail: barbara.pantera@unifi.it 

Cerato-platanin (CP) is a moderately hydrophobic protein 

abundantly secreted and localized in the cell wall of Ceratocystis 

platani. CP is assumed to play a role in host-plant interaction, 

since it induces production of H 2 O 2 and NO, programmed plant 

cell death, overexpression of defence genes, phytoalexin synthesis 

and restriction of conidia growth. Therefore, CP appears to act as 

PAMP able to activate effective primary defence mechanisms. 

Moreover, CP is the founder of the “cerato-platanin family”, 

whose members are secreted proteins involved in the microbehost 

interaction acting as phytotoxins, elicitors of plant defence 

responses or human allergens. The proteomic project that we are 

setting up has the aim to investigate the role of CP in the physiology 

of the fungus and in the physiopathology of the disease. 

Conidia of C. platani were harvested from fruiting cultures and 

suspended in PDB (2×10 5 conidia ml -1 ). Aliquots of 100 µl 

droplets were then applied to plane leaves and in empty Petri 

dishes as controls. All samples were maintained in a moist chamber 

at room temperature. After 48 h the mycelium was removed 

and lyophilized, and leaves were put at -80°C for further analysis. 

An aliquot of 0.05 g of dry mycelium was re-suspended in an 

acidic extraction buffer containing dodecyl-maltoside as a detergent. 

Results from 2D-gels have highlighted: (i) proteins expressed 

in the mycelium grown in PDB vs that grown on plant 

leaves, and (ii) proteins extracted from the treated leaves vs proteins 

from control leaves. 

EPIDEMIOLOGICAL AND MOLECULAR ASPECTS OF 

ALFALFA MOSAIC VIRUS OCCURRENCE IN LAVANDULA 

VERA CROPS OF LIGURIA. G. Parrella 1 , C. Cavicchi 2 , G. Zama 

2 , M.G. Bellardi 3 . 1 Istituto per la Protezione delle Piante del 

CNR, Via Università 133, 80055 Portici (NA), Italy. 2 Plesso Didattico 

G. Scarabelli, Università degli Studi di Bologna, Viale G. Ascari 

15, 40026 Imola (BO), Italy. 3 Dipartimento di Scienze e Tec- 

nologie Agroambientali, Sezione di Patologia Vegetale, Università 

degli Studi, Viale Fanin 42, 40127 Bologna, Italy. E-mail: giuseppe. 

parrella@ipp.cnr.it 

From 2008 to 2010, a study was carried out on the occurrence 

of Alfalfa mosaic virus (AMV) in some Lavandula vera crops of Albenga 

(Liguria, northern Italy). This non-persistent aphid-borne 

virus represents one of the most dangerous and economically important 

pathogens of lavander affecting both plant growth and appearance. 

In 2008 and 2009 numerous inspections were made at 

several lavender producers with accurate examination of potgrown 

plants before blooming. AMV was detected by DAS- 

ELISA and PCR in association with stunting and yellow mosaic 

symptoms. Considering that L. vera is propagated by shoots and 

that mother-plants (10- to 12-year-old) are grown at the borders of 

pot-plant crops, in 2010 Ligurian propagation materials were 

checked serologically. Some L. vera mother plants showing leaf 

mosaic proved to be infected by AMV. One AMV isolate from 

symptomatic mother plants was characterized molecularly and 

compared with previously characterized Italian lavender isolates 

of the same virus, including an isolate recovered in 2010 from L. 

vera cv. None Blue in Emilia-Romagna. Results showed that AMV 

isolates from Liguria and Emilia-Romagna belong to subgroup I, 

according with restriction profiles of the coat protein gene and 

phylogenetic relationships with other AMV isolates of subgroups I 

and II. As prevention measure, mother plants could be selected by 

periodic visual inspections and serologically tested for AMV presence. 

Virus-free shoots used as propagation material and crop rotation 

are the most effective type of control. 

ATTEMPTS TO CONTROL PSEUDOMONAS VIRIDIFLA- 

VA IN RANUNCULUS ASIATICUS. C. Pasini and G. Boeri. 

CRA, Unità di Ricerca per la Floricoltura e le Specie Ornamentali, 

Corso degli Inglesi 508, 18038 Sanremo (IM), Italy. E-mail: c.pasini@istflori.it 

Pseudomonas viridiflava is the prevailing bacterial pathogen in 

ranunculus-growing areas of the Italian Riviera, where it can 

cause serious damages to commercial flower production. Infections 

appear during autumn and winter, and consist of necrotic 

spots on leaves and stems. To develop a defence strategy, a series 

of trials were undertaken to evaluate the effects against P. viridiflava 

of: (i) chemical dressing by immersion of rhizomes in a suspension 

of various active ingredients; (ii) dry heat thermotherapy 

of dry rhizomes at 60 and 65°C, for different times; (iii) fungicide 

sprays with acibenzolar-S-methyl, Bacillus subtilis, dithianon, 

harpin protein, phosethyl-Al + copper oxychloride and several 

copper salts to ranunculus potted plants grown in a climatic cell 

at 15°C, and on cut flowers stored at 6-8°C. Results showed that 

thermotherapy inhibited rhizome germination. Dressing with 

phosethyl-Al, monopotassic phosphate and copper hydroxide 

provided a moderate activity, while several concentrations of ammonium 

salts and commercial bleach solutions were phytotoxic. 

Sprays with acibenzolar-S-methyl, different copper salts and 

phosethyl-Al + copper oxychloride reduced the infection both on 

emerged plants or on cut flowers. By applying the biocontrol 

agent B. subtilis a partial and inconsistent protective action was 

observed. 

INTERACTIONS BETWEEN ENDOPHYTIC STRAINS OF 

ALTERNARIA sp. AND VITIS VINIFERA PLANTS. R. Polizzotto, 

S. Grisan, R. Osler, R. Musetti. Dipartimento di Biologia e 

Protezione delle Piante, Università degli Studi, Via delle Scienze


208, 33100 Udine, Italy. E-mail: rachele.polizzotto@uniud.it 

The genus Alternaria is characterized by a large spectrum of 

plant-host interactions. It is therefore important to recognize and 

discriminate the pathogenic forms from the saprophytic or endophytic 

ones. It was reported that fungal endophytes recovered 

from symptomless natural hosts could cause severe symptoms 

when inoculated to other plant species. Since Alternaria endophytes 

from grapevine were effective in the biocontrol of Plasmopara 

viticola, both in pre- and post-infection treatments, the 

scope of this study was to investigate the behaviour of 12 Alternaria 

grapevine endophytic strains when artificially inoculated in different 

tissues of Vitis vinifera. Pathogenic assays were carried out 

using tomato (Solanum lycopersicum) as a nonhost test plant. A 

conidial suspension of each Alternaria obtained from single-spore 

cultures was sprinkled over six wounded leaves of grapevine and 

tomato seedlings. Grapevine and tomato berries were artificially 

inoculated by immersion (grapevine) or by wounding (tomato), 

using the Alternaria conidial suspension as above. Symptoms were 

evaluated 4 weeks after infection on leaves and one week on fruits. 

Re-isolation rate from inoculated tissues was estimated. Alternaria 

did not induce symptoms in inoculated leaves of both plant 

species, but was pathogenic (dark brown or black circular necrotic 

lesions) to tomato berries. Re-isolation frequency was 7% in 

grapevine and 41% in tomato. Results showed that Alternaria 

strains behave differently on the different plant tissues inoculated, 

as they are true endophytes of grapevine whereas they can cause 

severe damage to tomato berries. 

INFLUENCES OF CULTURAL PRACTICES ON BUNCH 

ROTS AND OCHRATOXIN A CONTAMINATION IN 

WINE. S. Pollastro 1 , C. Dongiovanni 2 , R.M. De Miccolis Angelini 

1 , P. Natale 2 , D. Perrelli 2 , F. Faretra 1 . 1 Dipartimento di Protezione 


Studi “Aldo Moro”, Via Amendola 165/A, 70126 Bari, Italy. 2 Centro 

di Ricerca e Sperimentazione in Agricoltura “Basile Caramia”, 

Via Cisternino 281, Locorotondo (BA), Italy. E-mail: stefania.pollastro@agr.uniba.it. 

Grapevine bunch rots are a complex of diseases and alterations 

caused by several microrganisms affecting the quality and 

safety of wine, as in the case of Aspergillus carbonarius. This fungus 

is the main responsible for ochratoxin A (OTA) contamination 

of grape and wine in the Mediterranean area. OTA is a mycotoxin 

frequently found in foods and beverages. The Reg. (EC) 

N. 123/2005 of 26.1.2005 established in 2.0 mg kg -1 the maximum 

tolerable limit of OTA in wine and other grape-juice derivatives. 

To improve the efficacy of IPM strategies in limiting bunch 

rots and OTA contamination, the role of some cultural practices, 

with particular regard to canopy and soil management, was evaluated 

on cv. Bombino nero in southern Italy. When shoot topping 

and tying-up were done at bunch closure, and were followed by 

leaf removal 20 days before vintage, all bunch rots were reduced, 

especially grey mould (100%), followed by A. carbonarius (50%) 

and OTA contamination (50%). Opposite results were obtained 

when canopy was abundant and almost free to grow. In this case, 

bunch rots, especially grey mould (+30%), A. carbonarius 

(+33%) and OTA contamination (+47%) were higher. The severity 

of bunch rots was high when grass-cut soil was in-between 

vine rows. A. carbonarius (+33%) and OTA contamination 

(+30%) also increased when ploughing between rows was carried 

out 20 days before vintage. Such findings suggest that a careful 

integrated management of both cultural practices and crop protection 

in vineyards is essential for reducing the risk of OTA contamination 

of wine. 

OBTAINMENT, CHARACTERIZATION AND FIRST AP- 

PLICATIONS OF A PHAEOMONIELLA CHLAMYDOSPO- 

RA BENZIMIDAZOLE-RESISTANT MUTANT IN EPI- 

DEMIOLOGICAL STUDIES. S. Pollastro, W. Habib, F. Faretra. 

Dipartimento di Protezione delle Piante e Microbiologia Applicata, 


70126 Bari, Italy. E-mail: faretra@agr.uniba.it. 

Studies on the fungal mycoflora of grapevine propagation materials 

showed a very high frequency of Phaeomoniella chlamydospora, 

which was also detected at different stages of the nursery 

propagation process. The availability of P. chlamydospora isolates 

carrying genetic markers easily detectable on semi-selective media 

can be helpful for clarifying: (i) the possible role of vine propagation 

steps in the contamination of vine materials; (ii) the transmission 

of Esca disease to new vineyards; (iii) the effectiveness of 

control measures. Preliminarly, the response to benzimidazole 

fungicides (benomyl, carbendazim and thiophanate methyl) of 10 

wild-type isolates of P. chlamydospora was evaluated in colonygrowth 

and conidial-germination assays. Colony growth of all isolates 

was inhibited by 0.3-1 µg ml -1 of fungicides whereas conidial 

germination was poorly affected, although germ tubes appeared 

deformed and bent from 0.3 µg ml -1 a.i. Resistant mutants were 

selected on a medium additioned with 1 µg ml -1 benomyl or carbendazim 

(mutation frequency: 2.1-2.5×10 -8 and 1.4-2×10 -8 , respectively). 

Generally, mutants showed high resistance. The mutant 

C1.A43.2 (EC 50 >100 µg ml -1 ) was selected and used in preliminary 

experiments. Grapevine propagation material was artificially 

inoculated at different stages of the nursery process with 

conidia of the mutants and its ability to colonize wood tissues was 

verified 1-3 months after inoculation. The mutant was always 

reisolated from inoculated rootstocks and scions, even far away 

from the inoculation point, confirming the capability of P. 

chlamydospora to move in xylem vessels. The traceable mutant is 

being now applied in large-scale epidemiological studies on Esca 

disease. 

PRODUCTION OF AN ANTISERUM SPECIFIC TO CIT- 

RUS LEAF BLOTCH VIRUS. O. Potere 1 , M. Guardo 2 , A. De 

Stradis 3 , A. Caruso 2 , D. Boscia 3 . 1 Dipartimento di Protezione 


Moro”, Via Amendola 165/A, 70126 Bari, Italy. 2 CRA, Centro 

di Ricerca per l’Agrumicoltura e le Colture Mediterranee, Corso 

Savoia 190, 95024 Acireale (CT) Italy. 3 Istituto di Virologia Vegetale 

del CNR,UOS Bari, Via Amendola, 165/A, 70126 Bari, Italy. 

E-mail: o.potere@agr.uniba.it 

Different Citrus species used as ornamentals and some trifoliate 

rootstocks were recently found infected by Citrus leaf blotch 

virus (CLBV), the type species of the genus Citrivirus, family 

Betaflexiviridae. This virus is currently detected by RT-PCR following 

nucleic acid extraction. For a faster virus detection from 

crude plant extracts, the feasibility of raising a CLBV-specific antiserum 

to be utilized for serological testing was investigated. To 

this aim, virus purification protocols were compared using several 

kinds of tissue (leaves, petioles and cortical scrapings) collected 

from Nagami kumquat and citron ‘Etrog’ in different seasons. 

Virus concentration was highest in spring, decreasing progressively 

as temperature arose. The successful mechanical transmission 

of CLBV to some herbaceous plants enabled the use of N. 

cavicola as host for virus propagation. Concentrated preparations 

of CBLV were evaluated by electron microscope before injecting 

them into a rabbit for immunization. The antiserum obtained 

decorated isolate ISA-10-CT-I particles used as inject antigen at a 

dilution of 1:20. Immunoglobulins (IgGs), purified from crude


antiserum preabsorbed with healthy plant extracts and conjugated 

with alkaline phosphatase, were comparatively used in Western 

blotting (WB) and DAS-ELISA. Specific detection of coat 

protein bands was obtained only in WB of infected Citrus and 

Nicotiana extracts. The test was successfully extended to ornamental 

Citrus trees, such as Calamondin (Citrus microcarpa) and 

Fortunella spp., from nurseries and commercial groves. 

PRESENCE OF DEOXYNIVALENOL AND NIVALENOL 

CHEMOTYPES OF FUSARIUM CULMORUM ISOLATED 

FROM DURUM WHEAT IN SOME ITALIAN REGIONS. A. 

Prodi 1 , D. Salomoni 2 , D. Alkadri 1 , S. Tonti 1,3 , P. Nipoti 1 , A. 

Pisi 1 , D. Pancaldi 2 . 1 Dipartimento di Scienze e Tecnologie Agro- 

Ambientali, Università degli Studi, Viale G. Fanin 40, 40127 

Bologna, Italy. 2 Dipartimento di Protezione e Valorizzazione Agro- 

Alimentare, Università degli Studi, Viale G. Fanin 46, 40127 

Bologna, Italy . 3 Ente Nazionale Sementi Elette, Via Cà Nova 

Zampieri 37, 37057 San Giovanni Lupatoto (VR), Italy. E-mail: 

antonio.prodi@unibo.it 

Durum wheat production in Italy is of great economical importance. 

Fusarium Head Blight (FHB) is an important and 

widespread disease of wheat that, in Italy, causes serious damages 

in terms of yields and quality of grains. Fusarium culmorum, one 

of the main causal agents of FHB, is spread in all the Italian regions, 

but especially in the center-northern areas. It produces mycotoxins, 

such as deoxynivalenol (DON), nivalenol (NIV) and 

zearalenone (ZEA), representing potential health hazards for 

both humans and animals. This work investigated a population of 

F. culmorum strains isolated from durum wheat grains collected 

from some Italian regions, mainly Emilia Romagna and Tuscany, 

but also Basilicata, Umbria and Marche. A multiplex PCR assay, 

based on primers derived from the Tri3 and Tri7 genes of the trichothecene 

gene cluster was used to assign a strain to one of the 

following trichothecene chemotype profiles: NIV, 3acetyldeoxynivalenol 

(3-ADON) and 15-acetyldeoxynivalenol 

(15-ADON). Almost the totality of the strains belonged to the 3- 

ADON chemotype, only a few to NIV, while 15-ADON chemotype 

strain was not found. This work allowed us to better determine 

the presence of F. culmorum chemotypes. Knowledge on the 

distribution of F. culmorum chemotypes is quite important, on a 

regional basis, to predict the possible contamination by mycotoxins 

in food and feed. 

OCCURRENCE OF MUTANTS DEFECTIVE IN BIOCON- 

TROL ACTIVITY IN PSEUDOMONAS CHLORORAPHIS 

subsp. AUREOFACIENS STRAIN M71. G. Puopolo 1 , A. 

Russo 1 , V. Battaglia 1 , A. Zoina 1 . 1 Dipartimento di Arboricoltura, 

Botanica e Patologia Vegetale, Università degli Studi di Napoli 

“Federico II”, Via Università 100, 80055 Portici (NA), Italy. Email: 

puopolo@unina.it 

The production of phenazines (PHZ) plays a key role in the 

biocontrol activity of several members of Pseudomonas chlororaphis 

subsp. aureofaciens (Pca). The biosynthesis of these molecules 

is regulated by a Quorum Sensing (QS) mechanism, that relies 

on the production of N-acyl homoserine lactones (AHL). In 

fluorescent pseudomonads, the GacS/GacA two-component system 

allows to perceive and respond to environmental stimuli by 

activating QS. Mutations in the genes gacS and/or gacA determine 

the loss of AHL and PHZ production in Pca members. Occurrence 

of mutants defective in the production of phenazines 

has been shown in Pca strain M71. These mutants, named M71a 

and M71b, had a siderophore production higher than that of the 

wild type strain. Strain M71b did not produce PHZ and AHL, 

while strain M71a was able to produce these metabolites only 

when grown in complex media. Furthermore, strain M71a and 

M71b were impaired in the colonization and persistence on 

tomato roots and in the control of Fusarium oxysporum f. sp. radicis-lycopersici. 

The cosmids pEMH97 (carrying gacS) and 

PME3066 (carrying gacA) were moved by triparental mating in 

the mutants in order to assess the loss of a functional GacS/GacA 

system. Restoration of wild type properties in M71b was achieved 

by introducing a functional copy of gacS, while the introduction 

of PME3066 (gacA) in M71a restored the wild type phenotype. 

To our knowledge, these data represent the first case where a mutation 

in gacA did not totally abolish the production of AHL and 

PHZ in a Pca member. 

COMPARISON OF BIOCONTROL FEATURES OF THREE 

BACTERIAL ANTAGONISTS BELONGING TO 

PSEUDOMONAS CHLORORAPHIS subsp. AUREOFA- 

CIENS. G. Puopolo 1 , V. Battaglia 1 , A. Russo 1 , L. Cozzolino 1 , A. 

Zoina 1 . 1 Dipartimento di Arboricoltura, Botanica e Patologia Vegetale. 

Università degli Studi di Napoli “Federico II”, Via Università 

100, 80055 Portici (NA), Italy. E-mail: puopolo@unina.it 

Members of the subspecies Pseudomonas chlororaphis aureofaciens 

(Pca) share several physiological features exploitable in the 

biological control of phytopathogenic fungi. The production of 

phenazines (PHZ), antibiotic molecules with a broad host range 

activity, is the main mechanism through which these bacteria control 

important fungal pathogens. PHZ production relies on the 

synthesis of N-acyl homoserine lactones (AHL) mediated by synthase 

enzymes belonging to the LuxI family. Once signal molecules 

reach a threshold density, they bind transcriptional factors 

of the LuxR family, responsible for the transcription of the operon 

involved in PHZ production. This molecular mechanism has 

been termed Quorum Sensing. In this study, three Pca strains 

were analysed for their biocontrol aptitudes, paying particular attention 

to the production of PHZ and AHL. These strains were 

also evaluated for their ability to control Fusarium oxysporum f. 

sp. radicis-lycopersici (Forl) attacks on young tomato plantlets and 

for their ability to persist on tomato roots. Pca strains 30-84, 

AZ10C2 and M71 were able to produce proteases, lipases, 

siderophores and to synthesize PHZ and AHL in complex 

growth media. Interestingly, M71 was the only strain capable of 

producing PHZ and AHL in minimal growth media. Pca strains 

were effective in the protection of tomato plantlets against Forl 

reducing up to 50% the incidence of the disease. Furthermore, 

these bacterial strains showed a good aptitude to colonization of 

tomato roots, but Pca M71 was the only strain that persisted on 

tomato roots up to three months in field trials. 

PRELIMINARY TRIALS ON THE USE OF VOLATILE OR- 

GANIC COMPOUNDS FOR THE POSTHARVEST CON- 

TROL OF GREY MOLD ON GRAPE. M. Quaglia, G. Linoci, 

A. Zazzerini. Dipartimento di Scienze Agrarie e Ambientali, Università 

degli Studi, Borgo XX Giugno 74, 06121 Perugia, Italy. Email: 

mara.quaglia@unipg.it 

Several plant species, including grapevine, are rich in a wide 

range of volatile organic compounds such as terpenes and their 

derivatives (caryophyllene, linalool, nerolidol etc.) that can act as 

defence against herbivores and pathogens. Botrytis cinerea Pers. 

is also able to produce a variety of terpenes including botrydial,


which is the primary phytotoxic compound of the fungus but can 

also have a limiting effect on its growth. Botrydial biosynthetic intermediates 

appear to be cyclization products of caryophyllene. 

Terpenes are already present in the food chain and do not display 

significant human toxicity, thus their use for the postharvest control 

of fruit and vegetable diseases could be interesting. However, 

only few studies deal with the effect of pure terpenes against phytopathogenic 

fungi. For this reason, we investigated the effectiveness 

of caryophyllene, linalool, nerolidol and ±-pinene against B. 

cinera. Nerolidol and linalool reduced significantly fungal growth 

in vitro when used at concentrations ranging from 1000 to 2500 

µl/l and from 1500 to 2500 µl/l, respectively. In particular, 

linalool completely inhibited the fungal growth at the highest 

concentration. In vivo, linalool (2500 µl/l) reduced significantly 

infection percentage when applied by dipping the berries in a solution, 

while it was not effective when applied by evaporation. 

Nerolidol (2500 µl/l) was not effective in both cases. Unfortunately, 

the two compounds exerted a strong phytotoxic activity 

when applied by dipping, causing browning of the berries. In further 

investigations, other terpenes and other strategies of application 

will be tested. 

HOST PECTIN METHYLESTERASE PLAYS A ROLE IN 

THE SUSCEPTIBILITY TO NECROTROPHIC 

PATHOGENS. A. Raiola 1 , V. Lionetti 2 , I. Elmaghraby 1 , F. Cervone 

2 , D. Bellincampi 2,3 . 1 Dipartimento Territorio e Sistemi Agro- 

Forestali, Università degli Studi di Padova, Viale dell’Università 

16, 35020 Legnaro (PD), Italy. 2 Dipartimento di Biologia Vegetale, 

Università degli Studi “La Sapienza”, Piazzale Aldo Moro 5, 00185 

Roma, Italy. 3 Dipartimento di Chimica, Università degli Studi “La 

Sapienza”, Piazzale Aldo Moro 5, 00185 Roma, Italy. E-mail: 

alessandro.raiola@unipd.it 

The host cell wall is a primary target during growth of 

necrotrophic pathogens. During the first stages of infection, 

pectin, one of the main components of the plant cell wall, is degraded 

by pectinolytic enzymes produced by the majority of fungal 

and bacterial pathogens. Some evidence indicates that variation 

of the pectin structure and composition may cause an altered 

disease response upon infection with pathogens. Pectin is synthesized 

and secreted into the cell wall in a highly methylesterified 

form and, soon thereafter, deesterified in muro by pectin 

methylesterases (PMEs). The action of PME makes pectin susceptible 

to degradation by enzymes such as endo-polygalacturonases 

(PGs) and pectate lyases (PELs). Endogenous PME activity 

is controlled through the interaction with the pectin 

methylesterase inhibitor (PMEI). PMEI over-expression and 

PME knockout have been used to stably increase pectin 

methylesterification in Arabidopsis plants. We have shown that 

the increase of pectin methylesterification and the lack of a specific 

PME activity correlate to a decreased susceptibility of Arabidopsis 

to the necrotrophic pathogens Pectobacterium carotovorum 

and Botrytis cinerea. The reduced symptoms of transformed 

plants have been related to the inability of the pathogens to take 

advantage of host PMEs and to their impaired ability to grow on 

methylesterified pectins. 

FIRST REPORT OF ANTHRACNOSE OF HOYA CARNOSA 

IN ITALY. G.L. Rana 1 , I. Camele 1 , F. Marziano 2 . 1 Dipartimento 

di Biologia, Difesa e Biotecnologie Agro-Forestali, Università degli 

Studi della Basilicata, Via Ateneo Lucano 10, 85100 Potenza, Italy. 

2 Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, 

Università degli Studi di Napoli “Federico II”, Via Università 100, 

80055 Portici (NA), Italy. E-mail: gianluigi.rana@unibas.it 

During spring 2009 some plants of Hoya carnosa (wax plant, 

Asclepiadaceae) with leaves showing sunken dry lesions 1-2 cm in 

diameter, bordered by a slightly raised rim, were observed in 

Molfetta territory (Bari province, southern Italy). The above lesions, 

as it was shown by stereomicroscopic and microscopic observations 

of entire and dissected symptomatic leaves, contained 

numerous, untidely arranged acervular conidiomata. The micromycete 

present in infected organs, tentatively considered as the 

aetiological agent of the leaf alterations, was isolated in pure culture 

in Petri dishes on PDA then used for: (i) studying colony, 

conidioma and conidium development at 24-26°C on PDA, OMA 

and SNA; (ii) DNA extraction, amplification with primers 

ITS4/ITS5 and sequence analysis and (iii) artificially inoculating 

leaves of healthy wax plants. Results showed that the microfungus 

under study was Colletotrichum gloeosporioides (Teleomorph: 

Glomerella cingulata, complex species). Its DNA sequence (547 

bp) was compared with those of the same fungus present in 

GeneBank with accession No. AJ301907 and EU326191 and 

showed 100% and 99% similarity, respectively. One of the sequences 

obtained was deposed at the European Molecular Biology 

Laboratory (EMBL) with accession code FN868840. In pathogenicity 

tests, inoculated leaves of H. carnosa showed symptoms 

very like those observed on naturally infected plants. Acervular 

conidiomata were also formed subepidermally inside necrotic foliar 

tissues. C. gloeosporioides, previously unreported on H. carnosa in 

Italy, was found infecting wax plant only in USA in 1984. 

A NEW PROTOCOL FOR THE RAPID DETECTION AND 

CHARACTERIZATION OF CITRUS TRISTEZA VIRUS ISO- 

LATES BY SEQUENTIAL ELISA-CE-SSCP. D. Raspagliesi 1,2 , 

G. Licciardello 2 , A. Lombardo 2 , S. Rizza 1 , M. Bar-Joseph 3 , A. 

Catara 1,2 . 1 Dipartimento di Scienze e Tecnologie Fitosanitarie, Università 

degli Studi, Via S. Sofia 100, 95123 Catania, Italy. 2 Laboratorio 

di Diagnosi e Biotecnologie Fitosanitarie, Parco Scientifico e Tecnologico 

della Sicilia, Blocco Palma I, Z.I., 95121 Catania, Italy. 3 The 

S. Tolkowsky Laboratory, ARO, The Volcani Center, Bet Dagan 

50250, Israel. E-mail: glicciardello@ pstsicilia.org 

Management and control of emerging Citrus tristeza virus 

(CTV) epidemics is based on the ability to rapidly detect infected 

trees and to differentiate between prevailing isolates. Previously, 

ELISA and CE-SSCP were used separately for CTV detection 

and for isolate differentiation. Here we combined these two diagnostic 

methods into a continuous process allowing the detection 

of infected trees among unknown samples and the rapid analysis 

of the genetic structure of samples positive to ELISA, thus saving 

both time and resources. Crude extracts from different samples 

were tested by DAS-ELISA, positive wells were washed, RNA 

eluted and used as template for cDNA synthesis and subsequent 

CE-SSCP analysis. Different susceptible citrus species, as sour orange, 

Etrog citron and Mexican lime, were used for preliminary 

tests using the p18 gene. A total of 41 plants inoculated with five 

CTV isolates (Tapi, TDV, RPC3, SG29, M1), or naturally infected, 

were analysed and successfully compared with stored profiles. 

Size, conformation, data point and migration of the two strands 

using this new protocol were clear cut and reproducible, demonstrating 

its robustness and validity. Tapi, TDV and M1 profiles 

were almost identical in relative migration (68.17 fw and 72.25 

rev), and different from SG29 profile (70.05 fw and 74.43 rev). 

RPC3 gave two peaks for the forward strand (70.86 and 72.93) 

and two for the reverse strand (74.2 and 76.16) due to a crossbreed 

of two strains. The procedure offers considerable practical 

advantages in CTV genetic characterization.


DETECTION AND CHARACTHERIZATION OF CITRUS 

TRISTEZA VIRUS IN THE NATIONAL GERMPLASM 

COLLECTION OF AFGHANISTAN. S. Rehman 1 , J. Ahmad 1 , 

C. Lanzoni 2 , C. Rubies Autonell 2 , C. Ratti 2 . 1 Aga Khan Foundation-Afghanistan, 

Wazir Akbar Khan, Road 13, H43, Main road - 

Kabul, Afghanistan. 2 Dipartimento di Scienze e Tecnologie 

Agroambientali, Sezione di Patologia Vegetale, Università degli 

Studi, Viale Fanin 40, 40127 Bologna, Italy. E-mail: shams-u-rahman.shams@ 

akdn.org 

Reviving horticultural industry is a government priority in 

Afghanistan. To the same purpose, EC supported programmes 

specifically focus on increasing the access to improved and appropriate 

planting material, in order to increase the quantity and 

quality of more competitive horticultural products. The availability 

of a biotechnology laboratory and of its services for quality 

control and management along the horticulture supply chains 

were considered an essential support to horticulture development. 

This laboratory started screening the health state of the 

Afghan Germplasm National Collection not only to ensure multiplication 

of the best selected varieties or ecotypes, but also to 

avoid production and distribution of virus-infected trees. During 

surveys of citrus at the National collection experimental farm in 

Jalalabad, plants from accessions showing vein flecking, yellowing 

and decline symptoms were sampled and analyzed by ELISA. 

Four accessions, kumquat cv. Margarita, orange cv. Mahali, mandarin 

cv. Fruter and rough lemon cv. Mahali, proved to be infected 

by Citrus tristeza virus (CTV). Known to be transmitted by 

aphids, CTV causes a serious disease that can rapidly spread and 

destroy orchards. In order to preserve the citrus national collection 

from CTV infection, a study for the characterization of virla 

isolates was undertaken. A 655 nt long fragment, corresponding 

to the major coat protein gene, was amplified by RT-PCR from all 

ELISA-positive samples. Preliminary analysis revealed sequence 

identity ranging from 91 to 100% within Afghan CTV isolates 

and high similarity with GenBank isolates from Angola, India 

and USA. 

SERIOUS DAMAGES BY IMPATIENS NECROTIC SPOT 

VIRUS IN ZANTEDESCHIA AETHIOPICA. D. Rizzo 1 , S. 

Lazzereschi 2 , B. Nesi 2 , A. Grassotti 2 . 1 Agenzia Regionale per lo 

Sviluppo e l’Innovazione nel Settore Agricolo-Forestale (ARSIA), 

Laboratorio di Diagnostica Fitopatologica, Via dei Fiori 8, 51012 

Pescia (PT), Italy. 2 CRA, Unità di Ricerca per il Vivaismo e la 

Gestione del Verde Ambientale ed Ornamentale, Via dei Fiori 8, 

51012 Pescia (PT), Italy. E-mail: sara.lazzereschi@entecra.it 

Calla lily (Zantedeschia aethiopica, family Araceae) has become 

one of the most popular cut flowers worldwide because of its ornamental 

characteristics, such as spathe beauty and post-harvest 

shelf-life. Calla lily is mainly used for cut flower, but also for potted 

plants production. Tospoviruses (family Bunyaviridae) are 

agriculturally important as they cause severe economic damage to 

various crops and flowers. Tospovirus diseases, induced by Impatiens 

necrotic spot virus (INSV) mainly affect a lot of ornamental 

plants, i.e. cineraria, cyclamen, prairie gentian and many others, 

including perennials. INSV causes yellow or brown ring spots, 

round brown black or white spots, brown or black stem sections, 

black or brown necrosis at the leaf base, stunting, etc. Symptoms 

vary with the plant species, cultivar and age. In calla lily the effect 

of infection are chlorotic or yellow spots radiating from the 

midrib towards the edge on the leaf. ELISA is a routine method 

for diagnosis of plant viruses including tospoviruses and RT-PCR 

has been widely used as a highly sensitive and specific detection 

method. In the present work the collection of calla lily plants of 

the CRA-VIV was analysed in collaboration with the phytopatological 

laboratory of ARSIA. INSV was detected by the immunocromatographic 

‘Lateral Flow’ tecnique and a protocol for 

double step RT-PCR was performed, using different amplification 

profiles. 

BIOSTIMULANT PRODUCTS FOR THE CONTROL OF 

POWDERY MILDEW ON ZUCCHINI. R. Roberti, M. Fabbri, 

I. Portillo, A. Veronesi, A. Brunelli. Dipartimento di Protezione 

e Valorizzazione Agroalimentare, Università degli Studi, 

Viale Fanin 46, 40127 Bologna, Italy. E-mail: roberta.roberti@ 

unibo.it 

Powdery mildew, caused by Podosphaera xanthii, is the most 

common disease of cucurbits and is mainly controlled by fungicides, 

the most recent and effective of which can induce resistance 

in pathogen populations, thus losing their efficacy. Therefore, 

the knowledge of the effects of alternative substances 

against the disease is useful in order to manage an integrated disease 

control. The following biostimulant products were tested on 

zucchini cv. Consul in greenhouse: Alga Special, Algaton, Algavital, 

Biosaral and Chelal Alga (based on seaweed extracts, Ascophyllum 

nodosum mainly), Bioactive (blood meal), Fungiplan and 

Oidium (fertilizers), Vitaflow (borlande), Ekoprop (Trichoderma 

spp. Glomus sp., Pseudomonas sp.) and Salavida (P. trivialis). Almost 

all all products were applied on the leaves and in the soil, in 

separate experiments, except Ekoprop and Salavida that were applied 

to soil only. The pathogen was artificially inoculated (50,000 

conidia/ml) on cotyledonary leaves two days after each treatment 

and disease symptoms were evaluated as percentage of infected 

leaf area, eight days after inoculation. Among leaf treatments, the 

best effect was obtained with Bioactive, Biosaral, Fungiplan and 

Vitaflow, which gave a control ranging from 31% to 37% with 

respect to the inoculated control. Overall, the products were 

more active when applied by soil irrigation, with Biosaral, Alga 

Special and Chelal Alga that reduced the disease by more than 

50% and Ekoprop and Salavida by 80%. We conclude that plenty 

of the tested products may be considered good partners of 

chemicals in integrated control systems. Further greenhouse and 

field experiments are in progress. 

ANTIFUNGAL ACTIVITY OF VOLATILE COMPOUNDS 

PRODUCED BY PENICILLIUM sp. ISOLATE R82 

AGAINST POSTHARVEST FUNGAL PATHOGENS. W. 

Rouissi, P. Bertolini, M. Mari. Centro per la Protezione e Conservazione 

dei Prodotti Ortofrutticoli “G.C. Pratella”, Università degli 

Studi di Bologna, Via Gandolfi 19, 40057 Cadriano (BO), Italy. 

E-mail: wafa.rouissi@studio.unibo.it 

The thiabendazole (TBZ) sensitive Penicillium R82 isolate, 

was grown in PDB for 10 days at 20°C. The liquid culture (LC) 

was lyophilized, resuspended in distilled water (1:10, 1:100, 

1:1000 v/v) and sterilized. Its influence on in vitro growth of 

Penicillium expansum, Monilinia laxa, Botrytis cinerea and Colletotrichum 

acutatum was evaluated by measuring the decrease of 

mycelium dry weight (DWM) and conidia germination. The LC 

of R82 reduced significantly the DWM of all pathogens tested, 

while it increased the length of the germ tubes compared to the 

control, however an abnormality in mycelium growth was observed. 

In vivo assays were performed on apples and pears. 

Wounded fruits were treated with a conidial suspension of R82 

(10 3 conidia/ml), inoculated with two isolates of TBZ-resistant 

P.expansum and stored for 10 days at 20°C. All fruit were rotted.


However lesions were produced only by isolate, R82 since no 

fungal growth was observed on malt extract agar plates amended 

with TBZ (400 mg/g) and inoculated with small pieces of rotted 

tissues. To explain the mode of action of isolate R82, a double 

Petri-dish assay was performed. Antifungal activity was probably 

due to the production of volatile substances whose identification 

through Gas Mass technique is in progress. In conclusion, diffusible 

substances generated by Penicillium R82 isolate and present 

in culture filtrate showed activity against the above listed 

postharvest pathogens. A possible application of these natural 

substances could be the postharvest biofumigation of fruit in 

storage room, to prevent losses through the distribution chain. 

THE ROLE OF PLANT GENOTYPE IN THE BENEFI- 

CIAL INTERACTION BETWEEN TOMATO AND THE 

BCAS TRICHODERMA spp. M. Ruocco 1 , M. Tucci 2 , L. De 

Masi 2 , M. de Palma 2 , S. L. Woo 3 , F. Vinale 3 , S. Lanzuise 3 , M. 

Nigro, A.M. El-Tabey Eid 3 , M. Lorito 3,1 . 1 Istituto di Protezione 

delle Piante del CNR, Via Università 133, 80055 Portici (NA), 

Italy. 2 Istituto di Genetica Vegetale del CNR, Via Università 133, 

80055 Portici (NA), Italy. 3 Dipartimento di Arboricoltura, Botanica 

e Patologia Vegetale, Università degli Studi di Napoli “Federico II”, 

Via Università 100, 80055 Portici (NA), Italy. E.mail: ruocco@ 

ipp.cnr.it 

Selected fungi of the genus Trichoderma have the ability to interact 

simultaneously with plants and pathogens. These antagonists 

can trigger systemic and localised resistance to diseases and 

promote plant growth and development. Additional effects include 

the suppression of deleterious soil microflora/fauna, degradation 

of toxic compounds, direct stimulation of root development, 

by producing hormone-like compounds or affecting plant 

synthetic pathways, and/or promotion of water and nutrient uptake. 

However, the occurrence of the above benefits depends on 

the specific plant genotype, although this concept has been poorly 

investigated. In this work we have studied the effects of two 

Trichoderma species (T. harzianum T22 and T. atroviride P1) on 

several cultivated and wild tomato genotypes (Solanum lycopersicum 

and S. habrochaites) in terms of promotion of seed germination 

and plant development, protection against pathogens and 

transcriptional modifications of response genes. Strong differences 

in the response to the symbiotic interaction with the two 

Trichoderma strains were found among different tomato varieties. 

In fact, the effect of T. harzianum T22 and T. atroviride P1 on the 

growth and systemic resistance against B. cinerea depended 

strongly on the genotype tested. Our data on the induction of defence 

response pathways may help the selection or breeding of 

tomato lines with enhanced ability to benefit from the interaction 

with Trichoderma, which may support a correct application of Trichoderma-based 

bio-pesticides and bio-fertilizers in agriculture. 

ACTIVITY OF ELECTROLYZED OXIDIZING WATER 

GENERATED BY DIAMOND THIN FILM ELECTRODES 

IN REDUCING PATHOGENIC MICROBIAL POPULA- 

TION IN PACKINGHOUSE WASH WATER. S.M. Sanzani 1 , 

F. Fallanaj 1 , C. Zavanella 2 , A. Ippolito 1 . 1 Dipartimento di Protezione 


Studi “Aldo Moro”, Via Amendola 165/A, 70126 Bari, Italy. 

2 Adamant Technologies SA Eplatures-Grise 17, 2300 La Chaux-de- 

Fonds, Switzerland. E-mail: simona.sanzani@agr.uniba.it 

Electrolyzed Oxidizing Water (EOW), generated by electrolysis 

of a salt solution, recently gained attention for its possible use 

as eco-friendly alternative method for the inactivation of the 

pathogenic microflora of fresh fruit and vegetables. Aim of the 

present investigation was to determine if EOW generated by Diamond 

Thin Film Electrodes has potential for use in commercial 

packinghouses to control the main pathogens of harvested fruits. 

EOW was generated with Diacell, an electrolytic cell based on 

boron-doped diamond electrodes (Adamant Technologies SA, 

CH) that are known for their outstanding electrochemical properties, 

including the production of a wide range of oxidising 

species. Spores of Penicillium expansum were suspended in EOW 

produced either in the presence or absence of 1 g/l of NaCl to increase 

conductivity. Furthermore, the suspension itself was subjected 

to direct electrolysation. Fungal growth was assessed after 

24-48 h incubation at room temperature. When NaCl was used, 

EOW exhibited nearly 100% inhibition of P. expansum spore 

germination, whereas in the absence of NaCl a significant although 

lower efficacy (around 60% reduction) was recorded. 

The efficacy of disinfection increased to 98% after 75 min of direct 

electrolysis. On the basis of the results obtained a further trial 

was conducted on the wash water from a local packinghouse, 

which was subjected to electrolysis in presence of NaCl for 3 h. A 

visible improvement of water clearness and a significant reduction 

of total microbial population was obtained after only 30 min 

treatment. Trials on sweet cherries treated with EOW in commercial 

packinghouses are in progress. 

PROFILING OF SMALL RNAs POPULATIONS DERIVED 

FROM SOUR ORANGE SEEDLINGS SHOWING CROSS- 

PROTECTION AGAINST SEEDLING YELLOWS 

STRAINS OF CITRUS TRISTEZA VIRUS. M. Saponari 1,4 , H. 

Doddapaneni 2 , G. Loconsole 3 , A. Giampetruzzi 3 , P. Saldarelli 1 , 

R.K. Yokomi 4 . 1 Istituto di Virologia Vegetale del CNR, Via Amendola 

165/A, 70126 Bari, Italy. 2 Carver Center for Genomics, Department 

of Biology, University of Iowa, 52242 Iowa City, IA, 

USA. 3 Dipartimento di Protezione delle Piante e Microbiologia Applicata, 


70126 Bari, Italy. 4 USDA, Agricultural Research Service, 93648 

Parlier, CA, USA. E-mail: m.saponari@ba.ivv.cnr.it 

Cross protection against the stem pitting strain of Citrus tristeza 

virus (CTV) has been used to reduce losses to grapefruit and 

sweet orange. Recent studies have shown that cross-protection 

occurs between isolates of the same genotype, but its mechanism 

is unknown. Recently, endogenous small (s) RNAs were shown to 

play a role in plant stress response. During viral infections they 

originate either from the viral or plant genomes. High-throughput 

sequencing to assess sRNA profile is a powerful new technology 

for discovery of new approaches for disease control by understanding 

plant/virus interactions at the sRNA level. This technology 

was used to examine seedling yellows cross protection of 

CTV. Small RNAs fractions from a symptomless sour orange 

seedling (SO) infected with a mixture of T3, VT and non-standard 

CTV genotypes (S2) vs. SO infected with the VT component 

which resulted in severe seedling yellows (S1) were sequenced 

by Illumina Genome Analyzer II. The 21-24 nucleotide 

size classes dominated both libraries. Among the virus-derived 

sRNAs those from the 3’end genomic region, which includes the 

three gene silencing suppressors were predominant in both libraries, 

but with significant difference in the accumulation of one 

or the other suppressor. The largest class of the host-derived sR- 

NAs localized in the CTV resistance gene locus; specifically they 

derived from the Gipsy-like retrotransposone C. More comparative 

bioinformatics analyses are underway to correlate sRNA profiles 

with the phenotypes for a better understanding of the CTV 

and host genes involvement and regulation.


INTRASPECIFIC VARIABILITY OF ARMILLARIA MEL- 

LEA IN LOMBARDY. M. Saracchi, F. Rocchi, P. Sardi. Dipartimento 

di Protezione dei Sistemi Agroalimentare e Urbano e Valorizzazione 

delle Biodiversità, Università degli Studi, Via Celoria 2, 

20133 Milano, Italy. E-mail: marco.saracchi@unimi.it 

Within the Ticino River Regional Park root rots are among the 

factors that significantly contribute to the death of plants involved 

in oak decline. In most cases they are due to the action of 

basidiomycetes referable to the genus Armillaria. In this preliminary 

study 12 groups of carpophores, collected in the central part 

of the park and 23 fungal isolates obtained in vitro from the same 

samples were considered and tested for identification at the taxonomic 

level. Sequencing of the ITS regions and the subsequent 

comparison of obtained sequences with those contained in the 

EMBL-release and EMBL-update databases showed that all samples 

belonged to Armillaria mellea (Vahl) P. Kumm. A preliminary 

investigation on the intraspecific variability of A. mellea 

within the park was carried out, also for defining the PCR amplification 

protocols suitable for this purpose. PCR results with 

primers designed on the sequences of the two microsatellites 

(CAG) 5 , (GTGC) 4 and of the minisatellite M13 allowed identification 

of the different genomic profiles of the strains under study. 

The two microsatellites gave interesting results, generating respectively 

9 and 12 polymorphic bands, well defined by agarose 

gel electrophoresis. Primer M13 produced a larger number of 

bands but some, especially those less than 1000 bp in size, could 

not be easily defined. Cluster analysis of all profiles revealed a 

wide genetic variability between isolates, even when coming from 

woods not too far from each other. 

DETECTION OF OCHRATOXIGENIC ASPERGILLI ON 

GRAPE BERRIES DURING DRYING FOR PRODUCTION 

OF “VIN SANTO”. A. Scala 1 , N. Parissi 1 , C. Comparini 1 , F. 

Abiusi 1 , C. Fanelli 2 , M. Reverberi 2 , A. Ricelli 3 . 1 Dipartimento di 

Biotecnologie Agrarie, Università degli Studi, Piazzale delle 

Cascine, 50144 Firenze, Italy. 2 Dipartimento di Biologia Vegetale, 

Università degli Studi “La Sapienza”, Largo Cristina di Svezia 24, 

00165 Roma, Italy. 3 Istituto di Chimica Biomolecolare del CNR, 

Piazzale Aldo Moro 5, 00185 Roma, Italy. E-mail: aniello.scala@ 

unifi.it 

Aspergillus carbonarius, A. niger and A. tubingensis are known 

to produce ochratoxin A (OTA), a secondary metabolite with 

very dangerous effects to animals and humans. The International 

Agency for Research on Cancer has classified OTA as a possible 

carcinogen to humans (group 2B). As a consequence, the European 

Commission has imposed regulatory limits for the maximum 

tolerable presence of this toxin in different foodstuffs. After 

cereals, grape products are accounted as a considerable source of 

human OTA intake. Based on the fungal requirements of environmental 

temperature and relative humidity, it is easy to suppose 

that wines, as the Tuscan “Vin Santo”, obtained from grapes 

partially dried for several months to concentrate sugar content to 

at least 30% (w/v), are potentially at risk more than table wines. 

In the present work, done in the “Azienda Agricola Montepaldi” 

(San Casciano Val di Pesa, Florence), we isolated from grape 

berries about 6x10 7 CFU, divided into six major fungal morphotypes. 

Among these 24 were typical colonies of Aspergillus spp. 

These colonies were processed in parallel using: (i) conventional 

culture methods that allowed their morphological description; (ii) 

a molecular approach based on sequencing of the internal transcribed 

spacer (ITS) region comprising the ITS1, the ITS2, and 

the intervening 5.8S rRNA gene. Due to the known high intraand 

interspecific variability of the ITS region, it was not difficult 

to identify the fungal morphotypes isolated from the berries. The 

Aspergilli belonged to the species A. tubingensis. Analysis to find 

isolates producing OTA is ongoing. 

PHYTOPHTHORA PSEUDOSYRINGAE ON SWEET 

CHESTNUT TREES IN SARDINIA. B. Scanu, B.T. Linaldeddu, 

A. Franceschini. Dipartimento di Protezione delle Piante, 

Sezione di Patologia Vegetale, Università degli Studi, Via E. De 

Nicola 9, 07100 Sassari, Italy. E-mail: bscanu@uniss.it 

Phytophthora pseudosyringae is a well known soil-borne root 

pathogen of deciduous tree species in several European countries. 

We now report a new finding of P. pseudosyringae on mature 

trees of Castanea sativa in Sardinia. Infected trees showed 

classic symptoms of ink disease, such as microphylly and yellowish 

foliage as well as necrosis of collar and root flares. Isolations 

were made from infected roots and soil using green apples as 

bait. Small pieces of tissue were cut from the lesions that developed 

in the apples, plated on Phytophthora selective medium 

(SMA) and then subcultured onto carrot agar. Cultural and morphological 

features were in close agreement with those described 

for P. pseudosyringae. Identity was confirmed by analysis of the 

internal transcribed spacer region (ITS1-5.8S-ITS2) of rDNA. 

BLAST searches in GenBank showed 100% identity with reference 

sequences of P. pseudosyringae. Pathogenicity was demonstrated 

by both stem and root inoculations of 5-month-old chestnut 

seedlings grown in pots. Aggressiveness was also confirmed 

by inoculating living chestnut logs (1 m long × 10 cm diameter). 

This finding represents the first record of P. pseudosyringae on C. 

sativa in a woodland area. Previously, this pathogen had been reported 

as causal agent of stem necroses only on chestnut 

seedlings in a nursery in Spain. 

NATURAL COMPOUNDS FROM TRAMETES VERSICOL- 

OR INHIBIT GROWTH AND MYCOTOXIN BIOSYNTE- 

SIS IN DIFFERENT FUNGI. M. Scarpari 1 , A.A. Fabbri 1 , C. 

Fanelli 1 , P. Cescutti 2 , R. Rizzo 2 , Y. Herasimenka 2 , M. Punelli 1 , 

S. Zjalic 1 , A. Ricelli 3 , M. Reverberi 1 . 1 Dipartimento di Biologia 

Vegetale, Università degli Studi “La Sapienza”, Largo Cristina di 

Svezia 24, 00165 Roma, Italy. 2 Dipartimento di Scienze della Vita, 

Università degli Studi, Via L. Giorgieri 1, 34127 Trieste, Italy. 3 Istituto 

di Chimica Biomolecolare del CNR, Università degli Studi “La 

Sapienza”, Piazzale Aldo Moro 5, 00189 Roma, Italy. E-mail: 

marzia.scarpari@uniroma1.it 

The contamination of food commodities by mycotoxins, 

health hazardous and carcinogenic secondary metabolites produced 

by different fungi, has to be closely controlled to safeguard 

human and animal health. The strategies applied to achieve this 

aim often induce environmental pollution and are toxic to the 

end users. Trametes versicolor is a basidiomycete known for its 

therapeutic effects, mainly based on the production of several 

glycoproteins and exoglucans displaying anti-tumoral action and 

anti-oxidative effects. In the culture filtrate of this fungus two different 

components were identified, one polysaccharidic and one 

proteic. The exo-polysaccharidic component was analysed by 

chromatographic separation techniques (Sephacryl S-300) and 

1 H-NMR analysis showing the presence of highly complexed glucans 

(alpha- and beta-), with a MW of ~20 kDa. It can be hypothesised 

that these fungal polysaccharides can act as non-self 

signals able to modulate secondary metabolism in mycotoxigenic 

fungi. The proteic component has a clear antimicrobial effect 

which slows or completely blocks conidia germination at high


concentrations (2% w/v). Proteome maps of extracellular proteins 

of T. versicolor grown in two conditions, supporting or not 

the production of bioactive proteins, were analyzed by 2D gel 

electrophoresis identifying spots with qualitatively and quantitatively 

differences between the two growth conditions and subsequently 

characterised by MALDI-TOF. These results might be 

promising in view of the application of a more environmentally 

friendly strategy aimed at achieving an improved control of the 

different toxins which are often present in foods and feeds. 

INTRASPECIFIC VARIABILITY OF ERWINIA AMYLOVO- 

RA STRAINS FROM MOROCCO. G. Scuderi 1 , F. Valentini 2 , 

C. Platania 1 , A.M. D’Onghia 2 , M. Fatmi 3 , G. Cirvilleri 1 . 1 Dipartimento 

di Scienze e Tecnologie Fitosanitarie, Università degli Studi, 

Via S. Sofia 100, 95123 Catania, Italy. 2 Centre International de 

Hautes Etudes Agronomiques Méditerranéennes, Mediterranean 

Agronomic Institute of Bari, Via Ceglie 9, 70010 Valenzano (BA), 

Italy. 3 Département Protection des Plantes, Institut Agronomique 

et Vétérinaire Hassan II, Complexe Horticole d’Agadir, Morocco. 

E-mail: gcirvil@unict.it333 

Fire blight disease, caused by Erwinia amylovora, detected for 

the first time in Morocco in 2006, has spread rapidly throughout 

the main orchards areas. Surveys were carried out in these regions 

to evaluate the current situation of the disease in the country. In 

2009 the disease was observed in 71 farms of different regions, i.e. 

Meknes, El Hajeb, Sefrou, Ifrane, Taounate and Khenifra. In 

terms of infected area, more than 720 ha were recorded in 2009. 

To date, over 215 ha of pear, apple and quince have been destroyed 

(dug out and incinerated). A collection of 48 isolates obtained 

from pear, apple and quince trees showing clear-cut fire 

blight symptoms in 2006 and 2009 were identified as E. amylovora 

using morphological, biochemical and serological tests. In addition, 

classical PCR and real-time PCR were used to confirm identification. 

This collection of E. amylovora isolates and reference 

bacterium strains from several countries were examined by rep- 

PCR and fAFLP fingerprint. Bacterial strains showed 90% similarity 

in the rep-PCR analysis using ERIC and BOX primers. The 

fAFLP analysis with primer set MseI+C, EcoRI+A showed a higher 

variability than previously observed, allowing the differentiation 

of all E. amylovora strains, even if no clear grouping of strains 

was possible in terms of correlation between isolates, host plant, 

year and country of isolation. In this study fAFLP analysis proved 

to be the most useful tool for discriminating among strains of E. 

amylovora from different hosts and years of isolation. 

IDENTIFICATION AND PATHOGENICITY OF BOTRY- 

OSPHAERIA spp. ASSOCIATED WITH GRAPEVINE 

TRUNK DISEASES IN SARDINIA. S. Serra, B. Scanu, A. 

Schiaffino, A. Deidda, B.T. Linaldeddu. Dipartimento di Protezione 

delle Piante, Sezione di Patologia Vegetale, Università degli 

Studi, Via E. De Nicola 9, 07100 Sassari, Italy. E-mail: salvase@ 

uniss.it 

Grapevine spur, cordon, and trunk dieback constitute a serious 

economic problem in Sardinian vineyards. Recent studies 

have indicated that several grapevine trunk pathogens belong to 

the genus Botryosphaeria. Thus, during 2009 a field survey was 

carried out to study Botryosphaeria spp. involved in the aetiology 

of grapevine trunk diseases in several vineyards located in north 

Sardinia (Italy). Botryosphaeria spp. were the most common fungal 

species isolated from symptomatic tissues. Four species 

(Botryosphaeria australis, B. obtusa, B. parva and Lasiodiplodia 

theobromae) were identified on the basis of morphological and 

cultural features. The identity of B. australis was also confirmed 

by analysis of nucleotide sequences of the internal transcribed 

spacer region (ITS1-5.8S-ITS2) of rDNA. BLAST searches in 

GenBank showed 100% similarity with reference sequences of B. 

australis and confirmed differences in three nucleotide positions 

from sequences of Neofusicoccum luteum, a closely related 

Botryosphaeria species. Pathogenicity of all species was assessed 

by inoculation of excised green grapevine shoots of cvs Vermentino 

and Cannonau under controlled laboratory conditions. B. australis 

and L. theobromae were more virulent than the other 

species. Inoculated fungi were successfully reisolated from all infected 

tissues, thus fulfilling Koch’s postulates. These findings 

confirm the active role of Botryosphaeria spp. in the aetiology of 

grapevine trunk diseases. For each fungal species, except for B. 

obtusa, this represents the first record on grapevine in Sardinia. 

DIAGNOSTIC ANALYSES ON FOURTH RANGE PRO- 

DUCTION IN THE PROVINCE OF SALERNO. L. Sigillo, V. 

Senape, G. Serratore, V. Spina, R. Bravi. Ente Nazionale Sementi 

Elette, SS 18 km 77.700, 84091 Battipaglia (SA), Italy. E-mail: 

l.sigillo@ense.it 

The production of species for fourth range market has strongly 

increased in the last years. Important economical losses are reported 

due to the widespread pesence of bacterial and fungal diseases. 

In our laboratory, 73 samples of rocket (Dyplotaxis tenuifolia), 

65 of lettuce (Lactuca sativa), 19 of corn salad (Valerianella 

locusta), 9 of spinach (Spinacia oleracea) and many other minor 

species were analysed from 2005 to 2010. Among these, 108 and 

68 seed and symptomatic plant samples were analysed for detection 

of the most important pathogens. Rocket seed samples were 

contaminated by Xanthmonas campestris (!5%) and pathogenic 

strains of Fusarium oxysporum (8%), Instead, 50% and 20% of 

rocket plants were infected by F. oxysporum and X. campestris, respectively. 

F. oxysporum, Verticillium sp. and Rhizoctonia sp. occurred 

rarely on lettuce seeds or plant tissues (about 3%). Generally, 

corn salad, beet and spinach seeds were healthy. X. 

campestris was detected only in a sample of corn salad. In conclusion, 

an increase of F. oxysporum and X. campestris incidence on 

rocket has been observed in the last years. This increase is probably 

due to the diffusion of monoculture system and the use of 

contaminated seeds in the fourth range production. 

DISTRIBUTION OF THREE DIFFERENT ISOLATES OF 

CITRUS TRISTEZA VIRUS IN SOUTHERN ITALY. G. Sorrentino 

1 , S. Davino 2 , M. Davino 3 . 1 CRA, Istituto Sperimentale per 

l’Agrumicoltura, Corso Savoia 190, 95024 Acireale (CT), Italy. 2 Dipartimento 

di Scienze Entomologiche, Fitopatologiche, Microbiologiche, 

Agrarie e Zootecniche, Sezione di Patologia Vegetale e Microbiologia 

Agraria, Università degli Studi, Viale delle Scienze, 

90128 Palermo, Italy. 3 Dipartimento di Scienze e Tecnologie Fitosanitarie, 

Sezione di Patologia Vegetale, Università degli Studi, 

Via Santa Sofia 100, 95123 Catania, Italy. E-mail: davino@unipa.it 

The discovery of large concentrations of different citrus 

species affected by the Citrus tristeza virus (CTV) in southern 

Italy has provided the opportunity for investigating the distribution 

of three main viral isolates. The survey was carried out in different 

farms mainly in the Baè area (Catania province). All trees 

in each area were sampled yearly from 2001 to 2008 in May and 

September. Four young apical shoots were collected and analyzed 

by DAS-ELISA or DTBIA. Molecular tests (SSCP and p20 and


p23 protein sequencing) showed that isolates were mild (CTV- 

DS1, CTV-Tardivo di Ciaculli) and severe (seedling yellow-CTV 

DS2). Monitoring the aphid population confirmed the results of 

previous work in which A. gossypii represented 85-90% of the 

aphid population. Our results for infected tree percentages, are 

very similar to the non-linear predictive estimation of CTV 

spread reported in other citrus areas where the vector is A. 

gossypii, up the 7 th - 8 th year from the beginning of the infection 

when it increases exponentially. Afterwards, in our non-linear 

prediction model, the percentage of infected trees in all plots was 

about 98% after eleven years, while elsewhere this percentage is 

reached by the 14 th -15 th year. This could probably be linked to 

severe CTV seedling yellow isolate combined with the high susceptibility 

of Tarocco old line sweet orange grafted on sour orange 

rootstock. The unregulated traffic of propagation material 

by farmers and the high transmitability by aphids put Sicilian 

farming, which accounts for 50% of the Italian citrus industry at 

great risk. 

MOLECULAR CHARACTERIZATION BY IGS SEQUENC- 

ING OF FORMAE SPECIALES OF FUSARIUM OXYSPO- 

RUM PATHOGENIC TO LAMB’S LETTUCE AND ROCK- 

ET. D. Spadaro 1,2 , K. Srinivasan 1 , G. Gilardi 1 , M.L. Gullino 1 , A. 

Garibaldi 1 . 1 Centro di Competenza per l’Innovazione in Campo 

Agro-Ambientale (AGROINNOVA), Università degli Studi di 

Torino, 10095 Grugliasco (TO), Italy. 2 Dipartimento di Valorizzazione 

e Protezione delle Risorse Agroforestali, Università degli 

Studi di Torino, Via Leonardo da Vinci 44, 10095 Grugliasco (TO), 

Italy. E-mail: davide.spadaro@unito.it 

Twenty-nine isolates of Fusarium oxysporum collected from 

wilted lamb’s lettuce plants (Valerianella olitoria) and 36 Fusarium 

oxysporum isolates collected from wilted rocket plants (Eruca 

vesicaria L., syn. E. sativa, cv. ‘Rucola coltivata’), including ATCC 

strains, were examined for differences in the nucleotide sequences 

of the ribosomal DNA (rDNA) intergenic spacer (IGS) 

region, approx. 2.5 kb long in the isolates analyzed. The isolates 

were tested for pathogenicity on lamb’s lettuce or rocket in 

glasshouse. Results showed that the isolates were slightly, moderately 

and highly pathogenic except for four non-pathogenic isolates 

from lamb’s lettuce. Most of the isolates from wilted rocket 

and lamb’s lettuce plants collected in Italy were very similar to F. 

oxysporum f.sp. raphani. In conclusion, the analysis of the IGS sequences 

revealed that the isolates studied had different origins 

and that phylogeny and pathogenicity were related. Non-pathogenic 

isolates differed genetically from those with low, moderate 

and high virulence. To our knowledge, this is the first report of 

differentiation of formae speciales of F. oxysporum on rocket and 

lamb’s lettuce by IGS sequence analysis. 

USE OF MEDITERRANEAN PLANT ESSENTIAL OILS TO 

CONTROL POSTHARVEST ROTS CAUSED BY BOTRYTIS 

CINEREA AND PENICILLIUM EXPANSUM ON FOUR 

CULTIVARS OF APPLES. D. Spadaro 1,2 , J.G. Lopez-Reyes 1 , 

M.L. Gullino 1 , A. Garibaldi 1 . 1 Centro di Competenza per l’Innovazione 

in Campo Agro-Ambientale (AGROINNOVA), Università 

degli Studi di Torino, 10095 Grugliasco (TO), Italy. 2 Dipartimento 

di Valorizzazione e Protezione delle Risorse Agroforestali, Università 

degli Studi di Torino, Via Leonardo da Vinci 44, 10095 

Grugliasco (TO), Italy. E-mail: davide.spadaro@unito.it 

The efficacy of essential oils of different Mediterranean plants 

was evaluated on apple cvs Golden Delicious, Granny Smith, Red 

Chief, and Royal Gala, to control Botrytis cinerea and Penicillium 

expansum in postharvest. The essential oils of basil (Ocimum 

basilicum), fennel (Foeniculum sativum), lavender (Lavandula officinalis), 

marjoram (Origanum majorana), oregano (Origanum 

vulgare), peppermint (Mentha piperita), rosemary (Rosmarinus officinalis), 

sage (Salvia officinalis), savory (Satureja montana), 

thyme (Thymus vulgaris) and wild mint (Mentha arvensis) were 

tested at different concentrations. Fruits were artificially wounded 

and inoculated with a suspension at 1x10 5 conidia/ml of each 

pathogen. After 12 h, emulsions at 1% and 10% of each essential 

oil were introduced into each inoculated wound. Tebuconazole 

chemical control and an inoculated control were also included. 

All treated fruit were stored at 4°C. After 15 and 30 days, the diameter 

of rots was measured. Results showed that the efficacy of 

the essential oils tested was cultivar- and storage time-dependent. 

Treatments with essential oils from oregano, savory, and thyme 

showed significant efficacy in all apple cultivars tested. Treatments 

with essential oil emulsions at 10% were phytotoxic for all 

apple cultivars evaluated. 

TESTING THE EFFICACY OF A TRANSPOSON-BASED 

DOUBLE COMPONENT SYSTEM TO TAG PATHO- 

GENICITY GENES IN THE WHEAT PATHOGEN FUSAR- 

IUM CULMORUM. F. Spanu 1 , B. Scherm 1 , V. Balmas 1 , A. Marcello 

1 , M. Dufresne 2 , M.J. Daboussi 3 , Q. Migheli 1 . 1 Dipartimento 

di Protezione delle Piante, Unità di Ricerca Istituto Nazionale 

Biostrutture e Biosistemi, Università degli Studi, Via E. De Nicola 

9, 07100 Sassari, Italy. 2 Institut de Biologie des Plantes, Bâtiment 

630, Université Paris Sud 11, F-91405 Orsay Cedex, France. 3 Institut 

de Génétique et Microbiologie, Bâtiment 400, Université Paris 

Sud 11, F-91405 Orsay Cedex, France. E-mail: qmigheli@uniss.it 

As it has been already achieved for other economically relevant 

Fusarium species (i.e., Gibberella zeae, F. sporotrichioides and F. 

oxysporum), the genome of F. culmorum, incitant of crown and 

foot rot on wheat and type B trichothecene producer, is now being 

sequenced. Based on available data, the number of predicted 

genes present in this genome is estimated to exceed 10,000. For 

many genes the function is yet unknown and consequently there is 

a strong need for a high-throughput method for functional genomic 

analysis. We tested the efficacy of two double component 

systems based on defective transposable elements mobilised by a 

separate source of transposase. In both systems, the tagging element 

is inserted into the first intron of the A. nidulans niaD gene, 

and a phenotypic assay for excision allows recovery of excision 

events on minimal medium containing nitrate as the sole nitrogen 

source. The first system is based on a hop copy that has been engineered 

by insertion of the A. nidulans hph gene conferring resistance 

to hygromycin B. This defective element is mobilized by the 

hop transposase under the control of the constitutive A. nidulans 

gpdA promoter on a separate vector. The selectable marker facilitates 

the recovery of strains with a reinserted element on hygromycin 

B-containing medium. The second system is based on 

the ability of the impala transposase to transactivate mimp1, which 

shows features of MITEs. Preliminary experiments demonstrate 

that the excised hop element is unable to reinsert in the genome of 

F. culmorum, making this tool unsuitable for the pathogen. 

CHARACTERIZATION OF p20 AND p23 GENES IN COR- 

SICAN ISOLATES OF CITRUS TRISTEZA VIRUS. M. Tessitori 

1 , J. P. Thermoz 2 , M. Davino 1 , S. Davino 3 . 1 Dipartimento di 

Scienze Fitosanitarie, Sezione di Patologia Vegetale, Università 

degli Studi, Via S. Sofia 100, 95123 Catania, Italy. 2 Unité Gene-


tique et Ecophysiologie de la Qualite des Agrumes–GEQA, INRA, 

Route de l’INRA, San Giuliano-Corse, France. 3 Dipartimento di 

Scienze Entomologiche, Fitopatologiche, Microbiologiche, Agrarie e 

Zootecniche, Sezione di Patologia Vegetale e Microbiologia Agraria, 

Università degli Studi, Viale delle Scienze, 90128 Palermo, Italy. Email: 

mtessitori@unict.it 

Tristeza disease, caused by Citrus tristeza virus (CTV), is the 

one of the most important viral diseases of citrus species. Several 

strains of CTV have been studied all over the world. Two isolates 

have been previously identified in Corsica, the K strain from 

Marumi kumquat, known to determine no symptoms on Mexican 

lime, and the Cal-1 from calamondin that induces stem pitting on 

the same indicator. Few sequence data of both isolates are published 

or available in GenBank. Recently additional CTV strains 

were found in Corsica. The isolates LA5 and CO3 were detected 

in two different 40-year-old orchards of Clementine on sour orange 

that did not show decline or other tristeza symptoms. CTV 

was identified in young shoots collected in the field by RT-PCR 

using primers specific for the p20 and p23 proteins. The amplicons 

obtained were analyzed by SSCP, cloning and sequencing. 

Both CTV isolates showed the same SSCP patterns for both proteins 

which differed from control patterns (DS1, DS2 and T36). 

Sequences analysis diclosed a high similarity between the two isolates 

(98%) for both p20 and p23. Multiple alignment analysis of 

LA5 and CO3 showed a similarity of 98% with CTV isolates deposited 

in GenBank. Moreover, with respect to the already described 

groups of CTV isolates, mild, severe and atypical, phylogenetic 

analysis revealed a new cluster that includes both LA5 

and CO3 isolates 

SYNERGISTIC INTERACTION BETWEEN POTATO 

SPINDLE TUBER VIROID AND CUCUMBER MOSAIC 

VIRUS IN TOMATO. E.M. Torchetti, B. Navarro, F. Cillo, F. 

Di Serio. Istituto di Virologia Vegetale del CNR, Via Amendola 

165/A, 70126 Bari, Italy. E-mail: em.torchetti@ba.ivv.cnr.it 

Potato spindle tuber viroid (PSTVd), a nuclear replicating viroid 

included in the EPPO A2 list of quarantine pests, causes severe 

diseases to potato and tomato. In the last few years, this viroid 

has been detected in several symptomless ornamental solanaceous 

species in many European countries, producing a general 

alert on the risk of possible PSTVd outbreaks in horticultural 

crops. The recent report of symptomatic tomato plants infected 

by PSTVd in Italy has increased such concerns. Since tomato is 

also a natural host of Cucumber mosaic virus (CMV), its response 

to mixed infections by this virus and PSTVd was investigated. 

Here, we report the synergistic interaction of contemporary infections 

by a mild strain of PSTVd and the Fny strain of CMV in 

tomato cvs. Micro-Tom and UC82. Plants infected by both 

pathogens showed severe stunting, delay of flowering, and frequent 

systemic necrotic lesions that completely impaired plant 

growth. These symptoms were never observed in PSTVd and 

CMV single-infected plants, which only showed slight stunting 

and leaf deformation, respectively. Data about the accumulation 

levels of each pathogen in single- and mix-infected hosts will be 

discussed in the context of a possible role of RNA silencing in the 

synergistic interaction. These findings highlight the need of implementing 

control measures to restrain PSTVd spread in Europe. 

ALTERNARIA LEAF SPOT ON OKRA IN TANZANIA: 

PROSPECTS FOR BIOLOGICAL CONTROL IN SUSTAIN- 

ABLE AGRICULTURE. E. Turco 1 , M. Galla 2 , D. Bocciolini 3 , S. 

Tofani 3 , A. Ragazzi 2 . 1 Istituto per la Protezione delle Piante del 

CNR, Via Madonna del Piano 10, 50019 Sesto Fiorentino (FI), 

Italy. 2 Dipartimento di Biotecnologie Agrarie, Sezione di Protezione 

delle Piante, Università degli Studi, Piazzale delle Cascine 

28, 50144 Firenze, Italy. 3 Cooperativa Agricola di Legnaia, Via di 

Sollicciano 13a, 50142 Firenze. E-mail: e.turco@ipp.cnr.it 

The “Progetto Tanzania” was funded in 2006 by the Cooperativa 

Agricola di Legnaia, the oldest and largest agricultural cooperative 

in Tuscany. In 2007, the Faculty of Agriculture of the Universisity 

of Florence joined the project. Besides the humanitarian 

and economical assistance to “Villaggio della Speranza” in 

Dodoma and San Gaspare hospital in Itigi, the project is involved 

in improving and upgrade livestock and crop plants (mainly vegetables) 

of local communities. Okra (Abelmoschus esculentus L.) , 

a traditional food plant in Africa with a high nutritive value, is essential 

to the daily diet of local population, consisting almost exclusively 

of carbohydrates. Eco-compatible measures to control 

and eradicate fungal diseases are therefore required to manage 

sustainable agriculture and rural development. Periodical surveys 

of the health status of okra plantations in the area of Itigi (Manyoni 

discrict, 5°42’S 34°29’E, 1300 m a.s.l.) revealed leaf spots and 

blight caused by Alternaria alternata. The biological control of 

the disease by the application of Trichoderma viride and Epicoccum 

nigrum is the scope of this report. Field experiments were 

carried out during the rainy season of 2009. Leaf spots and 

blight, before and after treatment with the two fungal antagonists, 

were scored and DI (disease score index) using a three class 

scale was assessed over 8 weeks. DI above 2 was observed starting 

6 weeks after pathogen inoculation. Leaf symptoms caused by 

A. alternata were not significantly reduced by the presence of either 

T. viride or E. nigrum. The efficacy of fungal antagonists and 

validity of the experimental protocol are discussed in relation to 

the environmental and climatic conditions. 

PRELIMINARY RESULTS ON THE EFFECT OF GOSSY- 

POL ON FEW AUSTRALIAN FUSARIUM OXYSPORUM f. 

sp. VASINFECTUM ISOLATES. E. Turco 1 , A. Ragazzi 2 , B. 

Wang 3 , C.L. Brubaker 4 . 1 Istituto per la Protezione delle Piante del 

CNR, Via Madonna del Piano 10, 50019 Sesto Fiorentino (FI), Italy. 

2 Dipartimento di Biotecnologie Agrarie, Sezione di Protezione delle 

Piante, Università degli Studi, Piazzale delle Cascine 28, 50144 

Firenze, Italy. 3 Centre for Plant Biodiversity Research, CSIRO Plant 

Industry, Clunies Ross Street and Barry Drive, GPO Box 1600, Canberra 

ACT 2601, Australia. 4 Bayer CropScience, Technologiepark 38, 

B-9052, Gent, Belgium. E-mail: e.turco@ipp.cnr.it 

The Australian cotton industry is an important component of 

the Australian economy. Researchers and farmers are working to 

build sustainable production based on new cotton cultivars with 

high fibre quality and yield coupled with improved pest and disease 

resistance. Fusarium wilt of cotton (caused by Fusarium oxysporum 

f.sp. vasinfectum or FOV) appeared unexpectedly in Australia 

in 1995 and represents a considerable threat to the sustainability 

of cotton production. Therefore, considerable efforts are 

directed toward developing and obtaining new cotton varieties 

with enhanced levels of FOV tolerance. In some breeding programs 

attention as turned to native Australian Gossypium species 

with variable concentrations of gossypol, a terpenoid aldehyde 

with a suspected potential to improve resistance to Fusarium wilt 

in cotton cultivars. Because the Australian FOV isolates are genetically 

distinct from FOV isolates found outside Australia and the 

probable co-evolution pathogen and wild cotton cultivars, the susceptibility 

of FOV conidia to varying levels of gossypol was deter-


mined for fourteen isolates collected from different locations in 

the cotton growing regions of Australia. Four gossypol concentration 

(0, 4, 10 and 20 mg/l) and five FOV isolates (two from each 

vegetative compatibility group, VCG 01111 and VCG 01112, and 

a fifth isolate not yet assigned to a VCG) were here considered. 

Different conidial germination rates were observed over 8 h 

among the isolates tested. The results are discussed according to 

the vegetative compatibility group to which the isolates belong 

and to their different virulence on cotton germoplasm. 

OPHIOSTOMATOID FUNGI ASSOCIATED WITH IPS 

ACUMINATUS (COLEOPTERA: CURCULIONIDAE) IN 

THE ITALIAN ALPS. C. Villari 1 , A. Battisti 1 , P. Capretti 2 , M. 

Faccoli 1 . 1 Dipartimento di Agronomia Ambientale e Produzioni 

Vegetali, Università degli Studi di Padova, Agripolis, Viale dell’Università 

16, 35020 Legnaro (PD), Italy. 2 Dipartimento di Biotecnologie 

Agrarie, Sezione di Protezione delle Piante, Università 

degli Studi, Piazzale delle Cascine 28, 50144 Firenze, Italy. E-mail: 

caterina.villari@unipd.it 

Most bark beetles are associated with symbiotic fungi, assisting 

beetles in killing healthy trees. The Scots pine beetle Ips 

acuminatus is reported to be associated with Ophiostoma brunneo-ciliatum, 

O. ips and Ambrosiella macrospora. The first two 

species are weak blue-stain pathogens thought to be involved in 

lowering the critical threshold of beetle attack density. A. 

macrospora is a non-pathogenic fungus used as a direct food 

source for the larvae. This study aims at explaining the role of the 

fungi associated with I. acuminatus in the decline of alpine Scots 

pine stands, exploring the possible quantitative and qualitative 

variations of fungal flora. Epidemic and endemic beetle populations 

were sampled in early spring 2009 from three sites at each 

of six locations selected across the Italian Alps. Adult beetles 

were obtained from logs incubated in rearing cages and a subset 

was washed in 1% Tween 80. The washing solution was then 

plated on selective medium, in order to isolate and identify the 

beetle-associated fungi on morphological grounds. Ophiostoma 

brunneo-ciliatum and O. ips were isolated from both males and 

females at nearly all sites, while A. macrospora was never isolated, 

probably because it is difficult to culture. The insect pathogenic 

fungus Beauveria spp. was isolated with high frequency. No differences 

were found between epidemic and endemic populations. 

Molecular markers specific for each fungal species were developed, 

in order to simplify and speed up the species identification, 

A. macrospora included, and to compare the fungal communities 

associated with I. acuminatus among populations. 

HARZIANIC ACID, A SIDEROPHORE FROM TRICHO- 

DERMA HARZIANUM WITH ANTIFUNGAL AND PLANT 

GROWTH PROMOTING ACTIVITY. F. Vinale 1 , G. 

Flematti 2 , K. Sivasithamparam 3 , E.L. Ghisalberti 2 , R. Marra 1 , 

M. Ruocco 4 , S.L. Woo 1 , S. Lanzuise 1 , M. Nigro 1 , M. Lorito 1,4 . 

1 Dipartimento di Arboricoltura Botanica e Patologia Vegetale, Università 

degli Studi di Napoli ‘Federico II’, 80055 Portici (NA), 

Italy. 2 School of Biomedical, Biomolecular and Chemical Sciences, 

University of Western Australia, Australia. 3 School of Plant Biology, 

The University of Western Australia, 35 Stirling Highway, 6009 

Crawley, Australia. 4 Istituto di Protezione delle Piante del CNR, 

Via Università 133, 80055 Portici (NA), Italy. E-mail: frvinale@ 

unina.it 

Many strains of the fungus Trichoderma are well known producers 

of secondary metabolites having antibiotic activity. Their 

production varies in relation to: (i) the specific compound; (ii) 

the species and strain; (iii) the presence/absence of other microbes 

and; (iv) the balance between elicited biosynthesis and 

biotransformation rates. The involvement of secondary metabolites 

in the ability of Trichoderma spp. to activate plant defence 

mechanisms and regulate plant growth has recently been investigated. 

A T. harzianum strain, isolated from composted hardwood 

bark in Western Australia, produces a metabolite that showed antifungal 

activity in vitro against Pythium irregulare, Sclerotinia 

sclerotiorum and Rhizoctonia solani. The structure and absolute 

configuration of the fungal metabolite, harzianic acid, was determined 

by X-ray diffraction studies. The effect of harzianic acid 

on plant growth promotion was evaluated by treating seeds of 

canola (Brassica napus) with different concentrations of the purified 

Trichoderma metabolite and measuring the stem length of developing 

seedlings. Applications of the compound at concentrations 

of 100, 10 and 1 ng per seed, stimulated plant growth as indicated 

by an increase of 42%, 44% and 52% of stem length, respectively, 

compared with the untreated control (water). However, 

this same harzianic acid compound also inhibited plant 

growth up to 45% and 33% (stem length) when augmented concentrations 

of 100 and 10 µg, respectively, were applied to the 

seed. Additionally, we have discovered that this tetramic acid is 

capable of binding essential metals such as Fe 3+ with a good 

affinity, providing an important mechanism for iron solubilization, 

influencing nutrient availability in the soil environment to 

other microorganisms and to the plant. 

EVALUATION OF ANTIFUNGAL ACTIVITY OF “BIO- 

PROTEGE”, A MIXTURE OF SODIUM BICARBONATE 

AND SILICIUM DIOXIDE, AGAINST MAJOR POSTHAR- 

VEST FUNGAL PATHOGENS OF FRUIT AND VEGETA- 

BLES. K. Youssef 1 , P. Di Primo 2 , M. Coniglione 2 , A. Ippolito 1 . 

1 Dipartimento di Protezione delle Piante e Microbiologia Applicata, 

Università degli Studi “A. Moro”, Via Amendola 165/A, 70126 

Bari, Italy. 2 Decco Italia srl, Contrada Barriera, 95032 Belpasso 

(CT), Italy. E-mail: ippolito@agr.uniba.it 

In recent years, public demands to reduce pesticide use, stimulated 

by increased awareness of environmental and health issues 

associated with fungicide residues, as well as the development of 

fungicide-resistant strains of pathogens, have created the need to 

find and develop safe alternative control means. The effectiveness 

of a formulation based on a mixture of sodium bicarbonate and 

silicium dioxide (Bioprotege, Decco Italia) as a possible alternative 

to synthetic fungicides for controlling postharvest pathogens 

was evaluated. Bioprotege at increasing concentrations (0, 0.1, 

0.2, 0.3, 0.4, 0.5, 1, 2, 4, and 6%, w/v) mixed with potato dextrose 

agar (PDA) medium was tested. The concentration causing 

50% growth reduction (ED 50 , SAS probit analysis) and the minimum 

inhibition concentration (MIC) values, were also evaluated. 

The ED 50 was 0.11, 0.10, 0.08, 0.08, 0.25, 0.53, and 1.3% (w/v) 

for Penicillium digitatum, P. italicum, P. ulaiense, Monilinia laxa, 

Phytophthora nicotianae, Geotrichum candidum, and Botrytis 

cinerea, respectively. Complete growth inhibition of the same 

pathogens, except for B. cinerea, was achieved at 0.3, 0.3, 0.2, 0.2, 

2, and 2% (w/v), respectively. No MIC was found for B. cinerea 

since complete inhibition was not achieved till 6%. The effect of 

Bioprotege was primarily fungistatic because when fungal discs 

showing no growth were re-seeded onto fresh PDA, their growth 

resumed. Bioprotege confirmed its activity in in vivo trials performed 

on fuits of different citrus species. Overall, the results 

show the potential benefits of Bioprotege for controlling postharvest 

pathogens attacking fruit and vegetables.


RESISTANCE OF MUSKMELON TO FUSARIUM WILT. A. 

Zechini D’Aulerio, R. Roberti, F. Piattoni, G. Servidio. Dipartimento 

di Protezione e Valorizzazione Agroalimentare, Università 

degli Studi, Viale Fanin 46, 40126 Bologna, Italy. E-mail: aldo.zechinidaulerio@unibo.it 

In Italy, Fusarium oxysporum f. sp, melonis induces an important 

disease of muskmelon. Four races of this pathogen are currently 

known, i.e. 0, 1, 2 and 1-2. Varietal resistance is the only reliable 

approach for preventing wilt induced by race 0, 1 and 2, 

which were studied in this work. Investigations carried out between 

2008 and 2010 aimed first at verifying the resistance of the 

cultivars certified as “resistant” by seed companies. To this aim, 

three “resistant” cultivars (Bingo, Giusto and Sweetness) were 

compared with three susceptible cultivars (Cantalupo, Harper and 

Retato degli Ortolani), by infecting plants with each of the three 

pathogenic races (spore suspension: 10 6 conidia/ml). Among the 

“resistant” cultivars, one resulted totally resistant (Bingo), whereas 

Giusto and Sweetness showed a partial resistance (disease severity: 

10%). Similar differences were observed among the susceptible 

cultivars for disease severity was close to 100% in Retato whereas 

Cantalupo and Harper it ranged between 80 and 85%. Differences 

in pathogenicity were also observed among the pathogenic 

races tested: race 0 and 2 caused the highest disease severity in 

Bingo and Retato. Attempts were made to investigate the mechanisms 

involved in plant resistance. We found that root exudates 

did not affect neither conidial germination rate nor mycelial 

growth. Currently, studies on the development of promycelium 

are being carried out. Further investigations will aim at highlighting 

possible differences between resistant and susceptible cultivars, 

in post-infection root morphological modifications, by means 

of fluorescence microscopy techniques.

Journal of Plant Pathology (2010), 92 (4, Supplement ... - Sipav.org

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