INVESTIGATIONS ON PENICILLIUM spp. POPULATION DYNAMIC IN CITRUS PACKINGHOUSE

CHARACTERIZATION OF A PR-10 PROTEIN FROM VI-TIS RIPARIA. M. Alessandrini, A. Lovato, L. Bortesi, G. Zoc-catelli, S. Zenoni, M. Pezzotti and A. Polverari. Dipartimento diBiotecnologie, Università degli Studi, Strada le Grazie 15, 37134Verona, Italy. E-mail: [email protected]

We have started a project aimed at characterizing the expres-sion profile and protein activity of pathogenesis-related proteins ofthe PR-10 family in grapevine. According to a previous microarrayanalysis, the expression of several genes, annotated as similar toPR-10 proteins, is strongly increased in resistant Vitis riparia and,to a lesser extent, in V. vinifera, following infection with Plas-mopara viticola. PR-10 proteins are induced by pathogen infectionand abiotic stresses in a number of species; because they oftenshow ribonucleolytic activity it has been suggested that they maybe involved in antiviral defence; their antimicrobial activity hasbeen tested with controversial results. The coding sequence of ahomologous gene, denoted Vr-PR10, has been cloned from the re-sistant species V. riparia and the corresponding protein expressedin E. coli. The purified recombinant protein showed ribonucleolyticactivity in vitro and was used to raise antibodies which recognizedthe native protein from leaf tissue in Western blot analyses. Theprotein was tested in vitro and in vivo against P. viticola, Botrytiscinerea and Grapevine virus A (GVA), without showing any antimi-crobial or antiviral activity. The possibility that PR-10 proteins havedifferent or indirect roles in defense should be considered in fur-ther investigations.

DISTRIBUTION OF BOTRYOSPHAERIACEAE SPECIES ASSOCIATED WITH GRAPEVINE DECLINE IN WEST-ERN SICILY. A. Alfonzo, C. Conigliaro, V. Mondello and S. Bur-ruano. Dipartimento DEMETRA, Università degli Studi, Viale delleScienze 4, 90128 Palermo, Italy. E-mail: [email protected]

Recently, the pathogenicity of different Botryosphaeraceaespecies on grapevines has been reported from many Europeanand extra-European areas. During summer 2008, in a Sicilianvineyard located in the Marsala area (TP), we observed a declineinduced by Lasiodiplodia theobromae. The occurrence and quiterapid spread of the disease seemed to support the regional vari-ability of the species in question, which is retained a tropical andsubtropical pathogen to different trees. In the last two years, thedecline syndrome has been observed in the following othergrape-growing areas of western and central Sicily: Alcamo andSalemi (TP), Montevago (AG) and Milena (CL). From decliningplants showing typical external and internal symptoms of spurs,canes and trunks, fungal isolates were obtained in pure and/ormixed culture and were identified, by morphological and molec-ular analysis, as L. theobromae, Diplodia seriata and Neofusicoc-cum luteum. These results, even if preliminary, show the rapidspread of the grapevine decline by Botryosphaeraceae species inSicily confirming, at the same time, their different distribution inrelation to environmental conditions of the surveyed areas. Path-ogenicity tests are in progress to ascertain the role of these fungiin the evoking of the decline syndrome.

EVALUATION OF THE ANTIFUNGAL ACTIVITY OFMORPHOLOGICALLY DISTINCT COLONIES OF BACIL-LUS SUBTILIS STRAIN ET-1. A. Ambrico and M. Trupo.Unità Tecnica Tecnologie Trisaia, Laboratorio Biotecnologie, C.R.ENEA Trisaia, ss106 Jonica km 419.5, 75026 Rotondella (MT),Italy. E-mail: [email protected]

Bacillus subtilis differentiates into distinct adaptive subpopula-

tions of specialized cells that coexist within highly structured com-munities. Here we report how phenotypic dissociations in B. sub-tilis can induce a significant reduction of the secondary metabo-lites which are associated with antifungal activity. Strain ET-1 ini-tially has a rough, dense, colony morphology phenotype (R-form)with undulated edges, which dissociated into a S-form (smooth,soft, whitish) and into a M-form (slimy, translucent, amoeba-shaped colony). Correlations between colony forms, spore forma-tion and in-vitro antifungal activity of cell-free supernatants werestudied. Germinal inhibition over 95% of Penicillium digitatumconidia was induced only by S and R forms when cell-free super-natants were diluted 1:16 and 1:128 respectively. Biosynthesis ofantifungal molecules in broth was significantly reduced when anold R-form colony was used for inoculation because it was alreadydissociated in M and S phenotypic cells. After 48 h of growth inliquid medium a comparison of the spore-forming colony and anexamination by phase-contrast microscopy revealed a reducedability of the dissociant M-form to sporulate (104 CFU/ml). Acomplete spore release from the vegetative cells was observed onlyfor the R-form (109 CFU/ml), whereas the S-form contained amixture of endospores (108 CFU/ml) and few vegetative cells. Dif-ferent levels of Podosphaera fusca control on melon by the threeform colonies were obtained in in-vivo trials. To reduce secondarymetabolite production problems, the use, as starter, of a pasteur-ized spore suspension by young isolated R-form colony of the ET-1 strain is recommended.

AGROBACTERIUM-MEDIATED TRANSFORMATION OFPYRENOCHAETA LYCOPERSICI FOR INVESTIGATINGMOLECULAR DETERMINANTS OF VIRULENCE. M.Aragona, M.T. Valente and A. Infantino. CRA-PAV, Centro diRicerca per la Patologia Vegetale, Via C.G. Bertero 22, 00156 Ro-ma, Italy. E-mail: [email protected]

Pyrenochaeta lycopersici is a soil-borne fungus causing an eco-nomically important disease known as corky root rot on tomatoand other hosts. A gene coding for an extracellular endo-1,4-b-glucanase has recently been cloned and, in order to explore itsputative role in virulence, an ATMT (Agrobacterium tumefaciens-mediated transformation) system was established. P. lycopersicidoes not produce conidia in culture so the transformationmethod currently available for this species involves protoplasttransformation in presence of PEG. Despite of the low efficiency,a GUS-expressing transformant has been obtained and used forinvestigating the timing of infection and colonization of P. lycop-ersici inside tomato roots. To increase the transformation efficien-cy and to obtain a larger number of transformants for gene re-placement experiments, we tested both mycelium and protoplastsas recipients for an ATMT-based method. Preliminary resultsshowed that only the protoplasts but not the mycelium were suc-cessfully transformed by using the pRF-HU2 vector, harbouringpromoter and terminator genes from Aspergillus nidulans and hy-gromycin coding gene as fungal marker. The average transforma-tion frequency obtained with the first experiments was up to 30transformants per 107 protoplasts, all showing the DNA vectorinsertion. We are going to test the applicability of this techniquefor targeted gene replacement in P. lycopersici, after cloning theendo-1,4-b-glucanase sequences into the pRF-HU2 vector.

MONITORING FACTORS AFFECTING EPIDEMIOLOGYOF ALTERNARIA BROWN SPOT OF CITRUS. P. Bella1, M.Russo2, M. Tomasello2, V. Catara1 and A. Catara2. 1Dipartimentodi Scienze delle Produzioni Agrarie e Alimentari, Università degli

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Studi, Via Santa Sofia 100, 95123 Catania, Italy. 2Parco Scientificoe Tecnologico della Sicilia, Zona Industriale, Via V. Lancia, 95030Catania, Italy. E-mail: [email protected]

Alternaria brown spot of citrus, caused by Alternaria alterna-ta, causes considerable damage mainly to hybrids of Dancy tan-gerine. The fungus induces brown to black spots on leaves, fruitand young twigs resulting in reduced fruit yield and quality. Fol-lowing the studies carried out primarily in Florida, Brazil, Israeland Spain, disease epidemiology observations were performed inan orchard of cv. Fortune mandarin grafted onto Citrange insouthern Italy (38°09’06.82”N- 16°09’57.40”E). Surveys, carriedout over the last eight years (2003-2011), have shown that foreight months per year (March to November) temperatures aresufficient for infection, however the annual rainfall distributionof 600-1000 mm, favours infection in March-April (65-236 mm;mean 121 mm) and in September-October (121-462 mm; mean252 mm). Flower and fruit infections, occurring before fruit setand fruit growth, directly affect fruit yield and quality, whereasinfections of the leaves and twigs in September-October increasethe fungal inoculum, which produces heavier infections the fol-lowing year. In 2010, the percentage of fruit with symptoms was71% whereas in March 2011 it was 98%. After the heavy rains of2011 in the last two weeks of April (152 mm) the incidence of thedisease on twigs (20-25 cm) was 96% with a disease index (DI) of0.72. However, in cv. Fortune plants growing randomly in plotswith other citrus cultivars, only 53% of the shoots showed symp-toms and the DI was 0.16. The inoculum level, expressed as theaverage concentration of air-borne conidia, was 26 conidia/m3 inearly spring and decreased to 8 conidia/m3 in May.

FIRST REPORT OF IMPATIENS NECROTIC SPOT VIRUSINFECTING ISOTOMA AXILLARIS. M.G. Bellardi1, L. Cav-icchi2, M. Pirini Casadei2, V. Vicchi3 and G. Bozzano4. 1Diparti-mento di Scieze e Tecnologie Agroalimentari -Patologia Vegetale,Università degli Studi, Viale Fanin 42, 40127 Bologna, Italy.2Plesso Didattico “G. Scarabelli”, Università degli Studi di Bologna,Viale G. Ascari 17, 40026 Imola (BO), Italy. 3Servizio FitosanitarioRegione Emilia-Romagna, Via di Corticella 133, 40129 Bologna,Italy. 4Società Cooperativa “L’Ortofrutticola”, Via Dalmazia 169,17031 Albenga (SV), Italy. E-mail: [email protected]

Isotoma axillaris Lidt. (sin. Laurentia axillaris; also known asblue stars or rock isotome) is a perennial species of Campanu-laceae native to Australia, typically cultivated as annual in Liguria(northern Italy). Leaves are ovate or obvate in outline, with pin-natified lamina; solitary flowers blooming in April-May at the leafaxils. In 2011, severe symptoms resembling those caused bytospoviruses occurred in a farm located in the Albenga area(Savona). Small necrotic concentric rings and necrosis of the leaflamina were present in almost 7% of blooming plants grown out-doors. Twenty symptomatic plants collected during inspectionswere maintained in insect-proof greenhouse at Bologna Universi-ty. Virus was isolated by mechanical transmission to Chenopodi-aceae species. Symptomatic I. axillaris was negative for Tomatospotted wilt virus (TSWV) and positive for Impatiens necrotic spotvirus (INSV) in DAS-ELISA. To further confirm the presence ofINSV, total RNA was extracted and used for RT-PCR. Nucleotidesequences obtained were found to be closely related to publishedsequences (98% nucleotide identity with INSV, GenBank acces-sion Nos DQ523598, L20886, AB109100, and L20885). To date,INSV has been found in 1991 naturally infecting I. fluviatilis (sin.L. fluviatilis), another species of Isotoma, weed in Canada. Ourrecord is thought to be the first of natural occurrence of INSV inI. axillaris. Sueveys for the presence of thrips vectors of INSV on

the farm were conducted and many populations were found, sug-gesting that thrips are the main responsible for INSV spreadingand widening its natural host range.

FIRST REPORT OF HOSTA VIRUS X INFECTING HOSTAIN ITALY. M.G. Bellardi1, L. Cavicchi2 and S. Davino3. 1Diparti-mento di Scienze e Tecnologie Agroalimentari, Patologia Vegetale,Università degli Studi, Viale Fanin 42, 40127 Bologna, Italy.2Plesso Didattico “G. Scarabelli”, Università degli Studi di Bologna,Viale G. Ascari 17, 40026 Imola (BO), Italy. 3Dipartimento diScienze Entomologiche, Fitopatologiche, Microbiologiche agrarie eZootecniche, Sezione di Patologia Vegetale e Microbiologia Agraria,Università degli Studi, Viale delle Scienze edificio 5, 90128 Paler-mo, Italy. E-mail: [email protected]

Species of Hosta (Liliaceae) are herbaceous perennials moreoften grown for their foliage (blue, gold, green or variegated)than for their flowers. Virus diseases reported elsewhere in theword on cultivated hosta species include Hosta virus X (HVX),first described in USA in 1996, and considered to be the mainpathogen of this genus. In October of 2010, the following sys-temic virus-like symptoms were observed on the leaves of threevarieties cultivated in Emilia-Romagna (northen Italy), i.e. GoldStandard, Substance and Whirlwind: irregular green blotchesscattered on the yellow lamina, green bands running along theveins having an “ink-bleeding” look, malformations, different leafthickness. Electron microscopic observations revealed the pres-ence of virus particles ca. 560-600 nm in length, which in ISEMand PAS-ELISA tests clearly reacted with the antiserum to HVX(provided by University of Minnesota, MN, USA). Mechanicalinoculations carried out using symptomatic hosta leaf sap made itpossible to transmit HVX to Chenopodium murale L. (necroticpin-point lesions) and Gomphrena globosa L. (necrotic red spots2-3 mm in size). RT-PCR with specific primers HVXCP+ 5’-ATGGCAAGTGACGCACCAACTCCACC-3’ and HVXCP- 5’-TCAACTTGAGCCTTCCGGG-3’ amplified the expected frag-ment of 663 bp the coat protein gene. HVX appeared closely re-lated to a Polish isolate FJ821705 (98% nucleotide identity). Toprevent HVX spread, it is important to minimize mechanicalcontact between plants during cultivation, and immediately re-move those showing visible symptoms. Considering that two ofthe infected cultivars tested have been imported from TheNetherlands certified virus-free propagation material must be se-cured.

STREPTOMYCETES AS POSSIBLE BIOCONTROLAGENTS AGAINST SOIL-BORNE PATHOGENS. M. Bonal-di, A. Kunova, P. Sardi, P. Cortesi and M. Saracchi. Dipartimentodi Protezione dei Sistemi Agroalimentare e Urbano e Valoriz-zazione delle Biodiversità, Università degli Studi, Via G. Celoria 2,20133 Milano, Italy. E-mail: [email protected]

Streptomycetes are well known as soil and rhizosphere inhabi-tants. Their role as plant pathogens has been extensively studied,whereas their activity as endophytes, that could influence plantdevelopment and health, is less known. Streptomycetes can alsoact as plant growth promoters or as biocontrol agents againstsoil-borne pathogens. Our aim was to search for Streptomyces ac-tive against soil-borne pathogens. Twenty-six Streptomyces spp.strains were chosen among a collection of some hundreds actino-mycetes isolated from surface-sterilized roots of healthy plants.They were first tested in vitro against isolates of soil-borne phy-topathogenic fungi belonging to six genera: Alternaria, Botrytis,

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Colletotrichum, Fusarium, Rhizoctonia and Sclerotinia. The resultsshowed different patterns of antifungal activity, and the sevenmost bioactive strains, named AZ112I, CMJ58I, CMJ60I,ER08W, MRX13R, VT111I, ZEA17I, were used for seed-dressingof some species (e.g. onion, tomato, etc.). Their growth on seed-coats and root endophytic colonization were confirmed by micro-scopic observation and back isolation from surface-sterilizedroots. Treated seeds showed significant improved percent germi-nation, and lower saprophyte contamination and seedlings hadlonger main roots and more lateral roots than the untreated ones.

FAIRY RINGS BY KILLER FUNGUS AFFECT PLANT DI-VERSITY, SOIL QUALITY AND MICROBIAL FUNCTION-ALITY OF SPECIES-RICH GRASSLAND. G. Bonanomi1, G.Incerti3, A. Mingo1, S. Mazzoleni1 and M. Allegrezza2. 1Diparti-mento di Arboricoltura, Botanica e Patologia Vegetale, Universitàdegli Studi di Napoli Federico II, Via Università 100, 80055 Portici(NA), Italy. 2Dipartimento di Scienze Ambientali e delle Pro-duzioni Vegetali, Università Politecnica delle Marche, Via BrecceBianche, 60131 Ancona, Italy, 3Dipartimento di Scienze della Vita,Università degli Studi, Via Giorgieri 10, 34127 Trieste, Italy. E-mail: [email protected]

More than 50 species of basidiomycetes can produce the socalled fairy rings through networks of mycelia radially growing be-low ground. In this study we addressed the following questions:do the fairy rings by Agaricus campestris affect the spatial distribu-tion of coexisting plant species? Is the ring development related tochanges of soil physical, chemical, and microbiological properties?Fairy rings located in a species-rich grassland were monitored forplant biomass, species richness and composition. Seventeen soilparameters were analysed including physico-chemical (water hold-ing capacity, pH, electrical conductivity, organic C, P, total N,NH4

+, NO3-, hydrophobicity and cyanide concentration), and mi-

crobiological properties (enzymatic activity by FDA, community-level physiological profile by BIOLOG EcoPlates™, microbialbiomass, fungal mycelium, culturable actinomycetes, bacteria andfungi). A bioassay was performed to evaluate the response of threecoexisting species (Bromus erectus, Cynosurus echinatus and Cen-taurea ambigua) growing on soils collected from fairy ring under-neath four different concentric belts. Plant species compositiondramatically changed in response to fairy ring development withdisappearance of grassland species from the most external belt.Profound changes of soil properties were found after fungal pas-sage with consistent reduction of C and N contents and increasesof FDA and microbial functional diversity. Soil from the fungalzone showed remarkable increases of mineral N forms, electricalconductivity and hydrophobicity. The bioassay showed species-specific responses to different soil types. This study provides evi-dence that the spread of fairy ring fungi, by killing dominantperennial plants, creates empty niches for many short-livedspecies, thus allowing coexistence.

BIOCHEMICAL CHANGES OF ORGANIC MATTER EX-PLAIN FUNGAL SAPROPHYTIC ABILITY: EVIDENCEFROM SOLID STATE 13C-CPMAS NMR SPECTROSCOPY.G. Bonanomi1, M. Capodilupo1, G. Incerti2, V. Lanzotti3, F.Scala1 and S. Mazzoleni1. 1Dipartimento di Arboricoltura, Botani-ca e Patologia Vegetale, Università degli Studi di Napoli FedericoII, Via Università 100, 80055 Portici (NA), Italy, 2 Dipartimento diScienze della Vita, Università degli Studi, Via Giorgieri 10, 34127Trieste, Italy, 3Dipartimento di Scienza degli Alimenti, Universitàdegli Studi di Napoli Federico II, Via Università 100, 80055 Portici

(NA), Italy. E-mail: [email protected]

Soil-borne fungal pathogens are among the major limiting fac-tors for the productivity of agro-ecosystems. However, the ecolo-gy of the saprophytic life phase and the role of fungal ecologicalsuccession in the process of pathogenesis are still poorly under-stood. By a litter-bag decomposition experiment, we investigatedthe saprophytic ability of seven fungal species (Aspergillus niger,Botrytis cinerea, Ganoderma lucidum, Mucor sp., Pythium ulti-mum, Trichoderma harzianum and Umbelopsis sp.) grown over 45organic matter types decomposing for 180 days. Organic mattersubstrates were characterized throughout the decompositionprocess by classic proximate chemical analyses (total C and N, la-bile C, cellulose and lignin content) and, at molecular level, bysolid state 13C-CPMAS NMR. The saprophytic ability of allspecies decreased during the decomposition process over all or-ganic matter types, being more rapid for A. niger, B. cinerea andMucor sp.. This finding is consistent with the early disappearanceof such species during fungal succession. The biochemical qualityof organic matter progressively decreased during decomposition,both considering proximate parameters and C types correspon-ding to spectral regions from 13C-CPMAS NMR. The latter werealso consistently predictive of fungal growth over the 45 sub-strates. Our results support the hypothesis that changes in organ-ic matter quality controls the dynamics of fungal succession.

DEGRADATION OF ELASTO-MECHANICAL PROPRI-ETIES OF WOODEN POLES USED IN SOIL BIOENGI-NEERING STRUCTURES DUE TO WOOD-DECAY FUN-GI. L. Bosso1, G. Menegazzi2, M. Brigante3, G.B. Chirico4, G.Cristinzio1 and N. Romano4. 1Dipartimento di Arboricoltura,Botanica e Patologia Vegetale, Università degli Studi di Napoli“Federico II”, Via Università 100, 80055 Portici (NA), Italy. 2ParcoNazionale del Vesuvio, Via Palazzo del Principe, 80044 Ottaviano(NA), Italy. 3Dipartimento di Ingegneria Strutturale, Universitàdegli Studi “Federico II”, Via Claudio 21, 80125 Napoli, Italy. 4Di-partimento di Ingegneria Agraria e Agronomia del Territorio, Uni-versità degli Studi di Napoli “Federico II”, Via Università 100,80055 Portici (NA), Italy. E-mail: [email protected]

Wood is degraded by a variety of organisms. The major agentsfor wood degradation are the fungi, especially those belonging tothe “wood-decay” group. These fungi produce lignocelluloses en-zymes that degrade cell wall components causing a variation ofsome properties of the wood. This paper discusses the results ofan experiment carried out on chestnut wooden poles collectedfrom soil bioengineering structures in some areas of the VesuvioNational Park (Naples, Southern Italy). We analyzed the effectsexerted on the decay in wood physical and mechanical character-istics by fungi belonging to the following four families: Meruli-aceae, Strophariaceae, Polyporaceae and Corticiaceae. Significantcorrelations were found between the presence of wood-decayfungi (in percent), wood moisture content and ultimate stress.Specifically, conditions of large fungi attack and high wood mois-ture can affect even severely the strength of a wood element andhence the behavior of the entire structure.

FUNGI AND BACTERIA IN THE BIOLOGICAL CON-TROL OF CHESTNUT INK DISEASE. L. Bosso1, T. Marras2,C. Caprari2, G. Naclerio2 and G. Cristinzio1. 1Dipartimento di Ar-boricoltura, Botanica e Patologia Vegetale, Università degli Studi diNapoli “Federico II”, Via Università 100, 80055 Portici (NA), Italy.2Dipartimento di Scienze e Tecnologie per l’Ambiente e il Territo-

Journal of Plant Pathology (2011), 93 (4, Supplement), S4.25-S4.62 S4.27


rio, Università degli Studi del Molise, Contrada Fonte Lappone,86090 Pesche (IS), Italy. E-mail: [email protected]

Ink disease, one of the most dangerous diseases of chestnuthas been reported from many European countries, from Portugalto Romania. Two species of Phytophthora are responsible for inkdisease, P. cambivora and P. cinnamomi. In this work we carriedout some in vitro experiments, as reduction of radial growth andmycelial dry weight, of these two Oomycetes, using two fungi be-longing to genus Byssochlamys and two bacteria belonging togenus Bacillus, all isolated from agricultural and forestry soil. Ourresults showed a high reduction in radial growth and mycelial dryweight by both fungi and bacteria. The Byssochlamys strains in-hibited the radial growth of P. cambivora and P. cinnamomi by ca.80% and ca. 83%, respectively, whereas Bacillus inhibited P.cam-bivora and P.cinnamomi. by ca. 83% and ca. 73%, respectively.Mycelial dry weight reduction was also remarkable, i.e.Byssochlamys by ca. 90% for both Phytophthorae while Bacillusby ca. 60% for P. cambivora and ca. 50% for P. cinnamomi.

ENDOPHYTIC BACTERIAL DIVERSITY ASSOCIATEDWITH GRAPEVINE PLANTS AND THEIR POTENTIALAPPLICATIONS. D. Bulgari1, P. Casati1, F. Quaglino1, P.A.Bianco1,2 and F. Faoro1,2. 1Dipatimento di Protezione Vegetale-Pa-tologia Vegetale, Università degli Studi, Via Celoria 2, 20133 Mi-lano, Italy. 2Istituto di Virologia Vegetale del CNR, UOS Milano,Via Celoria 2, 20133 Milano, Italy. E-mail: [email protected]

Endophytic bacteria can be defined as those colonizing the in-terior of plants without inducing disease. Though their relation-ship with the host is not well understood, they may reduce dis-ease severity by activating systemic resistance, antibiosis, compe-tition of nutrients and niches. Due to these abilities, bacterial en-dophytes are candidates for biological control of plant diseases.To identify potential biocontrol agents of phytoplasmas, endo-phytic bacterial communities associated with healthy, phytoplas-ma-diseased and recovered grapevine were characterized by culti-vation-dependent and independent methods. Endophytes wereisolated directly from grapevine leaf metagenome by an opti-mized DNA extraction methods. Sequencing of 16S rRNA genelibraries highlighted the presence of five operational taxonomicunits (OTUs) showing best sequence matches with g-Proteobac-teria, family Enterobacteriaceae, with a dominance of the genusPantoea. Furthermore, the genera Bacillus, Stenotrophomonas,Methylobacterium, Pectobacterium, Enterobacter, Brevundimonase Agrobacterium, Brevibacillus, Staphylococcus e Sphingomonaswere identified by cultivation-dependent methods. Some of thesebacteria, particularly Pantoea agglomerans are already known asbiocontrol agents of several plant diseases. P. agglomerans was al-so localized in grapevine leaf tissues by fluorescence in situ hy-bridization (FISH) and immunomicroscopy. In detail, this bac-terium was detected in the phloem, xylem and parenchyma ofgrapevine leaf veins, where it could play a role in modulatingphytoplasma infection and recovery.

MYCOTOXIN REDUCTION IN MAIZE AND WHEAT: APREDECTIVE APPROACH. M. Camardo Leggieri1, V. Rossi1,A. Logrieco2 and P. Battilani1. 1Istituto di Entomologia e PatologiaVegetale, Università Cattolica del Sacro Cuore, Via Emilia Par-mense, 84, 29100 Piacenza, Italy. 2Istituto di of Sciences of FoodProduction, del CNR, Via Amendola 122/0, 70126 Bari, Italy. E-mail: [email protected]

MYCORED is a 7° FP large collaborative project funded byEC aimed at developing strategic solutions to reduce mycotoxincontamination in economically important food and feed chains.Wheat and maize are important crops considered in the projectdue to relevance for human and animal consumption and as theycause major mycotoxin safety concerns worldwide. In particular,deoxynivalnol (DON) and zearalenone (ZEA) are secondarymetabolites associated with Fusarium head blight (FHB), a wheatear disease caused by a complex of Fusaria. Fumonisins (FUM)and aflatoxins (AFs), whose main producers are respectivelyFusarium verticillioides and Aspergillus flavus, are crucial prob-lems in maize. A specific work package in MYCORED is aimedat developing and validate predictive models for these relevantfungi to forecast the risk of mycotoxin contamination in differentgeographic areas and years, mainly based on meteorological con-ditions. A rational approach was elaborated and adopted by allcountries involved (Italy, United Kingdom, Hungary, Russia,Egypt, Mexico, Nigeria, Argentina) in order to collect samplesand data useful for models validation. Specific protocols were de-veloped for crop sampling at harvest, appropriate questionnaireswere prepared to collect cropping system data, and suitable sta-tions were selected for meteorological data. A data base was pre-pared and data storage is ongoing. A web access to predictivemodels is under development. Based on the first 2-year data(2009-2010), FHB model validation gave encouraging results,with good predictions in all the countries involved. More effortsare requested regarding maize, because the contribute of meteor-ological data seems not sufficient for a reliable prediction ofFUM and AFs contamination at harvest.

This work was supported by EC KBBE-2007-222690-2 MY-CORED. Marco Camardo Leggieri carried out this work within thePhD School “Agrisystem” of the Università Cattolica del SacroCuore (Italy).

OCCURRENCE OF TOMATO SPOTTED WILT VIRUS ONTOBACCO IN CAMPANIA. R. Carrieri1, R. Sorrentino2, E.Lahoz1 and D. Alioto2. CRA, Unità di Ricerca per le Colture Al-ternative al Tabacco, Via P. Vitiello 108, 84018 Scafati (SA), Italy.2Dipartimento di Arboricoltura, Botanica e Patologia Vegetale,Università degli Studi di Napoli “Federico II”, Via Università 100,80055 Portici (NA), Italy. E-mail: [email protected]

During a survey on the sanitary status of tobacco carried outin Campania (southern Italy) in 2010, Tomato spotted wilt virus(TSWV) was detected on Burley tobacco cv. F 3119. The analyseswere performed by DAS-ELISA with a specific polyclonal anti-serum. Symptoms appeared as severe necrosis on the stem andleaves and death of the plant. Total RNA was extracted fromleaves with the SV total RNA isolation system kit and used astemplate for RT-PCR, employing primers specific to the TSWVnucleocapsid (N) and movement protein of the middle segment(NSm) genes. The amplified fragments (754 and 670 bp, respec-tively) were aligned and edited using BlastN. The analysis of the754 bp N fragment showed the closest identity withp105/2006RB strain (DQ915946), identified on pepper in Italy,in 2006 (99.9% nucleotide identity). The 670 bp fragment, corre-sponding to NSm gene, showed the closest identity with thePED-1 isolate identified on pepper in Campania in 2009 (99.4%nucleotide identity). Phylogenetic analysis, based on the multiplealignments of NSm gene sequences of the tobacco isolate fromCampania and those present in databases, revealed that the viralisolate under study belongs to A-like subgroup. A-like isolateswere also identified in TSWV-infected lettuce and chrysanthe-mum from Campania. These findings, together with the previous



report from pepper, seem to indicate a threatening spread ofTSWV isolates A-type in Campania, even though they do notseem to belong to the resistance-breaking (RB) biotype, as evi-denced by the analysis of amino acids at positions 118 and 120 ofNSm protein.

GENETIC CHARACTERIZATION OF SCLEROTIUMROLFSII ISOLATES FROM SEVERAL ORNAMENTALCROPS IN SICILY. R. Carrieri1, A. Carella1, R. Caiazzo2, G.Polizzi3 and E. Lahoz1. 1CRA, Unità di Ricerca per le Colture Al-ternative al Tabacco, Via Pasquale Vitiello 108, 84018 Scafati (SA),Italy. 2Tree Fruit Research and Extension Center, Washington StateUniversity 1100 N Western Ave, Wenatchee WA 98801, USA. 3Di-partimento di Gestione dei Sistemi Agroalimentari e Ambientali,Sez. Patologia vegetale, Università degli Studi, Via S. Sofia 100,95123 Catania, Italy. E-mail: [email protected]

Sclerotium rolfsii is one of the most devastating plant patho-genic fungi throughout the world. It causes southern blight dis-ease in a wide variety of agricultural and horticultural crops. Re-cently, southern blight has caused large losses in ornamentalplants and turfs in eastern Sicily (southern Italy). A previous re-port showed that 37 isolates of S. rolfsii, collected from variousornamental hosts in eastern Sicily over two years, varied consider-ably in some morphological features: growth rate at differenttemperatures, number of sclerotia per plate, dry and freshweight. Moreover, based on pairings among isolates to establishmycelial compatibility, five vegetative compatibility groups(VCGs) were found that showed significant differences in patho-genicity. On the contrary, polygalacturonase patterns showed novariations within the isolates. In the present work, the geneticvariability among the 37 Sicilian isolates is investigated. GenomicDNA was purified and analyzed by PCR and analysis of the se-quence of ITS1-5.8S-ITS2 ribosomal subunit region and elonga-tion factor-1α gene. The purpose of this study was to detect apossible relationship between genetic diversity and pathogenicityof specific VCGs of the analyzed S. rolfsii isolates. The study ofvariability of S. rolfsii strains in a particular geographical area isimportant for documenting the origin of isolates and the changesoccurring in the population.

INFECTION PATTERNS OF FUSARIUM OXYSPORUM f.sp. MELONIS IN MELON PLANTS. V. Catalano1, L. Luongo1,A. Haegi1, M. Scotton2, N. Ficcadenti3 and A. Belisario1. 1CRA,Istituto Sperimentale per la Patologia Vegetale, Via C.G. Bertero22, 00156 Roma, Italy. 2Dipartimento di Agronomia Ambientale eProduzioni Vegetali, Università degli Studi di Padova, Viale dell’U-niversità 16, 35020 Legnaro (PD), Italy. 3CRA, Unità di Ricercaper l’orticoltura, Via Salaria 1, 63030 Monsampolo del Tronto(AP), Italy. E-mail: [email protected]

Fusarium wilt caused by Fusarium oxysporum f. sp. melonis(FOM) is the most important and widespread infectious diseaseof melon. The four known races of this pathogen can be distin-guished only by infection on appropriate cultivars. At the mo-ment, no molecular tools are available to discriminate races, andthe disease progression is poorly understood. Resistance to races1 and 2 is controlled by a single dominant gene, whereas onlypartial polygenic resistance to race 1,2 has been reported. Theanalysis of the colonization patterns of FOM race 1 and FOMrace 1,2, the two main races present in Italy, was performed incompatible and incompatible binomials to identify differences incolonization along the stem in consideration to the type of resist-

ance. A systematic reisolation procedure on infected stems cou-pled with PCR assays allowed to delineate the fungal growthalong the stem. Moreover, a quantitative assay was set up in orderto estimate the amount of the pathogen both in compatible andincompatible host-pathogen combinations, e.g. with cv.Charentais FOM-2-inoculated as either FOM race 1 or race 1,2.

TEMPERATURE RESPONSE OF BOTRYOTINIA FUCKE-LIANA SUB-POPULATIONS. N. Ciliberti, S.E. Legler, T. Caffi,L. Languasco and V. Rossi. Istituto di Entomologia e PatologiaVegetale, Università Cattolica del Sacro Cuore, Via Emilia Par-mense 84, 29122 Piacenza, Italy. E-mail: [email protected]

Botryotinia fuckeliana (de Bary) Whetzel (anamorph: Botrytiscinerea Pers.:Fr.), the causal agent of grapevine grey mould, hastwo sub-populations (transposa and vacuma) with different com-binations of the transposable elements Boty and Flipper: trans-posa has three combinations (Boty+Flipper+; Boty+Flipper-; Bo-ty-Flipper+), and vacuma lacks transposable elements (Boty-Flip-per-). The effect of temperature (5 to 30°C) was determined for arepresentative isolate of each of the four combinations. The iso-lates were obtained from the culture collection of the Universityof Bari. Colony growth rate was lower for the vacuma isolate thanthe transposa isolates at 30°C but the opposite was true at 5°C.Between 10 and 25°C, colony growth was similar for all four iso-lates. Only the transposa isolates produced sclerotia between 5and 20°C, and produced more conidia than the vacuma isolate,with an optimum at 15 and 20°C. Conidial germination after 24 hwas lower for the vacuma isolate than for the transposa isolates at5 and 10°C, did not differ among isolates between 15 and 25°C,and was lower for the Boty+Flipper+ isolate at 30°C. Inoculationof bunches at different growth stages (inflorescence swelling,flowering, groat-sized berries, ripening, and maturation) indicat-ed that the incidence of vacuma isolate was low before floweringwhile the it was very high and similar for all isolates at flowering.Occurrence of latent infection in berries was also investigated.The observed differences suggest that different sub-populationsof B. fukeliana require different infection models.

SPREAD OF AN ATYPICAL POPULATION OFPSEUDOMONAS SAVASTANOI ON MYRTUS sp. IN SAR-DINIA. T. Cinelli1, V. Ligios2, G. Marchi1, L. Mugnai1 and M.Fiori2. 1Dipartimento di Biotecnologie Agrarie, Sezione di Pro-tezione delle Piante, Università degli Studi di Firenze, Piazzaledelle Cascine 28, 50144 Firenze, Italy. 2Dipartimento di Protezionedelle Piante, Facoltà di Agraria, Università degli Studi di Sassari,Via E. De Nicola, 07100 Sassari, Italy. E-mail: [email protected]

Virulent strains of the phytopathogenic bacteriumPseudomonas savastanoi induce parenchymatous galls (knots) atthe site of infection. Knots are mostly seen on the young stems,branches and twigs of different plants belonging to Oleaceae(Olea, Jasminum, Forsythia, Phillyrea, Ligustrum), Fabaceae (Re-tama), Rhamnaceae (Rhamnus), Myrtaceae (Myrtus) or to Apoci-naceae (Nerium, Mandevilla). On ash (Fraxinus spp.) P. savastanoicauses wart-like excrescences. During surveys carried out in thespring 2010 in six locations of north-western Sardinia (insularItaly) on different selections of myrtle (Myrtus communis) and al-so on spontaneous plants, canker-like lesions of about 1 cm longwere observed on branches and twigs. From the tissues below thebark, which appeared dark-brown, dried and cracked, a bacteri-um was constantly isolated that, based on the results of biochemi-cal tests and molecular typing methods, was identified as



Pseudomonas savastanoi. Pathogenicity tests were performed with2 isolates by placing a bacterial suspension containing 108

CFU/ml on wounds made in the bark of twigs of Myrtus commu-nis var. tarentina. After 4 to 6 weeks the centre of the wound wassurrounded by an irregular mound of new tissue. Occasionally af-ter 2-3 months these newly formed tissues had joined togetherover the surface of the wound. Typical “knots” were never ob-served. Further characterization is now in progress to better eval-uate the differences that exist between the myrtle population of P.savastanoi found in Sardinia and knot inducing strains isolatedfrom Myrtus sp. in other countries of the Mediterranean basin.

ARMILLARIA MELLEA HYPHAL WALL POLYSACCHA-RIDES ELICIT PHENOL ACCUMULATION AS A DEFEN-SIVE RESPONSE IN GENISTA MONOSPERMA. F.Clematis1, S. Fascella1, B. Cangelosi1, J. Tedeschini2, M. Dolci3and P. Curir1. 1CRA, Unità Ricerca Floricoltura e Specie Ornamen-tali, Corso Inglesi 508, 18038 Sanremo, Italy. 2Agriculture Univer-sity of Tirana, Durres, Albania. 3Dipartimento di Valorizzazione eProtezione delle Risorse Agroforestali. Università degli Studi diTorino, Via L. da Vinci 44, 10025 Grugliasco (TO), Italy. E-mail:[email protected]

Polysaccharides obtained as a crude wall extracts (CWE) fromArmillaria mellea hyphae grown in vitro proved their ability totrigger a defensive response in the host plant Genista monosper-ma. Experiments were performed on 3-month-old herbaceousplants treated for 48 h with various concentrations of depro-teinated CWE. This latter consisted of proteins, neutral carbohy-drates, amino sugars and ashes. G. monosperma tissues, elicitedby CWE, displayed a defensive response consisting both in aquantitative increase of vacuolar phenol accumulation and inthickening of parenchyma cell walls, which could be appreciatedby microscopic examination. Histochemical analyses disclosedthat phenolic acids (e.g. ferulic acid) were the main responsibleof plant cell wall thickening, due to a progressive molecule en-crustation by esterification. Our data confirm therefore the im-portance of phenolics in the plant response to fungal attacks and,moreover, they indicate that hyphal polysaccharides may behaveas elicitors of the G. monosperma defensive response.

A TRANSCRIPTOMIC APPROACH TO UNDERSTANDINDUCED RESISTANCE TO BACTERIAL CANKER OFSTONE FRUITS ELICITED THROUGH TREATMENTSWITH COPPER GLUCOHUMATES. D. Dallai1, L. Dondini.2and E. Stefani1. 1Dipartimento di Scienze Agrarie e degli Alimenti,Università degli Studi di Modena e Reggio Emilia, Via Amendola2, 42122 Reggio Emilia, Italy. 2Dipartimento di Colture Arboree,Università di Bologna, viale G. Fanin 46, 40127 Bologna, Italy. E-mail: [email protected]

Bacterial canker of stone fruits, caused by Xanthomonas arbori-cola pv. pruni, is a recurrent disease in Italian peach and plum or-chards. Several field and glasshouse trials on peach and plum weredone, with the aim to effectively control the disease by using somenovel molecules, such as glucohumates. The most remarkable re-sults were obtained with copper glucohumates (with a reductionof the disease by ca. 80%). Results are very promising and suggestthe possibility to implement effective control strategies, wherecopper compounds and novel molecules are both used in com-mercial orchards. In order to study and understand the effect ofthe biomolecules used, untreated and glucohumate-treated peachplants were subject to further molecular analyses in order identify

possible genes/sequences involved in the induction of disease re-sistance. A transcriptomic approach was developed for detectingthe transcripts present in plant tissues, after elicitation of an in-duced protection state. Total RNA was extracted, retro-tran-scribed and c-DNA-AFLP was done to identify different sequencefingerprints in the protected plant tissue. Discrimination of newlyexpressed sequences was performed with DHPLC, and compari-son of transcripts was done on the complete peach genome data-base in order to identify the genes or sequences involved in theelicitation of induced resistance. Preliminary data showed, intreated plants, the presence of nine putative genes like a putativesenescence protein, already described as being involved in the in-duced resistance to fire blight in other Rosaceae. Thus, copperglucohumates might be considered possible candidates for theelicitation of resistance to bacterial diseases.

ULTRASTRUCTURAL MODIFICATIONS BY CHALARAFRAXINEA IN COMMON ASH. E. Dal Maso, G. Fanchin, S.Mutto Accordi, L. Scattolin and L. Montecchio. DipartimentoTerritorio e Sistemi Agro-Forestali, Università degli Studi di Pado-va, Viale dell’Università 16, 35020 Legnaro (PD), Italy. Email:[email protected]

Chalara fraxinea, responsible for ash dieback, is present inmany European countries where it constitutes a severe threat notonly to forests, but also to urban areas and nurseries. To examinethe C. fraxinea infection strategies in common ash tissues, an ul-trastructural investigation (ESEM, TEM) was performed. Artifi-cial infections in wounded stems demonstrated that C. fraxineadevelops intracellularly, moving through the cells and colonizingeasily wood cortex, phloem, paratracheal parenchyma andparenchymatic rays. The possible involvement of enzymes or tox-ins in parenchymatic cells disruption are discussed.

SUPPRESSIVE EFFECT OF EXPLODED BIOMASS OFARUNDO DONAX AND WHEAT STRAW AGAINST SOIL-BORNE PLANT PATHOGENS. U. De Corato1, N. Sharma2,O. Maccioni2 and E. Viola3. 1Unità Efficienza Energetica dell’E-NEA, Servizio Agricoltura, Ufficio Territoriale di Bari, Via R. DaBari 119, 70122, Bari, Italy. 2Unità Tecnologie Trisaia dell’ENEA,Laboratorio di Biotecnologie, Centro Ricerche Trisaia, s.s. 106 Joni-ca Km. 419.500, 75026 Rotondella (MT), Italy. 3Unità TecnologieTrisaia dell’ENEA, Laboratorio di Biomasse e Solare Termico, Cen-tro Ricerche Trisaia, s.s. 106 Jonica Km. 419.500, 75026 Rotondel-la (MT), Italy. E-mail: [email protected]

A suppressiveness of exploded biomasses of Arundo donaxand wheat straw, a renewable energy sources commonly used forindustrial production of ethanol by steam-explosion, was testedin a greenhouse against soil-borne plant pathogens, with respectto exploded biomass of Miscanthus sinensis and commercial com-post. The tests were performed at three doses (10, 20 and 30% ofpotting mix) on seven horticultural pathosystems (cucumber/ Pythium ultimum, tomato/Phytophthora nicotianae, bean/Rhizoc-tonia solani, fennel/Sclerotinia sclerotiorum, eggplant/Verticilliumdahlie, melon/Fusarium oxysporum f.sp. melonis and lettuce/Fusarium oxysporum f.sp. lactucae). A disease suppression wasevaluated by index Ds=[(Np–Ncb)/Np]×100 and analyzed byTukey’s HSD test (P<0.05). Suppressiveness of M. sinensis rangedfrom 97% in eggplant/V. dahliae to 25% in lettuce/F. oxysporumf. sp. lactucae. It was higher of the compost and wheat straw in allthe pathosystems and of A. donax in two pathosystems. A sup-pressiveness of A. donax ranged from 85% in eggplant/V. dahliae



to 18% in lettuce/F. oxysporum f. sp. lactucae, and it was higherof the compost and wheat straw in five pathosystems. A wheatstraw suppressiveness ranged from 45% in eggplant/V. dahliae to13% in melon/F. oxysporum f. sp. melonis. It was lower of M.sinensis in all pathosystems, of A. donax in six pathosystems andof the compost in five pathosystems. In conclusion, we confirmthat the exploded of Miscanthus is the most suppressive on allpathosystems at the highest dose tested. Moreover, theeggplant/V. dahliae and lettuce/F. oxysporum f. sp. lactucae result-ed more and less sensitive to a suppressive effect, respectively.

ACTIVITY OF ELECTROLYSIS IN REDUCING MICRO-BIAL POPULATION AND CONTROLLING POSTHAR-VEST DISEASES OF CITRUS FRUITS. F. Fallanaj1, S.M. San-zani1, C. Zavanella2 and A. Ippolito1. 1Dipartimento di Biologia eChimica Agro-forestale ed Ambientale, Università degli Studi “Al-do Moro”, Via Amendola 165/A, 70126 Bari, Italy. 2AdamantTechnologies SA Eplatures-Grise 17, 2300 La Chaux-de-Fonds,Switzerland. E-mail: [email protected]

Electrolysis has recently gained particular attention in thefood industry as a novel technology for preventing fruit infectionin the postharvest environment. Furthermore, the application ofelectrolyzed oxidizing (EO) water as alternative to chemicalfungicides against fruit spoilage gave also promising results. Thepresent investigation was carried out to evaluate the potential ap-plication of electrolysis in reducing the main postharvest rots ofcitrus fruits. Preliminary laboratory-scale trials with wash waterfrom different packing houses were performed. The water wasadded with a conidial suspension of Penicillium digitatum (105

CFU/ml) and subjected to electrolysis. Microbiological evalua-tion of P. digitatum vitality in orange fruit wash water showednearly 100% inhibition of spore germination after 30 min, where-as, the complete suppression of the fungal population in lemonwash water was observed after 15 min of electrolysis. On the ba-sis of these preliminary results, further trials were conducted intwo packing houses located in Sicily (insular Italy). During thecommercial trials, fruits were dipped in electrolyzed water toevaluate its ability to prevent infections during storage and shelf-life. Treatment of wounded fruits with electrolyzed water com-pared with tap water, reduced the incidence of infection by 70%after 15 days storage at room temperature. These results suggestthat the application of electrolysed water in conventional post-harvest treatments may increase citrus fruit shelf-life, particularlygiven the current need for alternatives in the global context of or-ganic and integrated disease management strategies.

INFLUENCE OF LIGHT ON GROWTH, CONIDIATIONAND FUMONISIN PRODUCTION BY FUSARIUM VERTI-CILLIOIDES. F. Fanelli1, M. Schmidt-Heydt2, M.Haidukowski1, A. Susca1, R. Geisen2, A. Logrieco1 and G.Mulè1. 1Istituto di Scienze delle Produzioni Alimentari del CNR,Via Amendola 122/0, 70126 Bari, Italy. 2Max Rubner Institut, De-partment for Safety and Quality of Fruit and Vegetables, Haid-und-Neu-Strasse 9, 76131 Karlsruhe, Germany. E-mail: [email protected]

Light is a very important signal for fungi since it influencesmany different physiological responses. We analyzed the influ-ence of light of varying wavelength and intensity on the growth,conidiation and fumonisin B1 (FB1), B2 (FB2) and B3 (FB3)biosynthesis by Fusarium verticillioides ITEM 10027. Wave-lengths from both sides of the spectrum, from red (627 nm) to

blue (470-455 nm), had a stimulating effect on the growth and fu-monisin production, with an increase of up to 150% of total fu-monisins analyzed, compared with dark incubation. If the intensi-ty of the 455 nm blue light was increased, fumonisin biosynthesiswas reduced. Furthermore the incubation under a short waveblue light (390 nm) had an inhibitory effect on the fungal growthand the fumonisin production was reduced by 85%. White puls-ing light had an inhibitory effect on fumonisin production, with areduction of about 50%, but not on the fungal growth, compara-ble with the dark incubation. The regulation of fumonisin pro-duction was analyzed by real time RT-PCR, measuring the ex-pression level of fum1, fum21 and fvVE1 transcripts, that encodeprotein involved in fumonisin biosynthesis, revealing a correla-tion between gene expression and fumonisin production.

Work supported by EC KBBE-2007-222690-2 MycoRed

SERIOUS OUTBREAK OF BACTERIAL CANKER OFTOMATO IN SOUTHERN ITALY. A. Fanigliulo1, A.Viggiano1, G. Piegari1 and A. Crescenzi2. 1Bioagritest Srl, ZonaPIP lotto E2, 85010 Pignola (PZ), Italy. 2Dipartimento di Biologia,Difesa e Biotecnologie Agro-Forestali, Università degli Studi dellaBasilicata, Viale dell’Ateneo Lucano, Campus Macchia Romana3A310, 85100 Potenza, Italy. E-mail: [email protected]

Clavibacter michiganensis subsp. michiganensis (Cmm) is thecausal agent of canker and wilt disease of tomato. This diseasecauses severe economic losses that may become devastating totomato production. Cmm bacteria penetrate the plant throughwounds or natural openings, find their way into the xylem, anddevelop a massive systemic infection. Typical disease symptomsinclude wilting of the leaves, leaflet necrosis, development ofcanker lesions on the stem and “Bird’s eyes” on fruits (spots withraised brown centers surrounded by an opaque white halo). Dur-ing summer 2010, an outbreak of bacterial canker was observedin tomato fields in the main tomato-growing area of Apulia andBasilicata (south-east Italy). The disease was observed starting 20to 30 days from transplant and reached at the beginning of ripen-ing, a 100% incidence, with complete crop loss. Detection assayswere performed on symptomatic plants in order to ascertain theetiological agent. The diagnostic procedure comprised isolationfrom infected tissues, presumptive diagnosis with a rapid test,identification of presumptive isolates and determination of path-ogenicity (2005 OEPP/EPPO, Bullettin OEPP/EPPO Bullettin35, 271-273. PM 7/42). All symptomatic plants were infectedwith the bacterium under investigation. They all belonged to thetomato cv. Uno Rosso, deriving from the same tomato seed batch.This let us to believe that bacterial infection was associated withtomato seed.

EVALUATION OF THE EFFICACY OF TANNING PROD-UCTS ON THE PRODUCTS’ DISTRIBUTION ON THESEED SURFACE AND DISPERSION OF THE ACTIVESUBSTANCE AT VARIOUS STAGES OF MANIPULATIONOF TANNED SEED. A. Fanigliulo1, A. Viggiano1, V. Filì1 andA. Crescenzi2. 1Bioagritest Test Facility, Bioagritest Srl. Zona PIPlotto E2. 85010 Pignola (PZ), Italy. 2Dipartimento di Biologia,Difesa e Biotecnologie Agro-Forestali, Università degli Studi dellaBasilicata. Viale dell’Ateneo Lucano Campus Macchia Romana3A310, 85100 Potenza (PZ), Italy. E-mail: [email protected]; [email protected]

A study was carried out in 2010 by the Bioagritest test facility



with the purpose of evaluating the efficacy of tanning productson the product distribution on the surface of the seed and disper-sion of the active substance at various stages of the manipulationof tanned seed. Seeds under investigation were those of Zea mais,the tanning product was a formulate whose a.i. was Clothianidin.The formulate was compared with the standard Poncho 600 FS(treatment 1) or used with the addition of: (i) fungicide and adju-vant (ii) fungicide, (iii) fungicide, adjuvant and glue. An aliquotof 333 grams of tanned seed from each treatment, in 3 replicates,plus the untreated control were subjected to the following: themass of seeds was subjected to stripping at the amplitude of 1.20mm/g for three different time intervals (45, 90 and 180 min) inappropriate disposable containers. The amplitude of 1.20 mm/gsimulates very strong stripping conditions. This choise was madein order to simulate the extreme (overestimated) conditions towhich the seeds may be subjected after tanning. The powder wasthen re-suspended in an appropriate buffer and the amount ofactive ingredient released and/or dispersed was determined, us-ing a method developed in this study. This method allows thequantitative determination of clothianidin by LC-DAD (liquidchromatography with photodiode array detection) in plastic con-tainers (for tanned seeds): the sample extracted with appropriatesolvent and diluted in mobile phase was filtered and then ana-lyzed in LC-DAD in reversed phase, in a chromatography col-umn C18 type, and the signal was read at 266 nm. The work wasplanned, performer and recorded according to UNI CEI EN ISOIEC 17025 and EN ISO 900: 2008 standards. The treatmentswere applied using electronic vibrators, at programmed speedand times, in disposable containers. The three intervals analyzedwere the simulation of the three main phases of seed manipula-tion: packaging, transport and sowing by seeder with hopper.The choice of the three time intervals was also a tools to assessthe tightness of the tanning product on the surface of the tannedseed. For the evaluation of the distribution of the active ingredi-ent on the surface of tanned seed, 50 tanned seeds for each of the4 treatments, in 4 replicates, were used. The degree of uniformityof the product on the treated seeds was determined by image ac-quisition and analysis of the surface of the treated seed corn.Quantification of the active ingredient (Clothianidin) releasedfrom the seeds after tanning treatment (drift of the a.i.) and % ofthe surface of colored kernels of corn were assessed. Data werestatistically analyzed using the XLSTAT statistical software.

PRELIMINARY INVESTIGATION ON THE USE OF ES-SENTIAL OILS TO CONTROL ROSE POWDERYMILDEW. S. Fascella, B. Cangelosi, P. Lucido, P. Curir and C.Pasini. CRA, Unità Ricerca Floricoltura e Specie Ornamentali,Corso Inglesi 508, 18038 Sanremo, Italy. E-mail: [email protected]

The purpose of this study was to evaluate the efficacy of es-sential oils from five vegetable species (oregano, thyme, clove,geranium, carvacrol) and a commercial solution of fertilizerKH2PO3 (50% w/w) for the control of powdery mildew of roses(Podosphaera pannosa var. rosa). The experiments were carriedout on the rose cv. Top Meillandina, grown in pots and kept un-der shade. The essential oils were sprayed at the dose of 0.5 ml/land the mineral salt KH2PO3 at 3 ml/l. Rose plants were treatedalso with Nimrod (Bupirimate 23,8 % a.i.) at the dose of 3 ml/l,as a standard reference. Disease severity was assessed seven daysafter the fourth weekly application. All the treatments reducedpowdery mildew severity, in comparison to untreated roses. Thepreliminary results showed a remarkable antifungal action, inparticular the essential oils provided a 80% disease reduction.Further studies will be aimed at assessing the antifungal activitiesof these substances on cut rose cultivars, to provide a natural al-

ternative to synthetic products in the control of the pathogen. Nophytotoxic effect was observed with natural products.

MOLECULAR IDENTIFICATION OF XYLOPHAGUSFUNGI ASSOCIATED WITH QUERCUS ILEX IN URBANLANDSCAPES. A. Francini, E. Pellegrini and C. Nali. Diparti-mento di Coltivazione e Difesa delle Specie Legnose “GiovanniScaramuzzi”, Università degli Studi, Via del Borghetto 80, 56124Pisa, Italy. E-mail: [email protected]

Studies on wood-decay fungi and their putative relationshipswith pollution, availability of nutrients, mechanical injuries anddisturbances due to constructions have recently been improvedin order to help decisions related to landscape planning and man-agement. It is important to remember that decay does not neces-sarily mean immediate death of trees, as the process may extendover several years. In urban areas the risk of accidents involvingpeople or properties could be important. In this work, the pres-ence and identity of wood decaying fungi affecting Quercus ilexwere evaluated for setting up ready-to-use diagnostic protocols.The survey was carried out in 2011, in the municipality of SanGiuliano Terme (Pisa, central Italy). Morphological descriptionsof the fungi detected were done. Moreover, detection and identi-fication of four Basidiomycota (Ganoderma, Fomes, Phellinus andTrametes) and one Daldinia (Ascomycota) were performed byPCR-based methods using mitochondrial ribosomal DNA (rD-NA) loci. The ITS1 and ITS2 regions from fungal cultures wereamplified and sequenced. The nucleotide sequence of Fomes fo-mentarius was deposited in the GenBank (accession No.JF927882, 604 bp). Further developments will take into accountthe correlation between tree age, presence and identity of fungiand their degradative potential.

SOIL DISINFESTATION BY OZONE FOR CONTROLLINGVERTICILLIUM WILT OF ARTICHOKE. M. Gallo, C. Fu-marola, S.A. Rocca and F. Ciccarese. Dipartimento di Biologia eChimica Agroforestale e Ambientale, Università degli Studi “A. Moro”, Via G. Amendola 165/A, 70126 Bari, Italy. E-mail: [email protected]

According to the European legislation, which has revised andreduced the number of pesticides with the objective of stimulat-ing new tools with low environmental impact, the need to identi-fy effective and environmentally friendly technologies for soil dis-infestations has emerged. In the course of studies aimed at identi-fying alternatives to the use of methyl bromide, a procedure forsoil disinfestation by ozone (Patent No. 05017372.3) has been de-veloped. The results of a trial for soil disinfestation using ozonefor controlling Verticillium wilt of artichoke are reported. Thetrials were carried out in field naturally infested by Verticilliumdahliae on a 3-year-old artichoke plot cv. Violetto di Provenza.Ozonated water, at 5 ppm concentration, was applied before (oneday) and after (twenty days) transplanting with a sub irrigationsystem. Chemically treated (Azoxystrobin at 0.5 ml/m2 concentra-tion applied before and after transplanting), untreated andhealthy controls were included. Verticillium wilt severity was ex-pressed as McKinney index of external symptoms (IES) and vas-cular discolorations (IVD). Every year the yield was also assessed.IES and IVD on artichoke grown on infested and untreated soilwere 82.55% and 72.15%, respectively. Ozone applicationsproved effective in the control of Verticillium wilt before(IES=16,3%; IVD=5,6%) and after transplanting (IES=18,6%;IVD=5,8%). The application of Azoxystrobin in sub irrigation



confirmed its efficacy against Verticillium wilt. Compared withinfested and untreated control, ozone and Azoxystrobin deter-mined a yield increase that nearly matched that of healthy crops.

EFFICIENT ELIMINATION OF VIROIDS FROMGRAPEVINES BY SOMATIC EMBRYOGENESIS. G. Gam-bino1, B. Navarro2, R. Vallania1, I. Gribaudo1 and F. Di Serio2.1Istituto di Virologia Vegetale del CNR, UOS Grugliasco, Via L. daVinci 44, 10095 Grugliasco (TO), Italy.2Istituto di Virologia Vege-tale del CNR, UOS Bari, Via Amendola 165/A, 70126 Bari, Italy.E-mail: [email protected]

Five different viroids have been recorded from grapevines,frequently co-infecting the same plant in various combinations.Grapevine yellow speckle viroid-1 (GYSVd-1) and Grapevine yel-low speckle viroid-2 are the causal agents of a disease by the samename, while Hop stunt viroid (HSVd), Citrus exocortis viroid andAustralian grapevine viroid cause symptomless infections. Theirpresence is threatening anyhow, since infected vines may repre-sent inoculum sources for other susceptible crops. Viroid spreadcan be restrained by prophylactic measures based on the use ofviroid-free propagation material. This strategy, however, is notapplicable to grapevines because the traditional sanitation tech-niques based on thermotherapy and meristem tip culture, whichare effective against most, if not all viruses, do not work with vi-roids. With the aim of finding an appropriate procedure for get-ting rid of these pathogens, we have tested somatic embryogene-sis, a technique known for its efficiency against grapevine viruses,as a means for GYSVd-1 and HSVd elimination from four grapecultivars. While both viroids were detected by RT-PCR in em-bryogenic and non-embryogenic calli from anthers and ovaries ofinfected grapevines, somatic embryos differentiated from infectedcalli and embryo-derived plantlets were viroid-free. In situ hy-bridization, used for ascertaining the spatial distribution ofGYSVd-1 and HSVd within proliferating calli, showed that bothviroids had the expected nuclear localization. The present resultssupport the use of somatic embryogenesis as a proficient methodfor generating viroid-free material and for further investigatingplant-viroid and plant-virus interactions in grapevine.

CO-OCCURRENCE OF FUNGAL ENDOPHYTES ANDOOMYCETES IN FOREST ECOSYSTEMS AND THEIRROLE IN TREE DECLINE. B. Ginetti, A. Ragazzi and S.Moricca. Dipartimento di Biotecnologie Agrarie, Sezione Pro-tezione delle Piante, Università degli Studi, Piazzale delle Cascine2, 50144 Firenze, Italy. E-mail: [email protected]

In the Boscoincittà Park of Milano (northern Italy), trees ofAcer pseudoplatanus and Quercus display bark cankers, tarry exu-dates from the lower trunk, stunted growth, and crown decline.From these trees some fungi of the family Botryosphaeriaceaewere isolated, including pathogens such as Botryosphaeria doth-idea and Neofusicoccum parvum. Trees were also positive for thepresence of Phytophthora in serological tests performed in situ.Infected trees exhibited greenish- and/or reddish-brown flame-shaped blotches and stripes below the bark. From these areasvarious Phytophthora isolates were recovered on a V8A-PARPNH selective medium. The same agents also occurred inthe soil around the trees and in nearby water sources. Isolatesfrom the soil and water were recovered by an apple bait and cul-tured on a selective medium. To induce fruiting bodies, mycelialportions were transferred to frozen pea medium and then to fil-tered and sterilized pond water; they were found to belong to

species of Phytophthora. These species, which are more vital andactive in the winter season, may have impaired tree physiology,gradually weakening the trees and predisposing them to theBotryosphaeriaceae in the following spring and summer, whenthese endophytes become most active. The co-occurrence ofthese two fungi and oomycetes on the same host and their com-plementary pathogenic action may well represent a new patho-genic complex causing the decline of the trees in question.

INFLUENCE OF SUBSTRATE AND ECOLOGICAL CON-DITIONS ON ASPERGILLUS FLAVUS SCLEROTIASPORULATION. P. Giorni and P. Battilani. Istituto di Ento-mologia e Patologia Vegetale, Università Cattolica del Sacro Cuore,Via Emilia Parmense 84, 29122 Piacenza, Italy. E-mail: [email protected]

Aspergillus flavus is one of the most dangerous fungi to maizecrops as it produces aflatoxins (AFs), i.e. mycotoxins with hightoxicity for animals and humans and with immunosopressive andcancerogenic properties. Many studies have been conducted toimprove the knowledge on the infection cycle of this fungus,however little information is available on sporulation, the crucialstage for the beginning of fungal infection. Sclerotia represent themain source of inoculum in the field since they can resist for longtime in the soil or on debris also in extreme conditions of temper-ature (T) and water activity (aw). Both these ecological factors re-sult important for sporulation. An artificial inoculum with sclero-tia produced by a strain of A. flavus was conducted both on cornstalks pieces (3 cm long) and on Czapek agar (CZ). Substrateswere inoculated with a single sclerotium and incubated in differ-ent conditions of temperature (5-45°C, with 5°C step) and aw(0.85-0.99 aw, with 0.02 aw step). Sporulation was checked be-tween 2 and 20 days of incubation. Data were analysed as func-tion of T, aw and as daily sporulation rate. High variability wasrecorded among sclerotia regarding the amount of spores pro-duced. Nevertheless, sporulation was never observed at 5 and45°C, while spores were produced at 0.85 aw. The maximumamount of spores was obtained at 30-35°C and 0.99 aw after 8days incubation both on stalks and CZ media. The number ofconidia produced was higher on the artificial media than on stalkpieces.

This work was partially supported by EC KBBE-2007-222690-2MYCORED.

AN INSIGHT IN SOME POPULATION FEATURES OFXANTHOMONAS ARBORICOLA pv. JUGLANDIS. D. Giova-nardi1, D. Dallai1, S. Bonneau2, M. Le Saux-Fischer2, C.Manceau2 and E. Stefani1. 1Dipartimento di Scienze Agrarie edegli Alimenti, Università di Modena e Reggio Emilia, Via Amen-dola 2, 42122 Reggio Emilia, Italy. 2Plant Pathology Department,UMR PaVé-INRA Angers, France. E-mail: [email protected]

Xanthomonas arboricola pv. juglandis (Xaj) is the causal agentof bacterial blight of walnut, an emerging disease, which has thepotential to severely affect walnut orchards. An Italian strain col-lection of Xaj, obtained during the past 3 years from affected or-chards in Romagna, was first assayed with conventional PCR withXajF/XajR primer pair developed to confirm strain identity. Thepopulation structure of the collection of Xaj isolates, confirms thepresence of different genetic groups identified by rep-PCR (usingthe REP, BOX and ERIC primers) and by multilocus sequencetyping (MLST) and multilocus variable number analysis of tan-dem repeat (MLVA). Xaj and Xaj-like bacterial isolates from the



Italian collection are currently being analysed by MLSA (multilocus sequence analysis), using 7 primers for 7 different house-keeping genes, with the purpose to better characterise the Italianisolates for phylotyping. The study of copper resistance on a widecollection of over 150 Xaj strains frequently showed high resist-ance (up to 500 ppm Cu++): two strains have been further studiedconfirming the presence of chromosomal genes copA and copB in-volved in the general copABCD copper resistance structure, asdescribed for Pseudomonas syringae. Sequencing and comparingwith other Xanthomonads were done. The elucidation of Xajpopulation structure may help to deeper investigate some addi-tional aspects of the molecular epidemiology of the disease, thusallowing a better control strategy in the field.

A COMPREHENSIVE APPROACH TO STUDY THE IM-PACT AND THE EPIDEMIOLOGY OF THE INVASIVEFOREST PATHOGEN HETEROBASIDION IRREGULAREIN ITALY. P. Gonthier1, N. Anselmi2, P. Capretti3, F. Bussotti3,M. Feducci3, M. Garbelotto4, L. Giordano1, F. Guglielmo1, T.Honorati2, G. Lione1, N. Luchi3, V. Mancini3, S. Michelotti1, M.Michelozzi5, G. Nicolotti1, B. Paparatti6, M. Pollastrini3, S.Speranza6, and A.M. Vettraino2. 1Dipartimento di Valorizzazionee Protezione delle Risorse Agroforestali, Università degli Studi diTorino, Via L. da Vinci 44, 10095 Grugliasco (TO), Italy. 2Diparti-mento per l’Innovazione nei Sistemi Biologici, Agroalimentari eForestali, Università della Tuscia, Via S. Camillo de Lellis, 01100Viterbo, Italy. 3Dipartimento di Biotecnologie Agrarie, Universitàdegli Studi, Piazzale delle Cascine 28, 50144 Firenze, Italy. 4De-partment of Environmental Science, Policy and Management,Ecosystem Sciences Division, University of California at Berkeley,137 Mulford Hall, 94720, Berkeley CA, USA. 5Istituto di GeneticaVegetale, CNR, Via Madonna del Piano 10, 50019 Sesto Fiorentino(FI), Italy. 6Dipartimento di Scienze e Tecnologia per l’Agricoltura,le Foreste, la Natura e l’Energia, Università della Tuscia, Via S.Camillo de Lellis, 01100 Viterbo, Italy. E-mail: [email protected]

The North American forest pathogen Heterobasidion irregu-lare was introduced in Italy in 1944. The fungus is invasive and iscurrently distributed in pine stands over about 105 km of coastapproximately centred around Rome, often in association withsignificant mortality of Pinus pinea trees. Little is known on theimpact and the aggressiveness of this exotic pathogen on nativetree species, as well as on its epidemiology and interactions withthe closely related native pathogen Heterobasidion annosum. TheItalian Ministry of Education, University and Research, withinthe PRIN program, funded a research aimed at: (i) investigatingthe ecology and epidemiology of H. irregulare in Italy, includingits level of pathogenicity on native tree species, its spreading abil-ity, its interactions with abiotic factors or related fungal species,and (ii) defining, based on the above information, the contents ofa proposal for its control. Several sites outside the current area ofinvasion have been investigated for the presence of H. irregulare.Preliminary data indicate that a northward expansion of its rangein the last six years did not occur. Investigations on the role of in-sect vectors and pruning wounds in the spread of the disease arein progress. Comparative inoculation experiments using H. irreg-ulare and H. annosum genotypes failed to detect a disproportion-ate pathogenicity of the exotic pathogen on native pine species.However, preliminary results from pine logs inoculations indicatea higher saprotrophic ability of H. irregulare versus H. annosum,which may be consistent with the observed invasion process.

RECOMBINANT PLUM POX VIRUS ISOLATES RECOV-ERED IN EMILIA-ROMAGNA REGION. P. Grillini, A. R.Babini, A. D’Anniballe, P. Fini and V. Vicchi. Servizio Fitosani-tario Regione Emilia-Romagna, Via di Saliceto 81, 40128 Bologna,Italy.

Plum pox virus (PPV) is the causal agent of Sharka, the mostdangerous disease of stone fruits, reducing fruit quality and yield.In Italy Sharka has been detected since 1973 but in Emilia-Ro-magna (northern Italy) it became epidemic on peach only after1995. Both PPV-D (Dideron) and PPV-M (Marcus) strains arepresent in our region as well as the recombinant strain of both(PPV-Rec). Since 2007, the strains of 135 PPV isolates from dif-ferent host species and geographical areas of Emilia-Romagna,were characterized using PCR-RFLP targeting two genome re-gions (CP and P3-6K1). Twenty-seven isolates from 10 differentorchards were identified as PPV-Rec strain. To investigate theirgenetic diversity in more details, the (Cter)P3-6K1-(Nter)CI and(Cter)NIb-(Nter)CP regions of few isolates were partially se-quenced. Nucleotide sequence alignment showed that analysedisolates are closely related to previously characterised recombi-nant strain and share the same recombination breakpoint in the3’ terminal part of the NIb gene. PPV-Rec isolates were mainlyfound on European and Japanese plum varieties (Grossa di Feli-sio, Stanley, Black Top, Angeleno). Only one apricot cultivar(Caldesi 2) and one rootstock (MRS2/5) resulted infected.

CHARACTERIZATION OF CITRUS LEAF BLOTCH VIRUSISOLATES COLLECTED IN OLD ITALIAN CITRUS COL-LECTIONS. M. Guardo, T. Marletta, G. Sorrentino, G. Varricaand A. Caruso. Centro di Ricerca per l’Agrumicoltura e le ColtureMediterranee, Corso Savoia 190, 95024 Acireale (CT), Italy. E-mail: [email protected]

Citrus leaf blotch virus (CLBV), the type species of the genusCitrivirus, family Betaflexiviridae recently found in Italy, causes abud union disorder of Nagami kumquat (Fortunella margarita)and Calamondin (Citrus mitis or madurensis) grafted on trifoliaterootstocks. The virus is symptomless on sour orange rootstocks.A survey was carried out with the aim to investigate the presenceand the origin of CLBV in old Italian citrus collections. Samplesof different citrus species from field trees, were collected inPalermo (botanical garden and San Giovanni degli Eremiti gar-den) in Naples (botanical garden) in Florence (gardens ofMedici’s villas of Boboli and Castello) and in a private collectionof Pescia (Pistoia). The tests were carried out using one step RT-PCR with specific primers to amplify part of coat protein (CP)and the RNA-dependent RNA polymerase (RdRp) genes; eachpositive sample was analysed by single-strand conformation poly-morphism (SSCP) and samples with different electrophoretic pat-tern were cloned and sequenced. Among 61 samples only someNagami kumquat gave positive reaction to CLBV infection. Allpositive samples showed a predominant SSCP profile belongingto ISA 9-ME-I isolate, found in Sicily in 2006. This suggest thatthe infected Italian CLBV population could derive from a singleorigin and that virus introduction may have occurred recently.

TIME COURSE ANALYSIS OF THE EXPRESSION OFGENES POSSIBLY CORRELATED TO THE DIFFERENTSUSCEPTIBILITY OF UNRIPE AND RIPE STRAWBERRYFRUITS TO COLLETOTRICHUM ACUTATUM. M.Guidarelli, M. Maradeo, L. Zoli, P. Bertolini and E. Baraldi.CRIOF, Dipartimento di Produzione e Valorizzazione Agroali-



mentare, Laboratorio di Biotecnologie, Università degli Studi, ViaFanin 46, 40127 Bologna Italy. E-mail: [email protected]

Many fungal pathogens interact with fruits of their hosts atpre-harvest unripe stages and remain quiescent during ripening,causing severe economic losses for post-harvest fruit production.Colletotrichum acutatum causes anthracnose symptoms only onripe strawberry (Fragaria × ananassa) fruits, whereas, on whiteunripe fruits, it becomes quiescent as melanized appressoria. Aprevious microarray experiment revealed that ripe and unripestrawberries, interacting with C. acutatum, regulate differently theexpression of several genes. Among these genes, a few code forproteins with important regulatory roles in plant response topathogens. In this study, qRT-PCR was used to make a narrowtime scale analysis of the different activation of these genes inwhite and red fruits challenged with the pathogen. In particular,the expression of lectin, WRKY, brassinosteroid insensitve recep-tor kinase 1 (BR1), and phenylpropanoid and flavonoid genes wasmonitored in fruits after 8-16-20-24 h interaction with C. acuta-tum. Differently from phenylpropanoid and flavonoid genes, theexpression of lectin, WRKY and BR1 genes was regulated exclu-sively on white fruits at 24 h post-interaction, when the C. acuta-tum becomes quiescent. Our results suggest that these genes playa specific role in C. acutatum quiescence.

MULTIPLEX RT-PCR FOR DETECTING FIVE VIROIDSINFECTING GRAPEVINES. M. Hajizadeh1, B. Navarro2, N.S. Bashir1, E. M. Torchetti2 and F. Di Serio2. 1Plant ProtectionDepartment, University of Tabriz, 29 Bahman, 51664 Tabriz, Iran.2Istituto di Virologia Vegetale del CNR, UOS Bari, Via Amendola165/A, 70126 Bari, Italy. E-mail: [email protected]

Grapevine is the natural host of at least five viroid species, i.e.Grapevine yellow speckle viroid 1 (GYSVd-1), Grapevine yellowspeckle viroid 2 (GYSVd-2), Australian grapevine viroid (AGVd),Hop stunt viroid (HSVd) and Citrus exocortis viroid (CEVd).Some species (HSVd, GYSVd-1, GYSVd-2) are of worldwide oc-currence, whereas others (AGVd and CEVd) seem to be more re-stricted geographically. The lack of fast and reliable laboratoryprocedures for the contemporary detection of multiple viroidalinfections is an important limitation for studying the distributionand relative incidence of these viroids in grapevine-growing ar-eas. To fill this gap, a multiplex RT-PCR (mRT-PCR) assay hasbeen developed for the contemporary detection of the five viroidsand the simultaneous amplification of the cDNA fragment of ahost-derived mRNA (actine mRNA), as an internal positive con-trol. The method was validated testing 57 grapevine plants fromIran infected by one or more viroid species. Concordant resultswere obtained by our mRT-PCR protocol and single RT-PCR(sRT-PCR) tests conducted using primers previously reported foramplifying separately cDNAs of the monomeric full-length ge-nomic RNA of each targeted viroid. Negative samples were fur-ther assayed by Northern-blot hybridization. Except for CEVd,in this survey HSVd, AGVd, GYSVd-1, and GYSVd-2 were de-tected in 100, 95, 93 and 65% of the tested samples, respectively,confirming their wide distribution in Iran. The present mRT-PCRprotocol has the potential to be used routinely for large-scale sur-veys and certification programs.

PLANT DISEASES IN EXOTIC PURPLE PASSION FRUITIN THE ANDEAN ZONE L. Hoyos-Carvajal1, S. Castillo1, S.Benítez1, E. Ortiz1, L. Farfán1 and D. Riascos2. 1Laboratorio deSanidad Vegetal, Facultad de Agronomía, Universidad Nacional de

Colombia, Bogotá, Colombia. 2Facultad de Ciencias Agropecuarias,Universidad Pedagógica y Tecnológica de Colombia, Tunja, Colom-bia. E-mail: [email protected]

Several species of passion fruits are marketed as exotic fruitsin Europe and North America, which has a great impact on localeconomies in Andean zones. In Colombia, purple passion fruit(Passiflora edulis) in one of the most important passionfruits,whose diseases are not well known. Through sampling on mainproducing areas of the country (9 regions), different causal agentswere detected by microbiological, histological and moleculartools, yielding a collecting of ca. 200 strains of fungi and bacteria.Among these, Xanthomonas axonopodis detected by molecularmethods (specific primers) did not correspond to X. axonopodispv. passiflorae commonly reported in this family of plants andwidely distributed in other passionfruit-producing countries.Histopathological analysis showed systemic distribution in plantof bacterial masses, colonizing vascular tissues, leafs and fruits. X.axonopodis can attack other hosts such as P. ligularis, P. edulisflavicarpa, P. mollissima and P. maliformis. By gene sequencing(ITS, tef) from fruits, Cladosporium cladosporiodes, Col-letotrichum boninense and C. acutatum were detected as causalagents of diseases, but not C. gloeosporioides which is reported asa common pathogen of fruits. F. oxysporum and F. solani were de-tected, which cause diseases in different Passiflora, and may at-tack other hosts such as cape gooseberry (Physalis peruviana).The detection of these causal agents and knowledge of their biol-ogy allows the appropriate management of diseases in this crop,keeping it sustainable and safe to producers and consumers.

GENOTYPING OF CLAVIBACTER MICHIGANENSIS sub-sp. MICHIGANENSIS BY HOUSEKEEPING GENE SE-QUENCING AND HIGH RESOLUTION DNA MELT-CURVE ANALYSIS. G. Ialacci1, G. Licciardello2, P. Bella1, K.H. Garteman3, R. Eichenlaub3 and V. Catara1. 1Dipartimento diScienze e Tecnologie Fitosanitarie, Università degli Studi, Via SantaSofia 100, 95123 Catania, Italy. 2Parco Scientifico e Tecnologicodella Sicilia, Zona industriale. Blocco Palma I, 95131, Catania,Italy. 32 Lehrstuhl für Gentechnologie/Mikrobiologie, UniversitätBielefeld, 33594, Germany. E-mail: [email protected]

High-resolution melting (HRM) analysis is emerging as an ef-ficient and robust method for discriminating DNA sequence vari-ants. The objective of this study was to apply HRM analysis togenotyping Clavibacter michiganensis subsp. michiganensis, causalagent of tomato bacterial canker. The first approach was to iden-tify suitable gene fragments able to distinguish the strains at thesubspecies level. To this aim a number of C. m. subsp. michiga-nensis strains, isolated in Italy previously genotyped by BOX-PCR and f-AFLP markers, were characterised using the se-quences of internal fragments of five house-keeping genes (gyrB,sdhA, ligA, bipA, kdpA). Approximately 600-800 bp internalfragments of each gene were sequenced on both strands using anautomated DNA capillary sequencer for a total of about 100 se-quences. Nucleotide sequences were aligned in MEGA4 usingCLUSTALW. Sequences of C. m. subsp. michiganensis strainsfrom other countries were included in the in silico analysis. ThekdpA and sdhA gene fragment analysis allowed to discriminatethe highest number of strains and two primer pairs were designedon the highest variable regions to be used for HRM analysis. Theresults for HRM of the kdpA PCR products showed a better re-producibility. The C. m. subsp. michiganensis strains were clearlydistinguishable from reference strains belonging to other C.michiganensis subspecies. The analysis allowed also to detect in-frasubspecific variability. HRM analysis provided a resolving



power similar of the other fingerprinting methods and it is apromising approach to easily genotyping bacterial population ofplant pathogenic bacteria.

EFFECT OF COMPOST ON ENZYME ACTIVITY, MICRO-BIAL BIOMASS AND HEALTH OF SOIL IN AN ORGANICSYSTEM. G. Innocenti1, M. Montanari1, C. Ciavatta2 and S.Scagliarini3. 1Dipartimento di Protezione Valorizzazione Agroali-mentare, Università degli Studi, Viale Fanin 46, 40127, Bologna,Italy. 2Dipartimento di Tecnologie Agroambientali, Università degliStudi, Viale Fanin 40, 40127 Bologna, Italy. 3Centro AgricolturaAmbiente Giorgio Nicoli, via Argini Nord 3351, Crevalcore (BO)Italy. E-mail: [email protected]

The response of soil biota to compost amendment is a verycomplex phenomenon. There is little information available onmicrobial density, enzyme activity and suppressive ability of soilafter compost addition under field conditions. In particular, thereis a lack of sufficient information on compost-microbiota interac-tion in organic systems. The study site was an experimental long-term organic pear orchard located in the Po Valley near Bologna(northern Italy), on a sandy-loam soil. The soil was amended witha commercial municipal solid waste at 8 and 16 t ha-1 year-1 inMarch from 2006 to 2008. Control treatments consisted in a com-mercial Nitrogen organic product (Fertil) and no fertilisationtreatment. Each plot was 232 m2, with 29 plants, and each treat-ment was replicated four times in a randomised block design. Soilsamples were collected using a soil sampler (30 cm long, 8 cm di-ameter) in early autumn from 2006 to 2008. Numbers of cul-tivable fungi and bacteria, and enzymatic activity were deter-mined by plate count technique on selective media and FDA test,respectively. Soil health was assessed using the patho-systemPythium-Beta vulgaris. Physico-chemical parameters were alsomeasured. The soil amendment with the highest rate of compostincreased the microbial activity, and did not influence the densityof microbial groups. Probably the microbial groups were in equi-librium, and the compost as a source of nutrients, was able tostimulate their activity. The suppressive ability was favoured bythe highest rate of compost, even if differences with other treat-ments were not significant.

STAGONOSPORA NODORUM BLOTCH: AGRONOMICAND QUALITY DATA OF THREE DURUM WHEAT VARI-ETIES. A. Iori, A. Niglio, P. Cacciatori, C. Cecchini, C. Cristo-fori and M. Chierico. CRA, Unità di Ricerca per la ValorizzazioneQualitativa dei Cereali, Via Cassia 176, 00191 Roma, Italy. E-mail:[email protected]

Stagonospora nodorum (teleomorph Phaeosphaeria nodorum)causes stagonospora nodorum blotch on wheat. This pathogencan attack all aerial parts of the plant including the seeds, causingconsiderable yield losses. The objectives of this preliminary studywere to: (i) evaluate the resistant or susceptible behaviour at theadult stage of three durum wheat varieties (Ciccio, Svevo andSimeto) subjected to S. nodorum artificial inoculation in the fieldand (ii) examine the effect of the pathogen on some quantitativeand qualitative traits. Field trials were carried out on varietiesgrown in an experimental field at Montelibretti (Rome) with asplit-plot design. Different experimental conditions were used:untreated control, treated control with commercial fungicide,twice inoculated plants at different stages with S. nodorum sporesuspension (1×106 spore/ml), once inoculated plants with thesame fungal suspension. Disease severity was evaluated directly in

the field considering the percentage of area affected by S. nodor-um on flag leaf and ear. Grain yield, 1,000 kernel weight, hec-tolitric weight, protein content and SDS sedimentation test weredetermined on all samples. Results show that all the varieties atthe adult stage were susceptible to the isolate used. Generally theinoculated samples showed values of grain yield, hectolitricweight and 1,000 kernel weight lower than the treated control,while data of the untreated control were variable. Grain proteincontent was quite similar in all the samples. For SDS sedimenta-tion test no differences between inoculated samples and controlswere observed, except for the cv. Ciccio.

ECTOMYCORRHIZAL FUNGAL SPECIES AS SENTINELORGANISMS OF TREE HEALTH STATUS. E. Lancellotti, A.Schiaffino and A. Franceschini. Dipartimento di Protezione dellePiante, Sezione di Patologia Vegetale, Università degli Studi, ViaDe Nicola 9, 07100 Sassari, Italy. E-mail: [email protected]

Root colonization by ectomycorrhizal fungi (ECM) is strictlydriven by host tree metabolism. Therefore, the alteration of phys-iological status of the host tree, for instance as a disease conse-quence, may cause changes in the presence and distribution ofECMs. For this reason some ECMs could be selected as sentinelorganisms of tree health status. With this aim in mind a study wascarried out to evaluate the influence of tree health status on theECMs detected in a Quercus suber stand where many trees are af-fected, with different degree, by cork oak decline. The ECMguilds characterizing differently damaged cork oak trees wereevaluated, as well as the relationships among tree health statusand distribution of genera, phylogenetic assemblages of ECMsand single ECM. The results showed that: (i) Cortinarius and Se-bacina genera colonized trees in the worst health conditions; (ii)some ECM phylogenetic assemblages detected into Inocybe andTomentella genera were linked only to healthy trees or diseasedtrees; (iii) some ECMs (i.e. Cenococcum geophilum) colonizedmainly diseased trees, while other ECMs (i.e. Lactarius chrysor-rheus) the healthy ones.

EXPRESSION ANALYSIS OF DEFENSIN-LIKE GENES(DEFL) IN GRAPEVINE IN RESPONSE TO BOTRYTISCINEREA AND PLANT DEFENSE HORMONES. L. Lenzi1,L. Giacomelli1, C. Zadra2 and C. Moser1. 1Edmund Mach Foun-dation, San Michele all’Adige (TN), Italy. 2Dipartimento di ScienzeAgrarie ed Ambientali, Università degli Studi, Borgo XX Giugno74. 06121 Perugia, Italy. E-mail: [email protected].

Defensins are a class of small, basic, cysteine-rich peptidesfound in plant, insect and vertebrates, which share a common ter-tiary structure and show broad antimicrobial activity. Using acombination of HMM and BLAST searches to scan the Vitisvinifera cv. Pinot noir genome, 79 defensin-like sequences (DE-FLs) have been identified, which correspond to 46 putative de-fensin genes.To better understand the involvement of this genefamily in the signalling and defense against Botrytis cinerea, we in-vestigated the changes in expression of 14 grapevine defensins inberries and inflorescences following artificial inoculation with thefungus. In parallel, we measured the content of some jasmonates(jasmonate, methyl jasmonate, OPDA) and salicylic acid (SA) inberries infected with Botrytis cinerea and tested whether these de-fensins are modulated by treatments with these typical mediatorsof defense. Among the 14 DEFLs, five were significantly inducedupon B. cinerea infection either in inflorescences or in berries.Analytical measurements revealed that SA, JA and OPDA, but



not MeJA accumulated in infected inflorescences and berries.Preliminary data on berries treated with different hormonesshowed that none of the tested defensins was induced by treat-ment with MeJA and BTH (an analogue of salicylic acid), where-as one defensin gene was up-regulated after treatment withethephon, a precursor of ethylene. Our results suggest a role ofspecific defensin genes in the grapevine defense against B. cinereaand the involvement of both MeJA and SA in the plant response.Preliminary data on the regulation of defensin genes by hor-mones have also been acquired.

SANITARY SELECTION OF CALABRIAN GRAPEVINEGERMPLASM. G. Leo1, D. Luison2, A. Roschetti1, G. Al-banese1 and F. Faggioli2. 1Dipartimento di Gestione dei SistemiAgrari e Forestali, Università degli Studi Mediterranea, LocalitàFeo di Vito, 89122 Reggio Calabria, Italy. 2CRA, Centro di Ricercaper la Patologia Vegetale, Via C.G. Bertero 22, 00156 Roma, Italy.E-mail: [email protected]

Many old autochthonous grapevine varieties of high value arewidely spread in Calabria (southern Italy). The improvement ofthese cultivars requires the registration of qualified clones afteran accurate genetic and sanitary selection, according to Europeanand national rules. Grapevine sanitary selection, essential to ex-clude the presence of viral diseases, was conducted on the follow-ing cultivars: Arvino, Gaglioppo, Greco bianco, Greco nero,Guardavalle, Lacrima, Magliocco dolce, Mantonico bianco,Parmisana, Pecorello and on the rootstock 17-37. One hundredand eight-two biotypes of these cultivars, with optimal agronomicand productive characteristics, were identified in the bestgrapevine-growing areas of the region. Sanitary selection was car-ried out inspecting the plants, in spring and autumn, to detect vi-ral symptoms. Woody material, collected from symptomatic andsymptomless vines were tested by serological (ELISA) and molec-ular (Multiplex RT-PCR) analysis to detect the following viruses:Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV),Grapevine leafroll associated virus-1 (GLRaV-1), Grapevineleafroll associated virus-2 (GLRaV-2), Grapevine leafroll associatedvirus-3 (GLRaV-3), Grapevine virus A (GVA), Grapevine virus B(GVB), Grapevine fleck virus (GFkV). During field inspectionswidespread symptoms of leafroll and infectious degenerationwere found. The serological and molecular analysis revealed thepresence of viruses in 172 samples out of 182 tested (94%). In-fected samples showed mostly mixed infections of different virus-es (105 out of 172 equal to 61%). The incidence of the identifiedviruses was: 67%, 60%, 23%, 13%, 13%, 3% and 0.6% respec-tively for GVA, GLRaV-3, GFLV, GLRaV-1, GFKV, GLRaV-2and GVB.

GENETIC ANALYSIS OF STEM RUST RESISTANCE INELITE DURUM WHEAT GERMPLASM THROUGH ASSO-CIATION MAPPING. T. Letta1,4, I. Terracciano1, M. Maccafer-ri1, A. Badebo2, K. Ammar3, J. Crossa3, P. Oliveira5, Y. Jin6,M.C. Sanguineti1 and R. Tuberosa1. 1Dipartimento di Scienze eTecnologie Agroalimentari, Università degli Studi, Viale Fanin 44,40127 Bologna, Italy. 2Debre Zeit Agricultural Research Center,Debre Zeit, Ethiopia. 3CIMMYT, Int. Apdo Postal 6-641, MexicoD.F., Mexico 06600. 4Sinana Agricultural Research Center, Bale-Robe, Ethiopia. 5Department of Plant Pathology, University ofMinnesota, St. Paul, MN 55108, USA. 6USDA-ARS, Cereal Dis-ease Laboratory, St.Paul, MN 51108, USA. E-mail:[email protected]

Stem rust (SR), caused by Puccinia graminis f. sp. tritici, is ahighly destructive fungal disease. To identify resistance sources, apanel of 183 elite durum wheat cultivars was characterized for SRresponse in four consecutive seasons (off- and main-seasons 2009and 2010) under field condition in Ethiopia, a ‘hotspot’ site for SRepidemics. The panel was artificially inoculated using isolatescomprising Ug99 and a local mixture of urediniospores of durum-specific races prevalent in Ethiopia. The recorded maximum dis-ease severity scores were then converted into coefficient of infec-tion (CI). In average, CI in the off-seasons was markedly higherthan in the main-seasons. About 13 and 30 cultivars had CI values<10 in off-seasons and main seasons, respectively. The CI pheno-typic distributions indicate that the overall genetics of resistancein the panel is complex. The molecular profiles of the panel (323SSR and 900 DArT markers) were subjected to association map-ping analysis using mixed linear model taking into account popu-lation structure and familial relationships. Several chromosome re-gions putatively involved in SR response were identified. The fourregions with the largest effect (R2 values ranging from 2.3 to5.7%) were mapped on chromosones 1B, 5A, 6A (coincident withSr13) and 7B. Significant associations were also detected in otherchromosone regions not known to harbor SR resistance genes.Our study has identified a number of novel regions for resistanceto Ug99 and other Ethiopian races potentially useful to contributeto the improvement of SR resistance in durum wheat.

EFFECT OF SEVERE AND MILD CITRUS TRISTEZAVIRUS ISOLATES ON SELECTED GENES EXPRESSIONOF DIFFERENT CITRUS HOSTS. G. Licciardello1, M. Tessi-tori 2, R. La Rosa2, G. Nobile1 , A. Catara1 and S. Rizza2 . 1Labo-ratorio di Diagnosi e Biotecnologie Fitosanitarie, Parco Scientifico eTecnologico della Sicilia, Zona Industriale, Blocco Palma I, 95121Catania, Italy. 2Dipartimento di Scienze delle Produzioni Agrarie eAlimentari, Sezione Fitopatologia e Genetica Vegetale, Universitàdegli Studi, Via S. Sofia 100, 95123 Catania, Italy. E-mail: [email protected]

Seven genes, involved in plant defence response (metalloth-ionein, MT and peroxidase, PRX, alcool-dehydrogenase ADH),signal transduction (CONSTANS-like, CLP and ethylene-respon-sive binding protein, EREBP), aminoacid transport (aminoacidpermease, AP) and regulation of gene silencing (rgs-CaM), wereselected to evaluate their modulation in different citrus hosts inoc-ulated with Citrus tristeza virus (CTV) isolates. Plants with differ-ent susceptibility to CTV, as Mexican lime, sour orange, pineapplesweet orange, Duncan grapefruit and Etrog citron were tested 18months after inoculation with a severe isolate (SY568 like), where-as sour orange and Mexican lime seedlings were also challengedwith a mild isolate (T30 like) to compare relative expression lev-els. Real-time RT-PCR results showed that the selected genes ex-hibited different patterns of expression depending on the citrushost. ADH and PRX genes were down-regulated in all citrushosts. Rgs-CaM, AP and CLP were down-regulated in Etrog cit-ron, Duncan grapefruit and sweet orange but were over-expressedin sour orange. Moreover, seedlings of sour orange and Mexicanlime, the most susceptible hosts, showed a similar expression pat-tern for ADH, EREBP and PRX (down-regulated), as well as forMT, rgs-CaM and AP (up-regulated) when infected with eitherSY568 or T30 like isolates. Nevertheless, changes induced in rgs-CaM gene by the severe viral isolate were 11 and 6 times higherthan the mild isolate, in sour orange and Mexican lime, respective-ly. Over expression of rgs-CaM gene occurred only in highly sus-ceptible plants showing severe symptoms. This gene appears toact as host specific suppressor of RNA silencing, enabling CTV toevade host RNA silencing activity.



TRICHODERMA VIRIDAE MAY BE A PLANTPATHOGEN. M.G. Li Destri Nicosia, S. Mosca and L. Schena.Dipartimento di Gestione dei Sistemi Agrari e Forestali, Universitàdegli Studi Mediterranea, Località Feo di Vito, 89122 Reggio Calabria, Italy. E-mail: [email protected]

Trichoderma viridae is commonly known as a biocontrol agentof soil-borne pathogens and is frequently utilized as an active in-gredient in bio-formulations. It is also known as a causal agent ofpostharvest rots on stored lemons, however it has never been re-ported as a root and/or crown rot pathogen. A strain of T. viridae(Fv2) isolated from the basal trunk of young decaying Pinus nigratrees with symptoms of decline was re-inoculated on both cut-tings and eight-year-old trees of the same pine species. A plug ofPDA with actively growing mycelium of T. viridae was insertedunder the bark and caused extended necrotic lesions around theinoculation site on both cuttings and trees. On two-years-old pineseedlings the same strain, inoculated as previously described,caused the death of 80 and 90% of the plants in one and threemonths, respectively. T. viridae was always re-isolated from symp-tomatic tissues around the inoculation site. Further studies areneeded to evaluate the range of hosts species and the actual roleof this strain in causing the decline of pine trees in natural condi-tions.

EFFECTS OF ESSENTIAL OILS, BACILLUS AMILOLIQUE-FACIENS AND ACIBENZOLAR-S-METHYL ON BACTERI-AL WILT INCIDENCE AND RALSTONIA SOLANACEARUMPOPULATIONS IN TOMATO PLANTS. V. Ligios and M.Fiori. Dipartimento di Protezione delle Piante, Università degli Stu-di, Via E. De Nicola, 07100 Sassari, Italy. E-mail: [email protected]

The efficacy of 5 Sardinian essential oils from Helichrysum sp.,Mentha piperita, M. pulegium, Myrtus communis, Thymusherbabarona, thymol (Fluka), Bacillus amyloliquefaciens and aSAR inducer acibenzolar-S-methyl was evaluated in vitro and ontomatoes plants grown in greenhouse for bacterial wilt control.The soil was first infested with a mixture of an equal amount oftwo virulent Rs strains. After 1 h incubation, essential oils andthymol were incorporated into the soil. To evaluate the antagonis-tic activity of B. amyloliquefaciens, the suspensions of Rs were in-corporated into soil 24 h after the antagonist. Acibenzolar-S-methyl was applied on tomato plantlets as a foliar spray at a con-centration of 25 mg/l before transplanting and increased to 50mg/l after transplanting. The trials were repeated twice. Sevendays after essential oils treatments, Rs populations density weredetermined on SMSA. Wilted plants and latent infection, detect-ed by direct isolation and ELISA, were determined one monthafter transplanting. Bacterial wilt incidence and Rs concentration,compared with control, were reduced considerably on tomatoplants transplanted in soil treated with 1 g/l of essential oils, thy-mol and B. amyloliquefaciens. However, the effectiveness of oilsand thymol was reduced when application rates were lowered(0.5 g/l). At two concentrations (0.5 and 1 g/l), significantly morewilt occurred in tomato plants grown in soil treated with M.piperita oil. Plants treated with acibenzolar-S-methyl were appar-ently healthy but diagnostic tests revealed Rs systemic infection inall plants. The possibility to test essential oils, together with an-tagonists and SAR inducers are now in progress.

EVALUATION OF ACTIVITY OF LIQUID ORGANIC FER-TILIZERS, HEMOZYM NK4.5-6 AND HEMOZYM BIO N5,ON THE GROWTH OF BIOCONTROL AGENTS. A.M.

Ligorio, M. Ferrara, F. Nigro and A. Ippolito. Dipartimento diBiologia e Chimica Agro-Forestale ed Ambientale, Università degliStudi “Aldo Moro”, Via Amendola 165/A, 70126 Bari, Italy. E-mail: [email protected]

Hemozym NK4.5-6 and hemozym BIO N5 are liquid-nitro-gen based organic fertilizers, obtained by processing beef bloodfor pharmaceutical and food industry purposes. In this study, lab-oratory assays were conducted to determine the effects of bothfertilizers on the growth of several biocontrol agents (BCAs).Thegrowth rate of the fungal BCAs Trichoderma harzianum F178 andClonostachys rosea F114 was determined on agar plate containingglucose (0.5%) and amended with 0.5 and 5% NK4.5-6. After 6days incubation at 24±2°C, NK4.5-6 at 5% inhibited the growthof T. harzianum F178, whereas at 0.5% growth was enhanced andan abundant sporulation was observed. C. rosea F114 was not in-hibited by the two concentrations of NK 4.5-6 and at 0.5% thecolony diameter was greater as compared to the control. Bacillusmegaterium EL050, B. licheniformis EL90, and B. subtilis EL80were cultured in a minimal medium (MM) amended with 5% ofNK 4.5-6 and bacterial growth was monitored 4, 6, 24, and 36 hafter inoculation. B. licheniformis EL90 and B. subtilis EL80showed a significant increase of the growth rate as compared tothe control, whereas no significant variations were observed forB. megaterium EL050. The effects of NK4.5-6 and BIO N5, onthe growth of fungal BCAs F178 and F114 was also investigatedin the soil. After 5 days incubation at 25±2°C, the population ofF178 was higher in the soil amended with NK 4.5-6 and BIO N5as compared to the un-amended control. Based on the results ob-tained, the use of both formulations can be suggested to improvethe performance of antagonistic microorganisms artificially intro-duced and those naturally present in the soil.

BRANCH DIEBACK OF PHOENICEAN JUNIPERCAUSED BY DIPLODIA AFRICANA IN ITALY. B.T.Linaldeddu, B. Scanu, L. Maddau, S. Seddaiu and A. Franceschi-ni. Dipartimento di Protezione delle Piante, Sezione di PatologiaVegetale, Università degli Studi, Via E. De Nicola 9, 07100 Sassari,Italy. E-mail: [email protected]

Since autumn 2009, a progressive dieback of shoots andbranches of Phoenicean juniper has been observed in a naturalarea on the island of Caprera (Sardinia, Italy). Necrotic lesionsfrequently girdled the main stems, causing a rapid death of theupper crown. The fungal isolates obtained in culture from symp-tomatic tissues were identified according to morphological fea-tures as Diplodia africana. On PDA at 25°C, all isolates developeda moderately aerial mycelium, initially white then turning darkgrey from the centre to the margin after 5 to 6 days. All isolatessporulated well in culture and produced hyaline, aseptate, thick-walled, oblong to cylindrical conidia measuring 30.1±1.8×11.7±1.1 µm. Identification was confirmed by analysis of the ITSsequence of a representative isolate (DA1). BLAST searches inGenBank showed 100% similarity with reference sequences of D.africana including the ex-type isolate (CBS 120835). The repre-sentative sequence of isolate DA1, was deposited in GenBank(accession No. JF302648). Its pathogenicity was verified by inoc-ulation on stems of ten 3-year-old seedlings of Phoenicean ju-niper with PDA plugs colonized by mycelium. Two months afterinoculation, all seedlings displayed canopy dieback and innerbark necrosis in the stem, from which the pathogen was success-fully reisolated, thus fulfilling Koch’s postulates. Five controlseedlings remained symptomless. D. africana was originallythought to be associated with disease symptoms on stone fruittrees in South Africa. This is the first report of D. africana in



the northern hemisphere and also as a fungal pathogen ofPhoenicean juniper.

EPIDEMIOLOGICAL AND AETIOLOGICAL INVESTIGA-TIONS ON THE SEVERE OUTBREAKS OF OLIVE AN-THRACNOSE IN APULIA. A. Longo, M. Ferrara, I. Penti-mone, A. Ippolito and F. Nigro. Dipartimento di Biologia e Chim-ica Agro-forestale ed Ambientale, Università degli Studi “Aldo Mo-ro”, Via Amendola 165/A, 70126 Bari, Italy. E-mail: nigrof@ agr.uniba.it

Olive anthracnose, caused by Colletotrichum spp., is an im-portant disease responsible for major yield losses and poor oilquality. The severe epidemic outbreaks of the disease occurringin Apulia (south-east Italy) during the last years, induced the re-gional government to declare the emergency status for theprovinces of Lecce and Brindisi, in which the disease incidencewas 50% in average, frequently reaching 90%. To collect new da-ta on the disease, samples from various olive orchards located inthe provinces of Brindisi and Lecce, were analyzed. In October2010 the incidence of drupe latent infections ranged from 20%to 70%, depending on the cultivar. The epiphytic population ofColletotrichum spp. on leaves and flowers showed an increasingtrend from February to the end of April 2011; the infection rateon 1-year-old branches varied from 25% to 80%. Isolates of Col-letotrichum spp. (about 100) collected during the surveys werecharacterized by morphological characters and molecular assays.Although differences in spore morphology, growth rate andcolony colour do not allow a conclusive identification of thespecies, preliminary data from molecular analysis suggests thatthe C. acutatum species complex is more common than C.gloeosporioides. Data from restriction fragment profiles of ampli-fied Tub2 gene, showed a prevalence of C. acutatum subgroupA4. Isolates showing a restriction profile similar to the moleculargroup A6 were also found.

FIRST REPORT OF STEMPHYLIUM sp. ON OLIVETREES IN SICILY. S. Lo Piccolo, G. Conigliaro, V. Mondello,L. Torta and S. Burruano. Dipartimento DEMETRA Universitàdegli Studi, Viale delle Scienze 4, 90128 Palermo, Italy. E-mail:[email protected]

In the last five years, we have observed a new decline of Oleaeuropaea in north-western and southern Sicily (insular Italy). Thesyndrome consists in more or less extensive leaf chlorosis, some-times with irregular necrotic marginal or apical spots. Apical de-foliation of twigs showing cortical necrosis and withering has of-ten been observed. Isolations, carried out in 2007/08, yilded acomplex of fungal genera associated with the syndrome. Amongthese, Stemphylium sp., a well-known foliar pathogen of otherplants, was constantly isolated from all the alterations observed.In December 2010, single spore colonies of this fungus were usedfor artificial inoculations on healthy 3-year-old olive plants (cv.Biancolilla). Necrotic foliar spots, extending from the inoculumsite to the apex leaf, and twig cortical necrosis with darkening ofinternal tissues were, observed from 15/40 and 90/120 days, re-spectively, after inoculations. Colonies of Stemphylium sp. werealways re-isolated from all symptomatic organs, thus fulfillingKoch’s postulates. To our knowledge, this is the first report ofStemphylium sp. as a pathogen to olive trees in Sicily. Furthermorphological and molecular studies are in progress to achievespecific identification.

BACTERIAL CANKER OF KIWIFRUIT BY PSEUDO-MONAS SYRINGAE pv. ACTINIDIAE: DETECTION OFTHE BACTERIUM IN FRUITS AND POLLEN. S. Loreti, A. Gallelli, A. L’Aurora and S. Talocci. CRA, Centro di Ricerca per la Patologia Vegetale, Via C.G. Bertero 22, 00156Roma, Italy. E-mail: [email protected]

Bacterial canker of kiwifruit induced by Pseudomonas syringaepv. actinidiae (Psa) is causing serious damages to Actinidia deli-ciosa and A. chinensis in Italy. The severe outbreak of the diseaseraises important questions about trade of kiwifruit materials likepollen and fruit subjected to import-export among countries. Thepresence of Psa in asymptomatic fruits and pollen was investigat-ed from October 2009 to October 2010. Symptomless fruits andpollen samples were analysed by bacterial isolation and two con-ventional PCR protocols. The presence of the bacterium in fruitssamples was investigated, for its endophytic presence, in 50 fruitsin bulk. Psa was detected in 6 of 49 fruit bulk-samples by isola-tion, and in 10 of 49, by PCR. One bulk fruit sample, was PCR-positive. The PCR performed for each single fruit showed a faintsignal by both the PCR assays in 5-7 of 50 fruits assessed. Con-cerning pollen samples, Psa was detected in 8 of 11 pollen sam-ples by isolation, and in 10 of 11 samples by PCR. The obtainedresults showed that fruit and pollen of kiwifruit can harbour thisemerging bacterium. Psa detection from bulk fruit samples isprobably due to the summation of a low charge of Psa distributedover more fruits; as a consequence, the risk of introducing thebacterium in Psa-free areas could be negligible. The role of thepollen in the dissemination of the disease is object of discussionand of development of further studies.

TOWARDS THE IDENTIFICATION OF RESISTANCE-RE-LATED GENES IN VITIS RIPARIA: AGROBACTERIUM-BASED FUNCTIONAL GENOMICS. A. Lovato, L. Bortesi, S.Dal Santo, T. Pandolfini, M. Pezzotti and A. Polverari. Diparti-mento di Biotecnologie, Università degli Studi, Strada le Grazie 15,37134 Verona, Italy. E-mail: [email protected]

Establishment of functional genomics in Vitis spp. is still diffi-cult, due to the lack of reliable high-throughput tools forgrapevine transformation. We are presently approaching twoAgrobacterium-mediated strategies in Vitis riparia, which is resist-ant to Plasmopara viticola, to study the function of genes possiblyrelated to the defense response. Genes have been identified in aprevious microarray analysis, on the basis of their specific expres-sion in V. riparia at early stages of downy mildew infection. Thefirst strategy is based on vacuum agroinfiltration of grape leaves.The technique was optimized for the expression of the GUS re-porter gene and for the silencing of the grapevine phytoene de-saturase (PDS) gene. In the second strategy we used Agrobacteri-um rhizogenes hairy root production to test whether the silencingsignal triggered by expression of the anti-PDS hairpin constructcan spread systemically from hairy roots to the leaves, resulting indown-regulation of target genes. V. riparia plantlets were infectedwith A. rhizogenes ARQA1 transformed with a pRedRoot vectorcontaining a 35S-hairpin construct against the grapevine PDSgene and a marker gene encoding the red fluorescence protein(DsRed1) under the control of a constitutive root promoter. Thisstrategy should allow us to select plantlets with transformed rootsbased on red fluorescence emission and to observe the possiblechlorophyll degradation caused by PDS gene silencing, if any.



FUSARIUM HEAD BLIGHT RESPONSE QTLs IN DURUMWHEAT. M. Maccaferri1, G. Ferrazzano2, M. Pascale3, K. Am-mar4, M.A. Canè1, P. Mantovani2, A. Prodi1, P. Nipoti1, M.C.Sanguineti1 and R. Tuberosa1. 1Dipartimento di Scienze e Tecnolo-gie Agroalimentari, Università degli Studi, Viale Fanin 44, 40127Bologna, Italy. 2Produttori Sementi Bologna, Via Macero 1, 40050Argelato (BO), Italy. 3Istituto di Scienze delle Produzioni Alimen-tari, CNR, Via Amendola 122, 70126 Bari, Italy. 4CIMMYT, Int. Apdo Postal 6-641, Mexico City, 06600 Mexico. Email: [email protected]

Fusarium head blight (FHB) is a serious challenge for durumwheat production in environments subjected to recurrent epi-demics, such as northern and central Italy. Effectiveness of breed-ing for quantitative trait (e.g. FHB response) can be improvedthrough quantitative trait loci (QTL) mapping and subsequentmarker-assisted selection activities. The presence of QTLs forFHB response was investigated in the cv. Kofa x Svevo popula-tion which includes 249 recombinant inbred lines (RILs) withknown genotype at ca. 200 simple sequence repeat markers. In2010, the RILs were evaluated in three field trials carried out inItaly (Argelato and Cadriano, Bologna) and Mexico (El Batan).The trials were mist-irrigated and artificially inoculated with lo-cal, highly virulent strains of Fusarium graminearum. FHB inci-dence and severity, percentage of damaged kernels, grain weight,test weight and DON content were recorded. The final infectionlevels were always high with the susceptible parent Kofa consis-tently showing an FHB incidence score (70-100%) higher thanthe medium-resistant parent Svevo. Heritability of FHB inci-dence and severity ranged from 15 to 60%. Some RILs showedconsiderably lower FHB disease scores than Svevo. In total, tenQTLs for FHB response were identified. Two main QTLs affect-ing all the FHB-related traits were located in chromosomes 2BLand 3BS. Some minor QTLs (chromosones. 1A, 1B, 6A and 7B)showed specificity for FHB-responses, while others (chro-mosones. 2A, 2B, 4A and 4B) affected FHB-responses and eitherheading date or plant height. These results indicate the presenceof novel QTLs for FHB-response in the elite durum germplasm.

GENETIC CONTROL OF RESISTANCE TO SOIL-BORNECEREAL MOSAIC VIRUS IN DURUM WHEAT. M. Macca-ferri1, R. Francia1, C. Ratti1, C. Rubies Autonell1, A. Massi2, S.Stefanelli1, R. Tuberosa1 and M.C. Sanguineti1. 1Dipartimento diScienze e Tecnologie Agroalimentari, Università degli Studi, VialeFanin 44, 40127 Bologna, Italy. 2Produttori Sementi Bologna, ViaMacero 1, 40050 Argelato, Bologna, Italy. E-mail: marco.maccafer-ri@ unibo.it

Soil-borne cereal mosaic virus (SBCMV) is the causal agent of aviral disease which affects wheat in Europe. In durum wheat, thecultivated germplasm shows responses ranging from susceptibilityto almost complete resistance. Genetic analysis of SBCMV resist-ance was carried out using two recombinant inbred line (RIL) pop-ulations of 180 lines each, obtained from the crosses Meridiano (re-sistant)×Claudio (moderately susceptible) and Simeto (susceptible)×Levante (resistant). The RILs were characterized for SBCMV re-sponse (symptom severity scores and virus concentration in theleaves) in the field in 2007, 2008 and 2009 under severe and uni-form SBCMV infections and were genotyped with SSR and DArTmarkers. Transgressive segregation was observed for the disease re-sponse traits, with high heritability values, consistently >80%. Amajor QTL (QSbm.ubo-2BS) in the distal chromosome 2BS regionwas identified in both populations; it accounted for 60-85% of thephenotypic variation for symptom severity, 40-55% for virus con-centration and 15-30% for grain yield. The meta-QTL analysis car-

ried out using the entire dataset suggested that in both populationsSBCMV resistance is likely to be controlled by the same QTL, withthe favorable allele being contributed by cvs Meridiano and Lev-ante. Our results confine QSbm.ubo-2BS in a ca. 2 cM-wide intervalflanked by SSR markers that are being used for marker-assisted se-lection. Several QTLs with minor effects were also detected. Thepresence of QSbm.ubo-2BS was validated in a panel of 111 cultivat-ed durum wheat accessions. The reported results open to the pos-sibility to further characterize QSbm.ubo-2BS and suggest its feasi-bility for a positional cloning approach.

RESPONSE TO SEPTORIA TRITICI IN THE DURUMWHEAT GERMPLASM INVESTIGATED THROUGH AS-SOCIATION MAPPING. M. Maccaferri1, A. Ricci1, K.Ammar2, R. Talebi3, T. G. Mahmod3, G. Kema3, S. Corneti1, M.C. Sanguineti1 and R. Tuberosa1. 1Dipartimento di Scienze e Tec-nologie Agroalimentari, Università degli Studi, Viale Fanin 44,40127 Bologna, Italy. 2CIMMYT, Int. Apdo Postal 6-641, MexicoCity, Mexico 06600. 3Plant Research International B.V., 6700 AAWageningen, The Netherlands. E-mail: marco.maccaferri@ unibo.it

Durum wheat production in the Mediterranean basin isplagued by a range of biotic stresses. Among these, Septoria triticiblotch (Mycosphaerella graminicola) has become important insouthern Europe and north Africa. The high genome plasticity ofthe pathogen and its specialization features (durum vs. breadwheat) complicates the identification of valuable resistance genes(e.g. durable and effective across diverse regions). The geneticvariation for the response to Septoria and the chromosomal loca-tion of resistance factors were studied using a germplasm collec-tion of 183 durum accessions of diverse origin suitable for associ-ation mapping (Maccaferri et al., 2006, Plant Genetics Resources4: 79-85). The panel was evaluated for two consecutive years(2008 and 2009) in Tunisia (Beja), Mexico (Toluca) and Italy(Argelato and Ferrara). The accessions were then inoculated un-der controlled conditions with Mycosphaerella isolates collectedfrom durum wheat in a range of Mediterranean countries, as wellas with bread wheat isolates. The germplasm collection was geno-typed with ca. 300 simple sequence repeat (SSRs) markers ofknown map position and ca. 900 durum DArT markers. Highlydiversified marker-trait association patterns were obtained basedon the field and seedling (as % of necrotic leaf area and picnidiaproduction). A preliminary analysis highlighted some chromo-some regions consistently involved in Septoria resistance, particu-larly in chromosomes 1BL, 2AL and 4AL that accounted for asizeable portion of phenotypic variation among accessions.

DISSECTION OF THE POWDERY MILDEW-RESISTANTRESPONSE PRESENT IN THE DURUM WHEAT cv. CLAU-DIO. M. Maccaferri1, A. Ricci1, F. Bini1, F. Chen1, S. Stefanelli1,S. Vecchi1, S. Corneti1, A. Massi2 and R. Tuberosa1. 1Dipartimen-to di Scienze e Tecnologie Agroalimentari, Università degli Studi,Viale Fanin 44, 40127 Bologna, Italy. 2Produttori Sementi Bologna,Via Macero 1, 40050 Argelato, Bologna, Italy. E-mail: marco.mac-caferri@ unibo.it

Powdery mildew (Pm) is one of the main foliar diseases affect-ing durum wheat in southern Europe. To date, a few resistancesources have been characterized and mapped in durum wheat.Based on repeated field observations, the durum wheat cv. Clau-dio shows a resistance response consistent over the years. Toidentify the Pm resistance loci carried by cv. Claudio, a popula-tion of 181 recombinant inbred lines from the cross Claudio (re-



sistant) × Meridiano (susceptible) has been evaluated in tworeplicated field trials carried out under artificially inoculated con-ditions in 2007 and 2008 at Bologna (northern Italy). In parallel,a linkage map based on SSR and DArT molecular markers wasproduced. The frequency distribution of the data collected overtwo years showed that the genetic control of Pm response in thispopulation is rather complex. Two major quantitative trait loci(QTLs) have been located on chr. 6BL (QPm.ubo-6B) and 7BL(QPm.ubo-7B) with both resistance alleles contributed by cv.Claudio. The effect of QPm.ubo-7B against Pm infection declinedas the disease progressed, while QPm.ubo-6B showed an in-creased effectiveness. Additional minor QTLs with lower and lessconsistent effects across the years were found on chromosomes2BS, 2BL, 6AS and 7AS, with both parents contributing resist-ance alleles. QPm.ubo-7B is tagged by barc340, gwm146 andgwm344, possibly located in the homoeologous position of Pm1.Claudio is thus characterized by a polygenic resistance based ontwo main loci and several minor genes.

LACCASE ACTIVITY IN TRICHODERMA VIRENS. L.Mannella1, L. Guglielminetti2, G. Vannacci1 and M. Vergara3.1Dipartimento di Coltivazione e Difesa delle Specie Legnose “G.Scaramuzzi”, Sezione di Patologia Vegetale, Università degli Studi,Via del Borghetto 80, 56124 Pisa, Italy. 2Dipartimento di Biologia,Università degli Studi, Via Mariscoglio 34, 56124 Pisa, Italy. 3Scuo-la Normale Superiore, Piazza dei Cavalieri 7, 56126 Pisa, Italy. E-mail: [email protected]

Fungal laccases are involved in multiple functions, such aslignin degradation, pigments (melanin) synthesis and degrada-tion, detoxification and pathogenesis. Furthermore, they are use-ful biocatalysts for several biotechnological applications.The lac-case gene function was previously analysed in Trichoderma virens,an effective biocontrol agent: six genes were identified and one ofthem was deleted and shown to be involved in the mycoparasiticactivity against B. cinerea sclerotia. Laccase activity in some Tri-choderma spp. is also associated with the production of green pig-ment in conidial spores. Further investigations on the laccasegene family in T. virens were performed in order to exploremechanisms putatively involved in ligninolysis, conidiogenesisand industrial dyes decolorization. Laccase functions in lignocel-lulosic process and sporulation mechanisms were studied bygrowing T. virens on two different substrates: wheat straw liquidmedium, containing lignocellulose as the only carbon source, orsolid Hölker medium, formulated to induce spore formation. Inaddition, liquid cultures containing twelve commercial textiledyes were set up and T. virens efficiently decolorized three ofthem. Biochemical and expression analyses performed on thesedifferent experimental systems suggested different roles of therelative enzymes in regulating multiple mechanisms. In conclu-sion, information is gained about some properties of the T. virenslaccase gene family. Six genes are shown to be differentially in-volved in physiological processes in T. virens some of which areimportant for its fitness or antagonistic attitude while others areexploitable in biotechnological applications related to ligninolysisor to textile dyes decolorization.

BIOCONTROL OF PENICILLIUM DIGITATUM AND P.ITALICUM OF ORANGE BY AUREOBASIDIUM PULLU-LANS. C. Mantelli, A. Spadoni and M. Mari. CRIOF, Diparti-mento di Protezione e Valorizzazione Agroalimentare, Universitàdegli Studi, Viale G. Fanin 46, 40127 Bologna, Italy. E-mail: [email protected]

Penicillium italicum and Penicillium digitatum causing blueand green mould, respectively, are the most common and seriouspathogens of citrus fruits in Mediterranean climates. Althoughfungicides are commonly used worldwide to control citruspostharvest moulds, alternatives to chemicals such as biologicalcontrol agents (BCAs) are extensively investigated. The aim ofthe present work was to investigate the efficacy of yeast Aureoba-sidium pullulans, L1 and L8 strains, isolated from the surface ofhealthy peach as a putative BCA for citrus blue and green mould.In in vivo trials, cv. Valencia oranges were treated with washed orautoclaved cells (108 CFU ml-1) or with sterile filtrate of L1 andL8 and were subsequently inoculated with a conidial suspension(5x103 conidia ml-1) of each pathogen. After 10 days at 20°C, thewashed cells completely inhibited P. italicum and P. digitatum, nocontrol of decay was observed in fruits treated with sterile fil-trates, while autoclaved cells of both BCAs reduced the incidenceof disease (-50%) compared to the control. In in vitro experi-ments the production of volatile compounds by BCAs was evalu-ated by a dual culture technique. L1 and L8 strains reduced theP. italicum growth of 48% and 45%, respectively with respect tothe control, while P. digitatum was inhibited by 29% (L1) and28% (L8). In a previous work, L1 and L8 strains showed a goodefficacy towards Monilinia laxa, M.fructicola and M. fructigena at-tacks to peach and the present results confirm the broad spec-trum of activity of this BCA against fruit postharvest pathogens.

PRELIMINARY INVESTIGATIONS ON FUNGAL DIS-EASES OF GOLF COURSES IN NORTHERN ITALY. C.Martini, L. Mazzarolo and M. Mari. CRIOF, Dipartimento diProtezione e Valorizzazione Agroalimentare, Università degli Studi,Via Gandolfi 19, 40057 Cadriano (BO), Italy. E-mail:[email protected]

The aim of this study was to monitor the fungal diseases infour golf courses located in northern Italy and to characterize thefungal isolates by testing the resistance to fungicides commonlyused against turf diseases. The influence of temperature on resist-ance to propiconazole, tebuconazole and prochloraz of Sclero-tinia homoeocarpa isolates was also investigated. Over 200 dis-eased turf samples were collected during the 2008, 2009 and2010 summers. S. homoeocarpa was the main fungal pathogen iso-lated (33%) followed by Microdochium nivale (29%), Curvulariainaequalis (20%) and Curvularia coicicola (6%). The occurrenceof Colletotrichum graminicola, Rhizoctonia cerealis and Laetisariafuciformis was low compared to other fungi. Ninety isolates of S.homoeocarpa were tested for sensitivity to the demethylation-in-hibiting (DMI) fungicides (propiconazole and tebuconazole) andthe inhibiting ergosterol biosynthesis (BSA) fungicides (prochlo-raz). Both DMI and BSA fungicides are registered for turfgrass inItaly. The pathogen’s sensitivity to fungicides was assessed usingthe discriminatory concentrations of 0.2 mg/l for propiconazole,tebuconazole, and prochloraz. The EC50 was estimated on rela-tive mycelial growth on fungicide-amended medium confirmingthe presence of resistance to propiconazole, tebuconazole andprochloraz in Italian S. homoeocarpa isolates. In addition, the iso-lates were inoculated on medium amended with the EC25 fungi-cide concentration and incubated at different temperatures(10°C, 20°C and 30°C) for 72 h. At 30°C the reduction of fungalgrowth on the fungicide amended medium was higher than thatobserved at 20°C (60% against 20%). More investigation are inprogress to elucidate the molecular bases of this phenomenon.



SENSITIVITY TO TEBUCONAZOLE AND THIO-PHANATE METHYL OF MONILINIA LAXA FROM EMIL-IA ROMAGNA ORCHARDS. C. Martini1, G. Schnabel2 andM. Mari1. 1CRIOF, Dipartimento di Protezione e ValorizzazioneAgroalimentare, Università degli Studi, Via Gandolfi, 19, 40057Cadriano (BO), Italy. 2Department of Entomology, Soils, and PlantScience, Clemson University, Clemson SC 29634, USA. E-mail:[email protected]

Demethylation inhibitor (DMI) and to a lesser degree methylbenzimidazole carbamate (MBC) fungicides are used in many Eu-ropean production areas to control stone fruit diseases and toavoid economic losses. Symptomatic blossoms, twigs, and fruitswere collected in summer from stone fruit orchards in Emilia Ro-magna (northern Italy) between 2009 and 2010. Over 100 single-spore isolates were generated; 61 were Monilinia laxa, 34 were M.fructicola and 5 were M. fructigena. Sensitivity of M. laxa to DMIfungicide (tebuconazole) and MBC fungicide (thiophanatemethyl) was determined. Sensitivity was assessed with the spiralgradient endpoint method developed in 2004. The concentrationof the fungicide is highest at the center of the plate and lowest atthe edge of the plate. The EC50values were calculated using SGEsoftware. The range of EC50 values for tebuconazole was 0.004 to0.32 µg/ml and for thiophanate methyl 0.03 to 2.37 µg/ml. FourM. laxa isolates were medium resistant to tebuconazole with EC50values of 0.12, 0.16, 0.18 and 0.32 µg/ml and 57 isolates weresensitive to tebuconazole with EC50 values ranging from 0.004 to0.063 µg/ml. Six M. laxa isolates exhibited low resistance to thio-phanate methyl with EC50 values ranging from 0.88 to 2.37 µg/mland 55 isolates were sensitive to thiophanate methyl with EC50values ranging from 0.03 to 0.69 µg/ml. The sensitivity pheno-types of selected isolates were verified with discriminatory dosesof 0.1 and 0.3 µg/ml for tebuconazole and 1 µg/ml for thio-phanate methyl.

ACIDOVORAX AVENAE subsp. CATTLEYAE ON ORCHIDIN LIGURIA. P. Martini1, M. Odasso1, L. Repetto1, E. Biondi2,S. Mucini2 and U. Mazzucchi2. 1Istituto Regionale per la Flori-coltura, Via Carducci 12, 18038 Sanremo (IM), Italy. 2Dipartimentodi Scienze e Tecnologie Agroambientali, Università degli Studi, VialeFanin 40, 40127 Bologna, Italy. E-mail: [email protected]

In Liguria region (northern Italy), during 2007 pot-grownplants of Phalaenopsis hybrid, cv. Malibù Lagoon, with leaf spotssymptoms were observed in a commercial nursery at Albenga(SV). In 2010, the same symptoms were observed on Phalaenopsishybrid, cv. Musashino Moon, in a commercial glasshouse nearSanremo (IM). The leaves of both samples showed soft, water-soaked lesions which eventually turned brown or black. The old-er plants died. To isolate the causal agent, cut leaves tissue (1-3mm tips) of each sample exhibiting bacterial streaming were dis-infected for 1 min in 10% NaClO solution and rinsed for 1 minwith SDW; then the tissues were macerated into mortars contain-ing a sterile saline solution. Serial dilutions up to 1×10–4 weremade in sterile water; 10 µl of each suspension was plated ontoNA medium and incubated at 25°C for 3 days. Non-fluorescent,Gram-negative bacteria were constantly isolated from the lesions.For each sample, three pure cultures were identified phenotypi-cally. Using Biolog GEN III Microplates and the Microlog Sys-tem (data base 5.1.1; Biolog, Hayward, CA) the bacteria showed0.927-0.997 similarity with Acidovorax avenae subsp. cattleyae.The identity was confirmed by PCR using specific primers. Path-ogenicity tests were carried out on Phalaenopsis hybrid plants.The isolates caused typical symptoms and reisolations were suc-cessful. This is the first report of A. avenae subsp. cattleyae on

Phalaenopsis hybrids in Liguria, and the third in Italy.

This work was partially funded by the “Programma ALCO-TRA, obiettivo cooperazione territoriale europea Italia-Francia(Alpi) 2007/2013 - Progetto FIORIBIO.

EVIDENCE AGAINST PATHOADAPTATION OF ER-WINIA AMYLOVORA IN EMILIA-ROMAGNA AND VENE-TO INVOLVING AN HRP-LINKED PATHOGENICITYLOCUS, DSPEF. P. Minardi1, S. Mucini2 and U. Mazzucchi2.1Dipartimento di Scienze Mediche Veterinarie, Università degli Stu-di di Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia (BO),Italy. 2 Dipartimento di Scienze e Tecnologie Agroalimentari, Uni-versità degli Studi, Viale Fanin 42, 40127 Bologna, Italy. E-mail:[email protected]

In Emilia-Romagna and Veneto (northern Italy) a clone of Er-winia amylovora (Ea1994) gradually spread mostly on pear trees,starting from the primary 1994 foci. In recent years an unusualincrease of certified fire blight cases on apple trees was observed.The introduction of novel strains or the modification of virulencein the existing clonal populations were hypothesized. A pathoad-aptation to the niche of a different natural host might involve thedspEF locus of the pathogenicity island (PAI1) subjected to im-mune selective pressure of the host plants. Pathoadpatation hy-pothesis was tested by restriction fragment analysis of the dspEgene, by the dspF gene sequence analysis in strains with virulencehigher than Ea1994 and by dspE and dspF genes sequence analy-sis of Ea1994. The restriction profiles were indistinguishable.Moreover, sequence analysis revealed that the sequences of thedspE and the dspF genes correlated 100% with those of Ea321, awild-type North American strain, used for comparison. The re-sults confirmed that the dspEF locus is highly conserved in thegenome of the species. Moreover, our results showed that inEmilia Romagna and Veneto E. amylovora strains more virulentthan the one associated with the primary foci can be found ondifferent host plants and locations, and that the increase of fireblight cases in apple do not seem attributable to pathoadaptationinvolving changes in the dspE and dspF genes, without ruling outpossible modifications in their expression.

THE DSPF AMPLICON AS A RELIABLE DIAGNOSTICTRAIT FOR THE FIRE BLIGHT PATHOGEN, ERWINIAAMYLOVORA. P. Minardi1, S. Mucini2, C. Lucchese2 and U.Mazzucchi2. 1Dipartimento di Scienze Mediche Veterinarie, Uni-versità degli Studi di Bologna, Via Tolara di Sopra 50, 40064 Oz-zano Emilia (BO), Italy. 2Dipartimento di Scienze e TecnologieAgroalimentari, Università degli Studi, Viale Fanin 42, 40127Bologna, Italy. E-mail: [email protected]

Fire blight has gradually spread in Europe, becoming endemicin many areas. Indeed, in northern Italy, except for Valle d’Aosta,cases of fire blight have been ascertained in all regions. An accu-rate diagnosis of fire blight is essential to gather reliable evidenceof its appearance in new areas. Moreover, an additional assay forthe identification of E. amylovora (Ea) can be very useful in theanalysis of asymptomatic nursery material during the quiescentperiod. In Italy, the currently used diagnostic protocol was devel-oped about 15 years ago when the compulsory control decreewas laid down (DM 356, 1999) and needs now to be updated. Inthe most recent diagnostic protocol proposed by EPPO (2004)the analysis includes the use of PCR primers designed to amplifysequences of the pEA29 plasmid that until a few years ago was



believed to be present in all strains of Ea and essential for viru-lence. Recent studies have drawn attention to the existence of vir-ulent Ea strains free of pEA29. The risk of false negative reac-tions caused by the absence of this plasmid could not be exclud-ed. In this study, the development is described of a PCR assay forEa diagnosis based on the use of primers designed on the se-quence of the dspF gene which encodes the chaperon of the cru-cial effector DspE. Its reliability was tested in a collection of Eastrains, and in saprophytic and pathogenic bacteria isolated fromEa host plants. The results showed that the PCR primers basedon the sequence of the dspF gene produce an amplicon highly re-liable for the diagnosis of fire blight.

STUDY OF THE DSPE GENE ESPRESSION IN ERWINIAAMYLOVORA USING THE GREEN FLUORESCENT PRO-TEIN. P. Minardi1, S. Mucini2 and U. Mazzucchi2. 1Dipartimen-to di Scienze Mediche Veterinarie, Università degli Studi diBologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia (BO), Italy.2Dipartimento di Scienze e Tecnologie Agroalimentari, Universitàdegli Studi, Viale Fanin 42, 40127 Bologna, Italy. E-mail: [email protected]

In phytopathogenic bacteria, the study of gene expression isof great value to understand the mechanisms involved in patho-genicity. The use of fluorescent markers, such as GFP, allow toobserve the pathogen in planta and to better understand the host-pathogen interaction focusing on the temporal expression of thegenes involved. In E. amylovora (Ea), the expression of the genesof the pathogenicity island (PAI1) play a crucial role in the earlyplant-pathogen interactions.With this preliminary study we aimat determining the in vitro and in planta conditions and timingunder which the dspE gene expression occurs. To do so, the ref-erence Ea1994 strain, isolated from pear in 1994 and virulent topear trees, and 7 northern Italian strains more virulent thanEa1994 were electro-transformed with the pDFl 124.B1 plasmidcontaining the gene for the protein gfpIII GFP under the controlof the Ea dspE gene encoding the DspE effector essential forpathogenesis. As for the dspE gene expression in minimal medi-um, our preliminary data confirm that the dspE gene is expressedonly in conditions of nutrient deficiency. The absence of the ex-pression in rich media is associated with several factors includingpH and a high osmolarity, and the presence of carbon sourcesavailable for the pathogen. As for the dspE gene expression in im-mature pears, our results showed that Ea may need an initial peri-od of adaptation to the host niche since the dspE gene expressionwas observed between 24 and 48 h after penetration. However,the dspE gene expression in immature pears, in the first 24 h,concurs with the hypothesis that the dspE gene product is inject-ed into the host cell 3-4 h after penetration when the bacteriumhas not yet multiplied or has just completed its first division.

EVIDENCE AGAINST THE PRESENCE OF PSEUDO-MONAS SYRINGAE pv. ACTINIDIAE IN FRUITS OF AC-TINIDIA ORCHARDS AFFECTED BY BACTERIALCANKER IN EMILIA-ROMAGNA. P. Minardi1, C. Lucchese2,S. Ardizzi2 and U. Mazzucchi2. 1Dipartimento di Scienze MedicheVeterinarie, Università degli Studi di Bologna, Via Tolara di Sopra50, 40064 Ozzano Emilia (BO), Italy. 2Dipartimento di Scienze eTecnologie Agroalimentari, Università degli Studi, Viale Fanin 42,40127 Bologna, Italy. E-mail: [email protected]

In Emilia Romagna, kiwifruit is of great economic relevancefor the horticultural industry and large investments have been

made in production plants and commercial platforms. From Oc-tober 2009, the sudden appearance of sporadic cases of bacterialcanker and the high costs of diagnosis and control are threaten-ing the prospects of the entire industry which plays a relevantrole for the Italian agricultural export as a whole. Hence, to in-vestigate whether the fruits themselves can harbor the causalagent of bacterial canker of kiwifruit, Pseudomonas syringae pv.actinidiae (Psa) acting as a possible source of dissemination of thepathogen, is essential. Samples of fruits of Actinidia chinesis cv.Jin Tao were harvested from symptomatic plants affected by bac-terial canker and symptomless plants close to them before break-ing down. Two samples were harvested directly from the plantsand one sample from waste bins. Groups of 15 fruits were as-sayed for the presence of Psa on the surface and within the col-umellae. Isolation from these samples allowed to select 36 Psa-like cultures (19 epiphytes and 17 endophytes). PCR analysis us-ing Psa-specific primers, HR on tobacco leaves and pathogenicitytest on tobacco leaves and on Actinidia deliciosa seedlings exclud-ed the presence of Psa. Our results show that in the fruits ofplants affected by bacterial canker, or close to them, Psa is not as-sociated with either the surface or inside the fruit. However itcannot be ruled out that Psa cells present on the fruits might benumerically below the sensitivity threshold of the detection tech-nique.

DIGOXIGENIN-LABELED DNA PROBES APPLIED TOTHE SIMULTANEOUS DETECTION OF TEN ARTI-CHOKE VIRUSES. S.A. Minutillo1, T. Mascia2,3 and D. Gal-litelli2,3. 1Agritest S.r.L. Strada Provinciale per Casamassima 3,70010 Valenzano (BA), Italy. 2Dipartimento di Biologia e ChimicaAgroforestale ed Ambientale, Università degli Studi “Aldo Moro”,Via Amendola 165/A, 70126 Bari, Italy. 3Istituto di Virologia Vege-tale del CNR, UOS Bari, Via Amendola 165/A, 70126 Bari, Italy.E. mail: [email protected]

Artichoke Italian latent virus (AILV), Artichoke latent virus(ArLV), Artichoke mottled crinkle virus (AMCV), Bean yellowmosaic virus (BYMV), Cucumber mosaic virus (CMV), Pelargoni-um zonate spot virus (PZSV), Tomato infectious chlorosis virus(TICV), Tobacco mosaic virus (TMV), Tomato spotted wilt virus(TSWV) and Turnip mosaic virus (TuMV) are pathogernic to arti-choke, so new artichoke fields should be planted with certifiedvirus-free germplasm. Accurate assessment of the sanitary condi-tion of artichoke nursery productions can be carried out usingnucleic acid-based tests, which overcome some of the drawbacksof serology-based virus detection for this crop. We have devel-oped a protocol for the simultaneous detection of ten artichokeviruses by non-isotopic molecular hybridisation with a DNAprobe. All DNA probes had approximately the same size andwere labelled with DIG-UTP in a PCR reaction. The probe mixdetected all viruses with a sensitivity similar to that obtained us-ing individual probes. In addition, we evaluated the possible useof the tissue-printing as a sample preparation technique appliedto routine diagnosis of artichoke viruses. DNA probes were alsoused to detect AILV and ArLV in artichoke seed coats and cotyle-dons and for early detection in young explants produced duringsanitation of artichoke germplasm. The detection limit for eachindividual probe was determined by using the complementaryunlabeled plus-strand viral RNA added to artichoke sap extract-ed from healthy plants. The color-based detection of hybridsovercomes the needs of expensive equipments and makes thetechnique affordable also for small diagnostic laboratories.



SOURCES OF RESISTANCE TO THE TUBER NECROTICSTRAIN OF POTATO VIRUS Y (PVYNTN) IN WILD POTA-TO SPECIES. V. Miraglia1, R. Garramone1, C. Villano1, D.Carputo1 and D. Alioto2. 1Dipartimento di Scienze del Suolo, dellaPianta, dell’Ambiente e delle Produzioni Animali, Università degliStudi di Napoli, Via Università 100, 80055 Portici (NA), Italy. 2Di-partimento di Arboricoltura, Botanica e Patologia Vegetale, Univer-sità degli Studi di Napoli, Via Università 100, 80055 Portici (NA),Italy. E-mail: [email protected]

The tuber necrotic strain of Potato virus Y (PVYNTN) cancause tuber necrotic ringspot, one of the most devastating dis-eases of cultivated potato. The use of resistant varieties is the bestapproach to control this virus in the absence of effective controlmeasures. Source of resistance can be identified within the largegenetic variability of wild potato germplasm. The objective of thiswork was to assess the response to artificial PVYNTN inoculationof 21 genotypes belonging to 12 wild potato species. Plants ofeach genotype were grown under greenhouse conditions andwere mechanically inoculated with PVYNTN. Plants were visuallyinspected for symptom expression 15 and 30 days post inocula-tion. On the same day, tissue samples from upper non inoculatedleaves were collected for serological analysis by ELISA. ELISA-negative plants were re-tested by RT-PCR. The screening test wasreplicated twice to rule out any potential escapes. Four genotypesbelonging to Solanum bulbocastanum, S. etuberosum, S. cardio-phyllum and S. phureja were extremely resistant (no disease symp-toms and negative ELISA and RT-PCR reactions from all testedplants). Two genotypes showed partial resistance, in one the per-centage of resistant plants decreased over time, in the second sys-temic infection was delayed of some days. Moreover, the similari-ty indexes obtained from SSR analysis highlighted differencesamong genotypes. Genotypes identified here will be used inbreeding programs aimed at transferring the resistance into culti-vated potato.

SELECTION AND IN VITRO CHARACTERIZATION OFPOTENTIAL BIOCONTROL AGENTS TO PROTECTLAMB’S LETTUCE IN HYDROPONICS. S. Moruzzi, G. Fir-rao, and M. Martini. Dipartimento di Scienze Agrarie e Ambien-tali, Università degli Studi, Via delle Scienze 208, 33100 Udine,Italy. E-mail: [email protected]

Fungi of the genera Pythium and Rhizoctonia, particularlyadapted to wet environment, are among the most harmful patho-genic microorganisms in hydroponics. Biocontrol microorgan-isms can play an important role in suppressing these rootpathogens in soilless systems. In the present work, bacterial mi-croorganisms were isolated in order to select potential biocontrolagents. Fifty-one strains were isolated from 30 root samples, col-lected from healthy hydroponic lamb’s lettuce plants grown inFriuli Venezia Giulia (FVG, north-eastern Italy). According tothe results of preliminary antagonism tests 12 promising bacteriathat underwent to further investigations, were selected. The iso-lates were characterized by RFLP and sequence analysis of a PCRamplified 16S rDNA region. The results showed that 3 isolateswere closely related to Pantoea agglomerans, whereas 9 isolatesbelonged to the genus Pseudomonas. The selected strains weretested for their potential to inhibit in vitro the growth of 2 strainsof Pythium aphanidermatum (CBS 118745, CBS 116664) and 2strains of Rhizoctonia solani isolated from symptomatic hydro-ponic plants in FVG. Fungal mycelium was centrally placed inPetri dishes that had been previously inoculated with bacteriaand incubated 48 h. The plates were further incubated at 24°Cfor 9 days, and the fungal growth was measured daily. Among the

12 isolates, one strain of Pseudomonas fluorescens and one strainof P. agglomerans showed an average inhibition of mycelialgrowth of 89% and 70%, respectively. Trials to assess the poten-tial of the selected bacteria as biocontrol agent against lamb’s let-tuce root pathogens are currently in progress.

MOLECULAR AND CHEMICAL CHARACTERIZATIONOF THE PATHOSYSTEM FUSARIUM VERTICILLIOIDES-MAIZE. G. Mulè2, I. Lazzaro1, A. Susca2, A. Ritieni3, A. Lanu-bile4, J. Bernardi4, A. Marocco4 and P. Battilani1. 1Istituto di En-tomologia e Patologia Vegetale, Università Cattolica del SacroCuore, Via Emilia Parmense 84, 29100 Piacenza, Italy. 2Istituto diScienze delle Produzioni Alimentari, CNR, Via Amendola 122/O,70126 Bari, Italy. 3Dipartimento di Scienza degli Alimenti, Univer-sità degli Studi “Federico II”, 80055 Napoli, Italy. 4Istituto diAgronomia, Genetica e Coltivazioni Erbacee, Università Cattolicadel Sacro Cuore, Via Emilia Parmense 84, 29100 Piacenza, Italy. E-mail: [email protected]

A two-year study on the pathosystem F. verticillioides-maizewas conducted speculating on both in vitro and in planta per-spectives. The former studied the effects of temperature (T) andwater activity (aw) on fumonisin B (FBs) production and expres-sion of FUM2 and FUM21 genes in ITEM 10027 and 1744 F. ver-ticillioides strains grown up to 21 days. The latter monitoredwhich genes were differentially expressed in resistant and suscep-tible maize lines after 2-3 days infection by ITEM 1744. The invitro study showed that ITEM 10027 was the highest FBs pro-ducer, with predominance of FB1. The maximum level of FB wasregistered after 21 days in both strains. FUM2 and FUM21 wereexpressed in all studied conditions, with a 10X difference be-tween the former and the latter. The peak of transcription levelwas reached after 14 days of incubation for both FUM genes. Nodata were collected on cultures grown at fixed aw=0.900 becausethe fungus did not grow at all temperatures tested till 21 days ofincubation. The in planta study showed that resistant lines testedwere poorly infected by ITEM 1744. Genes differentially ex-pressed were divided into 11 functional categories and nearly10% was assigned to the class “cell rescue, defence and viru-lence”. Most of the pathogenesis-related genes were differentiallyactivated after fungal infection in relation to the resistance levelof maize genotypes. In the resistant kernels, defence-relatedgenes provided basal protection against the fungus, while in thesusceptible kernels, the same genes were induced specifically af-ter pathogen attack.

Work supported by the Ministry of University and Research,PRIN 2007. Irene Lazzaro carried out this work within the PhDSchool “Agrisystem” of the Università Cattolica del Sacro Cuore(Italy).

STUDY OF VENTURIA INAEQUALIS SENSITIVITY TOSTROBILURINS: CORRELATION BETWEEN MOLECU-LAR AND BIOLOGICAL APPROACHES. I. Nanni, C. Tu-ran, R. Fiaccadori, G. Alberoni, M. Collina and A. Brunelli. Cen-tro di Fitofarmacia, Dipartimento di Protezione e ValorizzazioneAgroalimentare, Università degli Studi, Viale G. Fanin 46, 40127,Bologna, Italy. E-mail: [email protected]

Scab caused by Venturia inaequalis (Cke.) Wint. is the mostimportant disease of apple trees worldwide and requires a highnumber of fungicide applications. The introduction, in the late1990s, of strobilurin fungicides led to improve disease control.



After a few years of excellent protection, several cases of reducedscab control caused by pathogen resistance to strobilurins werereported from many countries. Our previous studies pointed outthe occurrence in Italy of V. inaequalis populations resistant tokresoxim-methyl and trifloxystrobin, with the presence of G143Asubstitution in cytochrome b, which is the most common muta-tion in strobilurin resistance in plant pathogenic fungi. The aimof this study was to compare the sensitivity of several V. inae-qualis populations coming from distinct locations obtained byconidial germination tests (DE50, percentage of germination at thehigher concentration) and molecular analysis detecting presenceand frequency of G143A substitution (CAPS PCR and Real TimePCR). Preliminary results from samples collected in 2009 and2010, generally demonstrate a good but not completely clear cor-relation between these different approaches.

BIOPROSPECTING FUNGAL ENDOPHYTES FROMFOREST PLANTS AT THE ASTRONI NATURE RESERVEFOR ANTITUMOUR EXTROLITES BASED ON THEIRFUNGITOXIC PROPERTIES. R. Nicoletti1, A. Carella1, A. DeFilippis2 and E. Buommino2. 1CRA, Unità di Ricerca per le Col-ture Alternative al Tabacco, Via Vitello 108, Scafati (SA), Italy. 2Di-partimento di Medicina Sperimentale, Seconda Università degliStudi di Napoli, Via de Crecchio 7, Napoli, Italy. E-mail:[email protected]

Fungal endophytes represent an effective component of biodi-versity of natural ecosystems which is increasingly considered forits implications in plant protection and growth promotion. Interac-tions in the first field can be mediated by antibiotics and/or cyto-static compounds whose bioactivity is sometimes exploited in hu-man medicine. Therefore the search for new pharmaceuticals cantake advantage by the discovery of novel such products, and actu-ally our previous experiences concerning soil-borne fungal antago-nists showed that fungitoxic compounds may have interesting an-tiproliferative and/or pro-apoptotic properties on human tumourcells. On the occasion of the International Year of Biodiversity in2010, an investigation concerning fungal endophytes of forest treeswas carried out at the Astroni Nature Reserve near Naples, withthe aim to identify a number of strains to be further exploited as asource of new antitumour compounds. One hundred and 7 isolateswere recovered from 28 plant species, and screened for their anti-fungal properties by evaluating the interactions with a strain ofRhizoctonia solani AG-2-1/Nt in dual cultures. In addition to 11isolates which were able to develop mycoparasitically, 37 isolatesdisplayed some extent of inhibitory capacity. Liquid cultures ofthese isolates were prepared, and the resulting culture extracts as-sayed for inhibitory effects on hyphal growth on the same R. solanistrain. Five isolates were able to completely inhibit R. solani over aweek, while 28 isolates induced retarded growth and/or alterationsof the hyphal structure. Effective extracts will be evaluated on hu-man tumour cell cultures for their further characterization and theisolation of the bioactive compounds.

SILVER LEAF OF APRICOT: AN EMERGENT DISEASE INEMILIA-ROMAGNA. P. Nipoti1, A. Mirotti2, S. Tonti1,3, A.R.Babini2, E. De Paoli2, C. Montuschi2 and A. Prodi1. 1Dipartimen-to di Scienze e Tecnologie Agroambientali, Viale Fanin 44, 40127Bologna, Italy. 2Servizio Fitosanitario Regione Emilia-Romagna,Via di Saliceto 81, 40128 Bologna, Italy. 3Istituto Nazionale diRicerca per gli Alimenti e la Nutrizione, Via Ca’ Nova Zampieri 37,37057 S. Giovanni Lupatoto (VR), Italy. E-mail: [email protected]

Apricot (Prunus armeniaca) is an expanding crop in easternEmilia Romagna (northern Italy). Since last two years the charac-teristic symptoms of silver leaf were noticed on 2- to 3-year-oldapricot orchards planted with new cultivars (Faralia, Farbaly,Kioto). The main symptom is a partial or total silvery sheen onthe foliage. The pathogen penetrates through wounds, thebranches may gradually die back and, sometimes, the affectedplant dies. Infected branches appear brown stained in transversesection, whereas the fruits remain smaller and fewer in number. Asimilar disease caused by Chondrostereum purpureum (Pers.)Pouzar was described on peach 20 year ago in many Emilia Ro-magna orchards. The fungus can colonize different host plantslike apples, pears, cherries, plums, peaches, apricots, nashi,berryfruit, poplars, willows, silver birches, eucalypts and roses.During summer 2010, many samples were collected from sympto-matic apricot plants. Wood disks were cut from the samples andcultured on a semi-selective medium, malt extract agar amendedwith benomyl, neomycin and streptomycin sulphate (BNS). Thecolonies showed early aerial mycelium, white turning to cream,woolly to floccose, granular to felty at 6 week, azonate and notfruiting. The colonies analyzed by a specific PCR primer pair,were molecularly identified as C. purpureum. This disease appearsas a limiting factor in apricot fruit production particularly for thenew varieties. So, appropriate investigations are desirable for re-ducing the impact of this pathogen.

PHYSIOLOGICAL AND MOLECULAR ALTERATIONSINDUCED BY ALTERNARIA ALTERNATA IN TWO VARI-ETIES OF APPLE. C. Nobili1, D. Carbone2, A.A. Fabbri2, M.Scarpari2, M. Punelli2, A. Ricelli3, C. Fanelli2 and M. Reverberi2.1Unità Tecnica Innovazione Biotecnologie Agroindustriali ENEAC.R., Casaccia, Roma, Italy. 2Dipartimento di Biologia Ambientale,Università “Sapienza”, Roma, Italy. 3ICB-CNR, P.le Aldo Moro 5,00185 Roma, Italy. E-mail: [email protected]

Alternaria alternata pv. mali causes a foliar and fruit disease tosusceptible varieties of apple known as Alternaria blotch disease(ABD). In Italy, a new disease of apple always related to an Al-ternaria infection, showing symptoms very similar to those de-scribed for ABD, was recently observed. The appearance ofnecrotic lesions on leaves and fruits suggests that also these newItalian isolates of A. alternata (B19, T25) may produce as the pv.mali (T65) virulence factors like AM toxin. The aim of this studyis to evaluate the pathogenicity of two Italian isolates of Alternar-ia in comparison with A. alternata pv. mali, during the experi-mental infections of detached fruits and leaves of the apple cvsGolden and Gala. In particular, we have evaluated in fruits andleaves the effects of this disease on the induction of an hyper-oxi-dant status through the evaluation of the main reactive speciesformation and degradation in the response to fungal infection.Reactive species (RS) can prime cell death, consequently the acti-vation of PCD, particularly in the leaves, was monitored at differ-ent time intervals, 0, 6, 24, 48 and 96 h post inoculation. Theseresults were matched, with determination by citofluorimeter ofthe apoptotic process in infected leaves. The results indicate thatas far as the evidences obtained, it could be hypothesised that celldeath induction, probably also consequent to the release of a notyet identified virulence factor, may play an important part in theestablishing this new disease of apples in Italy.

ANTIMICROBIAL ACTIVITY OF CEREAL BIOACTIVECOMPOUNDS. F. Nocente1,2, S. Bellato1, K. Carbone1, E.Galassi1, L. Gazza3, L. Sereni1, F. Taddei1, M. Pasquini1 and D.



Sgrulletta1. 1CRA, Unità di ricerca per la valorizzazione qualitativadei cereali, Via Cassia 176, 00191 Roma, Italy. 2Università CampusBio-Medico di Roma, Via Alvaro del Portillo 21, 00128 Roma,Italy. 3CRA, Centro di ricerca per la frutticoltura, Via di Fioranello52, 00134 Roma, Italy. E-mail: [email protected]

The antimicrobial properties of natural compounds or syn-thetic peptides against economically important plant pathogenicfungi and bacteria have been studied for their utilization in cropprotection. Vromindolines, starch-bound proteins involved in theextra-softness of the oat endosperm, are structurally similar topuroindolines PIN-A and PIN-B and as PINs are characterizedby a triptophan-rich domain likely responsible for their antimi-crobial activity. Alkylresorcinols (ARs) are a group of phenoliclipids involved in a multitude of interactions with biologicalmembranes; their amphiphilic and phenolic nature has been asso-ciated with their biological activity, but the mechanism of actionis not yet explained. Cereal alkylresorcinols are found to be mix-tures of saturated, monoenoic and dienoic homologues withmainly saturated alkyl chains in the range of 15-27 carbon atoms.Analyses have been carried out to test the antibacterial and anti-fungal activity of the vromindolines and a synthetic peptide(TRP), resembling their specific tryptophan-rich domain. Whentested in vitro these compounds showed a 70% inhibition of thegrowth of Escherichia coli and a minor effect on a DON (de-oxynivalenol) producing strain of Fusarium graminearum. Theantifungal activity of AR extracts, isolated from wheat intactgrain with two different solvents (ethyl acetate and acetone) wastested against F. graminearum. The bioassays showed that thefungal growth was distinctly reduced by addition of 400 µg of ARextracts to nutrient agar, the various extracts exhibiting a differ-ent inhibitory ability. The results suggest the availability of novelcompounds that could be considered alternative natural fungi-cides.

CHARACTERIZATION OF THE HYPERSENSITIVE RE-SPONSE INDUCED BY THE C2 PROTEIN OF TOMATOYELLOW LWAF CURL SADINIA VIRUS IN NICOTIANAspp. E. Noris1, A.J. Love2, R. Lozsa1 and S. Matic1. 1Istituto diVirologia Vegetale del CNR, Strada delle Cacce 73, 10135 Torino,Italy. 2The James Hutton Institute, Invergowrie Dundee DD25DA, United Kingdom. E-mail: [email protected]

The begomovirus Tomato yellow leaf curl Sardinia virus(TYLCSV) causes a devastating disease of tomato plants all overthe Mediterranean basin. Several functions have been attributedto the C2 protein of different begomoviruses. A functional analy-sis of the TYLCSV C2 protein has been undertaken. Agrobacteri-um-mediated transient expression of C2 resulted in a localnecrotic hypersensitive response (HR) in Nicotiana benthamianaand N. tabacum, as confirmed with trypan blue and 3,3’-di-aminobenzidine staining, thus indicating that C2 plays a key rolein pathogenicity.

We analyzed the dependence of C2-induced HR by utilizingVIGS vectors to silence some of the HR-related genes, such asSGT1, RAR1, and MEK2; HR was influenced upon silencing ofall of them. We also observed that the severity of HR was temper-ature dependant, so that HR was more pronounced at 25°C thanat 20°C. Using NahG transgenic N. benthamiana lines which failto accumulate salicylic acid (SA), a key potentiator of HR, we ob-served a different HR in C2-infiltrated tissue at both tempera-tures, indicating that SA participates to regulate the intensity ofC2-dependent HR. However, TYLCSV natural infection is notnormally associated with HR, suggesting that the virus encodesfactors that counter this response. Upon co-agroinfiltration with

other viral non-structural proteins, C2-induced HR could be par-tially counteracted.

SEED HEALTH IN ORGANIC FARMING SYSTEM. L.Orzali1, G. Di Giambattista1, L. Ortolani2, A. Matere3, A. San-tori1, F. Quaranta3, M. Pasquini3 and L. Riccioni1. 1CRA, Centrodi Ricerca per la Patologia Vegetale, Via C.G. Bertero 22, 00156Roma, Italy. 2AIAB, Via Piave 14, 00187 Roma, Italy. 3Unità diRicerca per la Valorizzazione Qualitativa dei Cereali, Via Cassia176, 00191 Roma, Italy. E-mail: [email protected]

Seed is the basic unit of crop production, and seed healthplays a key role for successful production especially in organicfarming systems, where less efficient seed-treatments are availablefor managing seed-borne disease. Phytosanitary seed analysiswere carried out to verify the actual impact of seed-borne dis-eases in some organic farms distributed throughout Italy and toevaluate the health of seeds used in Italian organic agriculture.Samples of durum and common wheat, carrot, bean and chick-pea seeds of different typologies (organic commercial seeds, self-produced organic seeds and conventional not treated seeds al-lowed) were analysed. In addition, seeds and production of somedurum and common wheat varieties were analysed for two years,to verify a possible relationship between the health status of seedssown and production under organic farming systems. Seed healthtesting was performed using the standard blotter method de-scribed by the International Rules for Seed Testing, and a list ofthe fungal species found and identified on seeds was drawn.

AN INTEGRATED STUDY ON THE EFFECTS OFGRAPEVINE LEAFROLL-ASSCOCIATED VIRUS 1,GRAPEVINE VIRUS A AND GRAPEVINE RUPESTRISSTEM PITTING-ASSOCIATED VIRUS ON FIELD PER-FORMANCE AND BERRY QUALITY OF VITIS VINIFERAcv. NEBBIOLO. D. Pacifico1, M. Giribaldi2, M. Purrotti2, D.Santini1, F. Mannini1, P. Caciagli1, L. Rolle3, L. Cavallarin2, M.G.Giuffrida2 and C. Marzachì1. 1Istituto di Virologia Vegetale delCNR, Strada delle Cacce 73, 10135 Torino, Italy. 2Istituto di Scien-ze delle Produzioni Alimentari del CNR, Via Leonardo da Vinci,44, 10126 Grugliasco (TO), Italy. 3Dipartimento di Valorizzazionee Produzione Agro Alimentare, Microbiologia Agraria e TecnologieAlimentari, Via Leonardo da Vinci 44, 10126 Grugliasco (TO),Italy. E-mail: [email protected]

Viruses can affect grapevine yield and field performance, butthe impact of infection on the quality and safety of final productshas been scarcely investigated. This study reports the first analy-sis of agronomic performance, fruit texture and composition, andproteomic changes occurring in berries of virus-infected Vitisvinifera cv. Nebbiolo, grown in field conditions. The cv. Nebbioloclone 308 infected by Grapevine leafroll-associated virus 1(GLRaV-1), Grapevine virus A (GVA), and Grapevine rupestrisstem pitting-associated virus (GRSPaV), were compared tohealthy vines obtained following heat treatment of the original in-fected mother plant. The phytosanitary status of each plant wasassessed by RT-PCR, and GLRaV-1 and GVA concentration de-termined by quantitative reverse transcription-Real time PCR(qRT-PCR). Mean loads of GLRaV-1 and GVA in the infectedplants were 2.5 (SD=0.8) and 0.4 (SD=0.2) viral genomes/100GAPDH transcript copies, respectively. The distribution of theviral loads was uniform among the infected plants. The compari-son of field performance and fruit quality of healthy and infectedplants revealed similar agronomical behaviour, with significant



differences in titratable acidity, resveratrol content, and in sometexture parameters. The proteomic analysis of skin and pulp fromberries of healthy and virus-infected grapes disclosed significantdifferences for 12 pulp spots and 7 skin spots. Virus infection al-tered proteins involved in cell structure metabolism in the pulp,and proteins involved in the response to oxidative stress in theberry skin.

POTENTIAL ROLE OF ORGANIC CARBON FRACTIONSFROM VEGETABLE COMPOSTS, IN THE BIOCONTROLOF RHIZOCTONIA SOLANI. C. Pane1, R. Spaccini3, D. Vil-lecco1, G. Celano2, A. Piccolo3 and M. Zaccardelli1. 1CRA, Cen-tro di Ricerca per l’Orticoltura di Pontecagnano, Azienda Speri-mentale di Battipaglia. SS 18 n. 204, 84091 Battipaglia (SA), Italy.2Dipartimento di Scienze dei Sistemi Colturali, Forestali e dell’Am-biente, Università degli Studi della Basilicata, Viale dell’Ateneo Lu-cano n. 10, 85100 Potenza, Italy. 3Centro Interdipartimentale diRicerca sulla Risonanza Magnetica Nucleare per l’Ambiente, l’A-gro-Alimentare ed i Nuovi Materiali, Via Università 100, 80055Portici (NA), Italy. E-mail: [email protected]

Composts-mediated soil-borne plant pathogen suppressioncan be affected by the organic matter structure that influenceavailability of carbon nutrients for microbes associated with bio-logical control. In this work, four vegetable composts (VCs) ob-tained from tomato+endive (VC-TE), tomato (VC-T), artichoke(VC-A) and artichoke+fennel (VC-AF) residues, were assessedfor their ability to reduce damping-off caused by Rhizoctoniasolani (Kühn) on cress. A municipal waste compost (MWC) wasused as reference. Active biotic component of composts relievedby CO2-release, FDA-hydrolysis, Biolog analyses and microbialcounts, was indispensable for in vitro and in vivo pathogen con-trol, as demonstrated by loss of suppressiveness after sterilization.Cross-polarization magic angle spinning 13C-nuclear magneticresonance (CP-MAS-13 C-NMR) was used to analyze the molecu-lar distribution of organic C in composts. Strong absorbancearound peaks at 175, 150 and 130 ppm of resonance spectra sug-gest that the main components of the final mature compostedvegetable residues are lignocellulosic materials and hydrophobicalkyl moieties, that are commonly found in well-humified organicmatter (humic C). Rhizoctonia disease suppression levels, result-ed in the following order: VC-TE, VC-T, VC-A, VC-AF andMWC They were associated directly to the relative abundance ofcarboxylic C (region 195-165), phenolic C (region 165-145 ppm)and aromatic C (region 145-110 ppm) and indirectly to alkyl C(region 45-0 ppm), but not with carbohydrate structures (O-alkylC, 110-60 ppm). The described humic C fractions appear to bepotentially responsible for suppressiveness by interaction withcompost-niche inhabiting microbes.

CONTROL OF EARLY BLIGHT OF CARROT CAUSED BYALTERNARIA DAUCI AND A. RADICINA USING PHYTO-CHEMICALS. C. Pane, D. Villecco, F. Campanile and M. Zac-cardelli. CRA, Centro di Ricerca per l’Orticoltura di Pontecagnano,Azienda Sperimentale, SS 18 n. 204, 84091 Battipaglia (SA), Italy.E-mail: [email protected]

Alternaria dauci (J.G. Kühn) J.W. Groves & Skolko and Al-ternaria radicina Meier, Drechsler & E.D. Eddy, are relatedpathogens causing severe blights on leaves, petioles and stems ofcarrot (Daucus carota). A. radicina can cause a distinctive blackrot symptom, consisting in damping-off and rotting of roots,crowns and seedlings of carrots that become unmarketable. Con-

trol of these pathogens is currently entrusted to chemical fungi-cides, but the preference of the consumers for safe agriculturalproducts has stimulated the search of sustainable alternatives.The goal of the present study was to investigate the in vitro anti-fungal activity of plant-derived compounds against Alternaria dis-ease agents of carrot. In vitro assays indicate that A. radicina wasmore sensitive to essential oils and raw saponins than A. dauci. Infact, a large spectrum of plant essential oils (hyssop, oregano,basil, caraway, sage, marjoram and lemon balm) was able to sup-press black rot agent, whereas A. dauci was inhibited only byoregano and thyme oils. No effects were observed with rosemaryand verbena oils in both cases. In agreement, A. radicina showedincreased sensitivity to treatments with saponin-enriched tissuemeals derived from Solanum chilense and Aster sedifolius, and bytreatments with Brassica carinata seed meal containing glucosyno-lates.

USE OF PLANT-DERIVED COMPOUNDS TO INHIBITCAUSAL AGENTS OF BEAN ANTHRACNOSE ANDCHICKPEA ASCOCHYTA BLIGTH. C. Pane, D. Villecco, F.Campanile and M. Zaccardelli. CRA, Centro di Ricerca per l’Orti-coltura di Pontecagnano, Azienda Sperimentale, SS 18 n. 204,84091 Battipaglia (SA), Italy. E-mail: [email protected]

Colletotrichum lindemuthianum (Sacc. & Magnus) Lams.-Scrib. and Ascochyta rabiei (Pass.) Labr. are the causal agents ofanthracnose of common bean (Phaseolus vulgaris) and leaf blightof chickpea (Cicer arietinum), respectively. Management of theseseed-borne pathogens requires an integrated approach, which of-ten includes the use of certified disease-free seed, fungicide seedtreatments, cultivar resistance and timely application of foliarfungicides. Protectants that provide a barrier against pathogenspropagules could be useful since foliar fungicide applications arenot cost effective when disease severity is low and are incompati-ble with organic farming protocols. Moreover, bean and chickpearesistance against these pathogens is very erratic. In this work, wehave tested several phytochemicals, such as essential oils, veg-etable colorants, raw saponins and meals from Brassica carinatacontaining glucosinolates, for in vitro antifungal activity againstC. lindemithianum and A. rabiei. Results indicate that hyssop andoregano-derived essential oils inhibit the two pathogens at 1%concentration. Moreover, oils distilled from basil, caraway, sageand thyme, block mycelial growth of C. lindemuthianum, whileessential oils from lemon balm and verbena block mycelialgrowth of A. rabiei. No effects were observed with rosemary andmarjoram oils. Among over thirteen plant-extracted colorantstested, only chestnut cortex colorant showed partial inhibition ofthe radial growth of these fungi. In plate assays, Solanum chilenseand Aster sedifolius tissue meals, containing raw saponins, exhib-ited no fungitoxic effect. Finally, volatile compounds released byB. carinata seed meals were effective in the in vitro control ofboth pathogens.

NEW STRAINS OF BACILLUS SUBTILIS (17S AND 08C)INVOLVED IN BIOCONTROL OF LETTUCE DROPCAUSED BY SCLEROTINIA MINOR. C. Pane, D. Villecco, F.Campanile and M. Zaccardelli. CRA, Centro di Ricerca per l’Orti-coltura di Pontecagnano, Azienda Sperimental , SS 18 n. 204,84091 Battipaglia (SA), Italy. E-mail: [email protected]

Introduction and establishment of living natural enemies ofpathogens is viewed as a powerful alternative to chemical fungi-cides, meeting the consensus of consumers for sustainable prod-



ucts with few or no chemical residues. In this work, we have se-lected new thermophilic bacterial strains from suppressive soils,on the basis of their in vitro antifungal activity against Sclerotiniaminor. A culture collection was obtained by selection, fromcountable plates, of over 250 colonies. Results of qualitative platechallenge experiments indicated that 39% of the isolates pro-duced an inhibition zone between individual colonies andpathogen. On the basis of these data, quantitative plate challengeexperiments were performed with selected potential antagonisticbacteria. Successively, these bacteria were separated in geneticallydistinct groups by DNA polymorphic analyses (M13 sequenced-based PCR fingerprinting). Crossing DNA fingerprints with thequantitative data of in-vitro assays, two strains named 17S and08C, genetically different and with the highest in vitro antibiosisactivity against S. minor, were identified by biochemical (Bi-olog™) and molecular analyses (16S rRNA gene sequencing) asBacillus subtilis. Application of B. subtilis 17S and O8C strains toS. minor/Lactuca sativa pathosystem, showed a biocontrol efficacyof disease severity, respect to untreated control, of about 70 and56%, respectively. Results suggest that these two new B. subtilisstrains are biopesticides effective in the reduction of lettuce dropsymptoms.

DETECTION OF ALFALFA MOSAIC VIRUS BY IC-RT-PCRIN VIBURNUM OPULUS. G. Parrella1, L. Cavicchi2, S.Rosati2 and M.G. Bellardi3. 1Istituto per la Protezione delle Piantedel CNR, Via Università 13, 80055 Portici (NA), Italy. 2Plesso Di-dattico “G. Scarabelli”, Università degli Studi di Bologna, Viale G.Ascari 17, 40026 Imola (BO), Italy. 3Dipartimento di Scienze e Tec-nologie Agroalimentari, Patologia Vegetale, Università degli Studi,Viale Fanin 42, 40127 Bologna, Italy. E-mail: [email protected]

Viburnum opulus L. (“snowball”, Caprifoliaceae) is a decidu-ous shrubs used as decorative plants for its fragrant white flowersthat bloom in spring. In May 2011, snowball plants showing avirus-like disease were observed in two ornamental public gar-dens at Imola (Bologna, Emilia Romagna). Symptoms were dis-tributed only on part of the foliage, and consisted of chloroticrings and spots, line-patterns and, in some cases, vein clearing onyounger leaves. Since only Alfafa mosaic virus (AMV) and Cu-cumber mosaic virus (CMV) have been reported to naturally in-fect this Viburnum species, and considering that snowball couldbe an important source of virus infection, symptomatic plantswere examined b IC-RT-PCR. Tests were performed on leaf ex-tracts using two polyclonal antisera against an AMV isolate fromlettuce and a CMV isolate from tomato, respectively. Only AMVwas found in symptomatic V. opulus. The CPAMV1/CPAMV2specific AMV primer pairs were used in RT-PCR reactions. ADNA fragment of ca. 750 bp, covering the entire AMV coat pro-tein gene (CP), was obtained after IC-RT-PCR and the productwas gel purified and cloned in pGEMT Easy for sequencing.Comparison of the CP sequence of AMV-V. opulus with the CPreference sequences of some AMV isolates, revealed the maxi-mum nucleotide identities with subgroup I isolates. AMV hasbeen previously found infecting V. opulus in Italy in 1995, but theleaf symptomatology observed was vein yellowing alone.

FUNCTIONAL CHARACTERIZATION OF CERATO-PLA-TANIN BY GENE DELETION AND PROTEOMIC AP-PROACH. L. Pazzagli1, S. Luti1, F. Martellini1, C. Comparini2,P. Bettini3, I. Baccelli2, B. Pantera4, G. Cappugi1 and A. Scala2.1Dipartimento di Scienze Biochimiche, Università degli Studi, VialeMorgagni 50, 50134 Firenze, Italy. 2Dipartimento di Biotecnologie

Agrarie, Università degli Studi, Via della Lastruccia 10, 50019 Ses-to Fiorentino (FI), Italy. 3Dipartimento di Biologia Evoluzionistica,Università degli Studi, Via Romana 17, 50125 Firenze, Italy. 4En-tomon s.a.s., Via di Ripoli 25, 50126 Firenze, Italy. E-mail:[email protected]

Cerato-platanin (CP) is a protein abundantly secreted and lo-calized in the cell wall of Ceratocystis platani. CP induces produc-tion of H2O2 and NO, programmed plant cell death, overexpres-sion of defence genes, phytoalexin synthesis and restriction ofconidia growth. Therefore, CP appears to act as PAMP able to ac-tivate primary defence mechanisms. CP is the founder of the “Cer-ato-platanin family”, whose members are proteins involved in themicrobe-host interaction. In the present study the physiologicalrole of CP in the life style of C. platani and its role as PAMP in in-ducing defence responses are investigated by: (i) gene deletion ex-periments. A deletion cassette for the cp gene was obtained usingthe fusion PCR technique. To obtain the cassette, the determina-tion of a 1,368 bp region upstream of the ATG codon was per-formed by genome walking. The construct for disruption of the cpgene will be further used to transform conidia and/or protoplastswhich will be characterized for their ability to induce defences inhost and non host plants. (ii) Proteomic approach. Conidia of C.platani are used to treat plane leaves for 48 h in a moist chamber.Proteins were extracted by phenol/chloroform treatment. 2D-elec-trophoresis was used to visualize proteins extracted from the treat-ed leaves vs. proteins from control leaves. Other leaves were treat-ed with purified CP for 15 and 30 min and for 1, 6, 12, 24 and 48h and protein were extracted. SDS-PAGE and Western blot wereperformed to detect protein kinase activated upon CP interaction.

FIRST REPORT OF PHYTOPHTHORA CRYPTOGEA ONCOMMON CYPRESS. A. Pecchioli1, R. Danti1, A.M.Vettraino2 and G. Della Rocca1. 1Istituto per la Protezione dellePiante del CNR, Via Madonna del Piano 10/I, 50019 SestoFiorentino (FI), Italy. 2Dipartimento di Protezione delle Piante,Università degli Studi della Tuscia, Via S. Camillo de Lellis, 01100Viterbo, Italy. E-mail: [email protected]

The common cypress (Cupressus sempervirens) is widespreadthroughout the Mediterranean area where it plays an essential rolein the characterization of the landscape, and is widely used for itsaesthetic value and timber production, for protection of soils fromerosion and for windbreaks. In 2009 and 2010 in two young orna-mental plantations in Tuscany and one nursery in Umbria (centralItaly), some specimens of C. sempervirens cv. Bolgheri showedsymptoms of sudden yellowing of the crown starting from thebasal portion, followed by death of the plants. The incidence ofdiseased trees was around 10-15% at all sites. A Phytophthoraspecies was isolated from the rhizosphere and from roots of symp-tomatic and dead plants on selective medium PARPH. Morpho-logical characteristics were registered on 5-day-old cultures grownon carrot agar. Ribosomal DNA regions (ITS1, 5.8S rDNA andITS2) were amplified, sequenced and compared to sequences ofknown Phytophthora species from GenBank. Morphological andmolecular results identified this species as Phytophthora cryptogea.A pathogenicity test was conducted using 3-years-old ramets ofseveral clones of C. sempervirens grown in pots containing soil in-oculated with sterile millet seeds colonised separately by two iso-lates of P. crytpogea. For each P. cryptogea isolate twenty plantswere inoculated and 10 untreated plants were used as controls.Reduced growth of the roots and crown wilting followed by deathof the plants were observed, thus confirming the pathogenicity ofP. cryptogea on C. sempervirens. This is the first report of P. crypto-gea on C. sempervirens.



TESTING THE BENEFICIAL EFFECTS OF PLANTGROWTH PROMOTING RIZOBACTERIA ON PLANTFITNESS AND RESISTANCE TO BIOTIC STRESSES INTHE CONTEXT OF THE ORGANIC FARMING SYSTEM.O. Petruzzelli1, M. Ferrara2, F. Cillo1, F. Nigro2 and L.Stavolone1. 1Istituto di Virologia Vegetaledel CNR, via Amendola165/A, 70126 Bari, Italy. 2Dipartimento di Biologia e ChimicaAgro-forestale ed Ambientale, Università degli Studi “Aldo Moro”,Via Amendola 165/A, 70126 Bari, Italy. E-mail: [email protected]; [email protected]

The relationships between plants and beneficial microorgan-isms in the rhizosphere (e.g. mycorrhizas, fungi and bacteria) areessential for root development, plant nutrition, and growth en-hancement. The decline of agricultural soils fertility is due notonly to the agricultural practices but also to the reduction of or-ganic matter content, as well as to the depletion of the edaphicmicroflora. Plant growth promoting rhizobacteria (PGPR) werershown to stimulate plant growth and natural resistance towardsdiseases. Some bacterial bioformulations are available on themarket for the use in agriculture. In this study, the effects of anew PGPR mixture (based on Pseudomonas spp. and Bacillusspp. strains) on plant growth, yield and tolerance to pathogens ofseveral horticultural crops, were evaluated. The experimentswere conducted in a commercial field cultivated according to theorganic farming system. Plants treated with water and the biofor-mulate Sublic (Elep Biotechnologies) were used as controls. Theincidence of naturally occurring diseases caused by fungi andviruses was monitored. Moreover, PGPR-treated plants were alsomonitored in a greenhouse and challenged for resistance/toler-ance to selected pathogen infections by means of mechanical in-oculations of pathogenic viruses and fungi. Treatments with thenew developed PGPR mixture greatly enhanced plant growthand significantly antedated harvesting for some horticulturalcrops. The effectiveness of the new PGPR mixture on plant fit-ness and tolerance to viral and fungal diseases, as well as the dif-ferences with the commercial bioformulation, are discussed.

EFFECTS OF COPPER ON T. MELANOSPORUM-INFECT-ED PLANTS. F. Piattoni, M. Iotti, S. Boutahir and A. Zam-bonelli. Dipartimento di Protezione e Valorizzazione Agroali-mentare, Università degli Studi, Viale G. Fanin 46, 40127 Bologna,Italy. E-mail: [email protected]

The positive results obtained by truffle cultivation in Europeand Oceania has determined the extension of this activity also toagricultural areas subjected to intensive crop production systemsfor a long time. It is known that copper accumulation in agricul-tural soils has a long-term impact on a wide range of soil biota. Inthis work copper effects on Q. pubescens seedlings infected withT. melanosporum were investigated for the first time. Cupper wasadded as CuSO4·5H2O chelated with ETDA. After repeatedtreatments the Cu accumulation obtained in soil was 145 ppm oftotal Cu and 89.4 ppm of assimilable Cu. T. melanosporum-infect-ed plants showed reduced symptoms of copper toxicity in com-parison with the plants without mycorrhizae. The quantity of Cuaccumulated in roots and leaves was lower in T. melanosporum-infected plants. T. melanosporum mycorrhizae produced oxalateswhich might play a fundamental role in Cu soil detoxification. T.melanosporum mycorrhizae at high Cu concentrations becamedark and the formation of new mycorrhizae was inhibited. In Cu-treated plants the formation of T. melanosporum stroma along Q.pubescens roots was detected. These structures could be inter-preted as a quiescent, resistance phase of the fungus.

THE MELANIN BIOSYNTHESIS INHIBITOR TRICYCLA-ZOLE REDUCES SECONDARY INFECTION OF MAGNA-PORTHE ORYZAE IN RICE LEAVES. C. Pizzatti, J. Gómez-Ariza, A. Kunova and P. Cortesi. Dipartimento di Protezione deiSistemi Agroalimentare e Urbano e Valorizzazione delle Biodiver-sità, Università degli Studi, Via Celoria 2, 20133 Milano, Italy. E-mail: [email protected]

Melanin biosynthesis inhibitors are used as fungicides in agri-culture. Among them, tricyclazole (TCZ) has demonstrated toplay an important role against rice blast, one of the most destruc-tive diseases of rice worldwide, caused by Magnaporthe oryzae.One discussed point regards the capability of the fungus to com-plete its life cycle after being exposed to TCZ. The aim of thiswork was to analyze the capability of conidia originated frommycelium that had been exposed to TCZ to infect rice. TCZ wascompared with azoxystrobin, a strobilurin fungicide. Strains ofM. oryzae were grown on medium supplemented with differentTCZ or azoxystrobin concentrations in order to harvest conidiafor downstream microscopic and pathogenicity analyses. Conidiaamount, appressoria size and the number of infection points inrice leaves revealed that the exposure of M. oryzae mycelium toTCZ negatively affects conidia production and appresoria forma-tion and function, thus resulting in a reduction of the fungus vir-ulence. Azoxystrobin was less effective than TCZ. These new re-sults highlight the potential of TCZ to reduce secondary infec-tions lowering conidia formation on diseased treated areas andconidia virulence. These properties will reduce the need of re-peated treatments benefiting both the rice ecosystems andfarmer’s economy.

TRICHODERMA spp. IN INNOVATIVE SUBSTRATES FORORNAMENTAL PLANTS. D. Prisa1, S. Sarrocco2, M. Forti1,C. Piaggeschi2, G. Burchi1 and G. Vannacci2. 1CRA, Unità diRicerca per il Vivaismo e la Gestione del Verde Ambientale ed Or-namentale, Via dei Fiori 8, 51012 Pescia (PT), Italy. 2Dipartimentodi Coltivazione e Difesa delle Specie Legnose, Sezione PatologiaVegetale, Università di Pisa, Via del Borghetto 80, 56124 Pisa,Italy. E-mail: [email protected]

Trichoderma spp. are free-living fungi commonly widespreadin soil and root ecosystems. Recent findings show them as oppor-tunistic, avirulent plant symbionts as well as parasites of otherfungi. Some strains establish robust and long-lasting colonizationof roots by entering the first epidermal layers. Root colonizationfrequently results in the enhancement of growth and develop-ment, crop productivity or induction of resistance to abiotic andbiotic factors. Due to the reduction of peat in ornamental sub-strates, great attention is focuses on setting up new and innova-tive substrates for this market. In this work, Trichoderma spp. iso-lates were selected for endophytism in Limonium sinuatum, Cu-pressus sempervirens and Camelia sp. Fungal solates (162) wereused as inoculants of soil employed for germination and growingof both Limonium and Cupressus whereas 202 isolates were inoc-ulated in soil used for transplanting Camelia cuttings. Germina-tion rate and germination time, growth parameters and % of rootcolonization (endophytism) in inoculated thesis were submittedto ANOVA and compared to uninoculated controls (natural potmix). Ten isolates for Limonium, 9 for Cupressus and 8 forCamelia improved one or more parameters and were chosen forfurther analysis. All selected isolates were identified by a molecu-lar approach. Selected strains are actually under evaluation in or-der to confirm their endophytic fitness on all the three ornamen-tal species. In addition, the ability to induce resistance againstBotrytis cinerea in Limonium will be investigated.



Work supported by the Project “Messa a punto di substrati arti-ficiali innovativi per il florovivaismo”, MIPAAF.

FIRST REPORT OF GRAPEVINE BLACK ROT IN SAR-DINIA. V.A. Prota, S. Serra, L. Cogotzi, G. Serra and R. Garau.Dipartimento di Protezione delle Piante, Sezione di Patologia Vege-tale, Università degli Studi, Via E. De Nicola 9, 07100 Sassari,Italy. E-mail: [email protected]

Grapevine black rot disease is caused by Guignardia bidwellii.It originated from North America but is now widespread all overthe world. In Italy, it was reported only in northern regions likeFriuli Venezia Giulia, Veneto, Liguria and Piedmont where infec-tions are variable and rarely reach a high level of severity. In 2010in northern Sardinia (Oschiri) symptoms resembling black rotwere observed on young cv. Sultanina vines. The basal leaves ofsome shoots showed necrotic reddish brown spots with dark redmargins and small black dots which were sometimes concentric.The canes were asymptomatic and the affected plants were fewand contiguous. In spring 2011, the same symptoms were ob-served on cv. Cannonau in central Sardinia (Oliena). Infectionswere more severe and did not affect only the basal leaves butwere also present in the axis of green shoots, petioles and rachisof several plants in the same vineyard. Some spots on the leaveswere observed also in other vineyards in the same area. Micro-scopical observations revealed the presence of asexual fruitingstructures, i.e. pycnidia containing one-celled, hyaline ovoid coni-dia with an apical hyaline appendage that measured 5.4±0.66 x9.42±0.82 µm. All parameters were in accordance with those ofPhyllosticta ampelicida, anamorph of G. bidwellii. According totypical symptoms and microscopic characters the disease wasidentified as black rot. Further studies are in progress to charac-terize the fungus and to assess its pathogenicity.

SUPPRESSIVE EFFECT OF COMPOST AGAINST PHY-TOPHTHORA CINNAMOMI ON ORNAMENTALS. M.Pugliese, M.L. Gullino and A. Garibaldi. AGROINNOVA, Uni-versità degli Studi di Torino, Via L. Da Vinci 44, 10095 Grugliasco(TO), Italy. E-mail: [email protected]

Suppression of soil-borne plant pathogens with composts hasbeen widely studied. Composts have been found to be suppres-sive against several soil-borne pathogens in various cropping sys-tems. Ornamental plants are generally cultivated in pots, allowingthe use of suppressive substrates to control zoospore-producingpathogens, like Phytophthora spp. The efficacy of compostagainst basal and root rot in Skimmia japonica and Rhododendronazalea caused by Phytophthora cinnamomi was evaluated undergreenhouse conditions. A compost (ACM) produced through aforced type of composting for 3 months from municipal organicwastes and a compost (ACV) produced from green wastes weremixed with peat substrate to obtain application rates of 10, 20and 40% (v/v). Substrates were inoculated at 1g/l dosage ofwheat kernels infected by P. cinnamomi and, after one week, 15-20 plants were transplanted for each treatment in 2 liter volumepots and placed in greenhouse at 20°C. A chemical control(Metalaxil-M) was also used. Diseased plants were assessed week-ly after transplanting and above-ground biomass of plants was as-sessed at the end of the trials. Results showed a significant diseasecontrol when compost was used at 20-40% on S. japonica, with-out showing any phytotoxic effect. Disease suppression wasshown at 20% on R. azalea, while higher concentrations of com-post where slightly phytotoxic to plants. The use of compost sub-

strates can be a suitable strategy for controlling soil-borne dis-eases on ornamentals, but results depend also on alkalinity toler-ance of different species.

EFFECT OF CLIMATE CHANGE ON GRAPEVINE IN-FECTION BY DOWNY MILDEW UNDER CONTROLLEDENVIRONMENT. M. Pugliese, M.L. Gullino and A. Garibaldi.AGROINNOVA, Università degli Studi di Torino, Via L. Da Vinci44, 10095 Grugliasco (TO), Italy. E-mail: [email protected]

Plant responses to elevated CO2 and temperature have beenmuch studied in recent years, but effects of climate change onpathological responses are largerly unknown. The pathosystemsgrapevine (Vitis vinifera)-downy mildew (Plasmopara viticola) waschosen as model to assess the potential impact of increased CO2and temperature on disease incidence and severity under con-trolled environment. Potted grapevine plants were grown in phy-totrons under 4 different simulated climatic conditions: (i) stan-dard temperature (ranging from 18 to 22°C) and standard CO2concentration (450 ppm); (ii) standard temperature and elevatedCO2 concentration (800 ppm); (iii) elevated temperature (rangingfrom 22 to 26°C, i.e. 4°C higher than standard) and standardCO2 concentration; (iv) elevated temperature and CO2 concen-tration. Each plant was inoculated with a spore suspension con-taining 5x105 CFU/ml. Disease index and physiological parame-ters (chlorophyll content, fluorescence, assimilation rate) were as-sessed. Results showed an increase of the chlorophyll contentwith higher temperatures and CO2 concentration, to which corre-sponded a higher fluorescence index. Disease incidence of downymildew increased when both CO2 and temperatures were higher.Considering that the rising concentrations of CO2 and othergreenhouse gases will lead to an increase in global temperatureand longer seasons, we can assume that this will allow more timefor pathogens evolution and could increase pathogen survival, in-directly affecting downy mildew of grapevine.

A LIPIDOMIC APPROACH FOR UNCOVERING THEOXYLIPIN DETERMINANTS WHICH DRIVE AS-PERGILLUS FLAVUS-ZEA MAYS INTERACTION. M.Punelli1, M. Reverberi1, M. Scarpari1, E. Di Fabio1, E. Camera2,A.A. Fabbri1 and C. Fanelli1. 1Dipartimento di Biologia Ambien-tale, Università “Sapienza”, Largo Cristina di Svezia 24, 00165 Ro-ma, Italy. 2Laboratorio di Fisiopatologia Cutanea e Centro Integra-to di Metabolomica, Istituto Dermatologico San Gallicano IRCCS,Roma, Italy. E-mail: [email protected]

Aflatoxins are carcinogenic secondary metabolites producedby Aspergillus flavus and other closely related species. Many in-ternal and external factors, such as nutrition, environment andinteraction with the host (Zea mays) can affect aflatoxin biosyn-thesis. Host defence-related compounds, like oxylipins, may in-duce sporulation and modulate mycotoxins biosynthesis in sever-al pathogenic fungi, apparently by replacing fungal endogenousoxylipins, produced by lipoperoxidative processes of lipoxyge-nase (LOX) and dioxygenase enzymes. In A. flavus, these oxylip-ins are crucial signals for the regulation of mycotoxins biosynthe-sis, conidiogenesis and sclerotia formation. To study how inhibi-tion of oxylipins formation may affect the A. flavus metabolismand its relation with the host (maize kernels), we obtained a mu-tant (Af∆lox strain) presenting a knock-out of the lox1 gene, en-coding a homologue of the human arachidonate 15-lipoxygenase.Onto liquid and solid media, the mutant displayed a different



colony morphology in comparison with 3357 wild-type strain, i.e.absence of conidia, lack of aflatoxins biosynthesis and a lowerbasal activity of LOX enzyme. Interestingly, its conidiation andaflatoxins production was recovered by growing the strain ontomaize seeds. To better understand the involvement of the cross-talk Z. mays-A. flavus, we performed an oxylipin profile of thetwo strains (WT and Af∆lox) and of the maize seeds after hostcolonization using an LC-TOF based lipidomic approach fol-lowed by a PCA statistical analysis. The results will contribute tothe current knowledge on the role of lipid peroxidation governedby the lox1 gene in the morphogenesis, aflatoxins biosynthesis,and in host-pathogen interaction.

AN OVERVIEW OF THE GENOME OF THE BIOCON-TROL AGENT LYSOBACTER CAPSICI STRAIN PG4. G.Puopolo1,2 and A. Zoina1. 1Dipartimento di Arboricoltura, Botani-ca e Patologia Vegetale, Università degli Studi di Napoli “FedericoII”, Via Università 100, 80055 Portici (NA), Italy. 2Fondazione Ed-mund Mach IASMA-Research and Innovation Centre, Via Mach 1,38010 S. Michele all’Adige, Italy. E-mail: [email protected]

The genus Lysobacter encompasses nonfruiting gliding bacte-ria that exhibit remarkable lytic activity exploitable for the bio-logical control of phytopathogenic fungi. Recently, L. capsicistrain PG4 was proven to efficiently control in vivo tomato crownand root rot caused by Fusarium oxysporum f.sp. radicis-lycopersi-ci. The bacterial strain showed proteolytic and chitinolytic activi-ty and was able to produce antibiotic substances active againstGram-positive bacteria exclusively. In order to unravel the mech-anisms responsible for the biocontrol aptitudes of strain PG4, thegenome of this bacterium was sequenced through Illumina GAI-IX platform. The genome of strain PG4 consists of almost 7 Mbpwith a 63.96% GC content. Once assembled and annotated, thegenome has been analyzed for the presence of genes coding forlytic enzymes and for the synthesis of antibiotic compounds. In-terestingly, strain PG4 genome possesses a high amount of genes(sixty nine) coding for proteases, eight and four genes respective-ly coding for glucanases and chitinases. The presence of all theselytic enzymes is coupled with the production of antibiotics. A 9kb region showing 98% homology with the operon responsiblefor the biosynthesis of HSAF, an antibiotic compound producedby L. enzymogenes strain C3, has been found in the PG4 genome.The availability of the whole genome of strain PG4 represents asound basis for the development of new studies aimed at betterunderstanding how PG4 accomplishes the biological control ofplant pathogenic fungi.

SELECTION OF NEW FUNGAL STRAINS FOR THE BIO-LOGICAL CONTROL OF OROBANCHE RAMOSA ON TO-BACCO. G. Puopolo1,2 and A. Zoina1. 1Dipartimento di Arbori-coltura, Botanica e Patologia Vegetale, Università degli Studi diNapoli “Federico II”, Via Università 100, 80055 Portici (NA), Italy.2Fondazione Edmund Mach IASMA-Research and Innovation Cen-tre, via Mach 1, 38010 S. Michele all’Adige, Italy. E-mail: [email protected]

The species Orobanche ramosa (broomrape) regroups obligateroot parasite plants causing severe losses to tobacco. As theseweeds attack crop roots they cannot be easily controlled mechan-ically or by using synthetic herbicides. The exploitation of biolog-ical control agents able to parasitize broomrapes offers a promis-ing solution to this problem. At the moment many O. ramosapathogenic organisms have been isolated to potentially control

this holoparasitic weed. During this work several naturally dis-eased broomrape plants were collected in tobacco fields surveyedin Campania (southern Italy). Fifty five fungal isolates recoveredfrom broomrape-diseased tissues were identified and evaluatedfor their capacity to control O. ramosa. Culture filtrates of all thefungal isolates were evaluated for their aptitude to inhibit the ger-mination of broomrape seeds in vitro. Nine isolates, that drasti-cally reduced the percentage of seed germination, were furtherevaluated in vivo to verify their ability to attack broomrape tuber-cules developing on the roots of parasitized plants. Although ableto inhibit seed germination, strains belonging to genus Ulocladi-um and Chalara were not able to attack the tubercules. Only fourfungal strains of Fusarium solani and one strain of Fusarium oxys-porum actively attacked broomrape tubercules causing theirdeath. These fungal strains might represent good candidates forthe formulation of new products suitable for the biological con-trol of O. ramosa on tobacco.

ALTERNATIVE CONTROL APPROACHES AGAINSTGRAY MOLD OF PELARGONIUM ZONALE. M. Quaglia, C.Moretti, G. Canala and R. Buonaurio. Dipartimento di ScienzeAgrarie e Ambientali, Università degli Studi, Borgo XX Giugno 74,Perugia, Italy. E-mail: [email protected]

In collaboration with a nursery located near Aprilia (centralItaly), we started to investigate the use of antagonistic fungi of thegenus Trichoderma (T. atroviridae P1, T. harzianum T22 and T67, T.reseii T34, Trichoderma spp. 8009, T. hamatum T382 and a com-mercial product containing the isolates ICC012 of T. harzianumand ICC080 of T. viride) and resistance inducers in the control ofpelargonium gray mold. In dual-culture tests, each Trichodermaisolate caused a significant reduction in the growth of an isolate ofBotrytis cinerea collected in the nursery, which was iprodione-re-sistant as confirmed by in vitro assays. The effect was significantlyhigher when Trichoderma isolates were inoculated two days beforethe pathogen rather than simultaneously. Moreover, the isolates P1,T22, T34 and ICC012+ICC080 caused an abnormal hyphalbranching of the pathogen, while T67 formed coiling around them.Preliminary in vivo assays seemed to confirm the effectiveness of allthe antagonists with the exception of T22, as they reduced signifi-cantly the severity of gray mold. Among the resistance inducers,Biochikol, which contains chitosan, reduced the disease severity,while treatments with pure chitosan dissolved in 1% acetic acid(0.04%, w/v) or 0.3 mM acibenzolar-S-methyl were ineffective andphytotoxic. As expected, pelargonium plants treated with iprodi-one (34 ppm) were not completely protected from gray mold, con-firming the necessity for alternative control approaches.

Work supported by OIGA project FLORNORDS - “Strategie didifesa a basso impatto ambientale per l’ottenimento di specie flori-cole”.

THE LACK OF INHIBITION OF CLAVICEPS PURPUREAPOLYGALACTURONASES BY PvPGIP2 MAY EXPLAINTHE FAILURE OF TRANSGENIC WHEAT PLANTS TORESIST TO THE ERGOT PATHOGEN. A. Raiola1, C. Volpi2,M. Janni2, D.M. O’Sullivan3, C. Castiglioni1, F. Favaron1 and R.D’Ovidio2. 1Dipartimento Territorio e Sistemi Agro-forestali, Uni-versità degli Studi di Padova, Viale dell’Università 16, 35020 Leg-naro (PD), Italy. 2Dipartimento di Agrobiologia e Agrochimica,Università della Tuscia, Via San Camillo de Lellis, s.n.c., Viterbo,Italy. 3National Institute of Agricultural Botany (NIAB), Cam-bridge, UK. E-mail: [email protected]



Claviceps purpurea is a biotrophic fungal pathogen of cerealsand grasses, attacking exclusively young ovaries. C. purpureagrows intercellularly in rye (Secale cereale) ovaries by degradingthe pectic polymers and the fungal polygalacturonase (PG) hasbeen shown to be a pathogenicity factor. Two pg genes of C. pur-purea, cppg1 and cppg2, are responsible for this activity. Trans-genic plants expressing a bean polygalacturonase-inhibiting pro-tein (PvPGIP2) proved to be a valuable tool to increase resist-ance against PG-producing fungi. However, a stable transgenicPvPGIP2 wheat line, previously shown to be more resistant tothe fungal pathogens Bipolaris sorokiniana and Fusarium gramin-earum, exhibited only a very low reduction of symptoms after in-fection with C. purpurea. To understand whether this reducedprotection of PvPGIP2 in wheat transgenic plants was ascribableto a lack of inhibition against the fungal PGs, we tried to performinhibition experiments against the C. purpurea PG activity. Un-fortunately, this fungus does not produce any PG activity in cul-ture, thus it was necessary to express this activity in a Pichia pas-toris heterologous system. The two heterologous expressed PGs,when assayed against the PvPGIP2, were poorly affected by thisinhibitor, indicating that the lack of resistance in transformedwheat line may be due to the lack of recognition of the PGs of C.purpurea by PvPGIP2. This finding supports a role of PGIP inplant defence only when PG-PGIP interaction occurs. The ex-pressed PGs may be useful to identify more effective PGIPs by abroad screening of plant PGIPs.

NEW INSIGHTS INTO KIWIFRUIT-INFECTING VIRUSESIN ITALY. C. Ratti1, A.R. Babini2, C. Lanzoni1, C. PoggiPollini1, R. Credi1, A. Pisi1, G. Filippini1 and C. RubiesAutonell1. 1Dipartimento di Scienze e Tecnologie Agroambientali,Università degli Studi, Viale Fanin 40, 40127 Bologna, Italy.2Servizio Fitosanitario Regionale, Regione Emilia Romagna, Via diSaliceto 81, 40128 Bologna, Italy. E-mail: [email protected]

In Italy, for protecting kiwifruit (Actinidia spp.) productionby restraining the spread of new and harmful diseases on bothnurseries and orchards, a specific Decree has been issued by theItalian Ministry for Agriculture, Food and Forest Policies (De-cree 7th February 2011 – published in the Official Journal of theItalian Republic n. 69 25th March 2011). According to this De-cree, primary source material must be free, at least, from 6 differ-ent virus species: Actinidia virus A (AVA), Alfaalfa mosaic virus(AMV), Apple stem grooving virus (ASGV), Citrus leaf blotchvirus (CLBV), Cucumber mosaic virus (CMV), Ribgrass mosaicvirus (RMV) and also from the Stolbur phytoplasma (CandidatusPhytoplasma solani). Deeper information on biological and mo-lecular proprieties of kiwifruit viruses is therefore necessary toensure the development of diagnostic tools for a rapid, sensitiveand reliable analysis of kiwifruit vegetative propagation material.A CMV isolate (K35) has been recently identified in kiwifruitplants collected in Emilia Romagna (northern Italy). A deep mo-lecular characterization of this isolate has been done to establishthe virus origin. Moreover, agroinfectious K35 clones have beenproduced with the purpose to infect new kiwifruit plants onwhich to perform studies on natural transmission of the virus andevaluate performances of developed detection methodologies.New viral isolates have been identified during survey carried outin 2011. Molecular characterization is in progress, but these newreports suggest that viral infections occur more extensively thanexpected on Italian kiwifruit nurseries and orchards.

DETECTION OF PRUNUS NECROTIC RINGSPOT VIRUSIN ALMOND NATIONAL GERMPLASM OFAFGHANISTAN. S. Rehman1, J. Ahmad1, C. Lanzoni2, C. Ru-bies Autonell2 and C. Ratti2. 1Plant Biotechnology Laboratory,Aga Khan Foundation-Afghanistan, Wazir Akbar Khan, Rd 13,H43, Main Road, Kabul, Afghanistan. 2Dipartimento di Scienze eTecnologie Agroambientali Patologia Vegetale, Università degliStudi, Viale G. Fanin, 40, 40127 Bologna, Italy. E-mail: [email protected]

About 70% of Afghanistan’s population is dependent directlyor indirectly on agriculture and livestock. Horticulture is one ofthe most important sectors in Afghan agriculture that can play avital role in the overall recovery of Afghan economy. To strength-en the horticulture sector of Afghanistan, EC supporting PHDP(Perennial Horticulture Development Project), the main objec-tive of the project is to provide true to type/ecotype and healthyplanting materials, of local fruit varieties, to nursery growers andultimately for the rehabilitation and establishment of new or-chards. The plant biotechnology laboratory, located in Kabul, isproviding services to PHDP by analyzing each year the nationalcollections and motherstock nurseries of national germplasm.The national collection of almond had 102 variety plots (6plants/plot). To know the sanitary status of this germplasm, sam-ples (young leaves in spring season) were collected from PHDPresearch station of Mazar Sharif (northern Afghanistan). DAS-ELISA was used to assay for the presence of PPV (Plum poxvirus), PDV (Prune dwarf virus) and PNRSV (Prunus necroticringspot virus) in the collected samples. PNRSV was detected inthree accessions: 4016 Kaghazi, 6309 Lauranne and 6041 Non-pareil. Molecular analysis is in progress in order to characterizePNRSV isolate detected. To our knowledge these results identi-fied PNRSV in almond plants for the first time in Afghanistan.Moreover phytosanitary certification scheme is needed inAfghanistan for the production of quality planting materials todefine strategies able to control viral and other pathogens.

GRAPEVINE RUPESTRIS STEM PITTING-ASSOCIATEDVIRUS IN DECLINING SYRAH GRAPEVINES IN ITALY.S. Rizza, C. Oliveti and M. Tessitori. Dipartimento di Scienzedelle Produzioni Agrarie e Alimentari, Sezione Fitopatologia e Ge-netica Vegetale, Università degli Studi, Via S. Sofia 100, 95123Catania, Italy. E-mail: [email protected]

A “decline” of Syrah grapevines was first observed as anemerging disease in France and later it was reported from Cali-fornia. Recently a similar disease was observed in a viticulturearea of Tuscany but no data on presence of Grapevine rupestrisstem pitting-associated virus (GRSPaV) were reported. Recently afour year old Syrah vineyard (rootstock 1103 Paulsen) of 3344plants in the Ragusa province (Sicily, insular Italy) were signaledfor the presence of severe syndrome ascribable to virus infection.Symptoms observed during field inspection were: cler-cut leafreddening, swelling and cracking at the graft level, stem pitting ofthe wood, decline and death of plants. To ascertain the presenceof the main grapevine viruses, symptomatic samples were collect-ed and were tested for the presence of GRSPaV, Grapevine virusA (GVA), Grapevine virus B (GVB), Grapevine leafroll-associatedvirus -1, -2, -3 (GLRaV-1, -2, -3), Grapevine fanleaf virus (GFLV),Grapevine fleck virus (GFkV), Arabis mosaic virus (ArMV). TotalRNA, extracted from leaf stems using the RNeasy method, wasanalyzed by RT-PCR assay by using primers designed in high con-served regions of the genome for each virus. GRSPaV was detect-ed in the symptomatic samples whereas none of the viruses werefound in synptomless vines. Deeper investigation will include



virus genome sequencing in order to define the nature of the iso-late and the identification of other possible viruses associated tothe syndrome. In our knowledge this is the first report of the as-sociation of the GRSPaV to Syrah decline in Italy.

SYMPTOMS EXPRESSION AND GROWTH OF CITRUSVARIETIES ON CITRANGE ROOTSTOCKS INOCULAT-ED WITH DIFFERENT CITRUS VIROIDS. S. Rizza1, R. LaRosa1, M. Tessitori1, G. Albanese3 and A. Catara1,2. Dipartimen-to di Scienze delle Produzioni Agrarie e Alimentari, Sezione Fitopa-tologia e Genetica Vegetale, Università degli Studi, Via S. Sofia100, 95123 Catania, Italy. 2Laboratorio di Diagnosi e BiotecnologieFitosanitarie, Parco Scientifico e Tecnologico della Sicilia, Zona In-dustriale, Blocco Palma I, 95121 Catania, Italy. 3Dipartimento diGestione dei Sistemi Agrari e Forestali,Università Mediterranea,Località Feo di Vito, 89060 Reggio Calabria, Italy. E-mail: [email protected]

The Citrus tristeza virus (CTV) epidemics in Italy require thesubstitution of sour orange, a rootstock highly susceptible to CTVbut tolerant to citrus viroids (CVds), with citranges that are toler-ant to CTV. Among the CVds just two are responsible of citrusdiseases: Citrus exocortis viroid (CEVd) for exocortis and Hopstunt viroid (HSVd) for cachexia, respectively. CEVd causes barkscaling of trifoliate orange and its hybrids, whereas its perform-ance on citranges and other hybrids has been questioned severaltimes and it is still unclear. The behaviour of CEVd and/or differ-ent citrus viroids was evaluated on cv. Tarocco sweet orange andcv. Clementine comune grafted on Troyer and Carrizo citranges,respectively inoculated 28 and 15 years ago. Trees were monitoredevery year by field inspections for symptom observations, assayson Etrog citron, RT-PCR and real time RT-PCR analysis. CEVd-inoculated clementine trees (2×3 m density planting) showed nobark scaling but only size reduction, whereas those inoculatedwith a mixture of Citrus dwarfing viroid (CDVd), HSVd andCEVd showed reduced size, bud union line indentation and barkgumming. Tarocco sweet orange performed very well in size andyield despite CEVd infection similarly to healthy trees.

SURVEY OF VIRUSES INFECTING A LILIUM VARIETALCOLLECTION IN TUSCANY. D. Rizzo1, B. Nesi2, L. Stefani1,S. Lazzereschi2, S. Pecchioli2, M. Della Bartola3, A. Materazzi3and A. Grassotti2. 1Regione Toscana, Laboratorio Servizio Fitosan-itario Regionale, Via dei Fiori 8, 51012 Pescia (PT), Italy. 2CRA-VIV, Unità di Ricerca per il Vivaismo e la Gestione del Verde Am-bientale ed Ornamentale, Via dei Fiori 8, 51012 Pescia (PT), Italy.3Dipartimento di Coltivazione e Difesa delle Specie Legnose “G.Scaramuzzi”, Sezione di Patologia Vegetale, Via del Borghetto 80,56124 Pisa, Italy. E-mail: [email protected]

In the frame of a phytosanitary survey of a Lilium varietal col-lection, carried out by CRA-VIV at Pescia (PT), leaf sampleswere collected from 60 plants belonging to 20 varieties and testedby RT-PCR for the presence of Cucumber mosaic virus (CMV),Lilium symptomless virus (LSV) and Lilium mottle virus (LMoV).Three plants of each variety were sampled in late spring-earlysummer 2010. Total RNA was extracted with a modified protocolusing the “RNeasy plant mini kit” (Qiagen, USA) and retro-tran-scribed into cDNA. Diagnosis of CMV, LSV and LMoV was atfirst performed with specific protocols described in the litera-ture.These protocols were then uniformed to unique amplifica-tion conditions to allow for a faster singleplex detection of thethree viruses. Results obtained with the new amplification condi-

tions perfectly matched the results of the standard protocols,confirming the specificity and sensitivity of the tests. Of 60 sam-ples, 40 (66.7%), belonging to 17 varieties, were infected withCMV, 56 (93.3%), belonging to 20 varieties, resulted positive toLSV and 4 (6.7%), belonging to 2 varieties, were infected withLMoV. The same 60 samples were successively tested by RT-PCRfor the tospoviruses Impatiens necrotic spot virus (INSV) andTomato spotted wilt virus (TSWV), with negative results.

PHYTOSANITARY SURVEY ON POME FRUIT AU-TOCHTHONOUS GERMPLASM IN TUSCANY. D. Rizzo1,L. Stefani1, M. Paoli1, M. Della Bartola2 and A. Materazzi2. 1Re-gione Toscana, Laboratorio Servizio Fitosanitario Regionale, Viadei Fiori 8, 51012 Pescia (PT), Italy. 2Dipartimento di Coltivazionee Difesa delle Specie Legnose “G. Scaramuzzi”, Sezione di PatologiaVegetale, Via del Borghetto 80, 56124 Pisa, Italy. E-mail: [email protected]

During 2010, a survey was conducted for the prsence of someof the main virus, viroid and phytoplasma pathogens affecting theautochthonous germplasm of Malus and Pyrus from Tuscany.Samples, consisting of leaves, shoots and phloem tissues, werecollected during spring 2010. In detail, a total of 97 plants, 73 ofwhich belonging to 29 Malus varieties and 24 belonging to 13Pyrus varieties, were tested by RT-PCR and nested PCR for thefollowing pathogens: Candidatus Phytoplasma mali (Ca. P. mali),Candidatus Phytoplasma pyri (Ca. P. pyri), Apple chlorotic leafspot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stemgrooving virus (ASGV), Apple mosaic virus (ApMV), Apple dim-ple fruit viroid (ADFVd) and Apple scar skin viroid (ASSVd). Ofthe eight pathogens investigated, ACLSV and ASPV proved tobe the most widespread (both present in 67.0% of the testedsamples), while ASGV was present in 14.4% of the surveyedplants. In detail, ACLSV was detected on 59 Malus and 6 Pyrusplants, while ASPV was found on 54 and 13 accessions of Malusand Pyrus, respectively. As to ASGV, infections were detected in13 Malus and 1 Pyrus plants. The remaining pathogens (ApMV,ADFVd, ASSVd, Ca. P. mali e Ca. P. pyri) were not found in anyof the surveyed plants.

FURTHER DATA ON THE DISTRBUTION OFGRAPEVINE YELLOWS IN TUSCANY. D. Rizzo1, H.Bouyahia2, M. Della Bartola2, P. Braccini1, L. Stefani1, M. Carli1and A. Materazzi2. 1Regione Toscana, Laboratorio Servizio Fi-tosanitario Regionale, Via dei Fiori 8, 51012 Pescia (PT), Italy.2Dipartimento di Coltivazione e Difesa delle Specie Legnose “G.Scaramuzzi”, Sezione di Patologia Vegetale, Via del Borghetto 80,56124 Pisa, Italy. E-mail: [email protected]

Grapevine yellows (GY) are an important disease of Vitisvinifera caused by phytoplasmas. In Italy, GY are mostly repre-sented by Flavescence dorée (FD) and Bois noir (BN) associated,respectively, with infection by 16SrV and 16SrXII phytoplasmaribosomal groups. In the frame of a regional strategy aimed atmonitoring GY presence, an extensive survey was carried out inlate summer 2010, collecting 629 leaf samples from commercialvineyards. Samples were preferably collected from plants show-ing symptoms of GY diseases. Each sampled plant was markedand localised by global positioning system (GPS) to allow track-ing of FD-positive samples for uprooting. DNA extracts weretested by Real-time PCR for detection of BN and FD phytoplas-mas. The results confirmed that BN is by far the most importantGY in Tuscany, with an ubiquitous distribution. In fact, it was



present in almost every surveyed vineyard with an overall inci-dence of 53,6% (337 out of 629 plants tested). The presence ofFD was recorded from 39 samples distributed in 4 provinces in-cluding Massa-Carrara, Florence, Pistoia and Lucca. An alarmingsituation was observed in this latter province, where 23 out of 74plants (31,1%) were infected by FD. On the basis of these re-sults, further investigations on insect vectors, natural reservoirsand molecular characterization of grapevine infecting phytoplas-mas are needed for a better understanding of GY epidemiologyin Tuscany.

OCCURRENCE OF HYDRANGEA RINGSPOT VIRUS IN AHYDRANGEA. VARIETAL COLLECTION. D. Rizzo1, B.Nesi2, L. Stefani1, M. Antonetti2, S. Lazzereschi2, S. Pecchioli2,M. Della Bartola3, A. Materazzi3 and A. Grassotti2. 1RegioneToscana, Laboratorio Servizio Fitosanitario Regionale, Via dei Fiori8, 51012 Pescia (PT), Italy. 2CRA-VIV Unità di Ricerca per il Vi-vaismo e la Gestione del Verde Ambientale ed Ornamentale, Viadei Fiori 8, 51012 Pescia (PT), Italy. 3Dipartimento di Coltivazionee Difesa delle Specie Legnose “G. Scaramuzzi”, Sezione di PatologiaVegetale, Via del Borghetto 80, 56124 Pisa, Italy. E-mail: [email protected]

In the frame of a survey on 21 species/varieties of Hydrangeaspp. and on 2 species of the close genus Schizophragma carriedour by CRA-VIV in Pescia (PT), 44 plants were tested by RT-PCR for the presence of possible infections by Hydrangearingspot virus (HdRSV). Leaf samples were collected in latespring-early summer 2010 from plants showing crinkling, rollingand asymmetry of the leaves, decreased number of florets per in-florescence, severe dwarfing and ‘running out’. Total RNA wasextracted with a modified protocol using the “RNeasy plant minikit” (Qiagen, USA) and retro-transcribed into cDNA. RT-PCRanalysis was done according to protocols already described in theliterature. Results showed that 15 out of 44 plants (34.1%), be-longing to 11 cultivars of 5 different species of Hydrangea wereinfected with HdRSV. The amplicon obtained was then se-quenced and proved to share a high similarity with the HdRSVsequences present in GenBank. All plants of the genus Schizo-phragma gave negative responses.

MOLECULAR ANALYSIS OF ROOT ROT PATHOGENSIN ORNAMENTAL PLANTS. D. Rizzo1, L. Stefani1, M.Paoli1, A. Grassotti2 and A. Haegi3. 1Regione Toscana, Laborato-rio Servizio Fitosanitario Regionale, Via dei Fiori 8, 51012, Pescia(PT), Italy, 2CRA-VIV Unità di Ricerca per il Vivaismo e la Ges-tione del Verde Ambientale ed Ornamentale, Via dei Fiori 8, 51012Pescia (PT), Italy, 3CRA, Centro di Ricerca per la Patologia Vege-tale, Via C.G. Bertero 22, 00156 Roma, Italy. E-mail: [email protected]

The sanitary situation of the ornamental plant industry is verydynamic and changing due to globalized market, introduction ofnew plant genera, species or varieties and new cultural tech-niques (which can favour or repress different pathogens). Moni-toring this kind of phytosanitary situation prove somehow diffi-cult, so, with the ultimate aim of developing new diagnostic tech-nologies based on molecular tools, molecular analysis ofpathogens were performed in ornamental plant industries of Tus-cany. We focused on woody plants, either conifers (e.g. Cupressussp., Pinus sp. or Taxus sp.) or not (Acer sp., Viburnum sp. or Pit-tosporum) collected in 2010 as affected by root rot and/or tra-cheomycoses. A total of 85 symptomatic samples were collected

and analysed: methods were set up to extract DNA from woodymaterial and perform efficient PCR with various primers on ma-terial of different plant origin. PCR assays with ITS5/ITS4primers were performed to amplify fungi and with ITS6/ITS4primers to amplify oomycetes. The first couple of primer ampli-fied 33 samples of 85, whereas the second set amplified 41 sam-ples of 85. Some samples yielded multiple bands (two or more)indicating that there was more than one fungus either pathogenicor saprophytic. All samples were also amplified with primers spe-cific for the most common fungi, namely Phytophthora sp., Cylin-drocarpon sp. and Phomopsis sp. Some samples were also se-quenced to confirm PCR results or to unravel other fungi.

EFFECT OF GREENHOUSE DEHUMIDIFICATION ONDAMAGES CAUSED BY BOTRYTIS CINEREA TO POIN-SETTIA. D. Rizzo1, G. Burchi2, L. Stefani1, M. Antonetti2, A.Haegi3, A. Teani2 and A. Grassotti2. 1Regione Toscana, Laborato-rio Servizio Fitosanitario Regionale, Via dei Fiori 8, 51012 Pescia(PT), Italy. 2CRA-VIV Unità di Ricerca per il Vivaismo e la Ges-tione del Verde Ambientale ed Ornamentale, Via dei Fiori 8, 51012Pescia (PT), Italy. 3CRA, Centro di Ricerca per la Patologia Vege-tale, Via C.G. Bertero 22, 00156 Roma, Italy. E-mail: [email protected]

This work was carried out in the framework of a researchproject, funded by Mi.P.A.A.F., that studies application of air de-humidification by means of hygroscopic solutions for humidityand climate control and energetic management in greenhousefloriculture. Phytosanitary surveys were performed in two green-houses at Verdello (Bergamo, northern Italy), one containing thedehumidification system and the other used as a control, with theaim to evaluate the beneficial effects of dehumidification on phy-tosanitary control. Analyses were performed on poinsettia (Eu-phorbia pulcherrima) pot-grown plants cultivated in both green-houses from October to December 2010. Damages on severalplants, with symptoms of wilting that eventually led to plantdeath, were recorded. Eighty percent of symptomatic plants be-longed to the ‘control’ greenhouse (without dehumidification sys-tem). Symptomatic samples (27) were collected to study the etiol-ogy of the disease: visual observation and mycological isolationsrevealed the presence of Botrytis cinerea and other saprophyticfungi. To confirm these results, molecular protocols were opti-mized to screen for ITS4/6 (PCR end point), Thielaviopsis basico-la (PCR Real Time Taqman), Phytophthora spp (PCR end point)and B. cinerea (PCR Real Time Sybr Green). Results on 27 symp-tomatic samples showed that 100% (27/27) were positive for B.cinerea, 92.6% (25/27) were amplified with ITS4/6 (that amplifyoomycetes), 7.4% (2/27) were positive for Phytophthora spp. andno one was positive for T. basicola. Results show that in these en-vironmental conditions (greenhouse) the main pathogen of Poin-settia seedlings is B. cinerea and that dehumidification of the en-vironment can better control the pathogen.

A SURVEY FOR THE MAIN DISEASES OF LIMONIUM spp.D. Rizzo1, G. Burchi2, L. Stefani1, M. Paoli1, M. Antonetti2, A.Teani2, A. Haegi3 and A. Grassotti2. 1Regione Toscana, Laborato-rio Servizio Fitosanitario Regionale, Via dei Fiori 8, 51012 Pescia(PT), Italy. 2CRA-VIV Unità di Ricerca per il Vivaismo e la Ges-tione del Verde Ambientale ed Ornamentale, Via dei Fiori 8, 51012Pescia (PT), Italy. 3CRA, Centro di Ricerca per la Patologia Vege-tale, Via C.G. Bertero 22, 00156 Roma, Italy. E-mail: [email protected]



Main fungal diseases that affect Limonium spp. are induced byPeronospora statices, Botrytis cinerea and Colletotrichum gloeospori-oides. In the frame of a survey of a Limonium collection carried outby CRA-VIV at Pescia (PT), a screening was performed in May2010 to verify the presence/susceptibility to the above-mentionedfungi. Seventyfour samples of symptomatic and asymptomatic ma-terial (leaves and stems) were collected from 20 different clones ofLimonium, belonging to 5 different species and hybrids, including6 commercial cultivars. DNA was extracted from plant materialthrough a modified protocol based on CTAB 3%. Diagnosis wasdone with molecular biology protocols optimized in our laboratoryfor Colletotrichum gloeosporioides (PCR end point), Botrytis cinerea(PCR Real Time SybrGreen) and with primers ITS4/6 that amplifyoomycetes, to check for the presence of Peronospora statices. Thislatter assay was positive for 91.9% of the samples, whereas Botrytiscinerea was detected in 60.8%, and Colletotrichum gloeosporioidesin 38.2% of the samples. These results were confirmed by wetroom tests. As to the visible effects of pathogenic fungi, none of thetested varieties of Limonium showed Peronospora statices symp-toms; 12 clones were completely asymptomatic for Botrytis cinereaand Colletotrichum gloeosporioides (L. gmelinii, L. altaica L. serot-inum and hybrids between them); 10 clones showed symptoms dueto Botrytis cinerea (mostly L. latifolium, L. altaica, and only oneclone of L. gmelinii); and 4 clones showed strong symptoms ofboth Botrytis cinerea and Colletotrichum gloeosporioides (L. latifoli-um and L. latifolium x otolepis).

POSSIBLE INDUCTION OF SYSTEMIC RESISTANCE INZUCCHINI AGAINST POWDERY MILDEW FOLLOWINGTREATMENT WITH CYANOBACTERIA EXTRACTS. R.Roberti1, H. Righini1, P.L. Burzi2, C. Perez Reyes3, S. Galletti2,B. Garcia and G. Reina3. 1Dipartimento di Protezione e Valoriz-zazione Agroalimentare, Università degli Studi, Viale Fanin 40,40127 Bologna, Italy. 2CRA, Centro di Ricerca per le Colture In-dustriali, Bologna, Italy. 3Centro De Biotecnología Marina, Univer-sidad De Las Palmas De Gran Canaria, Islas Canarias, Spain. E-mail: [email protected]

A sustainable agriculture that prevents environmental pollu-tion needs alternative control means. Cyanobacteria are compo-nents of fertilizer compounds which may interfere with plantphysiology, making plants less susceptible to biotic and abioticstresses. The aim of this research was to study the possibility toinduce systemic resistance in zucchini plants by foliar applicationof cyanobacterium Nostoc sp. extracts. Two trials were carriedout under controlled conditions: the first one consisted in a bio-logical assay of Nostoc extract against Podosphaera leucotricha andthe second one in biochemical assays in order to test the involve-ment of induced resistance. Chitosan was used as positive con-trol, since it is a well-known resistance inducer. Nostoc extract (5g l-1) was applied by spraying one of the two cotiledonar leaves inboth experiments, while the pathogen was inoculated on the non-treated leaves (5×104 conidia per ml of water) in the biological as-say only. Disease symptoms were recorded as percentage of in-fected area on non-treated leaves 7-9 days post inoculation. Forbiochemical assays, total proteins were extracted following a non-denaturing method and the expression of some PR-proteins (PR-3, PR-5 and PR-9) was tested by spectrophotometric and by iso-electrofocusing methods. The biological assay revealed that theNostoc extract reduced the disease by 20%, similarly to chitosan,with respect to the inoculated control. Both spectrophotometricand isolectrofocusing methods showed differences of the enzy-matic activities tested between treated and non-treated plants,suggesting the ability of the Nostoc extract tested to induce sys-temic resistance in zucchini.

ACTIVITY OF BIOSTIMULANT PRODUCTS INTEGRAT-ED WITH SULFUR FOR THE CONTROL OF ZUCCHINIPOWDERY MILDEW. R. Roberti, M. Fabbri, A. Veronesi andA. Brunelli. Dipartimento di Protezione e Valorizzazione Agroali-mentare, Università degli Studi, Viale G. Fanin, 46, 40127Bologna, Italy. E-mail: [email protected]

The biostimulants, Biosaral, Vitaflow and Chelal Alga (mainlybased on seaweed extracts) and Salavida (Pseudomonas trivialis)were applied with preventative or post-inoculation timing, incombination with sulphur against Podosphaera xanthii on pot-grown zucchini under greenhouse conditions. The pathogen wasinoculated on cotyledonar leaves on which sulphur was sprayedat a low dose (56 g/100 l). In the preventative timing, productswere applied two days before inoculation, by spraying leaves, bydrenching the potting mixture or by both, depending on theproduct: Biosaral was applied on the leaves, on potting mixtureor on both with and without sulphur; Vitaflow was applied onthe leaves only with and without sulphur; Salavida and Chelal Al-ga were applied singly with and without sulphur. In the post-in-oculation timing Biosaral, Salavida and sulphur were considered:Biosaral was applied on the leaves seven and nine days post-inoc-ulation (10-15% and 40-50% leaf area with disease symptoms re-spectively) with single or combined treatments with or withoutsulphur; Salavida was applied in the potting mixture seven dayspost-inoculation with or without sulphur. Both in the preventa-tive and in post-inoculation timing all products, depending ontheir application (on leaves, potting mixture or both) showed ef-fectiveness against the disease and their integration with sulfurenhanced powdery mildew control. Generally, Biosaral andSalavida were the more active products. In conclusion, biostimu-lants, integrated with a low dose of sulphur, provide a good op-portunity to reduce powdery mildew of zucchini.

CURRENT STATUS OF RESISTANCE TO FUNGICIDES INBOTRYOTINIA FUCKELIANA (BOTRYTIS CINEREA) ONSTRAWBERRY IN BASILICATA. C. Rotolo1, M. Masiello1, D.Gerin1, S. Pollastro1, R.M. De Miccolis Angelini1, A. Caponero2

and F. Faretra1. 1Dipartimento di Biologia e Chimica Agro-Fore-stale ed Ambientale, Università degli Studi “Aldo Moro”, Via G.Amendola 165/A, 70126 Bari, Italy. 2A.L.S.I.A., Agenzia Lucanadi Sviluppo ed Innovazione in Agricoltura, via C. Levi 6/I, 75100Matera, Italy. E-mail: [email protected]

Botryotinia fuckeliana (Botrytis cinerea), is the causal agent ofgrey mould on a wide range of crops affecting quality and quanti-ty of the produce. The disease management frequently requiresnumerous seasonal fungicide sprays increasing the risk of resist-ance development in the pathogen’s populations. A two-yearmonitoring of B. fuckeliana response to anilinopyrimidines (AP),phenylpyrroles, hydroxyanilides and pyridine-carboxamides (SD-HI) was carried out during 2009 and 2011 through a germinationtest on conidia sampled from naturally-infected strawberry fruitsin a total of 25 farms located in the Metaponto area (southernItaly). Resistant conidia were distinguished from sensitive conidiaby their ability to yield colonies on media amended with singlediscriminatory concentrations of each fungicide: pyrimethanil (1mg l-1), fludioxonil (0.3 mg l-1), fenhexamid (0.4 and 4 mg l-1) orboscalid (10 mg l-1). AP-resistant isolates were detected in allmonitored fields, frequently at high frequency (13-96%). A slightincrease in the frequency of conidia resistant to phenylpyrrroleswas detected in 2011 (up to 17%) as compared to that recordedin 2009 (0-7%). Resistance to fenhexamid was detected with avery low frequency (3×10-5 to 13×10-2) in 5 of the 8 fields moni-tored in 2009 and in 16 of the 17 fields monitored in 2011. Resis-



tance to boscalid, which was applied in mixture with the QoIfungicide pyraclostrobin, was detected in all fields at variable fre-quency (3-73%). The results disclose a widespread incorrect useof fungicides against gray mould and stress out the importance ofappropriate anti-resistance strategies.

VARIABILITY AMONG PECTOBACTERIUM CAROTOVO-RUM STRAINS ISOLATED FROM ARTICHOKE INSOUTHERN ITALY. M. Russo1, G. Ialacci2, S. Loreti3, V.Catara2 and P. Bella2. 1Parco Scientifico e Tecnologico della Sicilia,Zona Industriale, Via V. Lancia, 95030 Catania, Italy. 2Dipartimen-to di Scienze delle Produzioni Agrarie e Alimentari, Universitàdegli Studi, Via Santa Sofia 100, 95123 Catania, Italy. 3CRA, Cen-tro di Ricerca per la Patologia Vegetale, Via C.G. Bertero 22, 00156Roma, Italy. E-mail: [email protected]

Pectobacterium carotovorum subsp. carotovorum, the causalagent of soft rot, has a broad host range and is distributed world-wide. In 2010, a severe outbreak of artichoke soft rot occurred inSicily. The characteristic symptoms were leaf wilting and internaldecay at the crown often accompanied by vascular discoloura-tion. Eventually, the plants wilted completely and died. Bacteriawere consistently recovered from the diseased plants on nutrientdextrose agar and identified as P. carotovorum (Pc) by biochemi-cal tests, Biolog identification system, PCR using specie-specificprimers Y1/Y2 and pathogenicity on potato tubers. Sicilianstrains were genotyped with other representative P. carotovorumstrains isolated from artichoke in different locations in southernItaly. Twelve Pc from artichoke and reference strains of P.c. subsp.carotovorum (Pcc), P.c. subsps. odoriferum (Pco), P. wasabiae (Pw)and P. atrosepticum (Pa) were analyzed by BOX-PCR and fluores-cent amplified fragment length polymorphism (fAFLP). ByBOX-PCR, a common core profile was identified in all Pc strainsfrom artichoke, however different haplotypes were detected evenin the same production site. Adequate numbers of fragments forfAFLP analysis were selected by in silico AFLP-PCR experimentson P. c. subsp. carotovorum PC1 genome (http:/www.in-silico.com). Four primer combinations were screened for theirreproducibility and number of polymorphic bands on four repre-sentative Pc strains. The dendrogram of distances obtained byanalysis of fAFLP data highlights that Pc strains from artichokeform two distinct clusters including also Pcc and Pco referencestrains, but clearly separated from Pw and Pa strains.

CYLINDROCARPON SPECIES ASSOCIATED TO KI-WIFRUIT AFFECTED BY ELEPHANTIASIS. A. Rustignoli,A. Prodi, M.A. Mir, S. Borsari, P. Nipoti and A. Pisi. Diparti-mento di Scienze e Tecnologie Agroambientali, Università degliStudi, Viale Fanin 42, 40127 Bologna, Italy. E-mail: [email protected]

Kiwifruit is an important fruit crop of temperate regions andItaly is the leader in production. An unusual disease, named ele-phantiasis, in kiwifruit (Actinidia deliciosa) cv Hayward orchards ofnorthern Italy, was observed since 2001. Trunk hypertrophy is themost typical symptom of elephantiasis. A complex mycoflora be-longing to different genera, mainly Phialophora-like taxa, Fusarium(mostly F. solani) and Cylindrocarpon spp., was isolated fromnecrotic wood of symptomatic trunks. The causal agent/s is/areunknown because pathogenicity field assays are still in progress.The aim of this study was to identify at the species level isolates ofCylindrocarpon, which is the genus until now less investigated.Cylindrocarpon isolates were identified morphologically, assayed by

a multiplex PCR protocol for the identification of C. liriodendri, C.pauciseptatum and C. macrodidymum - species usually associated toa severe grapevine disease (black foot) and further investigated us-ing sequencing and phylogenetic analyses of the partial ß tubulingene. C. macrodidymum and C. destructans var. crassum were iden-tified. To our knowledge, this is the first report of these species inActinidia deliciosa. The role of Cylindrocarpon spp. in elephantiasisneeds more investigations in order to understand the synergy withthe other fungi involved in this complex disease.

EFFICACY OF ZATARIA OIL ON POSTHARVEST CON-TROL OF RHIZOPUS ROT OF PEACHES. S. Alizadeh-Salteh1, K. Arzani1, R. Omidbeigi1, N. Safaie2 and P. Mohamma-di3. 1Department of Horticultural Science, Tarbiat Modares Univer-sity (TMU), Iran. 2Department of Plant Pathology, TarbiatModares University (TMU), Iran. 3Department of Plant Pathology,Tehran University, Iran. E-mail: [email protected]

The advantage of essential oils is their bioactivity in thevapour phase, a characteristic that makes them attractive as possi-ble fumigants for stored product protection. The aim of this in-vestigation was to evaluate the antifungal effects of the herbal es-sential oil of Shiraz Thyme (Zataria multiflora Boiss) against Rhi-zopus stolonifer, causal agent of Rhizopus rot of peach. In in vitrotrials, the activity of Zataria oil was tested on the growth of thepathogen’s mycelium. Essential oil was added to the medium(ranging from 120 to 360 µl l-1), or to a filter paper (ranging from1 to 48 µl l-1) inserted on the lid of Petri dish tobe assayed as bio-fumigant. In in vivo experiments, four concentrations of Zatariaoil (0, 120, 240 and 360 µl l-1) were tested by dipping and sevendoses (0, 50, 100, 200, 400, 800 and 1600 µl l-1) were applied asvapour phase on peaches cv J.H. Hale artificially inoculated withthe pathogen (106 conidia ml-1). Fruits were stored at 25°C or1°C for 20 and 45 days respectively. In in vitro results the concen-trations of 360 µl l-1 used in the medium and that of 24 µl l-1 usedas volatile showed the highest activity against Rhizopus growth. Inin vivo trials, the most effective concentration of essential oil was360 µl l-1 used by dipping and 24 µl l-1 used as biofumigant. Moreinvestigation are in progress to evaluate the practical applicationof Zataria oil against Rhizopus rot.

BIOFUMIGATION FOR THE CONTROL OF FUSARIUMROOT ROT IN ITALY. A. Santori1, S. Mocali2, S. Landi2 andA. Infantino1. 1CRA-PAV Centro di Ricerca per la Patologia Vege-tale, Via C.G. Bertero 22, 00156 Roma, Italy. 2CRA, Centro diRicerca per l’Agrobiologia e la Pedologia, Piazza D’Azeglio 30,50121 Firenze, Italy. E-mail: [email protected]

Crown and root-rot (CRR) is a disease of complex aetiology ofwheat in which several Fusarium species and Microdochium nivaleare involved. Attacked plants show browning and rotting of thecrown and roots and whitening of the heads containing shrivelledkernels of no commercial value. A field experiment for the con-trol of the disease by using green manure with Brassica juncea cv.Scala (SIS, Italy) was conducted at the CRA-PAV farm (Rome,Italy) during the 2010-2011 season. Four sub-parcels, each repre-senting a different treatment, were considered: (i) green manurefor two years in a row; (ii) green manure for one year; (iii) effectof green manure of previous year; (iv) plot without green manure.B. juncea was grown for three months in sub-parcel 1 and 2, thenmulched and incorporated into the soil. After ten days, durumwheat cv. Claudio was sown in all sub-parcels. For microbiologi-cal analyses, soil samples were collected in two different periods,



before sowing B. juncea and wheat, respectively. Incidence ofwhite heads was assessed at the hard dough growth stage by visu-al evaluation of 200 heads for each treatment. Bio-fumigationwith B. juncea for two following years (treatment 1) determinedthe lowest incidence (9.5%) when compared to that of the con-trol (32%). Intermediate results were obtained for treatment 2(15.9%) and treatment 3 (23.5%). Microbiological analyses ofsoil samples and of wheat stem and crowns are in progress. Thepreliminary results obtained confirm that green manure is prom-ising for the control of CRR of wheat.

PHYTOPHTHORA SPECIES ASSOCIATED WITH SUD-DEN DEATH OF YOUNG OLIVE TREES IN SARDINIA. B.Scanu, B.T. Linaldeddu, S. Seddaiu, L. Maddau and A. Frances-chini. Dipartimento di Protezione delle Piante, Sezione di Patolo-gia Vegetale, Università degli Studi, Via E. De Nicola 9, 07100 Sas-sari, Italy. E-mail: [email protected]

Since 2007, about 7.000 ha of agricultural land has beenplanted with olive (Olea europea) in Sardinia (insular Italy). Fol-lowing several reports of sudden death of young olive trees, a sur-vey was carried out in 2010 in three 1- to 4-year-old groves and ina commercial nursery, one of the sources of olive saplings. Symp-toms observed in the field were typical of Phytophthora infection,such as wilting and dieback of the crown, often associated withthe presence of necrotic lesions at the collar level and root rot.The aim of the present study was to isolate and identify thespecies of Phytophthora associated with symptomatic olive trees.Fine roots, bark tissues and soil samples were collected and isola-tions were made using the selective medium SMA. Phytophthoracolonies were then sub-cultured onto carrot agar to study theirmorphological and physiological properties. Identification of theisolates were confirmed by analysis of the ITS region (ITS1-5.8S-ITS2) of the rDNA. Sequences were compared with those pres-ent in GenBank database. Three different species of Phytophtho-ra were identified. P. palmivora was the most frequent species. Itwas isolated from all orchards and from nursery stock material.With less frequency, P. nicotianae and another non-identified Phy-tophthora sp. were isolated from soil samples collected around in-fected olive trees at one site. Pathogenicity of two P. palmivoraisolates obtained from different olive cultivars was verified by in-oculating 2-year-old olive seedlings. Further tests are underway toassess the aggressiveness of the other Phytophthora species.

CHARACTERIZATION OF A FUSARIUM GRAMINEARUMXYLANASE EXPRESSED DURING WHEAT INFECTIONAND KNOCK-OUT OF ITS ENCODING GENE. L. Sella1, K.Gazzetti1,3, F. Faoro2, A. Raiola1, R. D’Ovidio3, W. Schäfer4 andF. Favaron1. 1Dipartimento del Territorio e Sistemi Agro-Forestali,Università degli Studi di Padova, Viale dell’Università 16, 35020Legnaro (PD), Italy. 2Dipartimento di Produzione Vegetale, Uni-versità degli Studi, Via Celoria 2, 20133 Milano, Italy. 3Diparti-mento di Scienze e Tecnologie per l’Agricoltura, le Foreste, la Natu-ra e l’Energia, Università della Tuscia, Via S. Camillo de Lellis,01100 Viterbo, Italy. 4Biocenter Klein Flottbek, Molecular Phy-topathology and Genetics, University of Hamburg, Hamburg, Ger-many. E-mail: [email protected]

Fusarium graminearum is the causal agent of Fusarium headblight (FHB) of wheat, barley and other cereal grains. During theinfection process, this fungus secretes a large number of hydrolyt-ic enzymes acting on the plant cell wall. In particular, F. gramin-earum is able to degrade xylans, the main constituent in the cell

walls of monocot plants like wheat, through the coordinate actionof a group of extracellular enzymes; among them, endo-ß-1,4-xy-lanases hydrolyze the inner ß-1,4 glycosidic bond and could beimportant pathogenic weapons for (pathogens) attacking cerealplants. However, xylanases could also exert effects independent oftheir enzymatic activity: a Botrytis cinerea xylanase, namedXyn11A, was shown to contribute to virulence on tobacco andtomato leaves with its necrotizing activity and not with the xylanhydrolyzing activity. In the genome of F. graminearum there is axylanase-encoding gene (named Xyl1) whose deduced aminoacidic sequence has a 55% identity with the B. cinerea xylanaseXyn11A. Since the transcript of Xyl1 gene has been detected inwheat spikelets infected with F. graminearum, we cloned this genefor heterologous expression in Pichia pastoris. The purified xy-lanase was characterized by studying its enzymatic activity in vitroand its necrotizing activity on wheat tissue. Knock-out mutants ofthe corresponding encoding gene were also obtained by site-di-rected homologous recombination. Wheat infection experimentsaimed at evaluating the virulence of these mutants are in progress.

DIAGNOSTIC ANALYSES ON FOURTH RANGE PRO-DUCTION IN THE PROVINCE OF SALERNO. L. Sigillo, V.Senape, G. Serratore, V. Spina and R. Bravi. Ente Nazionale Se-menti Elette, Sezione di Battipaglia SS 18, km 77.700, 84091 Batti-paglia (SA), Italy. E-mail: [email protected]

The production of species for fourth range market has stronglyincreased in the last years. Important economical losses are report-ed due to the widespread of bacterial and fungal diseases. TheLaboratory for Phytopathological Analyses of ENSE (Battipaglia,Italy) is specialized on detection of the most important pathogensthat infect these species. From 2005 to 2010, 73 samples of rocket(Dyplotaxis tenuifolia), 65 of lettuce (Lactuca sativa), 19 of corn sal-ad (Valerianella locusta), 9 of spinach (Spinacia oleracea) and manyother minor species were analysed. Among these, 108 and 68 seedand symptomatic plant samples were analysed for the detection ofthe most important pathogens. Contamination by Xanthomonascampestris and pathogenic strains of Fusarium oxysporum of rocketseed samples was 15% and 8% respectively. Instead, 50% and20% of rocket plants were infected by F. oxysporum and X.campestris. F. oxysporum, Verticillium sp. and Rhizoctonia sp. oc-curred rarely on lettuce seeds or plant tissues (about 3%). General-ly corn salad, beet and spinach seeds resulted healthy. X. campestriswas detected in a corn salad sample. In conclusion, an increase ofF. oxysporum and X. campestris incidence on rocket has been re-ported in the last years. This increase is probably due to the diffu-sion of monocropping system and the use of contaminated seeds inthe fourth range production.

SEVERE OUTBREAKS OF LETTUCE BIG VEIN INLATIUM AND CAMPANIA. R. Sorrentino1, R. Carrieri2, G.Cirillo3, G. Parrella4 and D. Alioto1. 1Dipartimento di Arbori-coltura, Botanica e Patologia Vegetale, Università degli Studi diNapoli, Via Università 100, 80055 Portici (NA), Italy. 2Diparti-mento di Biologia e Difesa delle Piante, CRA-CAT, Via P. Vitiello108, 84018 Scafati (SA), Italy. 3Enza Zaden Italia srl, Ss. Aureliakm 96+710, 01016 Tarquinia (VT), Italy. 4Istituto per la Pro-tezione delle Piante del CNR, Via Università 133, 80055 Portici(NA), Italy. E-mail: [email protected]

Big-vein is a widespread disease of lettuce, transmittedthrough the soil by Olpidium brassicae. Two viruses are associatedwith the disease, Lettuce big-vein associated virus (LBVaV; genus



Varicosavirus) and Mirafiori lettuce big-vein virus (MLBVV; genusOphiovirus). The latter has been identified as the disease-causingagent also if the etiology of big vein is still confused, since LBVaValone was found in some symptomatic lettuces. In winter 2011, inthe vegetable growing areas of Fondi (Latium) and Angri (Cam-pania), big-vein symptoms appeared in field-grown lettuce, caus-ing losses from 15 to 100% according to the cultivar and cultiva-tion area. Symptoms of the disease appeared as chlorotic regionssurrounding the vascular tissues accompanied by plant size re-duction and head abort or retard. Lettuces from Fondi showedalso symptoms resembling those of Lettuce ring necrosis virus(LRNV). Five symptomatic plants for each variety (Iceberg,Canasta, Cassiopea, Azimut) from Fondi and two (Iceberg) fromAngri, were analyzed for the presence of LBVaV and MLBVV.Results of mechanical transmission were negative for all testedsamples, probably due to greenhouse temperatures above 18°C.Mixed infections with MLBVV and LBVaV were detected in allsamples analysed by DAS-ELISA using commercial kits. LBVaVpresence in all symptomatic lettuce plants was also confirmed byRT-PCR using LBVaV-specific primers. On the other hand, onlyfew samples tested positive by RT-PCR with the universal primersfor Ophioviruses and, in this case, the identity of MLBVV wasverified sequencing amplicons which shared 97% similarity withMLBVV sequences deposited in GenBank.

USE OF LAMIACEAE ESSENTIAL OILS TO CONTROLPOSTHARVEST ROTS OF STONE FRUITS. D. Spadaro,J.G. Lopez-Reyes, A. Garibaldi and M.L. Gullino. AGROINNO-VA, Centro di Competenza per l’Innovazione in Campo Agroambi-entale, Università degli Studi di Torino, Via L. da Vinci 44,Grugliasco (TO), Italy. E-mail: [email protected]

Essential oils can be used to control postharvest pathogens ofstone fruits, such as Monilinia laxa, M. fructigena and Botrytiscinerea. The efficacy of essential oils treatments was assessed onapricots cvs. Kyoto and Tonda di Costigliole, nectarines cvs. BigTop and Nectaross and on plums cvs. Italia and TC Sun againstBotrytis cinerea, Monilinia fructigena and M. laxa. The essentialoils from basil (Ocimum basilicum), fennel (Foeniculum sativum),lavender (Lavandula officinalis), marjoram (Origanum majorana),oregano (Origanum vulgare), peppermint (Mentha piperita), rose-mary (Rosmarinus officinalis), sage (Salvia officinalis), savory (Sat-ureja montana), thyme (Thymus vulgaris) and wild mint (Menthaarvensis) were tested at different concentrations. Fruits were arti-ficially inoculated with a spore suspension of each pathogen andessential oil emulsions were applied after 12 h incubation time.Treatment with savory essential oil was statistically more efficientto control the tested pathogens than the other treatments. Treat-ments with essential oils from basil and oregano were also effec-tive but mainly on apricots cv. Tonda di Costigliole. Essential oilstreatments at elevated concentrations permitted a high level ofdisease control after 15 days of storage, but they could induce se-vere damages on the carposphere of apricots and subsequent dis-ease development after 30 days of storage. The efficacy of essen-tial oils and their potential phytotoxicity on stone fruits dependsboth on species and carposphere strength.

IN VITRO BIODEGRADATION OF OCHRATOXIN A BYYEAST ANTAGONISTS. D. Spadaro, S. Patharajan, A. Lorè,M.L. Gullino and A. Garibaldi. AGROINNOVA, Centro di Com-petenza per l’Innovazione in Campo Agroambientale, Universitàdegli Studi di Torino, Via L. da Vinci 44, Grugliasco (TO), Italy. E-mail: [email protected]

Yeast biocontrol agents, previously selected to control differ-ent fungal pathogens, were screened initially for their ability todegrade ochratoxin A (OTA) in liquid medium amended withOTA (7.5 mg ml-1) and different concentrations of yeast cells at30°C. The highest OTA degradation was observed when the yeastcell concentration used was 108 cells ml-1. Out of six yeast strains,three strains [Metschnikowia pulcherrima (MACH1), Pichia guil-liemonodii (M8) and Rhodococcus erythropolis (AR14)] were se-lected for further studies. Yeast strains were evaluated for theirability to degrade OTA at different temperatures (10, 15, 20, 25,30 and 35°C) and 30°C resulted the optimal temperature foryeast growth and OTA degradation. The three strains were fur-ther evaluated to test their potential on OTA degradation usingdifferent concentrations of OTA (5 and 10 µg ml-1) at 30°C. Thethree yeast antagonists were further tested to confirm eitherdegradation or cell wall adsorption of OTA at different time in-tervals. Among the strains, MACH1 effectively degraded OTA(>80%) at 30°C after 15 days incubation compared to the otherstrains tested and a little amount of OTA adsorption was ob-served in the yeast cell wall. LC-MS studies revealed that no by-product like ochratoxin or phenylalanine were found during thedegradation process. Therefore, further studies are needed to un-derstand the mechanism of action of these yeast strains duringOTA degradation.

USE OF A NOVEL STRAIN OF METSCHNIKOWIA sp. TOCONTROL PENICILLIUM EXPANSUM AND PATULINCONTAMINATION ON APPLES IN POSTHARVEST. D.Spadaro, A. Lorè, A. Garibaldi and M.L. Gullino. AGROINNO-VA, Centro di Competenza per l’Innovazione in Campo Agroambi-entale, Università degli Studi di Torino, Via L. da Vinci 44,Grugliasco (TO), Italy. E-mail: [email protected]

Penicillium expansum is the agent of blue mould, the mostcommon form of post-harvest rot of pome fruits, which causesconsiderable economic losses. The pathogen is able to producepatulin, a lacton that may cause acute and chronic toxicity. Thebiocontrol efficacy of antagonistic yeasts against the agent of bluemould and its patulin production was evaluated on apples cvsGolden Delicious, Granny Smith, Red Chief and Royal Galastored at 21°C for 7 days and at 2°C for 42 and 56 days. The re-sults showed that a new strain of Metschnikowia sp. presented thehighest efficacy on the control of P. expansum, on apples storedboth at room temperature and at refrigerated temperature. Insome cases, its efficacy was higher than the chemical control. Atroom temperature, a higher patulin contamination was observedon fruits treated with the most effective biocontrol agents. On ap-ples stored at refrigerated temperature, the patulin concentrationon cv. Golden Delicious apples was very low, in cvs GrannySmith and Royal Gala the highest concentration was observed infruits treated with Metschnikowia sp. Considering the totalweight of the apples, patulin was detected in low concentrationsand the lowest amount was found on apples treated with the newstrain of Metschnikowia sp.

SPECIFIC PCR PRIMERS FOR THE DETECTION OF ISO-LATES OF ASPERGILLUS CARBONARIUS PRODUCINGOCHRATOXIN A IN GRAPEVINE. D. Spadaro, S. Pathara-jan, A. Lorè, A. Garibaldi and M. L. Gullino. AGROINNOVA,Centro di Competenza per l’Innovazione in Campo Agroambien-tale, Università degli Studi di Torino, Via L. da Vinci 44, Gruglias-co (TO), Italy. E-mail: [email protected]



Aspergillus carbonarius is involved in ochratoxin A (OTA)contamination of grapes and wine. Polyketide synthases are in-volved in OTA biosynthesis in Aspergillus species. The ketosyn-thase (KS) domain of an A. carbonarius gene encoding polyketidesynthase (pks) was isolated, cloned and sequenced. The nu-cleotide sequence showed high similarity to KS domains isolatedfrom other Aspergillus species. The sequence was used to designa new set of primers in order to identify potential producers ofOTA from isolates of A. carbonarius. The primers amplified allthe A. carbonarius strains tested specifically, and did not amplifyany other species of Aspergillus or Penicillium normally found ongrapes or involved in OTA biosynthesis. Further, gene expressionwas related to OTA production. Both gene transcription and lev-els of the mycotoxin were higher when A. carbonarius was grownon YES medium at 15°C and 30°C, whereas a low transcriptionand mycotoxin presence were observed when the fungus wasgrown on PDB medium at the same temperatures. No transcrip-tion or OTA production were observed when the fungus wasgrown on either YES or PDB at 10°C. The primers will be usefulto detect ochratoxigenic strains of A. carbonarius both in vine-yards and during wine production.

TRANSPOSITION OF THE MINIATURE INVERTED-RE-PEAT TRANSPOSABLE ELEMENT MIMP1 IN THEWHEAT PATHOGEN FUSARIUM CULMORUM. F. Spanu1,B. Scherm1, V. Balmas1, A. Marcello1, M. Pasquali2, M.Dufresne3, M.J. Daboussi4 and Q. Migheli1. 1Dipartimento diProtezione delle Piante, Unità di Ricerca Istituto Nazionale diBiostrutture e Biosistemi, Università degli Studi, Via E. De Nicola9, 07100 Sassari, Italy. 2CRP-Gabriel Lippmann, 41, rue du Brill,4422 Belvaux, Luxembourg. 3Institut de Biologie des Plantes, Bâti-ment 630, Université Paris Sud 11, 91405 Orsay Cedex, France.4Institut de Génétique et Microbiologie, Bâtiment 400, UniversitéParis Sud 11, 91405 Orsay Cedex, France. E-mail: [email protected]

The genome of Fusarium culmorum, incitant of crown andfoot rot on wheat and type B trichothecene producer, is now be-ing sequenced. The number of predicted genes is estimated to ex-ceed 10,000 and for many of them the function is still unknown.Consequently, there is a strong need for a high-throughoutmethod for functional genomic analysis. Our aim is to test the ef-ficacy of a two double component system based on the ability ofthe impala transposase to transactivate the miniature inverted-re-peat transposable element mimp1 of Fusarium oxysporum. In thissystem, the tagging element mimp1 is inserted into the first intronof the A. nidulans niaD gene, and a phenotypic assay for excisionallows recovery of excision events on minimal medium containingnitrate as the sole nitrogen source. Since mimp1 has no coding ca-pacity, its mobilization requires a trans-activating impala trans-posase under the control of the constitutive A. nidulans gpdApromoter on a separate vector. Similarly to what was previouslyobserved in Fusarium graminearum, mimp1 was shown to trans-pose in F. culmorum by a cut-and-paste mechanism into TA dinu-cleotides, which are duplicated upon insertion. Our results alsoshow that mimp1 reinserts in genic regions for 28% (i.e., 16 over58) of the flanking sequences analysed, spanning throughout theentire genome of F. culmorum. Evidence that the mimp1/impaladouble-component system is fully functional in the heterologousspecies F. culmorum makes it an efficient tool for gene tagging inthis pathogen.

DETECTION OF SCLEROTINIA ROT DISEASE ON CIT-RUS MEDICA IN A SOUTH ITALY AREA. M.C. Strano, M.Calandra, V. Aloisi, G. Cicciarello and A. Caruso. CRA, Centrodi Ricerca per l’Agrumicoltura e le Colture Mediterranee, CorsoSavoia 190, 95024 Acireale (CT), Italy. E-mail: [email protected]

Sclerotinia rot (cottony rot) is a serious disease induced by dif-ferent species of the ubiquitous phytopathogenic fungiSclerotinia, which cause economically important losses to severalspecies of plants. The disease is more common in temperate andsubtropical regions characterized by cool and wet climate. Sclero-tinia spp. rarely cause diseases to citron trees (Citrus medica), butunder specific environmental conditions and plant susceptibility,diseases can develop with a high incidence, with serious yield re-duction. Citron trees are specially cultivated in Calabria (south-ern Italy) for candied production. Frequent rainfall and low tem-perature occurred in this area during winter season of 2010-2011.This paper reports the isolation of Sclerotinia sclerotiorum frominfected twigs of citron trees, grown under net shade, whichshowed brown lesion shortly followed by a characteristic cottony,white mycelium on infected tissue and superficial hard blackstructures (sclerotia). Koch’s postulates were tested for the con-firmation of the fungus as the cause of the disease, morphologycharacteristics in vitro were studies and artificial inoculation tocitrus fruit were made.

DETECTION OF NEW SEQUENCE VARIANTS OFGRAPEVINE RUPESTRIS STEM PITTING ASSOCIATEDVIRUS. F. Terlizzi1, C. Li2, C. Ratti1, W. Qiu3, R. Credi1 and B.Meng2. 1Dipartimento di Scienze e Tecnologie Agroambientali,Sezione Patologia Vegetale, Università degli Studi, 40127 Bologna,Italy. 2Department of Molecular and Cellular Biology, College of Bio-logical Science, University of Guelph, Ontario, N1G 2W1 Canada.3Department of Agriculture, Missouri State University, MountainGrove, MO 65711, USA. E-mail: [email protected]

Grapevine rupestris stem pitting-associated virus (GRSPaV) is amember of the genus Foveavirus (Betaflexiviridae). It occursworldwide and is considered the causal agent of Rupestris stempitting, a component of the Rugose wood complex. GRSPaV iscomposed of a wide range of sequence variants and so far, thecomplete genomes of five sequence variants have been se-quenced. Quick and reliable detection of different GRSPaV vari-ants is a critical step in the control of GRSPaV. Previously,primers designed from various genomic regions have been usedin RT-PCR for the detection of GRSPaV variants. The efficiencyof RT-PCR varied widely, depending on the spectrum of theprimers that were used. In this study, we designed a pair of de-generate primers based on the consensus sequence of the genom-ic region encoding the highly conserved RNA-dependent RNApolymerase domain from five reference isolates of GRSPaV forwhich the genomes are available. We demonstrated that this setof primers is comparable to the broad-spectrum primersRSP13&14 in detecting multiples GRSPaV variants and, by usingthese new degenerate primers, we identified two new and distinctsequence variants. The 3’ terminal genomic region of one of thenew variants, GRSPaV-ML, spanning the 3’ part of ORF1,through the entire ORFs 2-4, and the 5’ region of ORF5, was se-quenced. Sequence comparison showed that GRSPaV-ML is dis-tinct from each of the five reference isolates.



A MULTIPLEX REAL TIME TAQMAN RT-PCR ASSAYFOR THE DETECTION OF GRAPEVINE FLAVESCENCEDORÉE AND BOIS NOIR PHYTOPLASMAS USINGCRUDE SAP EXTRACTS. F. Terlizzi, C. Ratti, C. Poggi Polliniand R. Credi. Dipartimento di Scienze e Tecnologie Agroambien-tali, Università degli Studi, Viale Fanin 40, 40127 Bologna, Italy.E-mail: [email protected]

Flavescence dorée (FD) and Bois noir (BN) are the most im-portant phytoplasma diseases of grapevine in Europe. Detectionof FD and BN phytoplasmas (16SrV and 16SrXII-A groups, re-spectively) is particularly difficult, as they are generally present inlow concentrations and unevenly distributed in the plant phloem.Many efforts recently focused on developing rapid and sensitivemethods for their detection. In this study a triplex real time Taq-Man RT-PCR methodology has been set up to simultaneously,but specifically, amplify a 16SrDNA gene fragment of the twophytoplasmas and a 18SrDNA gene region of the host plant usinga crude sap extract as template. The use of RNA as template, in-stead of DNA, takes advantage from the high ribosomal-RNAcopy number present in pathogen cells and provides evidence ofviability and metabolic activity of phytoplasma, which is not pro-vided by DNA-based detection. During our study, an efficientand rapid method for FD and BN phytoplasmas detection in dif-ferent host plants has been developed. Absence of nucleic acidextraction step allows to greatly decrease both costs and timenecessary to perform the assay. Therefore the method presentedis a rapid, specific and sensitive alternative to conventional nest-ed-PCR. We successfully applied this technique to the detectionof FD and BN phytoplasmas on more than 300 samples ofgrapevine, strawberry, tomato, wild plants and insect vectors.

GENETIC BASIS OF THE LEAF RUST RESISTANCE Lr14LOCUS IN DURUM WHEAT. I. Terracciano1, M. Maccaferri1,F. Bassi1, P. Mantovani2, M.C. Sanguineti1, A. Massi2, J.Kolmer3, H. Simkova4 and R. Tuberosa1. 1Dipartimento di Scien-ze e Tecnologie Agroalimentari, Università degli Studi, Viale Fanin44, 40127 Bologna, Italy. 2Società Produttori Sementi Bologna, ViaMacero 1, 40050 Argelato, Bologna, Italy. 3USDA-ARS, Cereal Dis-ease Laboratory, 55108 St. Paul, MN, USA. 4Institute of Experi-mental Botany, Sokolovska 6, 77200 Olomouc, Czech Republic. E-mail: marco.maccaferri@ unibo.it

Leaf rust (Puccinia triticina) is a main disease affecting durumwheat production in the Mediterranean region. Improving the re-sistance to leaf rust can effectively be accomplished through map-ping of the resistance loci from valuable sources. The leaf rust re-sistant allele Lr14-Creso from durum wheat cv. Creso is one of themost important leaf rust resistance sources present in the moderndurum wheat germplasm. Lr14 is located in the distal portion ofchromosome 7BL. The Colosseo (a Creso derivative) x Lloyd re-combinant inbred line population (176 lines) has been used toenrich the Lr14 region with new molecular markers derived fromwheat ESTs. Phenotypic data were collected in the field and alsoat the seedling stage. A high heritability of the disease responsewas observed in both cases (h2 > 0.80). The population allowedfor mapping the locus at a good resolution level (5 cM). New mo-lecular markers were developed and mapped within an interval of14 cM that includes the Lr14-QTL peak. In particular somemarkers were obtained by exploiting the conserved colinearitybetween the most distal portions of rice chromosone 6, Brachy-podium chromosone 1 and wheat chromosone 7BL. The resultsare supported by an independent association mapping study car-ried out using a panel of 164 durum elite accessions. The ongoingstudies will provide further insights on the genetic basis of Lr14.

The newly developed markers tagging the Lr14-Creso allele arepresently used in marker assisted selection activities.

MULTIPLEX REAL TIME RT-PCR FOR TOMATOCHLOROSIS VIRUS AND TOMATO INFECTIOUS CHLORO-SIS VIRUS IN TOMATO PLANTS AND WHITEFLIES. A.Tiberini1, A. Manglli1, V. Cavalieri2, C. Rapisarda2 and L.Tomassoli1. 1CRA, Centro di Ricerca per la Patologia Vegetale, ViaC.G. Bertero 22, 00156 Roma, Italy. 2Dipartimento di Gestione deiSistemi Agroalimentari e Ambientali, Università degli Studi, ViaSanta Sofia 100, 95123 Catania, Italy. E.mail: [email protected]

Tomato chlorosis virus (ToCV) and Tomato infectious chlorosisvirus (TICV), genus Crinivirus, family Closteroviridae, are plantviruses characterized by flexuous, filamentous virions inducingsymptoms often confused with nutritional or physiological disor-der: interveinal yellowing, red and/or brown necrotic flecking andbrittleness of older leaves. Firstly identified in the USA, TICV andToCV rapidly spread worldwide now being reported in theMediterranean basin, North America, South Africa and Asia. Bothviruses are transmitted in a semi-persistent manner by whitefly vec-tors belonging to Aleyrodidae (Homoptera). In particular, TICV istransmitted by Trialeurodes vaporariorum and ToCV by Bemisiatabaci, T. vaporariorum and T. abutilonea. Since similar symptomsare induced on plants by the two viruses, as well as by nutritionaldeficiencies, and different vectors are involved in their transmis-sion, a sensitive diagnosis and virus discrimination is essential fordisease control. For these reasons, a multiplex TaqMan RT-PCR as-say was developed for a simultaneous detection of the two viruses.Primer and MGB probe sets were specifically designed in coat pro-tein gene region according to reference nucleotide sequences ofTICV and ToCV published in GenBank. Plant internal control, de-signed in the cytochrome c oxidase gene (COX), and vector con-trol, designed in consensus region of whiteflies (rRNA 18S) werealso included. During the last tomato season, leaf samples andwhiteflies were collected in several commercial greenhouses in Sici-ly and Sardinia. The method proved successful in detecting theviruses simultaneously, both in plant and insect extracts, anddemonstrated to be useful for wider future monitoring and epi-demiological studies.

Research supported by MiPAAF in the framework of the projectARON- ARNADIA (DM 19738/7303/08 DEL 29/12/2008)

IDENTIFICATION AND CHARACTERIZATION OFCHRYSANTHEMUM STUNT VIROID INFECTING ARGY-RANTHEMUM FRUTESCENS IN ITALY. E.M. Torchetti1, B.Navarro1, V.N. Trisciuzzi2, M.E. Silletti2 and F. Di Serio1. 1Istitu-to di Virologia Vegetale del CNR, UOS Bari, Via Amendola 165/A,70126 Bari, Italy, 2Centro di Ricerca e Sperimentazione in Agri-coltura Basile Caramia, Via Cisternino 281, 70010 Locorotondo(BA), Italy. E-mail: [email protected]

Chrysanthemum stunt viroid (CSVd), a harmful pathogen af-fecting ornamentals, is listed in the II/AII Annex of the PlantHealth Directive in the European Union (2000/29/EC). Therefore,introduction and distribution of this viroid in European Countriesis banned in chrysanthemum (Dendranthema spp.) plants, whichcan develop stunting, leaf chlorosis and floral developmental disor-ders as a consequence of infection. Although in the past CSVd hasbeen occasionally reported in chrysanthemum in Italy, its spread inthis ornamental species has efficiently been restrained so far. We



now report for the first time in Italy, the occurrence of several culti-vars of Argyranthemum frutescens, an ornamental species, infectedby CSVd. Infections were detected in symptomless plants byNorthern–blot hybridization and by RT-PCR using specificprimers, and viroid identity was conclusively ascertained bycloning and sequencing the cDNA amplicons. Molecular charac-terization of CSVd from different cultivars showed viroid popula-tions consisting of RNA molecules with a uniform size of 354 ntand nucleotide sequences identical to CSVd variants AF394452and X16408 previously reported from D. grandiflora. These resultscall for a prompt extension of surveys for assessing the presence ofCSVd in A. frutescens and in other ornamentals, which could con-stitute hidden reservoirs of this pathogen. To this aim, a tissue-printing hybridization method for detecting CSVd in A. frutescenswas tested and validated. The epidemiological risk of these find-ings for chrysanthemum crops will be discussed.

MIXED VIROID INFECTIONS OF SOLANUM JASMI-NOIDES IN ITALY. E.M. Torchetti1, B. Navarro1, V.N. Trisci-uzzi2, M.R. Silletti2 and F. Di Serio1. 1Istituto di Virologia Vege-tale del CNR, UOS Bari, Via Amendola 165/A, 70126 Bari, Italy.2Centro di Ricerca e Sperimentazione in Agricoltura “BasileCaramia”, Via Cisternino 281, 70010 Locorotondo (BA), Italy. E-mail: [email protected]

Since 2006, Potato spindle tuber viroid (PSTVd), a quarantinepathogen, has been detected in symptomless Solanum jasminoidesand other ornamental solanaceous species in Europe, causinggeneral alert on the risk of outbreaks in susceptible horticulturalcrops, such as tomato and potato. This concern increased whennatural transfer of PSTVd from S. jasminoides to tomato was doc-umented in Italy. In the last few years, similar worries were raisedin the EU by the discovery in several symptomless ornamentalsolanaceous hosts of other pospiviroid species, i.e. Tomato apicalstunt viroid (TASVd) and Citrus exocortis viroid (CEVd), both ofwhich are pathogenic to tomato and potato. We now documentthe first record of CEVd in S. jasminoides in Italy, highlighting itsvery high incidence (close to 100%) in this ornamental species.We also report the first identification of S. jasminoides plants con-currently infected by PSTVd and CEVd, and by TASVd andCEVd. These viroids were identified by RT-PCR using specificprimers followed by sequencing and Northern blot hybridization.Identification of samples with mixed infections was not alwayssuccessful using RT-PCR with generic pospiviroid primers fol-lowed by amplicon cloning and sequencing. This may depend onthe lower concentration of both viroids in mix-infected samples,as confirmed by Northern-blot hybridization. Implications ofthese findings on detection and identification method efficiencyare discussed. The molecular characterization of PSTVd frommix-infected samples revealed the presence of unreported strainslargely differing from those so far isolated from S. jasminoides.

CERINOLACTONE, A HYDROXY-LACTONE DERIVATEFROM TRICHODERMA CERINUM. F. Vinale1,4, I.A.Girona2, M. Nigro1,4, P. Mazzei3, A. Piccolo3, M. Ruocco4, S.Woo1,4, C.L. Herrera3 and M. Lorito1,4. 1Dipartimento di Arbori-coltura Botanica e Patologia Vegetale, Università degli Studi diNapoli ‘Federico II’, 80055 Portici (NA), Italy. 2Instituto de Agri-cultura Sostenible C.S.I.C., 14080 Córdoba, Spain. 3Centro Interdi-partimentale di Spettroscopia di Risonanza Magnetica Nucleare,Università degli Studi di Napoli ‘Federico II’, 80055 Portici (NA),Italy. 4Istituto per la Protezione delle Piante del CNR, 80055 Porti-ci (NA), Italy.

Trichoderma strains are among the most studied and appliedfungal BCAs in industry and agriculture and secrete several sec-ondary metabolites with different biological activity. The analysis ofmetabolic profiles (the “metabolome”) of Trichoderma species iscomplex because of the wide range of compounds produced andthe molecular activities identified, including the recently deter-mined role in the activation of plant resistance and growth promo-tion. Metabolomic studies may provide new insights on the mecha-nisms that regulate the complex interactions between plants, fun-gal phyto-pathogens and microbial antagonists of the genus Tricho-derma, thus improving the usefulness of these beneficial agents. Anovel metabolite, 3-Hydroxy-5-(6-isopropyl-3-methylene - 3,4,4a,5,6,7,8,8a-octahydro-naphthalen-2-yl)-dihydro-furan-2-one, namedcerinolactone (i), has been isolated from culture filtrates of Tricho-derma cerinum together with three known butenolides containingthe 3,4-dialkylfuran-2 (5H)-one nucleus, harzianolide (ii),T39butenolide (iii) and dehydroharzianolide (iv). The structure ofcerinolactone was determined by spectroscopic methods, includingUV, MS, and 1D and 2D NMR analyses. In vitro tests with the pu-rified compound exhibited a significant antifungal activity againstthe soil-borne fungal pathogen Rhizoctonia solani.

INVESTIGATIONS ON PENICILLIUM spp. POPULATIONDYNAMIC IN CITRUS PACKINGHOUSE. K. Youssef1,2, A.Ligorio1, S.M. Sanzani1, F. Nigro1 and A. Ippolito1. 1Dipartimen-to di Biologia e Chimica Agroforestale ed Ambientale, Universitàdegli Studi “Aldo Moro”, Via Amendola 165/A, 70126 Bari, Italy.2Agricultural Research Center, Plant Pathology Research Institute,9 Gamaa St., 12619 Giza, Egypt. E-mail: [email protected]

The most serious postharvest fungal diseases of citrus fruit aregreen and blue moulds, caused by Penicillium digitatum and P.italicum, respectively. Numerous factors related to the fruit itself,the pathogen, and the postharvest environment influence the in-cidence and severity of these diseases. Among these factors, theinoculum density of Penicillium spp. in the packinghouse envi-ronment and on citrus fruit can play a role in subsequent rot de-velopment during storage and shelf life. Population dynamics ofPenicillium spp. on clementine fruit and in the packinghouse at-mosphere were investigated considering five sampling zonesalong the packing line: fruit arrival, bins emptying, fruit washing,waxing, and calibration. Population of Penicillium spp. on thefruits fluctuated along the different zones, being significantlyhigher in “bin emptying” area as compared to the other controlpoints, with a density exceeding 2.6 log10 CFU/g of fresh fruit.The environmental mycoflora was sampled according to thegravimetric method, using Petri dishes containing potato dex-trose agar medium left open for 10 min. The highest inoculum ofPenicillium spp. spores in the packinghouse atmosphere was de-tected in the area of “bins empting”, with a density exceeding 66CFU/plate. The incidence of Penicillium rot in clementines, tak-



en from the same sampling zones along the packing line, showedan increasing trend, with values ranging from 23% (bins empty-ing) to 40% (calibration). Results are discussed in relation to thepossible epidemiological significance.

ATTEMPTS TO INCREASE THE ACTIVITY OF SALTSAGAINST POSTHARVEST CITRUS DECAY BY COMBI-NATION WITH NATURAL SUBSTANCES. K. Youssef1,2, A.Ligorio1, S.M. Sanzani1, F. Nigro1 and A. Ippolito1. 1Dipartimen-to di Biologia e Chimica Agroforestale ed Ambientale, Universitàdegli Studi “ Aldo Moro”, Via Amendola 165/A, 70126 Bari, Italy.2Agricultural Research Center, Plant Pathology Research Institute,9 Gamaa St., 12619 Giza, Egypt. E-mail: [email protected]

The control of postharvest diseases is generally exerted bychemicals fungicides. However, the issues associated with theiruse as public increasing awareness about human health and envi-ronmental risks, pathogen resistance, costs for registration andre-registration of active ingredients, etc., have motivated thesearch for new and safer alternatives. Also the use of these con-trol means have a limited practical application because of the in-consistent activity, lack of preventive effect, limited persistence,risk of fruit injury and disposal of exhausted solutions. To over-come these problems and to attain commercially acceptable per-formance, the integration of alternative means has been recom-mended. Chitosan and xanthan gum are widely used as thicken-ing agents and have been found to be useful as film-forming poly-mers. The activity of several salts (Na2CO3, NaHCO3, K2CO3,KHCO3, CaCl2, and NH4HCO3, 2%), known for their effective-ness, and natural substances (chitosan, 1% and xanthan gum,0.05%), alone or in combination, against Penicillium rots on cv.Comune clementine is reported. A significant (P≤0.05) reductionof the percentage of rotted fruits was achieved with all tested saltsand chitosan when applied alone. Na2CO3 and chitosan were themost effective, with a performance statistically not different fromthe chemical control imazalil. The activity of salts was not im-proved when applied in combination with chitosan and xanthan

gum. Results demonstrate that the combination of alternativecontrol means are not always useful and worthy to pursue.

STUDY OF THE DEFENCE MECHANISMS OFMUSKMELON TO TRACHEOFUSARIOSIS. A. ZechiniD’Aulerio, F. Piattoni, R. Roberti and G. Servidio. Dipartimentodi Produzione e Valorizzazione Agrolimentare, Università degliStudi, Viale Fanin 46, 40127 Bologna, Italy. E-mail: [email protected]

In Italy, tracheofusariosis caused by Fusarium oxysporum f.spmelonis (FOM) causes serious damages to muskmelon. Once as-sessed the reliability of certified cultivars (resistant or susceptibleto FOM (race0–ISPAVE909-; race1–ISPAVE 1296-; race2-IS-PAVE1411-) we focused on root exudates (RE), substancesthought to be involved in plant-pathogen interactions, showingno inhibition on conidial germination, independently from plantage (14 and 21 days old), pathogen race, and cultivar. Subse-quently we used RE from the most resistant (Bingo) and suscepti-ble cultivars (Retato degli Ortolani) and the most pathogenic race(2) to further investigate mycelial growth, promycelium lengthand promycelia number per conidium. Mycelial growth, analyzedfor the cultivar effect, increases at any incubation time (from 0 to72 h) with RE addition; in case of cv. Retato the effect is higher.Considering the incubation time, independently from RE age, inthe test mycelia growth enhances constantly whereas with BingoRE it stops after 20 h; cv. Retato path is similar to the test.Promicelium length increases with RE from plants of both ages,evidently for cv. Bingo; no enhancement is observed for 14-day-old cv. Retato RE. In case of macroconidia, 14 and 21-day-old REincrease promycelia number per conidium whereas for micro-conidia such effect is present only with 21-day-old RE, althoughless clear-cut. We conclude that RE contain substances able to in-terfere with mycelial growth, but their role has to be assessed.Thus, further investigations on defence mechanisms like suberinand callose production in infected root tissues from susceptiblevs resistant cultivars are necessary.



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INVESTIGATIONS ON PENICILLIUM spp. POPULATION DYNAMIC IN CITRUS PACKINGHOUSE