Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 FIRST REPORT OF PHYTOPLASMAS INFECTING LIMONIUM spp. IN ITALY.A. Abagnale, M. Barone, A. Ragozzino and D. Alioto. Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università degli Studi di Napoli Federico II, Via Università 100, 80055 Portici (NA), Italy. E-mail: ragozzin@unina.it During field surveys in 2009, symptoms of witches’ brooms, leaf reddening and reduced leaf size resembling those caused by phytoplasmas, were observed on a high percentage (50-60%) of statice plants (Limonium spp.) in several crops of Campania (Southern Italy). Phytoplasmas were detected in all symptomatic plants by PCR using the universal primers P1/P7. No amplification products were obtained from symptomless plants. The phytoplasma detected in diseased plants was identified as a member of the Aster Yellow (AY) phytoplasma group (16SrI group) (“Candidatus Phytoplasma asteris”) by PCR using the specific primers R16(I)F1/R1. This disease of Limonium has been previously reported in other countries (Iran, Israel, Canada, Japan, Poland and Germany) as the cause of devastating and pervasive damages. To our knowledge, this is the first report of the occurrence of a phytoplasma disease of Limonium spp. in Italy. IDENTIFICATION OF NEW ‘CANDIDATUS LIBERIBACTER ASIATICUS’-RELATED STRAIN BASED ON 16S rRNA AND RIBOSOMAL PROTEIN GENES. C.R. Adkar-Purushothama1, F. Quaglino1, P. Casati1, J. Gottravalli Ramanayaka2 and P.A. Bianco1. 1Dipartimento di Produzione Vegetale, Sezione di Patologia Vegetale, Università degli Studi, Via Celoria 2, 20133 Milano, Italy. 2Molecular Phytodiagnostic Laboratory, Department of Studies in Botany, University of Mysore, Mysore 570 009, Karnataka, India. E-mail: piero.bianco@unimi.it Edizioni ETS Pisa, 2009 S4.45 portant disease of red onion ‘Tropea’ in Calabria (Southern Italy). Sclerotia are the only reproductive structures of this fungus, which lacks the perfect stage and any form of spores. Sclerotia accumulate in the soil as a consequence of repeated culture, causing yield reduction and a progressive dismission of onion crop. Typically, sclerotia are regular in shape, uniformly round with a black smooth rind and a diameter varying between 0.35 and 0.50 mm. During the last years, in the traditional production area of red onion ‘Tropea’, larger sclerotia, irregular in shape, often elongated, 5 to 15 mm in size, were occasionally found around the base of the stem of onion plants in association with typical sclerotia. Surface-sterilized large sclerotia of S. cepivorum germinated on culture media and formed colonies that produced sclerotia of normal size. The S. cepivorum form with large sclerotia has been reported on onion from Egypt, New Zealand, United States and Australia as well as on artificially infected onion bulbs from Great Britain. It has been suggested that these large sclerotia are a vestige of the perfect stage of S. cepivorum ancestors. To our knowledge, this is the first report of the S. cepivorum form with large sclerotia in field crops of onion in Europe. CHARACTERIZATION OF RHIZOCTONIA SPECIES ISOLATED FROM ORNAMENTAL NURSERIES. D. Aiello1, R. Caiazzo2, A. Carella2, E. Lahoz2, R. Nicoletti2, A. Vitale1, M. Hyakumachi3 and G. Polizzi1. 1Dipartimento di Scienze e Tecnologie Fitosanitarie, Università degli Studi, Via S. Sofia 100, 95123 Catania, Italy. 2CRA, Unità di Ricerca per le Colture Alternative al Tabacco, Via P. Vitiello 106, 84018 Scafati (SA), Italy. 3 Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, 501-1193 Gifu, Japan. E-mail: gpolizzi@unict.it FIRST REPORT OF THE SCLEROTIUM CEPIVORUM FORM WITH LARGE SCLEROTIA IN EUROPE. G.E. Agosteo1 and S. Rhizoctonia isolates were recovered for over 3 years from rotting crowns and roots of potted ornamental plants grown in several nurseries in Eastern Sicily (Italy). Forty isolates were characterized by morphology, nuclear condition, anastomosis grouping and sequence homology of rDNA-ITS. Of the forty isolates, 30 had multinucleate and 10 binucleate hyphal cells. Multinucleate isolates were identified as Rhizoctonia solani AG-4 by pairing isolates with tester strains. The ITS region of rDNA of all isolates was sequenced to confirm the genetic identity. RFLP analysis was made for typing subsets within AG-4 (HGs). The fragments sequenced were compared with both the corresponding sequences of tester isolates and those available in GenBank, and restricted with three different enzymes. The majority of isolates belonged to HG-I. As to binucleate isolates, 90% of them belonged to AG-G according to molecular data and hyphal anastomosis reaction. Pathogenicity tests were made using 2 to 6 month-old potted plants of Murraya paniculata, Lagunaria patersonii, Osteospermum hybrids, Arbutus unedo, Coprosma repens, C. lucida, Citrus aurantium, Thevetia peruviana, Viburnus tinus. All R. solani AG-4 isolates were pathogenic, but the appearance of symptoms varied from 5 days to 3 months after inoculation in relation to the host and the isolate considered. One binucleate isolate belonging to AG-G caused root and stem rot on all Viburnus tinus plants after 1 month. Rhizoctonia spp. could be a limiting factor to successful cultivation of some ornamental species in nurseries. Davino2. 1Dipartimento di Gestione dei Sistemi Agrari e Forestali, Università Mediterranea, Località Feo di Vito, 89122 Reggio Calabria, Italy. 2Dipartimento di Scienze Entomologiche, Fitopatologiche, Microbiologiche, Agrarie e Zootecniche, Sezione di Patologia Vegetale e Microbiologia Agraria, Università degli Studi, Viale delle Scienze, 90128 Palermo, Italy. E-mail: geagosteo@unirc.it INCIDENCE OF FUSARIUM OXYSPORUM ON COMMERCIAL SEEDS OF DIPLOTAXIS spp., ERUCA SATIVA, LACTUCA SATIVA AND VALERIANELLA LOCUSTA. I. Alberti1, S. Tonti1, White rot is a disease of Allium spp. with a worldwide distribution, caused by Sclerotium cepivorum Berk. It is the most im- M. Dal Prà1, P. Nipoti2 and D. Pancaldi3. 1Ente Nazionale Sementi Elette, Unità Organica di Verona, Via Cà Nova Zampieri 37, 37057 San Giovanni Lupatoto (VR), Italy. 2Dipartimento di Scienze Citrus Huanglongbing (HLB) is one of the most destructive and widespread disease of citrus, caused by ‘Candidatus Liberibacter’, a non-cultured, phloem-restricted alpha-proteobacterium. In the present study, a ‘Ca. Liberibacter’ was detected in citrus growing regions of Southern India through specific PCR-based amplification of 16S rRNA and -operon ribosomal protein gene sequences. Analysis of 16S rDNA nucleotide sequences revealed that ‘Ca. Liberibacter’, here identified, shared a high sequence identity (97-99%) with ‘Ca. Liberibacter asiaticus’-related strains. Alignments of 16S rDNA and ribosomal protein gene sequences from all known ‘Ca. Liberibacter asiaticus’-related strains showed that the ‘Ca. Liberibacter asiaticus’, reported in the present study, is characterized by specific single nucleotide polymorphisms (SNPs). These genetic markers could be used for identifying and differentiating South Indian ‘Ca. Liberibacter asiaticus’-related strain by RFLP analysis. In-depth studies will be carried out for determining possible relationships between the genetic markers, here identified, and the biological niches of ‘Ca. Liberibacter asiaticus’, including alternative host plants and insect vectors. Edizioni ETS Pisa, 2009 S4.46 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 e Tecnologie Agroambientali, Università degli Studi, Viale Fanin 44, 40127 Bologna, Italy. 3Dipartimento di Protezione e Valorizzazione Agroalimentare, Università degli Studi, Viale Fanin 46, 40127 Bologna, Italy. E-mail: sezione-verona@ense.it Alimenti, Università degli Studi Federico II, Via F. Delpino 1, 80100 Napoli, Italy. 4Dipartimento di Scienze dell’Educazione, Università degli Studi di Salerno, Via Ponte don Melillo, 84084 Fisciano (SA), Italy. E-mail: lorito@unina.it The economical importance of “ready to use” fruit and vegetable products (4th range), has greatly increased in the last ten years so that, currently, Lactuca sativa, Cichorium spp., Eruca sativa, Diplotaxis spp. and Valerianella locusta represent a great deal of the vegetable market. Quality of final products can be achieved using healthy seeds and appropriate agronomic techniques. One of the most important pathogens of these species is Fusarium oxysporum. Infected plants are usually stunted, leaves turn pale green to golden yellow and later wither, die and drop off progressively upward from the stem base. Browning occur in the vascular tissues (xylem) of roots and lower stem, while roots may decay and seedlings sometimes wilt and die. Surviving seeds can act as source of primary inoculum. In the present work more than 100 commercial seed lots of Diplotaxis spp., Eruca sativa, Lactuca spp. and Valerianella locusta have been analyzed, in order to assess the incidence of F. oxysporum. Our data highlight the very low frequency of F. oxysporum infection and the presence of other Fusarium species belonging to Gibberella fujikuroi complex. Numerous microorganisms, such as bacteria and fungi, are utilized in the agro-food industry as source of enzymes and secondary metabolites. The aim of this work was to set up a reliable fermentation process in order to produce a fungal-based liquid formulation containing enzymes capable of increasing fiber digestibility (soluble fraction) in animal feeds and effectively degrade frequently associated mycotoxins such as aflatoxin B1 (AFB1) and ochratoxin A (OTA) which represent a serious problem for food safety. Different fungal strains of Trichoderma (T. atroviride P1, T. reesei T67 and T. harzianum T22) were grown for 7 or 14 days in a salt medium (SM) amended with different carbon sources. Culture filtrates were analyzed and the enzymes (cellulases, xylanases and glucanases) were characterized. The highest combined enzymatic activity was obtained with T. harzianum strain T22 grown on SM with corn silage. Animal feed treated with Trichoderma culture filtrates showed a considerable improvement of product quality (increment of soluble fiber content). The Trichoderma mixtures were also able to degrade different concentrations of AFB1 and OTA standards by 100 and 37% at 1 ppb, 63 and 46% at 10 ppb, and 28 and 31% at 100 ppb, respectively for each mycotoxin. Moreover, the mycotoxin content in corn flour contaminated by adding 100 ppb AFB1 was reduced by up to 30% after treatment with fungal culture filtrates. The enzymatic mixture was separated by gel filtration and fractions with a molecular weight of approximately 100 kDa showed the maximum capacity of mycotoxin degradation. ANTAGONISTIC ACTIVITY OF BACILLUS SUBTILIS STRAIN AG1 AGAINST FUNGAL MICROORGANISMS ASSOCIATED WITH VITIS VINIFERA. A. Alfonzo, G. Conigliaro, L. Torta, S. Burruano and G. Moschetti. Dipartimento di Scienze Entomologiche, Fitopatologiche, Microbiologiche, Agrarie e Zootecniche, Sezione di Patologia Vegetale e Microbiologia Agraria, Università degli Studi, Viale delle Scienze, 90128 Palermo, Italy. Email: antonio.alfonzo@email.it Among the antagonistic bacteria, Bacillus subtilis is well known for its activity against some fungal pathogens of the grapevine (Armillaria mellea, Botrytis cinerea, Eutypa lata and Erysiphe necator). Recent investigations, carried out on esca disease in Sicilian vineyards, revealed the occurrence of B. subtilis strain AG1 in symptomatic wood tissues (black punctuation). Moreover, by direct and indirect assays, this strain showed antagonism towards the known grapevine tracheomycotic pathogens Phaeoacremonium aleophilum and Phaeomoniella chlamydospora. In this work, the activity of AG1 against several other grapevineassociated fungi (Alternaria alternata, Aspergillus carbonarius, A. ochraceus, B. cinerea, Fomitiporia mediterranea, Fusarium oxysporum, Lasiodiplodia theobromae, Penicillium verrucosum, Phoma spp. and Verticillium dahliae) was tested. Crude extract protein (CEP), isolated from culture supernatant fluid (ammonium sulphate precipitate), was employed using agar well diffusion and critical dilution assays. The results showed a variability in the inhibitory activity of B. subtilis against the tested fungi. Further studies are required to determine both the chemical composition of the CEP and its genetic characterization. USE OF A TRICHODERMA spp. ENZYME MIXTURE TO INCREASE FEED DIGESTIBILITY AND DEGRADE MYCOTOXINS. V. Aloj1, F. Vinale1, S. Woo1, R. Marra1, M. Ruocco1, S. Lanzuise1, A. Ritieni2, G. Campanile3, F. Scala1, P. Cavallo4 and M. Lorito1. 1Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università degli Studi Federico II, Via Università 100, 80055 Portici (NA), Italy. 2Dipartimento di Scienze degli Alimenti, Università degli Studi Federico II, Via Università 100, 80055 Portici (NA), Italy. 3Dipartimento di Scienze Zootecniche ed Ispezione degli RESISTANCE TO ANTHRACNOSE DISEASE CAUSED BY GNOMONIA LEPTOSTYLA IN JUGLANS spp. N. Anselmi1, M. Annunziati1, M. Gras2 and G. Mughini2. 1Dipartimento di Protezione delle Piante, Università degli Studi della Tuscia, Via S. Camillo De Lellis, 01100 Viterbo, Italy. 2CRA, Unità di Ricerca per le Produzioni Legnose Fuori Foresta, Via Valle della Quistione 27, 00100 Roma, Italy. E-mail:ansrlmi@unitus.it. Among fast-growing Juglans species, Juglans regia is an indigenous species of Eurasia, characterised by high quality wood, J. nigra is native to North America, and J. major is close to J. nigra. Anthracnose by Gnomonia leptostyla is one of the most important diseases of J. regia. In order to select genotypes resistant to this disease, the performance of two interspecific hybrids, NG23 (J. nigra N23 x J. regia) and MJ209 (J. major x J. regia), was compared with that of the pure species J. nigra and J. regia. The correlation between resistance and genotype growth was also taken into account. During summer 2002, characterized by a significant incidence of anthracnose, the average number of necrotic spots per cm2 of leaf area and the average percentage of leaf necrotic area were recorded for each species and hybrid in a experimental field of the CRA Forest Research Unit, in Rome. Results showed that J. regia is a highly susceptible species, although a wide variability was observed among genotypes, and that J. nigra is always resistant. The interspecific hybrids showed an intermediate behaviour. In particular, the behaviour of NG23 hybrid was similar to that of J. nigra, while MJ209 behaved like J. regia. A significant correlation between disease incidence and growth ability was found only in J. regia and NG23. In the presence of heavy infection, anthracnose disease can limit the growth of the most severely attacked trees of J. regia and hybrid NG23 and, consequently, affect their timber production. Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 S4.47 APPLICATION OF MICROBIAL CONSORTIA FOR THE BIOLOGICAL CONTROL OF LETTUCE DROP. V. Antignani, G. 22, 00156 Roma, Italy. E-mail: maria.aragona@entecra.it Bonanomi, M. Capodilupo, M. Lorito and F. Scala. Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università degli Studi di Napoli Federico II, Via Università 100, 80055 Portici (NA), Italy. E-mail: vincenzo.antignani@unina.it In the last years, the incidence of diseases caused by Fusarium complex on rice had a severe increase all over the most important Italian cultivation areas. The appearance and spreading of the disease appears to be genotype- and environment-dependent. Besides phytosanitary intervention on seed batches, the most effective way to control the disease is breeding of resistant rice varieties. The search for genetic resources has been conducted on a panel of 52 rice cultivars belonging to ssp. japonica of the temperate area. Genotypes were all evaluated in the field for resistance to Fusarium complex by establishing a field plot test, using a highly susceptible variety intercropped every 4 rows as a natural “spreader” of the disease. The Fusarium species isolated with highest frequency from symptomatic plants and seeds were Fusarium fujikuroi, F. graminearum and F. proliferatum. Screening for Fusarium resistance to these species was done in the greenhouse: 10 of 52 genotypes, including 2 Italian lines, selected for the low incidence of the disease in the field, were inoculated with one isolate of each of the three Fusarium species. A differentiated pathogen attack resulted in the inhibition of germination and/or seedling development. Results of the experiments showed a significant difference in the response to the different pathogens depending on the genotype. Some genotypes showed a reproducible significant resistance to Fusarium inoculum and may be considered as potential genetic sources of resistance in breeding programmes. Italy is one of the major lettuce (Lactuca sativa) producing countries in the world with about 22.000 ha given over to this crop. The intensive cultivation has increased the incidence of soil-borne pathogens such as Sclerotinia spp., the causal agent of lettuce drop. To provide a satisfactory control of the disease, the integrated use of chemical methods and cultural and physical techniques (solarization) is currently applied. However, alternative methods to control soil-borne pathogens, mainly based on the use of biocontrol agents, are being developed. A number of antagonistic fungi, including Coniothyrium minitans, Gliocladium virens, Sporidesmium sclerotivorum, Talaromyces flavus and Trichoderma harzianum, has been tested toward different Sclerotinia species, but reliable commercial products are not yet available. In this work we evaluated the possibility to control lettuce drop by using different beneficial microbes in combination. For this purpose, we assembled microbial consortia including Pseudomonas spp., Bacillus spp., Trichoderma spp., Gliocladium spp., C. minitans and mychorrizae (Glomus spp.). Results indicate that some combination of microbes produced a double beneficial effect, by improving plant growth and significantly reducing the disease. Mechanisms involved in plant-microbe and microbe-microbe interactions are being investigated. PLANT GROWTH-PROMOTING AND ANTAGONISTIC BACTERIA FROM COMPOST-AMENDED SUBSTRATES FOR CONTROLLING FUSARIUM OXYSPORUM f. sp. MELONIS AND MONOSPORASCUS CANNONBALLUS. M. Antonelli1, C. Sebaaly1, A.M. D’Onghia2, G. Chilosi1 and L. Varvaro1. 1Dipartimento di Protezione delle Piante, Università degli Studi della Tuscia, Via S. Camillo de Lelllis, 01100 Viterbo, Italy. 2Istituto Agronomico Mediterraneo, Via Ceglie 9, 70010 Valenzano (BA), Italy. E-mail: varvaro@unitus.it In the past decades the use of compost to suppress soil-borne plant pathogens has been extensively studied, and several microorganisms have been identified in compost-amended substrates as biocontrol agents. We isolated and quantified fluorescent pseudomonads and aerobic spore-forming bacteria from some compost-based substrates for controlling Monosporascus cannonballus (MC) and Fusarium oxysporum f. sp. melonis (FOM), the casual agents of collapse and vascular wilt of melon, respectively. The same bacterial groups were also isolated from the rhizosphere and the rhizoplane of melons grown in these substrates. Representative strains of these bacteria were characterized, and were tested in vitro for their antagonism toward MC and FOM isolates and for their plant-growth promoting (PGP) activity, in terms of phosphorous solubilization, siderophore and auxin production. Our data confirm the great potential of the use of compost-based media for the application of antagonists or other beneficial microorganisms in horticulture. SCREENING FOR DISEASE RESISTANCE TO FUSARIUM COMPLEX ON RICE IN ITALY. M. Aragona1, A. Santori2, A. Infantino2, S. Cavigiolo1 and E. Lupotto1. 1CRA, Centro di Ricerca sul Riso, Strada Statale per Torino Km 2.5, 13100 Vercelli, Italy. 2CRA, Centro di Ricerca per la Patologia Vegetale, Via C.G. Bertero This work was carried out in the framework of the project MICORID, financially supported by Regione Piemonte Det.Dirig. n.4, 03.10.2007, coordinated by AGROINNOVA, University of Turin (Italy). DIVERSITY OF FUSARIUM ISOLATED FROM NONCULTIVATED SOILS OF SARDINIA. V. Balmas1, Q. Migheli1, P. Ga- rau1, S. Kang2, K. O’Donnell2 and D.M. Geiser3. 1Dipartimento di Protezione delle Piante, Unità di Ricerca Istituto Nazionale di Biostrutture e Biosistemi, Università degli Studi, Via E. De Nicola 9, 07100 Sassari, Italy. 2USDA, Agricultural Research Service, National Center for Agricultural Utilization Research (NCAUR), 1815 N. University St., Peoria, IL 61604, USA. 3Department of Plant Pathology, The Pennsylvania State University, University Park, PA 16802, USA. E-mail: balmas@uniss.it Sardinia is well known for its high levels of vascular plants diversity and endemism. We hypothesized that Fusarium species would show similar patterns. To test this hypothesis, we isolated 263 Fusaria collected from 10 diverse noncultivated field sites throughout Sardinia. Multilocus DNA sequence haplotypes were determined for each isolate using markers previously used for multilocus sequence typing (MLST). Portions of the translation elongation factor 1-alpha and second largest RNA polymerase subunit genes were sequenced in all isolates. In addition, the intergeneric spacer (IGS) region of the nuclear ribosomal RNA gene repeat was sequenced in members of the Fusarium oxysporum species complex (FOSC). For each of the members of the F. solani species complex (FSSC) a portion of the nuclear ribosomal RNA gene repeat comprising the internal transcribed spacer (ITS) and part of the large nuclear ribosomal RNA subunit was sequenced. Fifty-eight multilocus haplotypes were identified, 48 of which were from FOSC and FSSC. Thirty-seven out of 48 haplotypes of these two species complex had not been previously observed. Newly discovered haplotypes occur in one or a few sites. All FSSC isolates represented new haplotypes from phylogenetic species (FSSC 5 and FSSC 9), which differ from the phylogenetic S4.48 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 species known to dominate soils worldwide. The other species isolated were: F. polyphialidicum, F. redolens, F. culmorum, F. chlamydosporum, and five phylogenetic species in the Fusarium incarnatum-equiseti species complex (FIESC). In addition, a putative novel species was isolated. GENETIC VARIABILITY OF MEDITERRANEAN CITRUS TRISTEZA VIRUS ISOLATES. L. Barbarossa. Istituto di Virolo- None of the assayed isolates, from either olive or herbaceous hosts, was assigned to the VCG1. The pathogenicity of 108 randomly selected isolates, belonging to the different VCGs, was tested on twigs of the susceptible cv. Leccino. After three weeks incubation in the dark at 21°C, olive twigs inoculated with isolates from VCG4B and VCG2A showed significantly higher symptom severity and colonization index than VCG2B isolates. No differences were found between VCG2A and VCG4B isolates, which were similarly aggressive. gia Vegetale del CNR, Unità Organizzativa di Bari, Via Amendola 165/A, 70126 Bari, Italy. E-mail: l.barbarossa@ba.ivv.cnr.it. In the last decade Citrus tristeza virus (CTV) isolates have been reported in most of the Mediterranean countries, affecting some citrus groves or isolated trees. Although the virus often does not have an epidemic behaviour, it is a serious threat to citrus industries due to the predominance of sour orange as rootstock. CTV is usually present in field trees as a mixture or complex of isolates that produce a variety of symptoms in different citrus hosts. Studies of genetic structure and diversity are important for understanding the mechanisms of virus spread and the evolutionary factors shaping CTV populations. In this study data are presented on the sequence diversity of 30 CTV isolates from several Mediterranean countries. Genotypic profiles were determined based on amplification of multiple molecular markers. The results showed that the T30 genotype is quite common and genetically stable, associated with a low incidence and severity of field symptoms, while markers for VT and T36 genotypes identified larger CTV populations, associated with severe field symptoms, suggesting an evolution under different selection pressure. The presence of T36 genotypes (quick decline, orange stem pitting-inducing strains) confirmed that the Mediterranean is an area not particularly preserved from the absence of destructive strains. The high CTV isolate variability between and within different countries currently present in the Mediterranean region may reflect the sources of viruses introduced as infected budwood and subsequently spread by propagation and aphid vectors, but also suggests that new variants may be evolving from mixed infections. VEGETATIVE COMPATIBILITY GROUPS AND PATHOGENICITY TESTS OF VERTICILLIUM DAHLIAE ISOLATES FROM OLIVE TREES AND HERBACEOUS HOSTS. H. Barham, I. Pentimone, M. Ferrara, A. Ippolito and F. Nigro. Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. Email: nigrof@agr.uniba.it One-hundred-ten Verticillium dahliae Kleb. isolates, originating from different areas in Italy and in the Mediterranean basin, mainly from infected olive trees, were analyzed for vegetative compatibility groups (VCGs), using complementary auxotrophic nitrate-nonutilizing (nit) mutants. About 382 nit mutants were generated from 79 isolates (72%), whereas 31 isolates (28%) did not give any mutant. Among the generated mutants, a higher frequency of the nit1/nit3 type (71%) was observed, as compared to the nitM type (29%). Isolates that gave nitM mutants were tested individually against 8 international tester strains of V. dahliae, belonging to the most widespread VCGs. As to isolates from olive trees, 29% of them was assigned to the VCG2A, 7% to VCG2B, and 34% to VCG4B; whereas, 30% was not compatible with any of the 4 VCGs testers used in these trials. Instead, almost all isolates from herbaceous hosts were assigned to VCG2B (87%) and a few proved incompatible with the available testers (17%). POTATO TUBER NECROTIC RINGSPOT DISEASE: SEVERE OUTBREAKS IN CALABRIA. M. Barone1, R. Carrieri1, F. Car- damone2, A. Ragozzino1 and D. Alioto1. 1Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università degli Studi di Napoli Federico II, Via Università 100, 80055 Portici (NA), Italy. 2Collaboratore esterno. E-mail: raffaele.carrieri@unina.it In October 2008, potato tubers showing superficial and/or deep sunken necrotic rings were observed on potatoes cvs Monalisa and Vivaldi in two fields in Calabria (Southern Italy). The disease interested an area of 2 ha and its incidence varied from 20-30% (cv. Monalisa) to 40-50% (cv. Vivaldi). Symptoms resembled those of potato tuber necrotic ringspot, a severe disease with worldwide distribution, causing economical losses to processing and fresh market potatoes. The disease elicited by the tobacco veinal strain of Potato virus Y (PVYN), sub-group tuber necrosis (PVYNTN) had already been reported in several areas of northern Italy in 1998. Symptomatic and symptomless potato tubers of the two cultivars were analysed for the presence of PVY by sap inoculation, DAS-ELISA using a commercial kit (Loewe Biochemica, Germany) and by RT-PCR using the universal primers for the genus Potyvirus, M4T/M4/S1. Positive samples were further tested to discriminate PVY strains by RT-PCR using the specific primers, PVYc3/f, PVY3+/3- and CP2+/1-. PRELIMINARY RESULTS ON FUSARIUM CULMORUM-SOFT WHEAT INTERACTIONS IN STEM BASES AND ROOTS. G. Beccari1, L. Covarelli1, M.A. Thomsett2 and P. Nicholson2. 1Dipartimento di Scienze Agrarie e Ambientali, Università degli Studi, Borgo XX Giugno 74, 06121 Perugia, Italy. 2Department of Disease and Stress Biology, John Innes Centre, Colney Lane, NR4 7UH Norwich, UK. E-mail: lorenzo.covarelli@unipg.it An isolate of F. culmorum expressing the green fluorescent protein (GFP) was used to examine the infection process and host response during the early stages of the infection of the stem base and roots of wheat (cv. Genio). Following inoculation of seedlings, disease progress in leaf sheaths and roots was assessed macroscopically by visual symptom observations, microscopically by confocal laser scanning microscopy (CLSM) and by gene expression analysis of fungal and wheat genes by real time RT-PCR. The fungus sequentially penetrated though the first, second and third basal leaf sheaths and produced trichothecenes (expression of TRI5) very early in infection. Expression of TRI5 decreased later in colonisation. CLSM observations revealed a host response in uninfected areas on the internal leaf sheaths in advance of fungal infection. Differential expression of the wheat genes Wheatwin 1-2, PR1, Chitinase, PAL, LOXC, WIR1 and OPR1 was recorded in the stem base tissues depending on infection stage and leaf position. Interestingly, most of these genes were expressed in tissues ahead of fungal colonisation. In the roots, F. culmorum consistently expressed TRI5 (24, 48 and 96 h post-inoculation). CLSM investigations showed an initial intercellular Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 and a subsequent intracellular colonisation by fungal hyphae, epidermal layer and cortex invasion and the absence of the fungus in the stele. Expression of the host defence-associated genes increased early in infection and decreased in later stages. FURTHER SPREAD OF ALTERNARIA ALTERNATA BROWN SPOT ON TANGERINES AND THEIR HYBRIDS IN ITALY. P. Bella1, G. Ialacci1, A. Catara1,2 and V. Catara1. 1Dipartimento di Scienze e Tecnologie Fitosanitarie, Università degli Studi, Via Santa Sofia 100, 95123 Catania, Italy. 2Parco Scientifico e Tecnologico della Sicilia, Zona Industriale, Stradale G. Agnelli angolo V. Lancia, 95030 Catania, Italy. E-mail: patrizia.bella@unict.it. The introduction of new citrus cultivars and hybrids had an impact on the occurrence of emerging diseases in Italy. Among them, Alternaria brown spot is a well known limiting factor for production of tangerine and its hybrids worldwide. The causal agent of the disease is the “tangerine pathotype” of Alternaria alternata, that produces the host-specific ACT-toxin, involved in symptom expression. Infected young fruits and leaves often fall and mature fruits are unmarketable due to the lesions. In Italy this disease has been observed since 2000 on ‘Fortune’ mandarin. Surveys periodically carried out revealed severe outbreaks especially in humid areas and in the years characterized by favourable climatic condition such as extended wet periods. Symptoms include necrotic lesions on the leaves, twigs and fruits, surrounded by a yellow halo in young leaves. During the survey, fruit losses of up to 100% were recorded in ‘Fortune’ mandarin. The disease was also found with a lower incidence on the hybrids ‘Nova’ and ‘Winola’ and on ‘Valencia’ orange. Sixty single-spore fungal isolates, obtained from leaves and fruits collected in Sicily and Calabria (Southern Italy), were identified as A. alternata based on the morphological characteristics of conidia. Inoculations of young detached leaves of ‘Dancy’ tangerine and ‘Fortune’, reproduced the typical symptoms of the disease. No symptoms were observed in control leaves inoculated with sterile distilled water. Identification of the fungus was confirmed by PCR using speciesspecific primers designed on the AKT1 gene sequence of the Japanese pear pathotype of A. alternata and by partial endopolygalacturonase sequencing. FIRST REPORT OF ALFALFA MOSAIC VIRUS INFECTING LAVANDULA STOECHAS. M.G. Bellardi1, L. Cavicchi2, G. Boz- zano3 and G. Parrella4. 1Dipartimento di Scienze e Tecnologie Agroambientali, Area di Patologia Vegetale, Università degli Studi, Via G. Fanin 42, 40127 Bologna, Italy. 2Facoltà di Agraria, Università degli Studi di Bologna, Plesso Didattico G. Scarabelli, Viale G. Ascari 15, 40026 Imola (BO), Italy. 3Società Cooperativa L’Ortofrutticola, Via Dalmazia 169, 17031 Albenga (SV), Italy. 4Istituto per la Protezione delle Piante del CNR, Unità Organizzativa di Napoli, Via Università 133, 80055 Portici (NA), Italy. E-mail: mariagrazia.bellardi@unibo.it Lavandula stoechas L. (family Lamiaceae) also known as French lavender or Spanish lavender, is an attractive perennial shrub with pinkish-purple flowers produced on spikes at the top of slender leafless stems. Actually, this ornamental is one of the most important Lavandula species grown in the Albenga area (Liguria, Northern Italy). In 2008-2009, some L. stoechas potted plants were found to be naturally infected by Alfalfa mosaic virus (AMV). Symptoms consisted of a yellow mottling that appeared first on the oldest leaves. Flowering was apparently normal. AMV was first identified by PAS-ELISA in diseased plants. A few S4.49 herbaceous hosts were manually inoculated with sap expressed from symptomatic tissues but only Ocimum basilicum (cv. Gigante) developed necrotic line-patterns followed by a bright systemic yellow mosaic. Symptomatic basil was used as starting material for further mechanical transmissions to Vigna unguiculata ‘Black eye’, Vicia faba and Phaseolus spp. (local brown necrotic lesions), Chenopodium quinoa and C. amaranticolor (systemic chlorotic mosaic and malformations). Restriction profile obtained after RT-PCR-RFLP with BamHI endonuclease of a 750 bp fragment, comprising the viral coat protein gene, revealed that the L. stoechas isolate of AMV belongs to soubgroup I. This study confirms the spreading of AMV subgroup I in other “new” ornamental Lavandula (true species or hybrids) hosts, as recently reported for Lavandula x allardii. DIFFERENT PHYTOPLASMAS INFECTING PURPLE CONEFLOWER IN ITALY. A. Bertaccini, S. Paltrinieri, N. Contaldo, B. Duduk, S. Nahdi, A. Benni and M.G. Bellardi. Dipartimento di Scienze e Tecnologie Agroambientali, Area di Patologia Vegetale, Università degli Studi, Viale Fanin 42, 40127 Bologna, Italy. Email: bertaccini_a@cib.unibo.it Echinacea purpurea (L.) Moench. (family Asteraceae), purple coneflower, is an ornamental species widely cultivated as medicinal plant for its nonspecific immunostimolatory and wound healing properties. Symptoms, including stunting, virescence, phyllody and abnormal flower bud proliferation, have been described in the USA, Canada and Europe, with which different phytoplasmas belonging to diverse subgroups (16SrI-A, 16SrI-B and 16SrIC) in the ‘Candidatus Phytoplasma asteris’ group, are associated. During surveys carried out in May-June 2008, symptoms possibly related to phytoplasma presence were observed in two areas of Emilia-Romagna (Northern Italy). Almost 50% of purple coneflowers obtained by seeds and grown in the open field in the Herb Garden of Casola Valsenio (Ravenna province), showed virescence and stunting while in the herb garden of the Faculty of Agriculture of Bologna severe symptoms including phyllody, proliferation of axillary shots and flower abnormalities were observed in one plant out of ten. Based on RFLP analyses of the 16S rDNA PCR products, phytoplasmas belonging to subgroups 16SrI-B and 16SrIX-C were identified in Casola Valsenio, but not in mixed infection. In samples from Bologna only 16SrIX-C phytoplasmas were identified. This is the first report of these phytoplasmas in purple coneflower. 16SrIX-C phytoplasmas were also identified in symptomless samples of Taraxacum and Amaranthus collected in vineyards in Modena province, while in malformed Amaranthus samples collected near infected purple coneflower plants the 16SrIX phytoplasmas detected differed from 16SrIX-C for a TaqI restriction profile. Further phytoplasma characterization to verify their epidemiology is in progress. ELECTRONIC NOSE AS INNOVATIVE TOOL FOR THE DIAGNOSIS OF CROWN GALL TUMOURS. E. Biondi, S. Blasi- oli, I. Braschi, C.E. Gessa, U. Mazzucchi and C. Bazzi. Dipartimento di Scienze e Tecnologie Agroambientali, Area di Patologia Vegetale, Università degli Studi, Viale Fanin 44, 40127 Bologna, Italy. E-mail: erbiondi@tin.it The electronic nose (E-nose) is an innovative device developed for microbiological, food/drink and environmental safety, and medical applications. E-nose comprises appropriate sensors, able to detect and discriminate several volatile organic compounds from different sources. In the last decade, E-nose has S4.50 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 been applied to plant disease diagnosis. In grapevines, tumourigenic strains of Agrobacterium vitis cause crown gall disease which may give serious problems in nurseries and vineyards. For the first time, a portable E-nose (PEN3) was used to discriminate galled from healthy vines, experimentally inoculated with two different strains of A. vitis and water, respectively. All data were processed with principal component analysis (PCA) and linear discriminant analysis (LDA). PCA was performed to provide a bidimensional visualization of the data. A discrete separation of the samples into two groups was found, according to the presence or absence of disease symptoms. Moreover, the LDA model allowed a correct classification of healthy and galled vines with a percentage of 71.4%. Tumours caused by the two strains of the pathogen were not discriminated. Although a larger number of grapevine samples need to be analyzed to create a more robust model, our results provide important information on the diagnostic potential of this innovative system by applying multi-dimensional chemometric techniques. INTENSIVE SOIL CULTIVATION IMPAIRS SOIL FERTILITY AND AFFECTS RHIZOCTONIA DISEASE SUPPRESSION. G. Bonanomi1, V. Antignani1, M. Capodilupo1, L. Cozzolino1, R. D’Ascoli3, R. Marzioli3, G. Puopolo1, R. Scotti2, L. Gianfreda2, M.A. Rao2 and A. Zoina1. 1Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università degli Studi di Napoli Federico II, Via Università 100, Portici 80055 (NA), Italy. 2Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali, Università degli Studi di Napoli Federico II, Via Università 100, Portici 80055 (NA), Italy. 3Dipartimento di Scienza Ambientali, Seconda Università di Napoli, Via Vivaldi 43, 81100 Caserta, Italy. E-mail: giuliano.bonanomi@unina.it In the last decades a decrease of soil fertility has been observed as a consequence of irrigation- induced salinity, heavy metal pollution, reduction of soil organic carbon and loss of natural soil suppressiveness. In this multidisciplinary study we compared soil properties of 25 commercial farms selected in Campania (Southern Italy) classified either as intensive (IMR, i.e. protected crops under plastic cover) and extensive (EMR, i.e. tree orchards) management regime. In each farm, 28 properties were studied including physico-chemical (bulk density, field capacity and texture, organic C, N, P, K, Ca, Mg, Na, EC, CSC, limestone), enzymatic (arylsulfatase, -glucosidase, dehydrogenase, phosphatase, urease), microbiological (BiologTM; potential respiration; microbial biomass; fungal mycelium; cultivable actinomycets; bacteria; pseudomonads; fungi and bacterial diversity assessed by DGGE fingerprinting of 16S-rRNA sequences) and a bioassay aimed at evaluating the soil suppressiveness on the Rhizoctonia solani-Lactuca sativa pathosystem. IMR cultivation negatively affected chemical, biochemical and biological fertility. Reduction of soil organic C, microbial biomass, population and diversity of the microbial communities and a marked decrease of soil enzymatic activities were recorded. Suppression of R. solani was not influenced by EMR and IMR, but significant differences were observed among farms. None of the measured parameters alone was able to correctly predict R. solani suppression, except soil EC that showed a strong positive correlation. These results suggest a high sensitivity of R. solani to soil EC, but only in the presence of a competitive native microbial community. INTERACTIONS BETWEEN HETEROBASIDION ABIETINUM AND ABIES ALBA IN SOUTHERN ITALY. E. Boncaldo, G. Sicoli and G. Bruno. Dipartimento di Biologia e Patologia Ve- getale, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. E-mail: e.boncaldo@libero.it In Southern Italy, Heterobasidion abietinum Niemelä et Korhonen (Hab) is a widespread necrothophic fungus in natural and artificial stands of silver-fir (Abies alba Mill.) where it can cause root and butt rot in single or in groups of trees. Inoculation were carried out with 18 H. abietinum isolates collected from stumps and seedlings of nine different silver fir woods from Apulia, Calabria, Campania and Basilicata (Southern Italy). Three-year-old seedlings (obtained from seeds collected in a silver-fir stand in the Pollino mountains) were inoculated at the collar with a block of silver fir wood colonized by each Hab strain. The block was attached to the bark after or without wounding the seedling. Fourmonth-old silver fir seedlings [obtained from seeds collected in Serra San Bruno (Vibo Valentia province), “Pollino” and Laurenzana (Potenza province) forests] were inoculated by dipping roots and stem into a mycelial and conidial suspension. Seedlings were maintained in test tubes with a Murashige and Skoog basal salt mixture solution. Differences among strains were recorded as symptoms on leaves and woody tissues following fungal invasion and decay. Hab strains from Serra San Bruno induced a mild infection of root tissues and did not cause rot on a number of seedlings. A white decay was generally observed five months after inoculation. A NEW AGENT OF WOOD ALTERATION OF POPULUS NIGRA. L. Bosso1, G. Carleo2, G. Cristinzio1, A. Testa1, A.P. Garonna2 and G. Viggiani2. 1Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università degli Studi di Napoli Federico II, Via Università 100, 80055 Portici (NA), Italy. 2Dipartimento di Entomologia e Zoologia Agraria “Filippo Silvestri”, Università degli Studi di Napoli Federico II, Via Università 100, 80055 Portici (NA), Italy. E-mail: cristinz@unina.it Megaplatypus mutatus, an exotic insect introduced from the neotropical region, is an emerging pest in Southern Italy. The platypodid beetle is a xylomicetophagous species (= Ambrosia beetle). It may cause dramatic damage to poplar (Populus spp) plantations. In a P. nigra stand infested by M. mutatus in Campania (Southern Italy) a fungus responsible of a chromatic wood alteration was observed. Wall tissues were sampled from M. mutatus galleries coated with a firm mycelium. Several fungal colonies with a red mycelium and a strong coloration on PDA were isolated. All the red fungal strains belonged to the genus Verticillium. This fungus is a physical contaminant of the beetle and is massively present in the brood galleries. To our knowledge, this is a new association the possible role of which is unknown. A typical symbiontic species recorded for M. mutatus is an ascomycota of the genus Raffaelea. Differences in environmental conditions and ecological interactions may explain the presence of the Verticillium. Biological investigations for characterizing the beetle-fungus association are in progress. OCCURRENCE OF GRAFT INCOMPATIBILITY IN YOUNG VINEYARDS IN TUSCANY. H. Bouyahia, A. Materazzi and E. Triolo. Dipartimento di Coltivazione e Difesa delle Specie Legnose “G. Scaramuzzi”, Sezione di Patologia Vegetale, Università degli Studi, Via del Borghetto 80, 56124 Pisa, Italy. E-mail: hbouyahia@agr.unipi.it In 2006, symptoms of graft incompatibility and decline were observed in newly planted commercial vineyards in Civitella in Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 Val di Chiana (Tuscany, Central Italy). Affected grapevines cv. Marselan clone ENTAV-INRA 980 grafted on 110 Richter showed leaf reddening, swelling and cracking at the graft-union. Since the first year, many plants showed stunting and an early reddening of the leaves followed by scorching symptoms at the end of summer and, ultimately, by death. Foliar symptoms were associated with local excessive tissue growth at the graft union, with internal necrosis and incomplete connection between the conductive elements of the rootstock and scion. With the objective of studying the cause of the symptoms described, mature canes from 50 survived plants were sampled and analysed by ELISA for the presence of 9 viruses associated with major detrimental diseases: Grapevine virus A (GVA); Grapevine virus B (GVB); Grapevine leafroll-associated virus 1 (GLRaV-1), -2 (GLRaV-2), -3 (GLRaV-3) and -7 (GLRaV-7); Grapevine fleck virus (GFkV); Arabis mosaic virus (ArMV); Grapevine fanleaf virus (GFLV). None of the above viruses were detected, excluding their association with these symptoms. Successively the same samples were tested by RT-PCR for the presence of Grapevine rupestris stem pitting-associated virus (GRSPaV) and, interestingly, a high incidence of this virus was found (74%). Obviously, our data are not sufficient to put forward theories on the aetiology of declining cv. Marselan, even more since GRSPaV is known to affect grapevines with high incidence worldwide. Nevertheless, it is important to underline that GRSPaV was suspected to be associated with Syrah decline, where similar symptoms were observed on plants grafted on 110R sensitive rootstock. EPIDEMIOLOGICAL STUDY ON VERTICILLIUM WILT OF OLIVE IN SOUTHERN ITALY. G. Bubici1, F. Nigro2, M. Fer- rara2 and M. Cirulli1. 1Dipartimento di Biologia e Patologia Vegetale, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. 2Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. E-mail: giovabubi@email.it In a two-year study, the progress of Verticillium wilt symptoms, the frequency of Verticillium dahliae re-isolation from olive tissues, and the soil inoculum density were monitored in a commercial olive grove near Cerignola (Apulia, Southern Italy) consisting of 600 trees of the highly susceptible cv. Bella di Cerignola. Observations were made monthly in February, May, August and November 2008 and in February and May 2009. External disease symptoms were scored using a 0-8 empirical scale. At each observation time, a symptomatic branch was sampled from each diseased tree. Presence of V. dahliae was ascertained by conventional agar-plating method and real-time PCR, and in the soil by dryplating, wet-plating and real-time PCR. Verticillium wilt incidence increased from 3% at the beginning to 10% at the end of the investigation. Based on agar-plating, V. dahliae re-isolation frequency was higher (73%) in May than in the other months (15%), and lower in 1-year-old (13%) than in the older tissues (36%). Results of the real-time PCR assays agreed with those of the conventional detection methods, but were less erratic. V. dahliae re-isolation from branches was variably successful, while from leaf petioles was successful just in one out of 5,865 chips plated, and failed from flower petioles and seeds. Variations over time of soil inoculum density were not observed, probably because of the very low concentration (0.1-0.7 microsclerotia g-1 of soil). Real-time PCR and wet-plating were more sensitive than dry-plating. LOCALIZATION OF PANTOEA AGGLOMERANS IN GRAPEVINE TISSUES BY FLUORESCENCE IN SITU HY- S4.51 BRIDIZATION (FISH). D. Bulgari1, P. Casati1, F. Quaglino1, P.A. Bianco1,2, M. Iriti1 and F. Faoro1,2. 1Dipartimento di Produzione Vegetale, Università degli Studi, Via Celoria 2, 20133 Milano, Italy. 2Dipartimento di Biologia, Università degli Studi, Via Celoria 2, 20133 Milano, Italy. 3Istituto di Virologia Vegetale del CNR, Unità Organizzativa di Milano, Via Celoria 2, 20133 Milano, Italy. E-mail: franco.faoro@unimi.it Endophytes are ubiquitous endosymbionts found in all plant species studied so far. Alhough their relationship with the host is in most cases not well understood, they may benefit plants by preventing pathogen colonization through different mechanisms: competition, production of allelochemicals and induction of systemic resistance (ISR). Recently, the population of bacterial endophytes associated with grapevine leaf tissues was analyzed by both cultivation and cultivation-independent methods. Isolation of bacteria by culturing revealed the presence of members of the genera Curtobacterium, Bacillus and Enterococcus. Sequence analysis of 16S rRNA gene libraries underscored a dominance of the genus Pantoea. Previous studies emphasized the potential of P. agglomerans as a biocontrol agent against a range of plant pathogens through the secretion of antibacterial molecules and/or by the activation of plant induced systemic resistance (ISR). In this study P. agglomerans distribution in grapevine leaf tissues was assessed by fluorescence in situ hybridization (FISH). For this purpose, a probe to target bacterial 16S rDNA was labelled with a fluorophore emitting in the far-red (i.e. CY5) to avoid interference with autofluorescence of the abundant phenolic compounds present in grapevine tissues. Vibratome cut slides of leaf midribs, 70-80 µm thick, were incubated with the probe and examined with a confocal microscope. Putative endophytes were detected mainly in phloem and xylem parenchymas. The possibility of localizing endophytic bacteria inside grapevine tissues represents a valuable aid in studying their interaction with grapevine pathogens, particularly viruses and phytoplasmas. FIRST OBSERVATIONS ON THE INTERACTION BETWEEN LASIODIPLODIA THEOBROMAE AND EPICOCCUM PURPURASCENS, TWO ENDOPHYTES IN GRAPEVINE BUDS. S. Burruano, A. Alfonzo, G. Conigliaro, V. Mondello and L. Torta. Dipartimento di Scienze Entomologiche, Fitopatologiche, Microbiologiche, Agrarie e Zootecniche, Sezione di Patologia Vegetale e Microbiologia Agraria, Università degli Studi, Viale delle Scienze, 90128 Palermo, Italy. E-mail: santella@unipa.it Among the several species belonging to the genus Botryosphaeria occurring in the grapevine, Lasiodiplodia theobromae, the teleomorphic stage of Botryosphaeria rhodina, is the only one which was recently reported from Sicily. This fungus is associated with reduced bud burst or bud death, retarded growth, spur dieback and wood necrosis. Since the epidemiology of Botryosphaeria diseases of grapevines is little known, observations were carried out to ascertain the possible overwintering of L. theobromae in the buds. Samples of non-sprouting buds, collected during grape sprouting, were sectioned in half and divided in two groups. One group was immediately used for isolations on a solid medium, while the other was subjected to a clearing and staining procedure to detect the pathogen inside bud tissues by microscopic observations. From isolation tests many colonies of L. theobromae and a few of Epicoccum purpurascens were obtained. When both species were isolated from the same sample, the growth of L. theobromae was limited. Microscopic observations, detecting mycelial structures of the two fungi, allowed to confirm their antagonistic interaction. In fact, E. purpurascens, which was usually localized in the veins, could grow between host S4.52 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 cells, reaching the intercellular hyphae of L. theobrome, surrounding and necrotizing them. This is the first report on the overwintering of L. theobromae in grape buds and on the antagonistic activity (in vitro and in vivo) of the endophyte E. purpurascens against the pathogen. Studies on the development of both endophytes are in progress. TEMPERATURE EFFECT ON THE FORMATION AND MATURATION DYNAMICS OF ERYSIPHE NECATOR CLEISTOTHECIA. T. Caffi, V. Rossi and S.E. Legler. Istituto di Ento- mologia e Patologia Vegetale, Università Cattolica del Sacro Cuore, Via Emilia Parmense 84, 29100 Piacenza, Italy. E-mail: tito.caffi@unicatt.it Erysiphe necator is the causal agent of grapevine powdery mildew. In many areas its primary inoculum consists of ascospores produced in cleistothecia, which develop on the affected tissues and are dispersed by rain splashes to the bark, where they overwinter. The fungus is heterotallic, i.e., fruiting bodies develop when two opposite mating types combine. Once initiated, the cleistothecia reach maturity in three stages: initially they are white, then turn yellow, then brown, and finally, at maturity, become black. Temperature affects the growth of cleistothecia but data on the relationship between temperature and their maturation are not available to date. Grapevine cuttings of cv. Barbera were grown under controlled conditions until 5-6 leaves were completely unfolded. Leaves were inoculated with fresh conidia from naturally infected vineyards. When the first white globular cleistothecium was observed, cuttings were separated and placed at 10 to 30°C with high humidity and a 12 h photoperiod. Cleistothecia formed on the leaves were counted and classified as yellow, brown, or black every second day for 5 weeks. The experiment was done twice. Cleistothecia formed at each temperature tested but did not reach maturity at 10 or 30°C. The maximum density of cleistothecia occurred at 15°C but the most rapid maturation occurred at 20°C. At this temperature, the first yellow cleistothecium appeared after 4 days and the first black cleistothecium after 11 days. These results were used for designing a model that predicts production and maturation of E. necator cleistothecia. tiorum on Brassica oleracea L. var. acephala. Genetic analyses, performed on isolates collected from different crops, showed that S. Slerotiorum isolates are homogenous with limited variability in the sequences of ITS region (identity of sequence not less than 98%). Both isozyme and molecular analysis showed intra-specific variations among the isolates tested. The variability observed with molecular and biochemical techniques wasn’t related neither with morphological features nor with the specific host. Differences in virulence were also evaluated in relation to the variability observed. PRELIMINARY RESULTS OF BIOCONTROL AGENT AGAINST FUSARIUM OXYSPORUM WILT OF LENTIL LANDRACE “USTICA”. V. Campanella and C. Miceli. Ente Nazionale Sementi Elette, Viale Regione Siciliana S-E 8669, 90121 Palermo, Italy. E-mail:sezione-palermo@ense.it “Ustica” landrace is a microsperma dark-brown lentil, grown mainly in organic crops of Ustica, an island off the Northern coast of Sicily (Southern Italy). In this work, the preliminary results of treated “Ustica” seeds with Streptomyces griseoviridis K61 (Mycostop) are reported. Trials were conducted in 2006-2007 in the island, in a randomized block design with three replication of 30 m2 each. Seed treatment was performed before sowing. Cultivation technique was the same normally used in the cropping area. The disease was monitored during all growth stages and, after harvest, seeds were analysed. Data showed an increase in the number of plant per m2 (13%) and in the yield (18.6%), compared to the control. Both treated and untreated plants, analysed at the flowering stage, showed the presence of Fusarium oxysporum in high percentages, 60% and 75%, respectively. The pathogen, however, was not detected in any of the seed lots analysed. Thus, S. griseoviridis seems to improve the productive performance of “Ustica” lentil but does not seem to act directly on F. oxysporum. Further research is in progress to understand the mode of action of the antagonist. DURUM WHEAT ROOT AND FOOT ROT COMPLEX IN SICILY. V. Campanella and C. Miceli. Ente Nazionale Sementi Elette, Viale Regione Siciliana S-E 8669, 90121 Palermo, Italy. Email:sezione-palermo@ense.it GENETIC CHARACTERIZATION OF SCLEROTINIA SCLEROTIORUM ISOLATES FROM SEVERAL CROPS IN CAMPANIA. R. Caiazzo, A. Carella, F. Raimo and E. Lahoz. CRA, Cen- tro di Ricerca in Agricoltura, Via Vitiello 108, 84018 Scafati (SA), Italy. E-mail: ernesto.lahoz@entecra.it Sclerotinia sclerotiorum (Lib.) De Bary is one of the most devastating and cosmopolitan plant pathogen. The fungus infects over 400 species of plants worldwide including important crops and numerous weeds. In 2008 for the first time damages were observed on “torzella” (Brassica oleracea var. acephala) a typical crop of Campania (Southern Italy). Other crops such as lettuce, curly endive and cauliflower were also affected by this fungus, suffering heavy yield losses. In the present work a study on the intraspecific variability of 30 isolates of S. sclerotiorum collected in Campania from different crops was carried out by means of molecular and biochemical analysis. Genomic DNA was investigated by random amplified polymorphic DNA (RAPD) markers, amplification with a chromosomal telomere sequence-based primer, PCR analysis and RFLP analysis of the sequence of the ITS15.8S-ITS2 ribosomal subunit regions, and electrophoretic analysis of polygalacturonase isozymes. This is the first report of S. sclero- The recurrence of root and foot rot complex on durum wheat crops in Sicily (Southern Italy) was investigated. The survey was conducted on durum wheat plants at the 30 Zadoks scale in three different districts (Enna, Palermo and Trapani), during 20082009. Samples from each area were thoroughly rinsed under tap water, surface-sterilised by dipping in 3% sodium hypochlorite (NaOCl), thoroughly rinsed in distilled water and dried on paper towels. Small pieces of tissue were plated on PDA (potato dextrose agar, 37g/l) amended with streptomycin sulphate (1g/l), and neomycin sulphate (0.12g/l). Fungal identification was based on morphological characters of the isolates on tissues and on slides. Isolates of Fusarium species were sub-cultured using the single spore method and identified by morphological characteristics. Bipolaris sorokiniana (30%), F. acuminatum (10%), Rhizoctonia cerealis (10%) in the root and F. avenaceum (30%) in the stem were the predominant species found in Enna. B. sorokiniana (20%), F. culmorum (40%) in the root, F. acuminatum (20%) both in root and stem and F. avenaceum (10%) and F. crookwellense (10%) only in the stem, were the species found in Palermo. Microdochium nivale (40%), F. acuminatum (10%), B. sorokiniana (10%) in the root and F. crookwellense (10%) in the Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 stem were the species found in Trapani. Our results show how the wrong cultural practices (e.g. use of untreated seed or monogrowing) and the favourable whether conditions, that prevailed during the season, may determine the damages observed on durum wheat crops in Sicily. S4.53 ried out using both purified isolates and the reference strain PD 3389, showed that Pectobacterium carotovorum subsp. carotovorum Hauben et al. was the causal agent of the observed disease. The pathogen identity was confirmed by Biolog system. In addition, isolations made on CVP from soil of infected lettuce plots gave typical colonies of the pathogen, while isolations made from irrigation water and from soil of healthy plots from the same farm, did not produce any typical colony. USE OF ESSENTIAL OILS OF THYME AND OREGANO AGAINST PHYTO-PATHOGENIC FUNGI AND BACTERIA. F. Campanile, D. Villecco and M. Zaccardelli. CRA, Centro di Ricerca per l’Orticoltura, Azienda e Laboratori di Battipaglia, Strada Statale 18 n° 204, 84091 Battipaglia (SA), Italy. E-mail: francitech@libero.it Oregano (Origanum vulgare) and thyme (Thymus vulgare), family Lamiaceae, are aromatic plants widely used both as food source and for phytotherapy. Moreover, they can be potentially used as biocide, because their essential oils are toxic for phytopathogenic fungi and bacteria. The composition of essential oils is subjected to variations of the active compounds, partially due to the use of heterogeneous populations of the source plants and to environmental conditions. Aim of this work was to determine the effectiveness on the growth of phytophatogenic fungi and bacteria of essential oils extracted from oregano and thyme plants grown in a climatic chamber, in a substrate amended with different fertilizers (mineral, Brassica carinata seed meal, sunflower seed meal and compost). Antimicrobial activity of essential oils were measured by determining growth inhibition of fungi and bacteria at different concentrations. Oregano oil was more active than thyme oil, at both the highest (0.1%) and lowest concentrations (0.01%). However, with both oils, bacteria were more inhibited than fungi. The greatest inhibition occurred with oils form oregano and thyme fertilized with compost. In this case, oregano oil at 0.01% concentration inhibited all bacteria tested except for Pectobacterium carotovorum and Pseudomonas syringae pv. tomato, whereas among fungi, only Verticillium spp. was inhibited. In conclusion, different fertilizers influenced the antimicrobial activity of oregano and thyme oils. Fertilization with compost showed the highest activity. Studies in open fields will be performed. PECTOBACTERIUM CAROTOVORUM subsp. CAROTOVORUM AS THE CAUSAL AGENT OF LETTUCE PITH SOFT ROT IN APULIA C. Cariddi and A. Ippolito. Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. E-mail: corrado.cariddi@agr.uniba.it At the beginning of autumn 2008, a severe outbreak of pith soft rot occurred on lettuce plants, cv. Iceberg, in fields located in province of Bari (Apulia, Southern Italy), previously cultivated four year in a row with lettuce and potato. First symptoms of infection, observed approximately 30 days after transplanting, consisted of leaf chlorosis and wilting of older leaves, followed by death of the plants. Peculiar symptoms were observed in the stem pith, which showed at first a water soaked, pink-brownish discoloration, then it rotted and developed a greenish-dark colour. During the observations carried out in mid-October, in two contiguous crops, disease incidence was higher (approximately 90%) in transplants made in mid-August than in transplants made ten days later (about 40%) when the temperature had dropped. Isolations from rotted pith on the semi-selective crystal violet pectate (CVP) medium, after 48 h at 27°C, yielded only Pectobacterium species. Biochemical, nutritional and pathological tests, car- DIPLOCARPON MESPILI, A NEW PATHOGEN OF QUINCE IN APULIA. C. Cariddi, A. Ippolito and C. Vovlas. Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. E-mail: corrado.cariddi@agr.uniba.it For the past four years, symptoms were observed on quince plants (Cydonia oblonga Mill.), from late spring to early summer in various cool and moist areas of Apulia (Southern Italy). Circular reddish-brown spots, 1-4 mm in diameter, were scattered on the upper leaf surface, which eventually coalesced producing large necrotic areas. The leaves turned yellow or reddish and fell prematurely. Fruits displayed rounded black spots, 1 to 5 mm in diameter, which became slightly depressed with time. In autumn, subcuticular black acervuli, present in the centre of the spots on leaves and fruits, oozed conidia in a creamy-white mass during wet weather. Conidia were hyaline, cruciform, 4-celled, with the lateral cells smaller than the two central ones. All cells, except for the apical one, had a bristle-like appendage. The symptoms and the unusual morphology of conidia identified the causal agent as Entomosporium mespili (DC.) Sacc. (syn. Entomosporium maculatum Lév.) teleomorph Diplocarpon mespili (Sorauer) Sutton [(syn. Fabraea maculata (Lév.) Atk]. E. mespili grows very slowly in culture, being thiamine-deficient, therefore its isolation and growth on common media is rather difficult. The fungus was more easily isolated from leaf than fruit spots. Isolation and conidia germination trials on different media, showed that the most suitable was glucose casein agar plus 1 µg/ml thiamineHCl, whereas, maximum sporulation and growth occurred on sucrose casein medium plus 1 µg/ml thiamine-HCl under appropriate incubation conditions. RECENT ADVANCES IN STUDIES ON CHERRY CHLOROTIC RUSTY SPOT DISEASE. R. Carrieri, M. Barone, A. Abagnale and D. Alioto. Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università degli Studi di Napoli Federico II, Via Università 100, 80055 Portici (NA), Italy. E-mail: raffaele.carrieri@ unina.it Cherry chlorotic rusty spot disease (CCRS) occurring in Campania (Southern Italy) in sweet and sour cherry, causes small chlorotic spots turning rusty on the leaves of both species, but deformation and colour alteration of the fruits of sweet cherry only. Consistently associated with the disease are: (i) 10-12 double-stranded RNAs (dsRNAs) corresponding to the genome of mycoviruses of the genus Totivirus, Chrysovirus and Partitivirus; (ii) two circular RNAs (cscRNAs); (iii) mycelium-like structures probably belonging to a fungus. Up to now, the putative mycete has not been isolated and identified. PCR assays using primers EF4-EF3, which amplify a conserved region of the fungal 18S rDNA gene, have shown the presence of a fungus belonging to the order Diaporthales in symptomatic tissues of infected cherry leaves. These findings are congruent with the observation of fruiting bodies with morphological characters of Apiognomonia S4.54 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 erythrostoma (Diaporthales, Gnomoniaceae), on leaves ready to fall (pycnidia) and on fallen leaves (perithecia). However, sequences of the 18S rDNA gene, amplified from CCRS-affected sweet and sour cherry leaves, do not cluster with those of fungi belonging to Gnomoniaceae but, rather, cluster with sequences of fungi of Diaporthaceae and Cryphonectriaceae. ECOPHYSIOLOGICAL RESPONSE OF MELISSA OFFICINALIS TO OZONE. M.G. Carucci, E. Pellegrini, A. Campanella and C. Nali. Dipartimento di Coltivazione e Difesa delle Specie Legnose “Giovanni Scaramuzzi”, Università degli Studi, Via del Borghetto 80, 56124 Pisa, Italy. E-mail: cristina.nali@agr.unipi.it Ozone (O3) is widely considered the major phytotoxic air contaminant. Sensitive plants exposed to ambient O3 levels suffer significant growth reduction, premature senescence, as well as relevant alterations of secondary metabolism. Medicinal plants are poorly studied as far as toxicity of air pollutants is regarded. So, we investigated the response of Melissa officinalis to O3, under controlled conditions (i.e. fumigations in the form of a single square wave of 200 ppb for 5 h). Treated plants showed an alteration in both fast and slow kinetics of chlorophyll a fluorescence, like a decrease of Fv/Fm (-2% compared to controls) and energy dissipation (qP +40% and qNP -10%). O3 induced changes in the pigment complex [increase of neo-, viola-, zea- and anteraxanthin (+43, 34, 74 and 85% in comparison to controls) and decrease of the ratio chlorophyll (a/b) of about 40%]. There was a difference in the de-epoxidation index, which represents the xanthophyll cycle-dependent energy dissipation process for photoprotective mechanism (+100% in treated leaves). -carotene also increased (+94%), suggesting the importance of the antioxidant activity of this pigment. After 48 h, it was possible to observe a macroscopical response, in the form of severe minute rounded dark-blackish necrosis, localized in the interveinal area of the adaxial surface of the leaves. A ∆KU70 MUTANT OF TRICHODERMA VIRENS AS A TOOL FOR EFFICIENT GENE TARGETING. V. Catalano1, M. Ver- gara2, G. Vannacci1, C. Kubicek3 and V. Seidl3. 1Dipartimento di Coltivazione e Difesa Specie Legnose “G. Scaramuzzi”, Sezione di Patologia Vegetale, Università degli Studi, Via del Borghetto 80, 56124 Pisa, Italy. 2Scuola Normale Superiore, Piazza dei Cavalieri 7, 56126 Pisa, Italy. 3Institute of Chemical Engineering, Research Area Gene Technology and Applied Biochemistry, Working Group Molecular Biotechnology, Technical University, Getreidemarkt 9, 1060 Vienna, Austria. E-mail: vcatalano@agr.unipi.it The antagonistic ability of species of the genus Trichoderma make them ideal candidates for the biocontrol of phytopathogenic fungi in integrated pest management. Strains of Trichoderma spp. (including the T. virens I10 isolate) have proven to be effective mycoparasites of important plant-pathogenic fungi. An I10 GFP transformant was exploited for monitoring the sclerotia colonisation in Sclerotinia spp. Gene targeting by homologous recombination (HR) is a powerful technique that can be applied to studying gene functions and to alter interesting properties of fungal strains. However, in filamentous fungi this is often hampered by very low frequencies of HR and it is, therefore, not trivial to target a gene of interest to a specific genomic locus or to delete an endogenous gene. To increase the recombination rate in T. virens transformation, the orthologue of human ku70, which is required for the nonhomologous and joining (NHEJ) pathway and is responsible for ectopic DNA integration, was identified and deleted. The effect on gene targeting of the absent ku70 in T. virens was tested by deleting a laccase gene. Efficiency of gene targeting was 90% in the I10 ∆ku70 strain, which is a significant increase compared to the parental strain (non-ku70 deleted) where only a 10-15% gene knock-out frequency was observed. The generated T. virens ∆ku70 mutant can now be exploited for functional gene studies. The double mutant T. virens ∆ku70/∆lac will be used to study the physiological role of the laccase gene product including a potential function in mycoparasitism against sclerotial fungi such as B. cinerea or S. sclerotiorum. ARABIDOPSIS THALIANA AS A MODEL FOR THE ANALYSIS OF INTERACTION BETWEEN PLANT AND FLORAL SYMPTOMS-INDUCING PHYTOPLASMAS. E. Cettul and G. Firrao. Dipartimento di Biologia e Protezione delle Piante, Università degli Studi, Via delle Scienze 208, 33100 Udine, Italy. E-mail: firrao@uniud.it Despite the significant progresses in the genomic and molecular biology of phytoplasmas, it is still unknown how these pathogen manipulate plant host physiology to induce a complex, diverse, and unique array of symptoms. In order to provide a reliable plant-phytoplasma interaction model for the study of floral symptoms, we developed a high efficiency infection protocol for Arabidopsis thaliana, the plant for which floral development is best understood at the molecular level. Infection of different phytoplasma strains [“Candidatus Phytoplasma pruni” strain Italian Clover Phyllody (ICPh) phytoplasma and “Ca. P. asteris” strain Chrysanthemum yellows (CY) phytoplasma], on different A. thaliana ecotypes (Col-0, Ms-0, Loh-0, Bu-6, Ler) in different growing stages and conditions, resulted in different symptom patterns. By inoculating Col-0 at 30 days after germination with ICPh phytoplasma in short day (9 h light/15 dark), flowers were produced with increasingly severe symptoms for two months. Gene expression analysis showed strong down regulation of some, but not all homeotic genes involved in flowering and genes involved in the gibberellin metabolism that allowed elaboration of a preliminary hypotesis on the molecular mechanism underlying floral symptoms. Common morphological features were detected in phytoplasma-infected wild type plants and mutants impaired in the gene functions that were found to be down regulated. Infection of knock out and overexpressing mutant A. thaliana lines were also carried out to support a hypothesis that phytoplasmas interfere with the maintenance of meristem identity. INNOVATIVE TECNIQUE FOR THE CONTROL OF VERTICILLIUM DAHLIAE IN ARTICHOKE. F. Ciccarese1, N. Sasanelli2, C. Fumarola1 and M. Gallo1. 1Dipartimento di Biologia e Patologia Vegetale, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. 2Istituto Protezione delle Piante del CNR, Via Amendola 122/D, 70126 Bari, Italy. Email: fciccare@agr.uniba.it Artichoke (Cynara cardunculus var. scolymus) is one of the most ancient and typical vegetable crops grown in Italy. In the last years there was a reduction of cultivated areas attributed primarily to the alarming spread of Verticillium wilt caused by Verticillium dahliae Kleb. The disease is quickly spreading to all areas of artichoke growing, because of the general susceptibility of the crop. Control means are limited and, in any case, have a high environmental impact. This work reports the results of a trial, in a field naturally infested by V. dahliae, in which a fungicide (Azoxystrobin) and the biological control agent Aphanocladium album Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 were used against V. dahliae. The chemical and the biological control agent A. album, isolate MX-95, were applied in pre and post transplant via sub-irrigation at the doses of 3 ml/m2 (commercial formulation) and 1.3x107 CFU/ml, respectively. Disease severity was evaluated according to an empirical scale of four classes (0-3) based on external symptoms. Vascular discolorations were assessed according a scale from 0 to 3 on cross-sectioned stems. Plants grown on untreated soil showed severity indices of external symptoms and vascular browning of 2.4 and 2.8 respectively. Azoxystrobin via sub-irrigation significantly reduced both the severity of external symptoms (0.7) and vascular browning (1.2). A. album, isolate MX-95, significantly decreased the severity of Verticillium wilt. Severity of external symptoms and vascular browning were 1.2 and 1.7, respectively. OZONE IN THE CONTROL OF PYRENOCHAETA LYCOPERSICI AND MELOIDOGYNE INCOGNITA ON TOMATO. F. Ci- ccarese1, N. Sasanelli2, C. Fumarola1, M. Gallo1 and M. Ridolfi3. di Biologia e Patologia Vegetale, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. 2Istituto Protezione delle Piante del CNR, Via Amendola 122/D, 70126 Bari, Italy. 3Dipartimento di Ecologia, Sezione di Micologia, Università degli Studi, Via S. Epifanio 4, 27100 Pavia, Italy. Email: fciccare@ agr.uniba.it S4.55 caused by Nectria galligena, Phomopsis mali and Erwinia amylovora. Symptoms are elongated cankers on trunks and branches where dark picnidia are formed, which release conidia when the humidity is high. Thirty-two isolates from Emilia Romagna, Lombardia and Veneto and 14 Japanese strains of V. ceratosperma were morphologically described and genetically characterized by sequencing the ITS1-5.8S-ITS2 regions. Pathogenicity assays were also performed on apple and pear fruits. The morphology of Italian isolates was similar, i.e. white or pale yellow mycelium with patchy margins and forming numerous black picnidia. In contrast, the Japanese isolates showed a white or black mycelium and only five of them produced picnidia. Three different ITS types were found among Italian isolates and specific primers were developed for a rapid detection of the pathogen. The Italian isolate sequences were monophyletic with Valsa spp. GenBank accessions from China, Iran and Spain and they were phylogenetically separated from the Japanese isolates. All the Italian isolates were pathogenic to pear fruits whereas only two Japanese strains infected pear. None of the isolates infected apple fruits. 1Dipartimento Combined attacks of Pyrenochaeta lycopersici and Meloidogyne incognita are frequent in tomato-growing areas. The relationship between the fungus and the nematode has shown a positive correlation between gall index and severity of corky-root, confirming that M. incognita infections enhance attacks of P. lycopersici. A soil disinfection trial using ozone was carried out in a greenhouse naturally infested with P. lycopersici and M. incognita. Gaseous ozone treatments were applied by sub-irrigation. In the trial 3 different treatments were considered: (i) Dazomet; (ii) gasseous ozone applied for 10 h in pre-transplant and (iii) gasseous ozone applied in pre- and post-transplant for 10 and 4.5 h, respectively. Untreated soil served as control. Severity of corkyroot and gall index induced by the nematode were assessed according to an empirical scale from 0 to 5. Fungal and nematode population densities were monitored before and after ozone treatments. Severity of corky root on control roots (untreated soil) was 2.7 and 3.4 on the main and secondary roots, respectively. Ozone treatments significantly reduced disease severity. No statistical difference was observed between pre and post transplant ozone treatments and Dazomet. Gall index on untreated tomato roots was 4.1, whereas it was significantly lower in all other treatments. Soil nematode population density and fungal communities were significantly decreased by ozone treatments. EVALUATION OF A NATURAL COMPOUND AGAINST BOTRYTIS CINEREA ON CUT ROSES. F. Clematis, C. Pasini and P. Curir. CRA, Unità di Ricerca per la Floricoltura e le Specie Ornamentali, Corso Inglesi 508, 18038 Sanremo (IM), Italy. Email: f.clematis@istflori.it Botrytis cinerea causes major postharvest losses to rose flowers and other cut flower crops during storage and shipment. At present, many rose growers dip cut flowers in fungicides to prevent postharvest development of B. cinerea infections but this practice leaves unsightly residues on the plants. Recent studies on induction of systemic acquired resistance (SAR) in plants have shown that salicylic acid is one the SAR-inducing compounds. A component of Eucalyptus spp. phenolic pool, ellagic acid [2, 3, 7, 8Tetrahydroxy-chromeno (5, 4, 3-cde) chromene-5, 10-dione] has a chemical structure comparable to that of salicylic acid. In this work ellagic acid was evaluated for its potential SAR-inducing activity against B. cinerea on cut roses. An alcoholic mother solution of ellagic acid was prepared. In vitro studies were performed to understand the mode of action of this compound. In in vivo trials, cut roses were dipped in tap water additioned with mother solution of ellagic acid and B. cinerea was inoculated by spraying conidial suspension on flowers. Lethal dose 50% was recorded (4.2 g/l) confirming no direct antifungal action but a potential SAR-inducing activity. In in vivo studies, ellagic acid applied at 85 mg/l showed efficacy values (ranging between 22 and 77.4%) comparable with those recorded when ciprodinil plus fludioxonil (Switch) was applied (efficacy ranging between 37 and 52%). Uptake of ellagic acid and its degradation in rose plants will be evaluated in further experiments. BIOLOGICAL AND MOLECULAR CHARACTERIZATION OF ITALIAN ISOLATES OF VALSA CERATOSPERMA ON PEAR. E. Cicognani, M. Iotti, G. Alberoni, M. Collina, A. Zambonelli and A. Brunelli. Dipartimento di Protezione e Valorizzazione Agroalimentare, Università degli Studi, Viale G. Fanin 46, 40127 Bologna, Italy. E-mail: mcollina@agrsci.unibo.it The Ascomycete Valsa ceratosperma (Tode ex Fr.) Maire [anamorph Cytospora sacculus (Schwein.) Gvritischvili = C. vitis] is the causal agent of a bark canker also known as Valsa canker, one of the most important disease of apple trees in East Asia. In 2001 it was reported on pear in the Emilia Romagna (Northern Italy) for the first time and since then reports of the disease have increased. Valsa cankers can be confused with other cankers PEPPER LEAF CURL DISEASE CAUSED BY TOMATO YELLOW LEAF CURL SARDINIA VIRUS ON PEPPER IN SOUTHERN ITALY. S. Comes, A. Fanigliulo, R. Pacella and A. Crescen- zi. Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, Università degli Studi della Basilicata, Viale dell’Ateneo Lucano, Campus Macchia Romana 3A310, 85100 Potenza, Italy. E-mail: aniello.crescenzi@unibas.it An outbreak of pepper leaf curl disease (PLCD) was observed in pepper crops under plastic tunnels in the Ionian coast of Basilicata region in summer and autumn 2008. Diseased plants showed stunting, flower abortion, light mosaic, leaf distorsion, S4.56 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 marginal leaf chlorosis and upward leaf curling. The disease reached, in some instances, a 50% incidence. Large Bemisia tabaci populations were observed on the crop. Assays for the presence of Tomato yellow leaf curl sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV) were performed to idendtify the etiological agent. About 200 symptomatic samples were collected from different locations and assayed by DASELISA using a combined reagent detecting TYLCV, TYLCSV and other Begomoviruses, 176 of which reacted positively. To discriminate among TYLCSV, TYLCV or other Begomoviruses, 15 ELISA-positive samples were analyzed by PCR using a couple of synthetic oligonucleotides allowing the amplification of the whole coat protein (CP) gene. RFLP analysis of the 1008 bp long PCR product showed the presence of only TYLCSV in all assayed samples. The molecular characterization of the sequenced CP gene revealed that this isolate shares a similarity of 97% with the corresponding sequence of a tomato TYLCSV isolate from Sicily and is almost identical with the pepper isolate CAB-It recovered in the same area in 2007. The wide and further spreading of PLCD in the monitored area is to be connected with the presence of tomato crops, of weed hosts alternative to pepper, and with the strong presence of the B biotype of B. tabaci. STUDY OF THE VARIABILITY OF AN ENDOPHYTIC ACREMONIUM POPULATION IN SYMPTOMLESS GRAPEVINES. G. Conigliaro1, S. Lo Piccolo1, A. Alfonzo1, S. Burruano1, B. Salerno2 and E. Carra2. 1Dipartimento di Scienze Entomologiche, Fitopatologiche, Microbiologiche, Agrarie e Zootecniche, Sezione di Patologia Vegetale e Microbiologia Agraria, Università degli Studi, Viale delle Scienze, 90128 Palermo, Italy. 2Dipartimento di Biologia Cellulare e dello Sviluppo, Sezione Biologia Molecolare, Università degli Studi, Viale delle Scienze, 90128 Palermo, Italy. E-mail: gaeconigliaro@unipa.it The endophytism of genus Acremonium in symptomless grapevines of several cultivars, grown both in pots or in the field, is reported. The no-tillage management of tested grapevines and the lack of chemical treatments facilitated the investigation of the ecological plant-fungus interaction. The endophyte was found in all the sampled organs: buds, leaves, petioles and shoots. Acremonium byssoides strain A20 showed strong in vivo and in vitro antibiosis and hyperparasitism towards Plasmopara viticola, and in vitro antagonistic activity against Botrytis cinerea, Phemoniella clamidospora, Pheoacremonium aleophilum, Phoma tracheiphila, Phytium spp., Rhizoctonia solani and Verticillium dahliae. Since host- or organ-specificity is the typical feature of most endophytes in healthy plants, possible variability of the Acremonium population in the field was studied. Endophytic fungal isolates, collected monthly from different organs of a single field-grown cv. Insolia vine, were identified by conventional means. The isolates were differentiated at the strain level by vegetative compatibility assay and randomly amplified polymorphic DNA (RAPD) analyses, using a set of four oligo-nucleotide primers. The endophytic Acremonium population in the healthy grapevine object of the survey seemed to show a low variability; all the techniques agreed in grouping the Acremonium strains in three different clusters and no relationships were observed among them. TRANSFORMABILITY OF PSEUDOMONAS CORRUGATA BY TRANSGENIC DNA FROM PLANTS AND CELL LYSATE OF SOIL BACTERIA. L. Cozzolino1, A. Zoina1 and A. Raio2. 1Università degli Studi di Napoli Federico II, Via Università 100, 80055 Portici (NA), Italy. 2Istituto per la Protezione delle Piante del CNR, Via Madonna del Piano 10, 50019 Sesto Fiorentino (FI), Italy. E-mail: lucia.cozzolino@unina.it Pseudomonas corrugata, the causal agent of tomato pith necrosis, can survive in infected plant debris and in soil for more than 5-6 months. Plant DNA released during the degradation of plant tissues can persist in a biologically active status for significant periods of time, and thus soil or plant-associated bacteria may acquire that free DNA. The ability of P. corrugata to uptake and integrate exogenous DNA into its genome was investigated. P. corrugata chr::_nptII, carrying a deleted nptII gene, was able to capture exogenous DNA and integrate it by homologous recombination. Restoration of nptII originated kanamycin-resistant transformants, that were detected when transformation was performed using plasmid DNA, plant DNA, and cell lysates of P. corrugata, Acinetobacter baylii and Pseudomona stutzeri carrying the functional gene nptII. DNA transfer was not detected when P. corrugata wild type strain (without _nptII gene) was used as control. P. corrugata chr::_nptII strain was used in greenhouse experiments to verify a possible transformation of P. corrugata by DNA of genetically modified tomato plants during an in vivo interaction but no transformants were detected. EFFECT OF ANTAGONISTIC SACCHAROMYCES CEREVISIAE ON THE EXPRESSION OF THE PKS GENE IN OCHRATOXIN A-PRODUCING ASPERGILLUS spp. L. Cubaiu1,2, A. Abbas3, A.D.W. Dobson3, M. Budroni1 and Q. Migheli2. 1Dipartimento di Scienze Ambientali Agrarie e Biotecnologie Agroalimentari, Viale Italia 39, 07100 Sassari, Italy. 2Unità di Ricerca Istituto Nazionale di Biostrutture e Biosistemi e Dipartimento di Protezione delle Piante, Via E. De Nicola 9, 07100 Sassari, Italy. 3Microbiology Department, University College, Cork, Ireland. E-mail: qmigheli@uniss.it The biocontrol effect of Saccharomyces cerevisiae strains involved in wine fermentation was tested against Aspergillus ochraceus and A. carbonarius, two fungal species responsible for the accumulation of ochratoxin A (OTA) in grape and wine. Some of the S. cerevisiae strains were able to significantly reduce OTA content in culture filtrates when co-cultivated with ochratoxigenic Aspergillus spp. strains in a conducive (YES) medium. A selected strain of S. cerevisae (coded 1182) was tested for its capacity to inhibit both OTA production and pks (polyketide synthase) gene expression in A. carbonarius and A. ochraceus. In order to examine a possible correlation between OTA production and the expression of the pks gene regulated by the yeast antagonistic activity, A. carbonarius and A. ochraceus were co-cultured with living cells of S. cerevisiae 1182 or exposed to its supernatant. S. cerevisiae 1182 was able to inhibit both fungal growth and OTA production when co-cultured in YES medium. The transcription of the pks gene was monitored using a reverse transcription PCR-based approach, and OTA production was measured in parallel by HPLC. As expected, pks gene transcript levels tightly correlate with OTA production. Moreover, the pks gene was down-regulated in the presence of both living cells of S. cerevisiae 1182 and by its supernatant, while no effect on the expression of the housekeeping genes calmodulin and beta-tubulin was detected. These data suggest that, besides direct competition, alternative biocontrol mechanism may take place during the interaction between antagonistic wine strains of S. cerevisiae and OTA-producing Aspergilli. Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 POPULATION STUDIES OF XANTHOMONAS ARBORICOLA pv. PRUNI AND NEW STRATEGIES FOR CONTROL IN PEACH ORCHARDS. D. Dallai1, N. Parkinson2, D. Giovanardi1 and E. Stefani1. 1Dipartimento di Scienze Agrarie e degli Alimenti, Università di Modena e Reggio Emilia, Via J.F. Kennedy 17, 42100 Reggio Emilia, Italy. 2The Food and Environment Research Agency (FERA), Sand Hutton, York YO41 1LZ, UK. E-mail: emilio.stefani@ unimore.it The population structure of Xanthomonas arboricola pv. pruni (Xap), the causal agent of the bacterial canker/leaf and fruit spots of stone fruits, was investigated in order to achieve a deeper insight in this taxon, thus enabling an improvement of the current diagnostic protocols and a more effective control of the disease. We confirmed the homogeneity of a vast collection of strains (over 100), isolated from stone fruit orchards during the last decade in Romagna (Northern Italy). Protein profiling and repPCR genetic fingerprints showed no significant differences among the strains: population homogeneity was also confirmed by phylotyping analysis. Copper resistant strains are present, but not widespread among the whole population. Xap is regulated (Directive 2000/29/EC, Annex A, Part II, Section II) and is included in the EPPO A2 list. An effective control of it is based both on analysis of propagation material and use of appropriate control strategies in the field. Based on available DNA sequences, we designed primers and attempted to developed a PCR protocol to be used in certification schemes. We also conducted field and glasshouse trials on peach, with the aim to effectively control the disease by using novel molecules, such as Glucohumates or antagonistic bacteria. Results are very promising and suggest the possibility to implement effective control strategies, where copper compounds and novel molecules are both used in commercial orchards. EFFECT OF SOME RARE EARTH ELEMENTS ON THE GROWTH AND LANTHANIDE ACCUMULATION IN DIFFERENT TRICHODERMA STRAINS. L. d’Aquino1, M. Mor- gana2, M.A. Carboni2, M. Staiano3, M. Vittori Antisari4, M. Re5, M. Lorito6, F. Vinale6, K.M. Abadi6 and S.L. Woo6. 1ENEA, Centro Ricerche di Portici, Via Vecchio Macello, 80055 Portici (NA), Italy. 2ENEA, Centro Ricerche di Trisaia, S.S. 106 Jonica Km 419.5, 75026 Rotondella (MT), Italy. 3Dipartimento di Ingegneria Aerospaziale, Università degli Studi Federico II, Piazzale V. Tecchio 80, 80125 Napoli, Italy. 4ENEA, Centro Ricerche di Casaccia, Via Anguillarese 301, 00060 Santa Maria di Galeria (Roma), Italy. 5ENEA, Centro Ricerche di Brindisi, S.S. 7 Appia Km 706, 72100 Brindisi, Italy. 6Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università degli Studi di Napoli Federico II, Via Università 100, 80055 Portici (NA), Italy. E-mail: luigi.daquino@ enea.it Accumulation of rare earth elements (REE) in the soil may be due to the use of REE enriched fertilizers and to contamination by REE containing wastes. Although widely used in China for soil and foliar dressing of crops, little is known about the effect of REE applications on the soil microbial community. The effect of REE on the growth of biological control strains of Trichoderma atroviride and T. harzianum was investigated in vitro using either a mix of different REE containing various amounts of lanthanum, cerium, praseodymium, neodymium, gadolinium nitrate and lanthanum nitrate alone in comparison to treatments with potassium nitrate and water. In plate tests, applied concentrations ranged from 0.1 mM to 300 mM for lanthanum and REE mix and from 0.1 mM to 900 mM for potassium. In liquid culture tests, applied concentrations ranged from 0.001 mM to 100 S4.57 mM for lanthanum and REE mix and from 0.003 mM to 900 mM for potassium. ICP-MS, TEM and TEM X-ray microanalysis were used to study the accumulation of REE in fungal biomass. All the Trichoderma strains showed a good tolerance to the presence of REE in the culture media. Some growth enhancing effects were observed in liquid cultures of T. harzianum strains but not in T. atroviride. Accumulation of REE in fungal biomass, both at intracellular level and in the extracellular matrix, was observed. ANALISYS OF A NATURAL POPULATION OF CUCUMBER MOSAIC VIRUS INFECTING MEDICINAL PLANTS. S. Davi- no1, A. Lombardo2, M. Davino3 and M.G. Bellardi4. 1Dipartimento di Scienze Entomologiche, Fitopatologiche, Microbiologiche, Agrarie e Zootecniche, Sezione di Patologia Vegetale e Microbiologia Agraria, Università degli Studi, Viale delle Scienze, 90128 Palermo, Italy. 2Parco Scientifico e Tecnologico della Sicilia, Stradale G. Agnelli angolo V. Lancia, 95030 Catania, Italy. 3Dipartimento di Scienze e Tecnologie Fitosanitarie, Sezione di Patologia Vegetale, Università degli Studi, Via Santa Sofia 100, 95123, Catania, Italy. 4Dipartimento di Scienze e Tecnologie Agroambientali, Sezione di Patologia Vegetale, Università degli Studi, Viale G. Fanin 42, 40127 Bologna, Italy. E-mail: davino@unipa.it Cucumber mosaic virus (CMV) is one of the most important viruses, infecting more that 1,000 species. During the last decade, CMV was detected in Italy in several medicinal species associated to severe symptoms that reduced the yield of the crops and of theirs commercial by-products. A preliminary molecular study was carried out to compare five CMV isolates recently found inthe Herb Garden of Casola-Valsenio (Emilia-Romagna, Northern Italy): Anchusa officinalis L., Dipsacus silvetris Miller, Echinacea purpurea Moench., Inula viscosa L., and Nepeta cataria L. For genetic analyses, the movement protein gene (with the two primers: MP+ 5’-CATGGCTTTC CAGGTACCAG-3’ and MP5’-CTAAAGACCGTTAACCACCTGC-3’ amplifying a fragment of 842 bp) of each CMV isolate, amplified from original material by one-step RT-PCR, was characterized by SSCP and determination of its nucleotide sequence. The generated phylogenetic trees classified CMV-D. silvestris and CMV-I. viscosa in subgroup II, and the other three in subgroup IA. Probabilistic Divergence Measures Phylogentic method indicated two recombination breackpoints (at 110 and 660 nucleotide positions of the alignment). The molecular diversity of these isolates can be explained as a possible natural reassortement of a CMV population. In particular, the efficient virus dissemination by aphids may have facilitated the transfer of CMV isolates from plant to plants (weeds and cultivated herbs) allowing an increase in interspecifc recombination rates and favouring the emergence of “new” sequence variants originating from the two subgroups detected. This natural reassortement of viral-segmented genomes, in combination with spontaneous mutations and RNA recombination, may be viewed as a major mechanism of Cucumovirus evolution. OUTBREAK OF TOMATO INFECTIOUS CHLOROSIS VIRUS IN A RELEVANT ARTICHOKE PRODUCING AREA OF SICILY. S. Davino1, L. Tomassoli3, A. Tiberini3, V. Mondello1 and M. Davino2. 1Dipartimento di Scienze Entomologiche, Fitopatologiche, Microbiologiche, Agrarie e Zootecniche, Sezione di Patologia Vegetale e Microbiologia Agraria, Università degli Studi, Viale delle Scienze, 90128 Palermo, Italy. 2Dipartimento di Scienze e Tecnologie Fitosanitarie, Sezione di Patologia Vegetale, Università degli Studi, Via Santa Sofia 100, 95123 Catania, Italy. 3CRA, Cen- S4.58 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 tro di Ricerca di Patologia Vegetale, Via C.G. Bertero 22, 00156 Roma, Italy. E-mail: davino@unipa.it THE ORIGIN OF PLANT-ASSOCIATED PINK YEASTS INFLUENCES THEIR BIODIVERSITY, BIOCONTROL EFFICACY AND ABILITY TO DEGRADE PATULIN. F. De Curtis1, R. During winter 2009, in a artichoke (Cynara scolymus L.) field between Siracusa and Ragusa provinces (Sicily, Southern Italy), interveinal yellowing was observed on the leaves of many plants of different varieties. Greenhouse whitefly (Trialeurodes vaporariorum) populations were present with high density on the crops. Young shoots and mature leaves from symptomatic plants were collected and analyzed for some of the viruses usually infecting artichoke. In particular, total RNA was extracted from five infected plants of each variety and tested by RT-PCR using primers specific for Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV) and two criniruses, Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV). These latter viruses are known to cause leaf yellowing and are particularly widespread on tomato in the area where the affected artichoke crop was surveyed. RT-PCR was positive only for TICV generating expected amplicons of 500 bp which were purified and sequenced. The pairwise similarity analysis confirmed the species identification showing a percentage value of 99%, with the HSP70 region of other Sicilian isolates of TICV. In Italy, only scattered artichoke plants affected by TICV were previously reported. To our knowledge this is the first important outbreak of TICV on artichoke crop. Because of the high number of infected plants present in the field, TICV can be viewed as a potantial threat to artichoke cultivation in Sicily. Castoria1, L. Palmgren2, G. Ianiri1 and S.A.I. Wright1,2. 1Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Università degli Studi del Molise, Via F. De Sanctis, 86100 Campobasso, Italy. 2Department of Plant and Environmental Sciences, University of Gothenburg, Göteborg, Sweden. E-mail: decurtis@unimol.it Research supported by MiPAAF in the framework of the project PROM (CIPE deliberating 17/2003). MONITORING OF PLANT PATHOGENIC FUNGI ON READY-TO-EAT SALADS IN ITALY. U. De Corato and M. Trupo. Dipartimento di Biotecnologie, Agroindustria e Protezione della Salute dell’ENEA, Centro Ricerche Trisaia, S.S. 106 Jonica Km 419.500, 75026 Rotondella (MT), Italy. E-mail: ugo.decorato@enea.it Ready-to-eat vegetables can be contaminated by several fungi which can incite spoilage processes. The aim of this work was to monitor the population of plant pathogenic fungi on ready-to-eat bagged samples of Lactuca sativa L. baby leaf and Diplotaxis spp. (rocket) during shelf life, in order to evaluate the effect of the length of storage and of the season of production on the spoilage of these products. A total of 108 samples collected from 10 trademarks located in Southern and Central Italy were stored into refrigerators at 8°C and analyzed by plate count at the 2nd, 5th and 8th day after packaging in spring and summer. Results showed a great variability of the yeast and mould count at the 1st day of sampling among the tested samples. The yeast and mould counts did not show significant variations in relation to the length of storage or the season of production. The rocket presented a 2 Log CFU/g increase in the level of moulds and yeasts with respect to lettuce. Some plant pathogenic fungi were constantly detected (Cladosporium fulvum, Sclerotinia sclerotiorum and Fusarium spp.). S. sclerotiorum did not contribute to spoilage because samples appeared fresh and without any decay symptoms. The other moulds detected belonged to the toxigenic genera Alternaria, Aspergillus, Fusarium and Penicillium. Aspergillus flavus and Penicillium spp. were constantly found in both types of salad in summer. Their growth at low temperatures represents a potential hazard for mycotoxin production. Pink yeasts belonging to the genera Sporobolomyces, Rhodosporidium, Rhodotorula and Sporodiobolus are natural inhabitants of many agricultural and wild plant species. Their natural role in the microbial ecology of the phylloplane has not been studied sufficiently. When isolated and purified, they have shown the ability to suppress plant diseases. Therefore, pink yeasts were isolated from the phyllosphere of agricultural and wild plants in various climatic zones, islands, coastal, hilly and mountain areas of Southern-Central Italy. The yeasts were purified and characterized by morphological and molecular methods. Subsequently, they were tested for biocontrol ability of blue mould of apple and for the ability to degrade patulin, a mycotoxin produced by Penicillium expansum, the causal agent of the disease. Among the climatic zones examined, the highest degree of yeast biodiversity was recorded on the Tremiti islands, probably because more species of plants were sampled, and because of the absence of an extensive agricultural activity. Of the 170 pink yeasts isolated, most belonged to the genus Rhodosporidium. More yeasts were isolated from apple and fig trees than from olive trees in the three locations compared. Fig trees had a more diverse epiphytic microflora than did apple and olive. Interestingly, apples appear to host predominantly two different types of pink yeasts, as shown by analysis of morphological traits and of RFLP of the ITS regions. The biocontrol of P. expansum and the ability to degrade patulin was assessed in function of the location and plant species from which the yeasts were isolated. ACTIVITY OF ANTAGONIST BACTERIA FOR THE BIOLOGICAL CONTROL OF RHIZOCTONIA SOLANI AND SCLEROTIUM ROLFSII ON TOMATO. F. De Curtis, D. Vitul- lo, G. Lima and V. De Cicco. Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Università degli Studi del Molise, Via F. De Sanctis, 86100 Campobasso, Italy. E-mail: decurtis@unimol.it Rhizoctonia solani and Sclerotium rolfsii are fungal pathogens responsible for severe crown and stem rot of horticultural crops worldwide. Chemical control of these pathogens involves technical, environmental and toxicological risks. To find eco-compatible and safer alternative control methods, the effectiveness of the newly selected bacterial isolates Burkholderia cepacia (T1A-2B) and Pseudomonas spp. (T4B-2A), originating from suppressive organic matrices, were tested on tomato plants grown under both controlled and field conditions. The potential antagonists were compared with two commercial biofungicides (Bacillus subtilis BSF4® and Trichoderma asperellum TV1®) and four synthetic fungicides (Tolclofos-methyl, Azoxystrobin, Fosetyl-Al and Fosetyl-Al + Propamocarb). In the rhizosphere of tomato plants treated by both antagonists, the total bacterial population was about 100-fold higher than that recovered from untreated control. Scanning electron microscope observations revealed that both biocontrol bacteria efficiently colonized the root surface and were capable of adhering to tomato roots by producing biofilm-like structures. In a 2-year field experiments, the novel selected biocontrol bacteria significantly reduced both incidence and severity of disease caused by the pathogens to tomato. The effectiveness of the antagonists was equal to that of T. asperellum Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 (TV1), better than that of B. subtilis (BSF4) and was also comparable to that of synthetic fungicides. The results of these investigation encourages further studies aimed at optimizing the novel biocontrol bacteria for application in controlling crown and root rot of horticultural crops. FIRST REPORT OF BOSCALID-RESISTANT MUTANTS OF BOTRYOTINIA FUCKELIANA (BOTRYTIS CINEREA) IN SOUTHERN ITALY VINEYARDS AND THEIR GENETIC CHARACTERIZATION. R.M. De Miccolis Angelini, C. Rotolo, S. Pollastro and F. Faretra. Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. E-mail: faretra@agr.uniba.it Resistance to the recently introduced fungicide boscalid in the ‘grey mould’ fungus Botryotinia fuckeliana (de Bary) Whetz. (Botrytis cinerea Pers.) was monitored in 2008 in Apulian vineyards (Southern Italy). Conidia sampled at harvesting from naturally infected berries were plated on a minimal medium amended with 1 mg/l boscalid and containing acetate as carbon source. Resistant isolates were detected in 5 out of 15 monitored vineyards with a frequency ranging from 5.4x10-5 to 2.3x10-2. The resistant mutants were recovered with the highest frequency when the fungicide was applied 1 to 3 times in the season, alone or in mixture with the QoI fungicide kresoxim-methyl. In colony growth test, sensitive wild-type isolates showed EC50 = 0.3 µg/ml and MIC = 10 µg/ml of boscalid, whereas all the characterized resistant isolates displayed EC50 and MIC values higher than 100 µg/ml. A strong inhibition of germ tube elongation was observed between 0.01-0.03 and 1-3 µg/ml in sensitive and resistant isolates, respectively. In addition, all field mutants showed double resistance to boscalid and QoI fungicides. The molecular bases of resistance to boscalid were investigated in representative resistant isolates. Gene sequence analysis of highly conserved regions of the iron-sulphur (Ip) sub-unit of the succinate dehydrogenase complex (the target-site of boscalid) revealed that point mutations were associated with the resistance. Mutations resulted in a histidine (CAC) to tyrosine (TAC) or arginine (CGC) replacement at position 272 (H272Y or H272R), previously found associated with boscalid resistance in laboratory mutants of B. fuckeliana. EVALUATION OF SUSCEPTIBILITY OF LEMON CULTIVARS TO MAL SECCO DISEASE BY A MOLECULAR METHOD. A. De Patrizio1, F. Raudino2, A. Pane2, Q. Migheli3, G. Magnano di San Lio4 and S.O. Cacciola5. 1Dipartimento di Scienze Entomologiche, Fitopatologiche, Microbiologiche Agrarie e Zootecniche, Università degli Studi, Viale delle Scienze 2, 90128 Palermo, Italy. 2Dipartimento di Scienze e Tecnologie Fitosanitarie, Università degli Studi, Via S. Sofia 100, 95123 Catania, Italy. 3Unità di Ricerca Istituto Nazionale di Biostrutture e Biosistemi e Dipartimento di Protezione delle Piante, Università degli Studi, Via E. De Nicola 9, 07100 Sassari, Italy. 4Dipartimento di Gestione dei Sistemi Agrari e Forestali, Università Mediterranea, Località Feo di Vito, 89122 Reggio Calabria, Italy. 5Dipartimento di Chimica Biologica, Chimica Medica e Biologia Molecolare, Università degli Studi, Viale Andrea Doria 6, 95125 Catania, Italy. E-mail: olgacacciola@unict.it Mal secco disease, caused by the mitosporic fungus Phoma tracheiphila, is a destructive vascular disease of lemon (Citrus limon) in the Mediterranean region. P. tracheiphila is a quarantine pathogen on the list of most regional plant protection organizations including the European and Mediterranean Plant Protec- S4.59 tion Organization (EPPO). According to the standard diagnostic protocol of EPPO, P. tracheiphila can be identified by both conventional and molecular methods. In 2005, a PCR-based assay for P. tracheiphila was published and a pair of specific primers (PtFOR2 and Pl-REV2) were designed on the consensus sequence obtained from the alignment of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. This PCR-based specific assay made it possible to detect the fungus in infected tissues of both symptomatic and symptomless citrus plants. In the last three years, this molecular method has been used to test the susceptibility of various lemon cultivars and hybrids to artificial infections with P. tracheiphila. The susceptibility to mal secco of steminoculated lemon accessions as evaluated on the basis of symptom severity correlated with the colonization rate of the xylem by the pathogen and the results of PCR assay were consistent with conventional isolations on artificial media. However, the molecular method proved faster and more sensitive than conventional isolation. In summer, symptomatic plants recovered and the pathogen was not detected in new vegetation flushes by both diagnostic methods, confirming that high temperature inhibits xylem colonization by the fungus. TRANSIENT EXPRESSION OF HCPro AND 2b RNA SILENCING SUPPRESSORS DOES NOT DEFEAT RNA-MEDIATED PLUM POX VIRUS RESISTANCE. E. Di Nicola-Negri, L. Salandri and V. Ilardi. CRA, Centro di Ricerca per la Patologia Vegetale, Via C.G. Bertero 22, 00156 Roma, Italy. E-mail: vincenza.ilardi@entecra.it RNA silencing in plant is a natural defence mechanism against viruses. Viruses encode proteins able to counteract this defence. HCPro protein of potyviruses and 2b protein of cucumoviruses are two well-characterised RNA silencing suppressors. Potato virus Y (PVY) HCPro suppresses RNA silencing in tissues where it was already established, whereas Cucumber mosaic virus (CMV) 2b binds both siRNAs and long double stranded RNA in vitro. Plum pox virus (PPV) is one of the most detrimental pathogens of stone fruits. In nature there are five PPV strain: M, D, Rec, EA and C. In previous studies, we transformed Nicotiana benthamiana plants with four PPV-M-derived RNA silencing constructs: 5’UTR/P1, P1/HCPro, HCPro and HCPro/P3. All constructs protected plants from PPV-D, PPV-M and PPV-Rec infection while only plants harboring the 5’ UTR/P1 sequence were also resistant to PPV-C and PPV-EA. Moreover, as expected on the basis of the molecular resistance mechanism, PPV-resistant plants were susceptible to PVY and CMV. Here we present data on the influence of virus-mediated expression of RNA silencing suppressors on the stability of the PPV-M-resistant trait following two distinct approaches. In the first, 5’UTR/P1 plants were challenged with CMV or PVY followed by PPV-M inoculation, while in the other P1/HCPro, HCPro and HCPro/P3 plants were challenged with PPV-C followed by PPV-M inoculation. All double challenged plants were resistant to PPV-M. This data indicates that virus-mediated expression of homologous or heterologous RNA silencing suppressors was not able to defeat PPV-M RNAmediated resistance. Work carried out within the frame of the Research Programme MiPAAF–CIPE “FRU.MED Project PRO VI SUD”. GUANO BIOFERTILIZER IN THE BIOLOGICAL CONTROL OF VERTICILLIUM WILT OF OLIVE PLANTLETS. A.M. D’Onghia1, A.M. Ismail1, T. Yaseen1, A. Ippolito2 and F. Nigro2. S4.60 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 1Istituto Agrononomico Mediterraneo, Via Ceglie 9, 70010 Valenzano (BA), Italy. 2Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. E-mail: donghia@iamb.it In the last two decades Verticillium wilt of olive, caused by Verticillium dahliae Kleb., has occurred with increasing frequency and severity in most olive growing areas of the Mediterranean basin. The disease affects olive trees starting from the nursery and microsclerotia are a critical factor for the epidemiology and control of the disease. Among the environmentally friendly alternatives to protect plantlets against V. dahliae infection, the use of organic amendments has received much attention. In this study, the effect of Guano bio-fertilizer on the inoculum density of V. dahliae microsclerotia, and on vegetative parameters of olive plantlets growing in organic medium, was assessed. Trials were carried out either on 8-month-old self-rooted and 18-month-old grafted olive plantlets of cv. Leccino. The organic growing medium was amended with different concentrations of Guano (0.5, 1%, 2%, 3% v/v). Before plants transplanting into pots, the growing medium was artificially inoculated with V. dahliae microsclerotia. A phytotoxic effect was observed on plantlets growing in the medium amended with 2% and 3% Guano. At these bio-fertilizer concentrations the inoculum density of V. dahliae microsclerotia was almost completely inactivated. These results were more evident on grafted than on self-rooted plantlets. Conversely, plantlets growing in 0.5% and 1% Guano-amended medium, showed a higher plant height than those growing in the non amended medium. Moreover, the inoculum density of V. dahliae microsclerotia in the medium amended with 1% Guano, was significantly lower than in the non amended control. PROTEOMIC ANALYSIS OF TRICHODERMA HARZIANUM IN RESPONSE TO CADMIUM AND MERCURY IONS. R. Faedda1, G. Petrone2, S.O. Cacciola3, V. Rappa3 and D. Sheehan4. 1Dipartimento di Scienze e Tecnologie Fitosanitarie, Università degli Studi, Via S. Sofia 100, 95123 Catania, Italy. 2Dipartimento di Scienze Agronomiche, Agrochimiche e delle Produzioni Animali, Università degli Studi, Via S. Sofia 98, 95123 Catania, Italy. 3Dipartimento di Chimica Biologica, Chimica Medica e Biologia Molecolare, Università degli Studi, Viale A. Doria 6, 95125 Catania, Italy. 4Department of Biochemistry, University College Cork, Lee Maltings, Mardyke, Cork, Ireland. E-mail: d.sheehan@ucc.ie Heavy metals represent a major environmental pollutant. Cellular metal toxicity may occour through interaction with protein sulphydryl groups, formation of reactive oxygen species or displacement of cations from metal binding proteins. Protein thiols are both highly-susceptible to oxidation and involved in cell signaling. Trichoderma harzianum Rifai is able to grow in presence of large amount of heavy metals, however the molecular mechanism underlying cellular tolerance to these metals, as well as in other fungi, has been little investigated. The aim of this study was to study the effect of either cadmium or mercury ions on the T. harzianum proteome to understand the metabolic response to heavy metal stress. Two-dimensional electrophoresis was carried out on total and thiol-containing proteins extracted from fungal mycelia grown with 10 ppm CdCl2 or HgCl2. Total protein analysis of T. harzianum grown with CdCl2 revealed 12 up-regulated and 11 down-regulated proteins, whereas the fungus grown with HgCl2 showed 11 up-regulated and 18 down-regulated proteins with respect to the control. Thiol-containing proteins of Cd-treated cultures showed 13 up-expressed and 5 down-expressed SHcontaining proteins, whereas Hg-treated cultures revealed 8 up- regulated and 9 down-expressed SH-containing proteins with respect to the control. These results support the idea that a thiolbuffer is a possible defence response to oxidative damage. MALDI-TOF analysis revealed several enzymes involved in the protection of cell from oxidative stress induced by heavy metals. G3PDH (a known redox sensor) and NMT1 (involved in biosynthesis of thiamine, a crucial coenzyme for pyruvate oxidation) are up-regulated in the presence of cadmium. RESULTS ON THE EFFICACY OF THE FUNGICIDE CYAZOFAMIDE IN THE CONTROL OF BREMIA LACTUCAE ON LETTUCE. A. Fanigliulo1,2, V. Filì1 and A. Crescenzi2. 1Bioagritest Srl, Centro Interregionale di Diagnosi Vegetale, Zona PIP lotto E2, 85010 Pignola (PZ), Italy. 2Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, Università degli Studi della Basilicata, Viale dell’Ateneo Lucano, Campus Macchia Romana 3A310, 85100 Potenza, Italy. E-mail: aniello.crescenzi@unibas.it; info@bioagritest.it During summer 2007 and 2008, trials were conducted according to EPPO guidelines and Principles of Good Experimental Practice, aimed at establishing the efficacy of Cyazofamide 25SC (Mildicut, Belchim) and Cyazofamide 400SC plus an organosilicone adjuvant containing polyalkyleneoxide modified heptamethyltrisiloxane (Ranman 400 SC, Belchim) for controlling Bremia lactucae in lettuce. The study was performed in Nocera Inferiore (Campania, Southern Italy), in a greenhouse with polyethylene cover. Experimental design consisted in randomized blocks with 3 replicates. Two different dosages of Mildicut (3.5 lt/ha and 4.5 lt/ha) were compared with the unique dosage 0.35 lt/ha of Ranman and a commercial formulate: 2.4% metalaxyl-M plus 40% copper administered at 4 kg/ha (Ridomil Gold R 46WP, Syngenta Crop Protection). Four foliage treatments were made at weekly intervals. The severity and spreading of the disease were evaluated on leaves together with possible phytotoxic effects. The extreme climatic conditions occurred during the course of study, with rather low temperatures and high humidity in the greenhouse, accompanied by high rainfall, favoured extremely serious attacks by Bremia lactucae, so as damage heavily the 3 control replicates. Statistical analysis was performed using XLSTAT data analysis and statistical software. Results showed the excellent efficacy of Cyazofamide (Mildicut and Ranman) reagadless of the dose employed. Also the standard formulate used in comparison showed to be effective in the control of the B. lactucae. No phytotoxicity was observed on the leaves of treated plants. BOTRYTIS CINEREA LACCASE ABOLISHES RESVERATROL TOXICITY AND REDUCES GRAPE PR PROTEIN SOLUBILITY. F. Favaron1, M. Lucchetta2, S. Odorizzi1, A.T. Pais da Cunha1 and L. Sella1. 1Dipartimento del Territorio e dei Sistemi Agro-forestali, Sezione Patologia Vegetale, Università di Padova, Viale dell’Università 16, 35030 Legnaro (PD), Italy. 2Centro Interdipartimentale per la Ricerca in Viticoltura ed Enologia, Università di Padova, Via XXVIII Aprile 14, 31015 Conegliano, Italy. E-mail: francesco.favaron@unipd.it Some grape proteins are structurally and functionally related to plant pathogenesis-related proteins. When challenging mature grape berries, Botrytis cinerea encounters these PR proteins as well the stilbenic phytoalexin trans-resveratrol and other grape polyphenols. To mimic these conditions, Botrytis cinerea was grown in vitro with proteins and polyphenols extracted from ma- Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 ture grapes and with trans-resveratrol. Results showed that in the presence of highly toxic amounts of trans-resveratrol, grape polyphenols and proteins allowed total and partial recovery of fungal growth, respectively. These resveratrol-polyphenol or resveratrol-protein combinations also induced a strong release into the medium of laccase activity, which is likely to be involved in the trans-resveratrol detoxification process. Most grape proteins quickly disappeared from the culture when polyphenols and trans-resveratrol were supplied together and similar protein patterns were obtained in vitro by incubating grape proteins with grape polyphenols and/or trans-resveratrol with a purified B. cinerea laccase. Under these conditions, most protein became insoluble. The grape protein pattern obtained from grape berries infected by B. cinerea strongly resembled that obtained in vitro by incubating the grape proteins and polyphenols with the fungal laccase. It seems that B. cinerea, in the presence of the berry polyphenols and through laccase activity, easily neutralizes the toxicity of grape stilbenic phytoalexins and makes the grape pathogenesis-related proteins insoluble. Whether changes of grape proteins solubility affect the expression of proteinase genes and the proteolytic activity of B. cinerea is currently under investigation. ACIBENZOLAR-S-METHYL INCREASES THE TRANSCRIPT LEVEL OF PAL AND CHS GENES IN OLIVE PLANTLETS. M. Ferrara, I. Pentimone, A. Ligorio, S.M. Sanzani, A. Ippolito and F. Nigro. Dipartimento di Protezione delle Piante e Microbiologia Applicata, Via Amendola 165/A, 70126 Bari, Italy. Email: nigrof@agr.uniba.it. Plants are continuously exposed to pathogen attacks, but they defend themselves with various mechanisms including induced resistance which involves rapid changes in gene expression. Induction of resistance can also be induced in plants by several substances. Acibenzolar-S-methyl (ASM), a functional analogue of salicylic acid (SA), was found to behave as an effective resistance elicitor, thus facilitating protection under field conditions against a wide range of pathogens and in several crops. Phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) are key enzymes in phenylpropanoid/flavonoid pathway, leading to production of compounds, including lignin, flavonoids and phytoalexins, extremely important in the plant defence strategy. A molecular method for monitoring PAL and CHS gene expression in the leaves of olive plantlets (cv. Leccino), in response to ASM application was developed. Specific primers, amplifying a 233 and 188 bp amplicons were designed to quantify the relative transcript level of PAL and CHS genes, respectively, by quantitative reverse transcription-PCR. Gene expression was normalised to the housekeeping -actin (act1) mRNA gene of Olea europaea. Plantlets exhibited a good tolerance toward the different amount (0.05÷0.5 g/l a.i.) of ASM tested. Results indicate that ASM up-regulates the expression of the target genes. In particular, PAL and CHS expression were strongly induced by ASM (0.25 g/l a.i.) within 6 h from application, whereas peak expression of CHS was maintained for a longer time than PAL. Moreover, the time-course of PAL expression revealed a persistence of genes up-regulation till the fifth day after treatment, as compared to the control. MOLECULAR CHARACTERIZATION OF ‘CANDIDATUS PHYTOPLASMA PRUNORUM’ ISOLATES IN CACOPSILLA PRUNI INSECT VECTOR. L. Ferretti1, A. Gentili1, C. Poggi- Pollini2, P. Ermacora3 and G. Pasquini1. 1CRA, Centro di Ricerca S4.61 per la Patologia Vegetale, Via C.G. Bertero 22, 00156 Roma, Italy. 2Dipartimento di Scienze e Tecnologie Agroambientali, Università degli Studi, Viale Fanin 44, 40127 Bologna, Italy. 3Dipartimento di Biologia e Protezione delle Piante, Università degli Studi, Via delle Scienze 208, 33100 Udine, Italy. E-mail: luca.ferretti@entecra.it ‘Candidatus Phytoplasma prunorum’ (16SrX-B subgroup), the causal agent of European stone fruit yellows (ESFY), occurs in all Italian stone fruit-growing areas. Recently, a molecular characterization based on the non ribosomal tuf gene of ‘Ca. P. prunorum’ detected in infected plants, revealed the presence of two different groups of isolates (‘type a’ and ‘type b’) with a defined geographical distribution. To verify whether this differentiation occurred also in phytoplasmas present in the vector Cacopsylla pruni and to correlate the presence of the two types of isolates in insects and infected orchards, a molecular characterization of phytoplasmas from C. pruni specimens was carried out. Individuals were captured in ESFY-affected orchards located in northern Italian regions (Lombardia, Trentino, Friuli Venezia Giulia, and Emilia Romagna) both on cultivated and wild Prunus species. Total DNA from 66 infected individuals was submitted to the amplification of a phytoplasma tuf gene fragment and then analyzed by RFLP. Single infections of both isolates were identified in C. pruni specimens confirming their differentiation also inside the vector. As to the geographical distribution of the ‘a’ and ‘b’ types, previously defined in the investigated areas, only ‘type a’ was found in insects collected from orchards in Emilia Romagna and Friuli Venezia Giulia, while both types were detected in individuals from Lombardia and Trentino. Moreover, in these last two regions, the percentage of C. pruni specimens infected by ‘type a’ was prevalent (92.3% and 80.0%, respectively), in agreement with the infection rate of the same isolates detected in the corresponding orchards. MOLECULAR CHARACTERIZATION OF CITRUS TRISTEZA VIRUS ISOLATES IN CALABRIA. L. Ferretti1, A. Fontana2, R. Schimio2, R. Sciarroni1, G. Albanese2 and M. Barba1. 1CRA, Centro di Ricerca per la Patologia Vegetale, Via C G. Bertero 22, 00156 Roma, Italy. 2Dipartimento di Gestione dei Sistemi Agrari e Forestali, Università Mediterranea, Località Feo di Vito, 89060 Reggio Calabria, Italy. E-mail: luca.ferretti@entecra.it The recent identification of new Citrus tristeza virus (CTV) outbreaks in different citrus growing areas of Calabria region keeps high the risk of a rapid field spreading of the disease with severe economic damages for the local citriculture. In these areas, the characterization of infecting CTV isolates appears particularly important in order to prevent, by a quick uprooting of the infected plants, a rapid spread of especially of severe isolates. Results of the molecular characterization of isolates collected in five different CTV-infected citrus orchards are reported. Thirty-nine CTV isolates were analyzed by single strand conformation polymorphism (SSCP) technique. Total RNA extracted from leaves was amplified by RT-PCR with primers amplifying a p20 CTV gene fragment. RT-PCR products were then denatured and separated by electrophoresis in a 8% acrylamide non-denaturing gel. Four different isolates: DS1SR (mild, Italy), T385 (mild, Spain), DS2CT (severe, Italy) and T388 (severe, Spain) were used as reference controls. SSCP analysis showed four different profiles named type I, II, III and IV. Out of 39 tested isolates, 10 showed the type I pattern, whereas the remaining samples had type II, III or IV profile. Since type I strains reacted positively with Mab MCA13, which is specific for the severe isolate, their SSCP patterns could belong to severe isolates even if they differed from the reference strains DS2CT and T388. By contrasts, other CTV S4.62 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 isolate types, which did not react with Mab MCA13, could be attributed to mild strain in spite of their differences with the controls. Further investigations on the molecular variability of the isolates are in progress. three layers feed-forward neural network and predict fumonisin content from the calculated indexes. The results showed a significant correlation between predictions from image analysis and fumonisin concentration. The method developed produces reliable contamination estimates within few minutes and can be readily used to assist lot selection in various steps of the maize processing chain. EVALUATION OF THE RESISTANCE OF PEACH GERMPLASM TO PLUM POX VIRUS STRAIN. M.L. Ferretti1, M. Barba1, A. Gentili1, I. Verde2, L. Conte 2, L. Campus1, S. Micali2 and G. Pasquini1. 1CRA, Centro di Ricerca per la Patologia Vegetale, Via C.G. Bertero 22, 00156 Roma, Italy. 2CRA, Centro di Ricerca per la Frutticoltura, Via Fioranello 52, 00134 Roma, Italy. E-mail: graziella.pasquini@entecra.it The use of tolerant or resistant genotypes could significantly aid the management of Sharka induced by Plum pox virus (PPV). In order to establish the level of tolerance/resistance, 122 interesting selections were evaluated by phenotypic analysis and diagnostic responses to PPV-M infection, after grafting onto GF305 peach seedlings and successive experimental inoculation with the PPV-M isolate GR0019. Most of the evaluated selections (92/122) derived from a cross between peach cv. Maria Aurelia and an F1 hybrid of P. persica x P. davidiana, displaying a complex pattern of quantitative inheritance. The remaining selections included wild Prunus spp. genotypes showing tolerance to PPV-D. Symptoms evaluation was performed according to a four- step arbitrary scale. A percentage of 17.2 (21/122) of the selections did not show any symptoms after two years of observations and 13.1% were symptomless also on GF305. All symptomless selections were tested by ELISA, RT-PCR and TaqMan real time RT-PCR for response classification. Twelve selections were positive for PPV in ELISA and RT-PCR (tolerant plant response). The remaining selections (9/122) were positive only by real time RT-PCR (resistant or highly resistant plant response) and two of them were negative for virus presence in the rootstock. Further investigations are in progress to develop the genomic map by microsatellite markers and the QTL analysis, together with the evaluation of the temporal stability of the observed responses. IMAGE ANALYSIS FOR THE RAPID ESTIMATION OF FUMONISIN CONTENT OF MAIZE. G. Firrao, E. Torelli, E. Gobbi and R. Locci. Dipartimento di Biologia e Protezione delle Piante, Università degli Studi, Via delle Scienze 208, 33100 Udine, Italy. E-mail: firrao@uniud.it Fumonisin contamination is a major concern to the maize industry in North East Italy. Several strategies have been explored in an attempt to reduce the severity of this problem, ranging from cultural practices to resistance, predictive models and postharvest processing. Nevertheless, due to the largeness, diversity and complexity of the production system, in conducive years severely contaminated lots are still introduced in the maize processing chain with the effect of lowering the general quality of the regional product. As chemical analysis is laborious, time consuming and equipment-dependent, an efficient method is urgently needed for the early identification of contaminated lots. Here we present a novel method based on image analysis that provides fast response with a minimal equipment and effort. Maize samples were ground and imaged at 1024x768 pixels resolution under 10 different LED lights centered at wavelength ranging from 720 to 940 nm. The digital images were converted into matrices of data to compare the relative shift intensity values at the different lights for each pixel and compute comparative indexes. A custom program written in C language was then used to train a PATHOGENIC FUNGI ASSOCIATED WITH BRANCH DIEBACK OF DECLINING CORK OAK TREES IN SARDINIA. A. Franceschini, B.T. Linaldeddu, B. Scanu and L. Mad- dau. Dipartimento di Protezione delle Piante, Sezione di Patologia Vegetale, Università degli Studi, Via E. De Nicola 9, 07100 Sassari, Italy. E-mail: afran@uniss.it Over the past two decades several studies have pointed out the preminent role played in the aetiology of oak decline by different pathogenic fungi inhabiting branch tissues. Many of these have been reported as causal agents of canker and branch dieback in various oak species. However, until now very few of these fungi have been associated with cork oak decline. In the present work, the results of surveys carried out in 19 sites of a declining cork oak forest located in South-Central Sardinia are reported. In each site, one 3- to 4-year-old symptomatic branch from seven declining trees was sampled and examined for the presence of fungal reproductive structures. The occurring fungi were isolated, identified and tested for their pathogenicity. Six species were found: Botryosphaeria corticola, Coryneum depressum, Diatrypella quercina, Phomopsis spp., Stuartella formosa and an unidentified ascomycete. Their frequency was highly variable, and only C. depressum occurred in all investigated sites in both anamorph and teleomorph (Pseudovalsa umbonata) state. Pathogenicity tests carried out on potted cork oak plants showed that all these fungi induced stem necrosis. B. corticola was the most virulent species causing sunken necrotic lesions and brown discoloration on the bark and xylem tissues. These results suggest that all isolated fungi, and in particular B. corticola, play an active role in cork oak decline. SURVEY OF FUNGAL SPECIES INHABITING ASPARAGUS TURIONS IN APULIA. S. Frisullo, F. Lops, V. Gentile, M.L. Raimondo, F. Cibelli, M. Guerrieri and A. Carlucci. Dipartimento di Scienze Agro-Ambientali, Chimica e Difesa Vegetale, Università degli Studi, Via Napoli 25, 71100 Foggia, Italy. E-mail: s.frisullo@unifg.it Results are reported of a three-year survey carried out on asparagus turions intended for fresh use and IV-gamma products from 22 fields in the provice of Foggia (Apulia, Southern Italy). The investigation consisted in colleting weekly 100 asparagus turions from each field during April-June 2007-2009. All turions were first inspected for external symptoms such as spots with purple or hydropic haloes and bent apices (“pastorale”) then, they were analysed mycologically. Visual observations detected symptoms associated with attacks by Stemphylium spp. (8.0 to 25.4% incidence) and Alternaria spp. (3.0 to 8.0% incidence). Turions with curved apices ranged from 0.4 to 2.3%. Isolations in culture showed that Fusarium spp. occurred with a frequency ranging from 5.8 to 42.7%, Alternaria spp. ranged from 3.75 to 38.7%, Stemphylium spp. ranged from from 2.35 to 8.9% and Phialophora spp. from 0.25 to 2.35%. Other fungal species belonging to the genera Cladosporium, Mucor, Aerobasidium, Plectosporium and Botrytis were isolated with variable frequencies. F. Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 proliferatum and F. oxysporum were the most frequent species recovered from turions. The authors want to thank the President, Mr. Antonio Ippedico, and the farms of the Società Cooperativa Agricola “Produttori Ortofrutticoli Associati” for providing turions and for the technical support. INFLUENCE OF SUSCEPTIBILITY TO FUSARIUM OXYSPORUM ON THE BIOFUMIGATION ACTIVITY OF SOME BRASSICAS. A. Garibaldi, G. Gilardi, P. Lu and M.L. Gullino. S4.63 quenced starting from the polyadenylated 3’ terminus. Genomic RNA consists of four open reading frames encoding, in the 5’ → 3’ direction, the replication-associated proteins (ORF1), a 42 kDa putative movement protein (ORF2), the 46 kDa coat protein (ORF3), and a 10 kDa protein with nucleic acid binding properties. The genome structure and organization of FLV-1 resembles that of members of the genus Trichovirus, family Flexiviridae and, indeed, the virus clusters with trichoviruses in phylogenetic trees contructed with the coat protein sequences. However, a distinct difference with other members of the genus rests with the size of the coat protein subunits (46 versus 22-27 kDa) and the presence of ORF 4, which occurs only in trichoviruses currently classified as tentative species. Centro di Competenza per l’Innovazione in Campo Agro-Ambientale, Università degli Studi di Torino, Via L. da Vinci 44, 10095 Grugliasco (TO), Italy. E-mail: marialodovica.gullino @unito.it A study was undertaken to investigated the possible negative side-effects of biofumigation caused by pathogens able to attack both plants used for biofumigation as well as agricultural crops. This study showed that many of the brassica crops used for biofumigation are susceptible to Fusarium oxysporum f. sp. conglutinans and/or to F. oxysporum f. sp. raphani. The simulation of green manure treatment was not able to reduce the incidence of both Fusaria on all the crops that were susceptible to one or both pathogens. On the contrary, the population of both pathogens increased as a result of the incorporation of tissues of the susceptible plants, and a good inverse correlation between disease index and biomass was observed. As a consequence, green manure simulation, carried out by growing different cycles of biocidal plants, did not reduce Fusarium wilts on susceptible brassica hosts. When brassica crops were resistant to the two f. sp. of F. oxysporum used in our study for soil infestation, green manure simulation reduced propagules of he pathogens, thus confirming its biocidal activity. The results obtained under our experimental conditions show that biofumigation is not applicable to crops susceptible to the same f. sp. of F. oxysporum affecting brassica species used for biofumigation, because in the presence of Fusarium wilt of the biocidal plants, the inoculum potential of their incitans will increase in the soil, with possible negative effect on the crops. FIG LATENT VIRUS 1, A NEW MEMBER OF THE FAMILY FLEXIVIRIDAE. G. Gattoni1, A. Minafra2, M.A. Castellano1, A. De Stradis2, D. Boscia2, T. Elbeaino3, M. Digiaro3 and G.P. Martelli1. 1Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. 2Istituto di Virologia Vegetale del CNR, Unità Organizzativa di Bari, Via Amendola 165/A, 70126 Bari, Italy. 3Istituto Agronomico Mediterraneo, Via Ceglie 9, 70010 Valenzano (BA), Italy. E-mail: martelli@agr. uniba.it A virus with filamentous particles with distinct cross banding, ca. 700 nm long, denoted Fig latent virus 1 (FLV-1) is widespread in Apulian (Southern Italy) fig orchards, in trees showing or not mosaic symptoms and in symptomless seedlings. FLV-1 was transmitted through seeds at a very high rate, and mechanically to a restricted range of herbaceous hosts, in which it multiplied with difficulty and did not induce apparent symptoms. A virus-specific antiserum proved useful for FLV-1 detection in fig leaf dips by immunosorbent electron microscopy. The cytology of infected cells was little affected. Bundles of filamentous particles were observed in the cytoplasm of parenchyma cells of infected fig trees and seedlings. The viral genome is a single stranded (+)RNA with an estimated size of ca. 8,000 nt, 6,620 nt of which have been se- PRELIMINARY STUDIES ON DISCULA PASCOE, A FUNGUS ASSOCIATED WITH ROTTED CHESTNUT FRUITS NEW TO ITALY. S. Gentile, I. Visentin, D. Valentino and G. Tamietti. Di- partimento di Protezione e Valorizzazione Agroalimentare, Sezione di Patologia Vegetale, Università degli Studi di Torino, Via L. da Vinci 44, 10095 Grugliasco (TO), Italy. E-mail: giacomo.tamietti@unito.it Since 2005, an uncommon and severe mummification of sweet chestnut fruits has been observed in Southern Piedmont (Northern Italy). In 2007-2008 a survey was carried out collecting flowers and fruits at different developmental stages in chestnut stands in 4 locations in the South of Cuneo. Small pieces of fruit stem, burrs, stigmas, styles and developing seeds were plated on malt agar, pH 4.75, and incubated at 25°C. A fungus was isolated with a variable frequency from stigmas and styles, constantly from the bark of flower/fruit stems, but not from the xylem and the inner layer of the hylum. It was isolated sporadically from fruits just after setting and in complete ripened fruits with frequency from 25 to 80% in 2007 and 4 to 84% in 2008. In the immature nuts the disease was symptoless, whereas in the ripening ones it caused small chalky areas in the endosperm outer layers, which progresses inwards in the kernel, then turned brown, leading to the complete mummification of the endosperm. On the pellicle and the shell the pathogen produced 122x85- 251x171 µm globose black pycnidium-like acervuli, that split open along the top releasing a slimy, globular mass of conidia. Conidia were pale brown, onecelled, ovoid-oblong, biguttulate, 6.1-7.32 x 2.44-2.68 µm in size. This fungus was constantly associated with the symptoms and reproduced the disease in artificially inoculated healthy nuts. Based on such characters, the pathogen was identified as Discula pascoe (teleomorph Gnomonia pascoe), a pathogen recently reported in the rainy areas of Australia and New Zealand. A DECISION SUPPORT SYSTEM FOR INTEGRATED MANAGEMENT OF WHEAT CROPS. S. Giosuè1, P. Meriggi1, T. Caffi2 and V. Rossi2. 1Horta srl, Spin off Company, Università Cattolica del S. Cuore, Via Emilia Parmense 84, 29100 Piacenza, Italy. 2Istituto di Entomologia e Patologia Vegetale, Università Cattolica del S. Cuore, Via Emilia Parmense 84, 29100 Piacenza, Italy. Email: s.giosue@horta-srl.com A DSS (Decision Support System) is described for guiding decisions about both strategic and tactical management of wheat crops. The DSS is available for registered users via the internet in an interactive way. It is based on: (i) a network of weather stations that measure environmental variables; (ii) a file repository that stores weather data; (iii) a set of mathematical models that use weather data and crop-specific information to predict the bi- S4.64 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 ological events relevant for decision making; (iv) a user interface that makes it possible to readily input crop-specific variables and obtain information for decision making; and (v) a register of the crop-management options already applied that could influence future decisions. Output provides information on: (i) current weather conditions; (ii) recommended crop rotation; (iii) suggested soil preparation for sowing; (iv) optimal sowing date and seeding rate; (iii) wheat growth and development; (iv) timing and amount of fertilization; (v) weed control timing and herbicides to be applied; (vi) risk indexes for the main wheat diseases, including rusts, powdery mildew, and Fusarium head blight; and (vii) the probable level of mycotoxin contamination of kernels at harvest. Different users can access information at different levels of complexity, depending on their role (i.e., policy makers, advisors, or growers). Each year, an experimental site is managed so as to compare advantages arising from using the DSS in comparison with the usual grower’s practice. RECENT ADVANCES ON THE EPIDEMIOLOGY AND CONTROL OF BACTERIAL BLIGHT OF WALNUT, INCITED BY XANTHOMONAS ARBORICOLA pv. JUGLANDIS. D. Giova- nardi1, D. Dallai1, E. Cozzolino2 and E. Stefani1. 1Dipartimento di Scienze Agrarie e degli Alimenti, Università di Modena e Reggio Emilia, Via J.F. Kennedy 17, 42100 Reggio Emilia, Italy. 2Azienda Agricola San Martino, Via Persiani 61, 47010 San Martino in Strada (FC), Italy. E-mail: emilio.stefani@unimore.it The bacterial blight of walnut caused by Xanthomonas arboricola pv. juglandis (Xaj) is an emerging disease, which can severely affect walnut orchards. Symptoms are visible on all aerial parts of the host, particularly on leaves and nuts. The disease develops more rapidly in spring, causing spots on the leaves and immature fruits, followed by the formation of small cankers on leaf petioles and twigs. Affected fruits fall down throughout the growing season, with a peak within mid-May and mid-June. The primary source of the inoculum is available very early in spring, since evasion is likely to occur from small overwintering cankers present on twigs as soon as the new vegetation busts. The primary inoculum is spread by wind-driven rain droplets and pollen. Our study confirms the spread by pollen, but catkins seems to become infected during their spring development from bacteria oozing from small twig cankers. Female flowers are not contaminated before pollination, and become infected during pollination and/or during spring rain. Copper resistance has been studied on a collection of over 150 strains isolated in Romagna during 20072009. A high percentage (83%) of the collection proved tolerant to copper, whereas 36% was highly resistant. Control strategies are difficult to implement and are based on the timely and effective use of copper compounds with emphasis on spring treatments. In order to bypass copper resistance the use of alternative molecules as resistance inducers is under evaluation, coupled with a reduced use of copper. BIOLOGICAL CHARACTERIZATION OF APRICOT LATENT VIRUS AND SURVEY IN THE CZECH REPUBLIC. L. Grimová1, M. Zouhar1, P. Rysánek1, A. Bazzoni2, F. Palmisano2 and V. Savino2. 1Department of Crop Protection, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences, Kamycká 129, 165 21 Prague 6, Czech Republic. 2Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. E-mail: lgrimova@seznam.cz Stone fruits are an important and widely grown in the Czech Republic. A number of viruses are known to occur in apricot, peach, plum and cherry trees in this country. The most common pathogens include Plum pox virus (PPV), Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV). Apricot latent virus (ApLV), a definitive species in the genus Foveavirus, family Flexiviridae is another recently discovered virus which has been recorded from Mediterranean countries like France, Italy, Turkey, Egypt and Lebanon. In the Czech Republic, the occurrence of this virus has not yet been ascertained. To determine the best source material and the most reliable period for ApLV detection, RT-PCR assays, using a virus-specific set of primers, were performed during the growing season. Flowers, pollen grains, fruits, leaves, petioles and bark tissues from ApLV-infected apricot tree cv. Tirynthos were used as a plant material in this study. In order to check the presence and distribution of ApLV in the Czech Republic, a survey was carried out in several commercial stone fruit orchards and germplasm collections. Samples from 200 apricots were collected randomly for RT-PCR analysis in 2005, 2006, 2009. Until now no infected trees have been found. This suggested that ApLV has limited distribution in this area. DISTRIBUTION OF CITRUS LEAF BLOTCH VIRUS IN A NAGAMI KUMQUAT PLANT. M. Guardo, G. Sorrentino, T. Marletta, A. Leonardi and A. Caruso. CRA, Centro di Ricerca per l’Agrumicoltura e le Colture Mediterranee, Corso Savoia 190, 95024 Acireale (CT), Italy. E-mail: maria.guardo@entecra Citrus leaf blotch virus (CLBV), a member of the family Flexiviridae, has a single-stranded, positive-sense genomic RNA 8747 nucleotides in size. CLBV causes a bud union disorder of Nagami kumquat and Calamondin grafted on trifoliate rootstocks, it is symptomless in sour orange. In this work the distribution of CLBV was monitored in 30-year-old field-grown Nagami kumquat plant grafted on sour orange. Leaf and green bark samples were separately collected from the quadrant of the tree every month for one year. CLBV was detected using RT-PCR with two set of primers specific for RNA polymerase and coat protein genes. Results showed that the virus is unevenly distributed in the tree canopy for it is detectable all year round but not consistently in each sample. CLBV was found in all leaves collected in June and September; and in all bark samples in May. Virus presence was also investigated in closed and open flowers, fruits (flesh, peel and juice), and in the roots. It was found only in flowers. Old and young leaves collects from 30 plants were tested for establishing the best tissue for CLBV diagnosis. Results showed that the highest virus titer was found in young leaves in spring and autumn. Research supported by the “MiPAAF” in the framework of the project: Innovazioni per il Raggiungimento della qualità Globale in Agrumicoltura – AGRUQUAL (Paper n. 36). EXPRESSION OF PHENYLPROPANOID PATHWAY GENES DURING THE COMPATIBLE INTERACTION OF COLLEC- TOTRICHUM ACUTATUM – FRAGRARIA X ANANASSA FRUITS AT DIFFERENT RIPENING STAGES. M. Guidarelli1, E. Baraldi1, P. Bertolini1, M. Mari1, C. Rosati2 and F. Mourgues2. 1CRIOF, Dipartimento di Protezione e Valorizzazione Agroalimentare, Laboratorio di Biotecnologie, Università degli Studi, Viale Fanin 46, 40127 Bologna, Italy. 2ENEA, Trisaia, Dipartimento di Genetica e Genomica, S.S. 106 Km 419+500, 75026 Rotondella (MT), Italy. E-mail: elena.baraldi@unibo.it Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 Colletotrichum acutatum is a major fungal pathogen of strawberry, causing severe economic losses. In this study, we investigated the variation of sensitivity to C. acutatum of strawberry fuits at different ripening stages. Red fruits are highly susceptible, with severe disease symptoms visible after 72 h from pathogen infection, whereas, after this time, no disease symptom is apparent on white fruits. Since conidia of C. acutatum germinate and produce appressoria on both these type of fruits, it may be inferred that this pathogen becomes quiescent on white fruits. In order to investigate the molecular mechanisms determining C. acutatum quiescence in white fruits, we analysed the variation of expression of genes encoding enzymes of the phenylpropanoid pathway, known to be involved in the biosynthesis of several antifungal compounds. By comparing white and red fruits both inoculated with C. acutatum or mock inoculated, we found that all the phenylpropanoid genes, except those encoding the enzymes involved in the antocyanidins biosynthesis, are upregulated in the red fruits as compared with to the white ones. No differences were found in inoculated versus mock-inoculated fruits. These data indicate a possible role for antocyanidins in determining quiescence of C. acutatum in white strawberry fruits. USE OF THE COMBIMATRIX MICROARRAY METHOD FOR GENE EXPRESSION ANALYSIS IN STRAWBERRY FRUITS INTERACTING WITH COLLECTOTRICHUM ACUTATUM. M. Guidarelli1, E. Baraldi1, P. Bertolini1, F. Carbone2, C. Rosati2 and G. Perrotta2. 1CRIOF, Dipartimento di Protezione e Valorizzazione Agroalimentare, Laboratorio di Biotecnologie, Università degli Studi, Viale Fanin 46, 40127 Bologna, Italy. 2ENEA, Trisaia, Dipartimento di Genetica e Gnomica, S.S. 106 Km 419+500, 75026 Rotondella (MT), Italy. E-mail: elena.baraldi@unibo.it In this study, the different phases of compatible interaction between the hemibiotroph fungal pathogen Colletotrichum acutatum and strawberry fruits were investigated by combining microscopical and colorimetric analysis of infected tissues. During its biotrophic phase, C. acutatum displays a completely different colonization strategy depending on the ripening stage of strawberry fruits. Both on white and red fruits, conidia germinate 16 h after inoculation, and after 20 h appressoria are apparent. At 24 h, most of the appressoria are melanized. At this time, only in red fruits primary hyphae penetrate in the epidermal layer. On the contrary, on white strawberries, no hyphae were visualized underneath the fruit surface, suggesting that, in these fruits, this pathogen becomes quiescent after the appressorium formation. The Combimatrix microarray technique was used to investigate the genes differently regulated at 24 h interaction of C. acutatum with red and white strawberry fruits. A total of 456 genes were found differently regulated (292 up- and 164 down-) both in red and white fruits upon pathogen interaction. Among these, genes encoding pathogenesis related (PR) proteins and proteins typically involved in plant responses to biotroph pathogens were found. Furthermore, 788 and 1499 genes were differently regulated by the pathogen only in red or white fruits, respectively. These genes could possibly be of great significance for elucidating the genetic mechanisms regulating pathogen quiescence. EFFECTIVENESS OF HOT WATER TREATMENT OF GRAPEVINE ROOTSTOCKS AGAINST PHAEOMONIELLA CHLAMYDOSPORA. W. Habib1,2, S. Pollastro1, F. Mannini3 and F. Faretra1. 1Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. 2Istituto Agronomico Mediterraneo, Via Ceglie 9, S4.65 70010 Valenzano (BA), Italy. 3Istituto di Virologia vegetale del CNR, Unità Organizzativa di Grugliasco, Via L. da Vinci, 44, 10095 Grugliasco (TO), Italy. E-mail: faretra@agr.uniba.it Phaeomoniella chlamydospora (W. Gams, Crous, M.J. Wingf. et Mugnai) Crous et W. Gams is supposed to be the main responsible of decline and dieback of young vines (Petri disease) and is involved in Esca disease of grapevine. The fungus can spread via infected propagation materials. In different countries, hot water treatment (HWT) is applied to control grapevine trunk pathogens, including P. chlamydospora, with variable results. Naturally infected rootstocks (140 Ru and 1103 P), ownrooted or grafted with cv Negroamaro scions, were submitted to HWT at 50°C for 45 min. Wood discoloration and P. chlamydospora occurrence were evaluated after the treatment and after one growing season. In addition, bud breaking and growth were periodically measured. The results of two-year observations were quite similar, showing that HWT did not affect the severity of wood discoloration, while the pathogen viability was drastically reduced. Soon after the treatment, the pathogen was detected in 29.4% of the untreated control rootstocks and only in 2.7% HWT rootstocks, with a reduction in the frequency of infected chips of wood up to 60% in the HWT material. At the end of one growing season, the fungus was recovered in 70.8% of the untreated rootstocks and in 38.0% of the HWT rootstocks. Bud breaking and growth were significantly (P = 0.05) reduced by the treatment independently of the rootstock. These results suggest that HWT is effective against P. chlamydospora. However, further research is in progress to evaluate the risk of re-infection of grape propagation material in the field by P. chlamydospora and other trunk pathogens. NURSERY PRACTICES INFLUENCE PHAEOMONIELLA CHLAMYDOSPORA INFECTIONS IN GRAPEVINE GRAFTED ROOTSTOCKS. W. Habib1,2, A. Pichierri1, S. Pollastro1 and F. Faretra1. 1Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. 2Istituto Agronomico Mediterraneo, Via Ceglie 9, 70010 Valenzano (BA), Italy. E-mail: faretra@agr.uniba.it Phaeomoniella chlamydospora (W. Gams, Crous, M.J. Wingf. et L. Mugnai) Crous et Gams. involved in brown streaking of rootstocks, Petri and esca disease of grapevine, is frequently detected in one-year-old vines. The presence of the fungus as environmental contaminant in nurseries was ascertained with several investigations, while it remains unclear in which way the fungus infects rootstocks. This study was aimed at evaluating the influence of several steps of the routine process for producing grafted rootstocks on P. chlamydospora infection. Specific actions were taken singularly or in combination, i.e. sterile water was used in pre-grafting or pre-callusing hydration, blades of grafting machines were cleaned with 4% sodium hypochlorite solution, and decontaminated soil was used to plant grafted-cuttings. At rootstock uprooting, nucleic acids were extracted from wood fragments from the crown and graft union of at least 150 rootstocks per treatment, and nested-PCR was used for P. chlamydospora detection. In combination, the above-cited actions lead to a significant (P = 0.05) reduction (57%) of infection frequency as compared with the normal nursery practice. The decrease of infection frequency due to single actions ranged from 8% (decontamination of grafting machines) to 62% (hydration of grafts and cuttings in sterile water). Such findings suggest that each investigated step can contribute significantly to P. chlamydospora infection of grafted rootstocks. Further studies are in progress for establishing the weight of each step on rootstock infection by P. S4.66 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 chlamydospora to prevent spreading of the pathogen to new vineyards. PARTIAL CHARACTERIZATION OF BERMUDA GRASS MOSAIC VIRUS, A TENTATIVE MEMBER OF THE FAMILY POTYVIRIDAE. A. Hosseini1, M. Kouhi Habibi1, K. Izadpanah2, G.H. Mosahebi1, C. Rubies-Autonell3 and C. Ratti3. 1Department of Plant Protection, College of Agriculture, Shiraz University, Iran. 2Department of Plant Protection, University College of Agriculture and Natural Resources, University of Tehran, Iran. 3Dipartimento di Scienze e Tecnologie Agroambientali, Sezione di Patologia Vegetale, Università degli Studi, Viale G. Fanin 40, 40127 Bologna, Italy. Email: concepcion.rubies@unibo.it Mosaic symptoms were seen on Bermuda grass (Cynodon dactylon) in many parts of Iran. Examination by electron microscopy of samples from diseased plants revealed elongated filamentous virus particles. The virus was mechanically transmitted to some gramineous plants. Virions of two isolates of this virus were purified from goose grass (Eleusine compressa) and field-infected Bermuda grass and a virus-specific polyclonal antiserum was raised. No serological relationships were found between these isolates and some members of the family Potyviridae that infect poaceous plants. The virus did not react with an univeral potyvirus group antiserum in serological assays. RT-PCR assays using potyvirus degenerate primers confirmed the serological results. General primers derived from the nuclear inclusion b (NIb) sequence of potyviral genome used in a RT-PCR assay yielded the expected amplification product of 1700 bp which was cloned and sequenced. The predicted amino acid sequence corresponded to part of the NIb protein and the capsid protein (CP), followed by an untranslated region and a polyadenylated tail at the 3’ end. The most similar sequence in the databases was that of Spartina mottle virus, a tentative member of family Potyviridae. The sequence had 76% nt identity to Spartina mottle virus in the 3’-terminal 1700 bp nucleotide sequence of the RNA genome. Serological and sequence comparisons suggest that the Bermuda grass filamentous virus should be regarded as a new species of the family Potyviridae, which is tentatively named Bermuda grass mosaic virus. PATHOGENICITY PHENOTYPES OF FUSARIUM spp. ASSOCIATED WITH CITRUS DRY ROOT ROT. A. Hussien1, T. Yaseen1, S. Vicario2, M. Santamaria2, C. Saccone2 and A.M. D’Onghia1. 1Istituto Agronomico Mediterraneo, Via Ceglie 9, 70010 Valenzano (BA), Italy. 2Istituto di Tecnologie Biomediche del CNR, Via Amendola 122/D, 70126 Bari, Italy. E-mail: y.thaer@iamb.it Many Fusarium spp. are supposed to be casual agents of citrus dry root rot, whose identification can be very problematic. The aim of this work was to characterize Fusarium spp. into non-pathogenic, mild, intermediate and severely pathogenic strains. Six Fusarium oxysporum and seven F. solani strains, representative of 79 isolates, collected from citrus rhizosphere that grouped according the sequence of β-tubulin and α-elongation factor loci into 9 phylogenetic clusters, were used in this study. Three pathogenicity characters were assessed: phytotoxicity of fungal excretions, capacity to colonize the surface of citrus host plant, and ability to colonize the internal tissues. All trials were carried out on Carizzo citrange. Symptoms of wilting were assessed over two months with a phytotoxicity of culture filtrate trial. Citrus plantlets grown in vitro under controlled conditions, were inoculated with Fusarium strains, and the ability of the pathogen to surface colonize and evoke alterations to the host were visually assessed. The ability of the pathogen to proliferate and colonize host tissues was estimated by CFU/g of plant fresh weight. The results highlighted the significant correlation between phytotoxic excretions and invasion ability. No correlation of this two traits with ability to colonize citrus plantlet surface was detected. This study also revealed the considerable variation of severity among Fusarium strains belonging to different phylogeny clusters. In conclusion both genotype and excretion phytotoxicity assay seem good predictor of aggressiveness. PRODUCTION OF HOMOSERINE LACTONES BY BACTERIA ASSOCIATED WITH OR PATHOGENIC TO CULTIVATED MUSHROOMS. N.S. Iacobellis1, S. Prashanth1, P. Lo Can- tore1, T.R.I. Cataldi2, G. Bianco3, S. Abate3 and M. Zeccola3. di Biologia Difesa e Biotecnologie Agro-Forestali, Università degli Studi della Basilicata, Viale Ateneo Lucano 10, 85100 Potenza, Italy. 2Dipartimento di Chimica, Università degli Studi, Via E. Orabona 4, 70126 Bari, Italy. 3Dipartimento di Chimica, Università degli Studi della Basilicata, Via N. Sauro 85, 85100 Potenza, Italy. E-mail: iacobellis@unibas.it 1Dipartimento In a great number of bacteria a major level of regulation exists, mediated by the production and response to signal molecules. Since this regulation is driven by a cell-density response it has been called Quorum Sensing (QS). In gram-negative bacteria a typical QS system is based on the production and response to acetylated homoserine lactones (AHLs). In plant pathogenic bacteria, QS has been shown to control the production of virulence factors, the Ti plasmid conjugal transfer in Agrobacterium tumefaciens as well as fitness-related characters. For these important features AHL-mediated-QS has been addressed as potential target for an alternative strategy for bacterial disease control based on its inactivation (Quorum Quenching, QQ). Bacterial diseases of cultivated mushrooms are limiting factors for Agaricus and Pleurotus spp. growing mainly because of the lack of suitable bactericides. Hence, new and alternative control measures appear necessary. The aim of this study was to evaluate the production of AHLs in strains of Pseudomona agarici, P. tolaasii, P. gingeri, P. reactans and Burkholderia gladioli pv. agaricicola and in bacteria associated with mushrooms in order to unravel the possible regulation of the disease process by AHLs-QS and to develop a potential mushroom disease control by QQ. Strains of P. agarici, P. gingeri and B. gladioli pv. agaricicola were shown to produce AHL analogs based on the biosensor response to diffusible molecules and LC-ESI-LTQ-FTICR MS. Among 234 bacterial isolates obtained from Agaricus bisporus, Pleurotus ostreatus and Pleurotus eryngii 43 were found to produce an array of AHLs. MOLECULAR CHARACTERIZATION OF CLAVIBACTER MICHIGANENSIS subsp. MICHIGANENSIS. G. Ialacci1, P. Bella1, K.H. Gartemann2, G. Licciardello3, R. Eichenlaub2 and V. Catara1. 1Dipartimento di Scienze e Tecnologie Fitosanitarie, Università degli Studi, Via Santa Sofia 100, 95123 Catania, Italy. 2Lehrstuhl für Gentechnologie/Mikrobiologie, Universität Bielefeld, 33594 Bielefeld, Germany. 3Parco Scientifico e Tecnologico della Sicilia, Zona Industriale, Stradale G. Agnelli angolo V. Lancia, 95030 Catania, Italy. E-mail: vcatara@unict.it The gram-positive plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) that causes bacterial wilt and canker of tomato, is an EU quarantine organism spread in all major tomato-growing areas. Recently the nucleotide se- Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 quence of the genome of strain NCPPB382 was determined. Although the genome has a high G+C content, many of the potential virulence genes are clustered in a low G+C-content chromosomal region (chp/tomA), which is essential for effective colonization. This strain contains two plasmids, pCM1 (27kb) and pCM2 (70kb) that carry genes essential for virulence on tomato. Thirty Cmm strains isolated in Italy were evaluated by molecular targeting of virulence factors and by PCR-based DNA fingerprinting. Virulence genes celA and pat-1, which are located on the plasmids pCM1 and pCM2, respectively, were detected by PCR in 30 and 25 strains, respectively. PCR amplification of the genes ppaA, chpC and tomA located on the chp/tomA chromosomal putative pathogenicity island was obtained from all strains. Restriction fragment polymorphism analysis using HhaI, HhaII, HaeIII, HpaII did not reveal any difference between the strains. After an on-line analysis of Cmm and C. michiganensis subsp. sepedonicus genomes at http:/www.in-silico.com, suitable restriction endonucleases and selective nucleotides were selected to perform fluorescent AFLP analysis. The core BOX-PCR and fAFLP profiles were clearly distinguishable from those of other C. michiganensis subspecies. Cmm strains revealed a low degree of variability. The differences were often limited to few polymorphic bands, however fAFLP proved to be most discriminatory allowing to differentiate a larger number of strains. CAN ORGANOSILICONE TREATMENT IMPROVE TOMATO RESPONSE TO CUCUMBER MOSAIC VIRUS INFECTION? V. Ilardi, L. Calandri and E. Di Nicola-Negri. CRA, Centro di Ricerca per la Patologia Vegetale, Via C.G. Bertero 22, 00156 Roma, Italy. E-mail: vincenza.ilardi@entecra.it Cucumber mosaic virus (CMV) is one of the most important viruses worldwide. Due to the difficulties in producing CMV resistant tomato plants by classical breeding and in accepting transgenic technology as an effective alternative approach to produce CMV resistant plants new strategies to control this economically important pathogen are needed. A potential not fully explored approach is to search for molecules that can induce or strengthen endogenous plant defences thus lessening virus symptomatology and increasing the yield. In the frame of a research program regarding the effect of polyamine treatment on CMV infection in tomato we used an organosilicone surfactant during foliar spraying. Organosilicone adjutants are designed to improve the spreading and wetting properties of chemical spray mixtures by lowering the surface tension of the solution. Three tomato cultivars (Corbarino, Galatino and Vesuviano) and one hybrid (Tomito) were treated with putrescine and spermidine as well as with the polyamine analogue guazatine plus the silicone surfactant at time T-1, T0, T+1, then weekly till the end of the experiment. CMV mechanical inoculation was made at time T0. Although the CMV concentration in the treated plants did not change, disease symptoms were attenuated. Unexpectedly, the highest yield for each tomato cultivar was obtained treating CMV-inoculated plants with H2O plus organosilicone. Work carried out within the frame of the Research Programme MiPAAF–CIPE “PROM”. OUTBREAK OF PHOMA VALERIANELLAE ON CORN SALAD IN CENTRAL ITALY. A. Infantino1, A. Caraffa2 and G. Di Giambattista1. 1Centro di Ricerca per la Patologia Vegetale , Via C. G. Bertero 22, 00156 Roma, Italy. 2Enza Zaden Italia srl., Strada Statale Aurelia km 96+710, 01016 Tarquinia (VT), Italy. E- mail: S4.67 alessandro.infantino@entecra.it Lamb’s lettuce [Valerianella olitoria (L.) Pollich = Valerianella locusta L.], also known as corn salad, is gaining more interest in Northern and Central Italy for ready-to-eat production. Several plants showing damping-off were observed in two protected crops of the Latina area (Latium, Italy), one in a single plastic tunnel (6x80 m) and the other in a multitunnel of 5000 m2. Initial symptoms were small black spot on the cotyledons and elongated red-brown lesions on the stem of seedlings at the soil level, that soon evolved into necrosis and collapse of the seedlings. Isolations on potato dextrose agar (PDA) amended with antibiotics consistently yielded fungal colonies of grey colour with small, single or aggregate pycnidia containing 1-celled conidia. Based on morphology, the fungus was identified as Phoma valerianellae Gindrat, Semecnik et Bolay. For pathogenicity test, seeds of cv Juvert were previously analyzed by the “deep freezing” blotter test in order to exclude the presence of seed-borne inoculum. Then, 30-day-old seedlings were sprayed with a suspension of fungal conidia (6.5x105 ml-1) in sterile water and covered for 48 h with plastic bags to maintain high humidity. After 5 days, symptoms similar to those observed in the greenhouse were observed and. P. valerianellae was always reisolated from all infested seedlings. A Phoma spp. has already been observed on corn salad in Italy in 1965. The continuous cropping of corn salad on the same field up to seven cycles per year and, probably, the use of improperly produced and conditioned seeds, could have determined the present outbreak. RECOMBINATION EVENTS IN RNA-2 OF GRAPEVINE FANLEAF VIRUS AND ARABIS MOSAIC VIRUS IN GRAPEVINES AFFECTED BY YELLOW MOSAIC. J. Jawhar1, A. Minafra2, P. La Notte2, C. Pirolo1, P. Saldarelli2, D. Boscia2, V. Savino1 and G.P. Martelli1. 1Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 BARI, Italy. 2Istituto di Virologia Vegetale del CNR, Unità Organizzativa di Bari, Via Amendola 165/A, 70126 Bari, Italy. Email: martelli@agr.uniba.it Grapevine fanleaf virus (GFLV) has strains associated with distorting (Mf) and chromogenic (YM) syndromes, which are indistinguishable physico-chemically and serologically, induce the same symptoms in herbaceous hosts and have the same vector (Xiphinema index). Sequences of the RNA-2A gene (homing protein) of Mf and YM isolates of Italian and foreign origin were compared for detecting possible regions of variability that would allow designing molecular tools (probes and/or primers) for their differential identification. Phylogenetic trees constructed with 2A sequences showed two major clusters, each comprising two subgroups. Sequence alignment of sub-group c (YM) and d (Mf) showed the presence in YM isolates of a short sequence fragment (164 nt) that differed markedly from that of the comparable region of Mf isolates. This region had 100% identity with a comparable fragment of two grapevine isolates of Arabis mosaic virus (ArMV) and no apparent homology with any of the sequenced GFLV isolates. Computer-assisted analysis of 2A sequences showed that subgroup c isolates are recombinants between GFLV and ArMV. STUDY OF THE GENETIC VARIABILITY OF FUSARIUM spp. ISOLATED FROM CITRUS. A. Khlij1, H. Barham1, M. Fer- rara1, T. Yaseen2, F. Nigro1 and A. Ippolito1. 1Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli S4.68 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 Studi, Via Amendola 165/A, 70126 Bari, Italy. 2Istituto Agronomico Mediterraneo, Via Ceglie 9, 70010 Valenzano (BA), Italy. Email: anis_khl@yahoo.com Understanding the genetic structure of Fusarium populations attacking citrus plants may provide new insights into their epidemiology and evolutionary potential, thus leading to an improvement of management strategies. Molecular techniques can be used to study population structure by examining many traits at once and providing information unobtainable by other methods. AFLP with a range of selective primer pairs, and DNA sequencing of ”nuclear translation elongation factor 1-alpha (tef)” and “betatubulin (benA)” genes, were used to assess the genetic variability between and within a natural population of Fusarium spp. isolated from citrus plants. Inter and intra-specific genetic similarity was detected. In particular, within species the major genetic similarity (62%) was found among F. oxysporum strains, followed by F. proliferatum (59%), whereas analysis of F. solani strains revealed a high genetic variability which reached 51% especially among Tunisian strains. Isolates of F. solani from the same field or nursery, despite of their genetic variability, shared some polymorphic markers which allowed them to be grouped together, with some exceptions. Comparing sequences of tef and benA genes, Fusarium isolates from citrus were clustered at species level. AFLP technique proved to be sensitive, allowing grouping and differentiation either at the species and intra-species level. A NEW BIOCONTROL STRAIN (NAP71) OF BACILLUS SUBTILIS WITH ISR ACTIVITY. S. Lanzuise1, M. Ruocco2, S.L. Woo1, R. Marra 1, V. Aloj1, R. Quarto1, R. Panza1, F. Vinale1 and M. Lorito1. 1Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Sezione di Patologia Vegetale, Università degli Studi di Napoli, Via Università 100, 80055 Portici (NA), Italy. 2Istituto per la Protezione delle Piante del CNR, Via Università 133, 80055 Portici (NA), Italy. E-mail: lanzuise@.unina.it The selection of biocontrol agents that control plant pathogens and stimulate induced systemic resistance (ISR) in the plant are criteria used in the search for new microbial biopesticides. Bacillus subtilis is effective for the control of a variety of pathogens due to direct antagonism and activation of the plant defence to attack. This work assesses the biocontrol activity of a new strain B. subtilis NAP71 and its ability to induce plant resistance. In vitro studies indicated that NAP71 antibiotic production is probably involved in the control of pathogenic bacteria (Agrobacterium tumefaciens, Pseudomonas syringe pv. tabaci) and fungi (Rhizoctonia solani, Sclerotinia spp., Fusarium spp., Botrytis cinerea), noted by the presence of a clear inhibition zone. In vivo tests on lettuce and tomato showed a high capacity of NAP71 to reduce disease development by Bremia lactucae and B. cinerea respectively. NAP71 treatments to lower leaves of tomato, followed by treatments with B. cinerea to upper leaves one day later, induced systemic resistance, reducing both the number and size of lesions. Applications with NAP71 were synergistic with Kendall (a synthetic resistance inducer), resulting in less disease than either treatment singularly or additively. The culture broth of NAP71 was analyzed by HPLC in order to identify and characterize the compounds that cause ISR. NMR and mass spectrometry identified two compounds as iturin-like lipopeptides (ILLs). These purified molecules showed antimicrobial activity in vitro against pathogenic fungi (B. cinerea, Fusarium spp.) and bacteria (A. tumefaciens), and stimulated ISR in tomato plants. SCREENING OF AMPELOMYCES STRAINS AS CANDIDATE AGENTS FOR THE BIOLOGICAL CONTROL OF GRAPEVINE POWDERY MILDEW. S.E. Legler1, T. Caffi1, L. Kiss2, A. Pintye2 and V. Rossi1. 1Istituto di Entomologia e Patologia Vegetale, Università Cattolica del Sacro Cuore, Via E. Parmense 84, 29100 Piacenza, Italy. 2Plant Protection Institute, Hungarian Academy of Sciences, P.O. Box 102, 1525 Budapest, Hungary. Email: saraelisabetta.legler@unicatt.it Powdery mildews are among the most important plant diseases worldwide. These diseases, caused by biotrophic, epiphytic fungi belonging to Erysiphales, account for the greatest part of the fungicides used in European agriculture and especially in vineyards. An attractive and environmentally safe alternative to fungicide application is the use of biocontrol agents (BCAs), including Ampelomyces spp., a group of hyperparasitic fungi affecting several powdery mildews of different cultivated and wild plants. This work was carried out within the EU-funded project “BCA-grape”, aimed at developing new biocontrol agents for powdery mildew on grapevines. Eighteen strains of Ampelomyces belonging to the culture collection of the Plant Protection Institute of the Hungarian Academy of Sciences, Budapest, were investigated for their ecological requirements and biological characteristics in order to identify candidate BCAs for grapevine powdery mildew. Mycelium growth rate, production of picnidia, and germination of conidia were determined in vitro under different environmental conditions. The mycoparasitic activities of the strains were tested in both controlled and natural environments using potted and field-grown grapevine plants, respectively. In these experiments, Ampelomyces strains were applied to powdery mildew colonies at different colony growth stages. Statistically significant differences were found among the strains and, interestingly, among some of these strains and the commercial product AQ10, which contains A. quisqualis. The preliminary results from this work are promising in that they demonstrate substantial potential for control of grapevine powdery mildew by Ampelomyces spp. MOLECULAR DETECTION OF FUSARIUM OXYSPORUM f. sp. CHRYSANTHEMI ON GERBERA JAMESONII, OSTEOSPERMUM spp. AND ARGYRANTHEMUM FRUTESCENS. Y. Li, M.L. Gullino and A. Garibaldi. Centro di Competenza per l’Innovazione in Campo Agro-ambientale, Università degli Studi di Torino, Via L. da Vinci 44, 10095 Grugliasco (TO), Italy. E-mail: yuanlibeijing@gmail.com A rapid and sensitive molecular diagnostic assay for Fusarium oxysporum f. sp. chrysanthemi, an economically important pathogen of ornamentals, was developed and tested on three hosts (Gerbera jamesonii, Osteospermum spp. and Argyranthemum frutescens). A specific primer set was designed on the basis of single nucleotide polymorphisms (SNPs) of the fow1 gene of F. oxysporum f. sp. chrysanthemi. This primer set was highly specific and able to distinguish F. oxysporum f. sp. chrysanthemi among different formae speciales of F. oxysporum. A 304 bp sequence was amplified from DNA of three isolates of F. oxysporum f. sp. chrysanthemi, no amplification was obtained from DNA of 16 other Fusarium spp. and f. sp. of F. oxysporum. In addition, this primer set was successfully used in nested PCR assays for detecting F. oxysporum f. sp. chrysanthemi on three host plants. The detection limit was as low as 10 fg/µl of genomic DNA. Moreover, nested PCR successfully allowed the detection of F. oxysporum f. sp. chrysanthemi in the three artificially infected sympomless host plants. The developed molecular method will make possible an early diagnosis of this pathogen. Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 RECOVERY OF A COLLETOTRICHUM ACUTATUM MUTANT WITH REDUCED PATHGENICITY BY TRANSPOSON TAGGING. M.G. Li Destri Nicosia1, S.O. Cacciola2, G.E. Agos- teo1, M.J. Daboussi3 and G. Magnano di San Lio1. 1Dipartimento di Gestione dei Sistemi Agrari e Forestali, Università Mediterranea, Località Feo di Vito, 89122 Reggio Calabria, Italy. 2Dipartimento di Chimica Biologica, Chimica Medica e Biologia Molecolare, Università degli Studi, Viale Andrea Doria 6, 95125 Catania, Italy. 3Institut de Genetique et Microbiologie, CNRS, UMR8621, University of Paris-Sud, 91405 Orsay, France. E-mail: gmagnano@ unirc.it In this study, impala, a DNA transposable element isolated from the phytopathogenic fungus Fusarium oxysporum, was shown to be active in Colletotrichum acutatum, the causal agent of olive anthracnose. A C. acutatum mutant impaired in nitrate reductase function was co-transformed with the plasmids pNI160, carrying the niaD::impala construction, and pAN7.1, that confers resistance to hygromycin. Seventy six hygromycin-resistant transformants were obtained and among them 55 impalatransformants were selected by PCR with impala-specific primers. The first evidence of impala transposition was obtained by selecting niaD+ revertants by the phenotypic excison assay. Two out of three co-transformants analysed gave rise to niaD+ revertants. Transposon excision from the niaD gene and its reinsertion in a new genomic site in revertants were shown by Southern blotting analysis. Further evidence of impala transposition was obtained by sequencing the typical transposon footprints left at the niaD site of excision. Twenty four mono-conidic revertant strains were tested for their pathogenicity and a mutant with reduced virulence on olive drupes was recovered. Isolation and analysis of impala flanking regions in this mutant revealed that the transposon was inserted in a coding region containing a conserved domain of the kinesin family, motor proteins associated with microtubules, thus suggesting a role of this putative kinesin in C. acutatum pathogenicity. Moreover, the mutant impaired in pathogenicity showed a peculiar morphology of condia, possibly as a a consequence of altered cell division. This is in agreement with previous studies indicating that kinesin mutations can affect cell division in filamentous fungi. AMPLIFICATION AND CHARACTERIZATION OF THE INTERGENIC REGIONS OF RIBOSOMAL DNA IN PENICILLIUM DIGITATUM, P. ULAIENSE AND P. ITALICUM. A.M. Lig- orio1, K. Youssef1,2, F. Nigro1 and A. Ippolito1. 1Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. 2Plant Pathology Research Institute, Agriculture Research Centre, Giza, Egypt. Email: ippolito@agr.uniba.it Penicillium digitatum, P. italicum and P. ulaiense, are responsible for the green, blue and whisker mould of citrus fruit, respectively. A more rational approach for controlling postharvest diseases rely on the early and univocal identification of the causal agents. In P. digitatum, P. italicum and P. ulaiense, the Internal Transcribed Spacer (ITS), the most widely utilised targets to develop molecular markers, are not sufficiently variable for designing species-specific primers. By convese, the IGS regions have great potential since they are multicopy and their length (1000–4000 bp) provides considerable space for species-specific markers development. In the present work, the IGS region of the three Penicllium species was amplified and sequenced using universal primers, designed by comparing IGS flanking regions from a large number of fungi. The total length of the amplified IGS region was 2850 bp and 2600 bp for P. ulaiense and P. digitatum, re- S4.69 spectively. A partial sequence, approximately 1800 bp, was also obtained for P. italicum. The sequenced IGS region showed tandem repeated units, five in P. ulaiense (two of 65 nucleotides and three of 35 nucleotides) and six in P. digitatum (59 nucleotides each). Species-specific primer pairs, Sppenula1F/Sppenula1R and Sppendig3F/Sppendig2R, designed on the most variable IGS regions, amplified amplicons of 352 and 480 bp in P. ulaiense and P. digitatum, respectively. Primer specificity was successfully tested in PCR assays using DNA from a range of fungal taxa as template. Studies are in progress to complete the IGS region sequence of P. italicum. INTEGRATION OF BIOCONTROL YEASTS AND NEW FUNGICIDES FOR CONTROLLIG PENICILLIUM EXPANSUM AND PATULIN CONTAMINATION IN APPLES. G. Lima1, A. Sgrignuoli1, A. Ritieni2, R. Ferracane2 and R. Castoria1. 1Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Università degli Studi del Molise, Via F. De Sanctis, 86100 Campobasso, Italy. 2Dipartimento di Scienza degli Alimenti, Università di Napoli Federico II, Parco Gussone, Portici (NA), Italy. Email: lima@unimol.it Penicillium expansum is the causal agent of blue mould, a major postharvest disease of pome fruits, and is also responsible for the production of the mycotoxin patulin, which contaminates infected fruits and derived products. Thiabendazole is currently used for postharvest treatment. The use of this benzimidazole fungicide, however, is nor deprived of toxicological risks and is often ineffective beacue of the raise and large diffusion of P. expansum benzimidazole-resistant strains in packinghouses. For these reasons, new effective fungicides and safer control strategies are needed. The objective of this study was to assay the combined application of two selected biocontrol yeasts, Rhodotorula glutinis LS11 and Cryptococcus laurentii LS28, with low dosages of thiabendazole or of more recently developed fungicides such as boscalid, cyprodinil and fenexamide, to control more efficiently blue mould on stored apples. Isolates LS11 and LS28 were compatible in vitro with high doses of boscalid and cyprodinil. In vitro assays also showed that several thiabendazole-resistant strains of P. expansum were strongly inhibited by cyprodinil. Further experiments performed in vivo on wounded apples treated with combinations of the biocontrol yeasts and cyprodinil showed that a more effective and prolonged control of blue mould was obtained by the biocontrol yeasts when applied together with cyprodinil at a low dose. This treatment also resulted in lower levels of patulin and fungicide residues in apples. Our data suggest that the integration of biocontrol yeasts with a low rate of cyprodinil may be an useful and safer strategy to control the attack of P. expansum to stored apples. TEMPORAL AND SPATIAL ENDOPHYTIC INCIDENCE OF BISCOGNIAUXIA MEDITERRANEA IN A DOWNY OAK FOREST IN SARDINIA. B.T. Linaldeddu, B. Scanu, P. Corda and A. Franceschini. Dipartimento di Protezione delle Piante, Sezione di Patologia Vegetale, Università degli Studi, Via E. De Nicola 9, 07100 Sassari, Italy. E-mail: ben@uniss.it Infections of Biscogniauxia mediterranea are considered one of the key factors in the aetiology of oak decline in Mediterranean countries. This pathogen can parasitize all plant organs symptomlessly as an endophyte, but colonizes also woody tissues causing branch dieback and typical “charcoal cankers” in trunks and branches. Apart from the events leading to the pathogenic ex- S4.70 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 pression of this fungus little is known on the dynamics of its endophytic behaviour. In this study the temporal and spatial variations of B. mediterranea endophytic infections were investigated in a downy oak forest located in North-Western Sardinia (Italy). An area with dead and declining trees showing charcoal cankers was delimited, plus three other sites 400, 850 and 2000 m away from it, comprising trees with no charcoal cankers. Five downy oak trees were selected in each site and ten apparently healthy leaves from each tree were collected at random throughout the growing season in order to detect the pathogen. Results showed that the isolation frequency (IF) of B. mediterranea rose with the age of the leaves in all sites. The highest IF values were found in trees located inside the declining site, whereas no significant differences were detected among trees of the other sites. Therefore B. mediterranea is able to greatly increase its inoculum density in infected areas, but the spatial spread of the inoculum is limited. Consequently the risk of epidemic outbreaks is strictly linked to the rise in susceptibility of plants in which the pathogen can develop its reproductive forms. DEVELOPMENT OF DEGENERATE PRIMERS FOR UNIVERSAL DETECTION OF OLIVE-INFECTING NECROVIRUSES BY RT-PCR. G. Loconsole1, M. Saponari2, V. Savino1 and G.P. Martelli1,2. 1Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. 2Istituto di Virologia Vegetale del CNR, Unità Organizzativa di Bari, Via Amendola 165/A, 70126 Bari, Italy. E-mail: giuliana.loconsole@agr.uniba.it Three necrovirus species, Olive latent virus 1 (OLV-1), Olive mild mosaic virus (OMMV) and Tobacco necrosis virus (TNV), infect olive trees. OMMV was initially identified as a strain of Tobacco necrosis virus D (TNV-D) based on its coat protein sequence. However, when the complete genome sequence became available, the putative RNA-dependent RNA polymerase (RdRp) was found to have high sequence homology with OLV-1 and TNV-A. Therefore, OMMV was suggested to be a new virus which had likely developed following gene exchange between OLV-1 (or TNV-A) and TNV-D. Since the protocol for the production of certified olive propagating material requires absence of OLV-1 and TNV/OMMV sensitive and reliable diagnostic methods are needed for unambiguous sanitary assessment. Based on the multiple alignments of the full genome sequences of OLV-1, TNV-D, TNV-A and OMMV, the RdRp gene was selected to design different universal primer pairs. Total nucleic acid extracts from herbaceous hosts infected with OLV-1, OMMV, TNV-A, and TNV-D were used to test the primers in RT-PCR reactions. Among the primers tested, the pair denoted NECf/r proved to be the most reliable. An amplicon of the expected size (385 bp) was obtained from all infected samples, while there was no amplification from healthy controls. The NECf/r-generated amplicons obtained from each single infected plant were sequenced, confirming to belong to OLV-1, OMMV, TNV-A, TNV-D, respectively. These results provide evidence that the newly designed NECf/r primers can detect all extant olive-infecting necroviruses using a single RT-PCR reaction. Twenty strains of Ralstonia solanacearum, the causal agent of bacterial wilt of solanaceous crops, collected during systematic monitoring of greenhouse tomato cultivations in 2007 and 2009 in Sardinia from tomato plants, soil and water, were characterized using different approaches. The disease occurred on ungrafted tomato plants of cvs Arawak, Cuore di Bue and Ikram, and on Cuore di Bue grafted onto the interspecific hybrids “Beaufort” and “He-Man”. Affected plants showed collapse and wilting, stunting and chlorosis and the presence of adventitious roots on the stem. Fluidal colonies were isolated on SMSA and Kelman’s tetrazolium media and selected for their typical morphology. The twenty selected strains were pathogenic to tomato and geranium plantlets, and were identified by Biolog, IFAS and by conventional PCR using the primers OLI-1 and Y-2. Phylotype characterization performed following Fegan and Prior (2005) scheme revealed that all strains belonged to phylotype II, which comprises strains belonging to biovar 1, 2 and 2T of R. solanacearum. It contains also R. solanacearum race 3, a of potato and tomato pathogen, and race 2, a banana pathogen. Phylotype II strains had considerable phylogenetic diversity, including 10 different sequevars. Amplification and sequencing of the endoglucanase gene (egl) of these strains and phylogenetic analysis performed with known egl sequence, showed that these Sardinian strains falls in sequevar 1. Further genomic characterization of these strains was performed by amplification and sequencing of the endoglucanase gene, by rep-PCR and by M13-PCR fingerprinting. These preliminary results showed similarity among R. solanacearum strains used in this work. DEVELOPMENT OF QUANTITATIVE REAL-TIME RT-PCR FOR THE DETECTION OF PEACH LATENT MOSAIC VIROID. M. Luigi1,2, M. Barba1 and F. Faggioli1. 1CRA, Centro di Ricerca per la Patologia Vegetale, Via C.G. Bertero 22, 00156 Roma, Italy. 2Dipartimento di Gestione dei Sistemi Agrari e Forestali, Università Mediterranea, Località Feo di Vito, 89060 Reggio Calabria, Italy. E-mail: francesco.faggioli@entecra.it Peach latent mosaic is a cosmopolitan disease caused by Peach latent mosaic viroid (PLMVd). The main symptoms are reduction of fruit quality and tree vigour and increased susceptibility to other biotic and abiotic stresses. Moreover, infected trees exhibit a delay in leaf emergence, flowering and fruit ripening, malformed fruits showing cracked sutures and enlarged pits, bud necrosis and early decline. A new primer pair and a probe were designed to perform a real-time RT-PCR assay based on TaqMan™ chemistry. The method was tested for its sensitivity, reproducibility and specificity. Sensitivity was evaluated using serial dilutions of in vitro transcripts from cloned whole PLMVd genome. Dilutions were used as standard for quantification of the viral template in infected samples. The method was also evaluated for its specificity using healthy peach, apricot, plum and pear controls and non-target viroids , i.e. Apple scar skin viroid, Hop stunt viroid, Pear blister canker viroid and Potato spinder tuber viroid. Reproducibility was evaluated using as template 20 different PLMVd isolates from peach, including three ‘calico’ variants. Moreover, four total RNA extraction methods were compared to determine the best extraction protocol to be used in the assessment of PLMVd quantification. PRELIMINARY GENOMIC CHARACTERIZATION OF RALSOLANACEARUM STRAINS ISOLATED FROM TOMATO IN ITALY. S. Loreti1, M. Fiori2, A. Gallelli1, V. Fiori2, STONIA V. Ligios2 and G. Perez1. 1CRA, Centro di Ricerca per la Patologia Vegetale, Via C.G. Bertero 22, 00156 Roma, Italy. 2Dipartimento di Protezione delle Piante, Università degli Studi, Via E. De Nicola, 07100 Sassari, Italy. E-mail: stefania.loreti@entecra.it HISTOLOGICAL STUDIES OF RFID-MARKED GRAPEVINES. A. Luvisi1, A. Panattoni1, A. Colosimo1, E. Rinaldelli2, M. Pagano2, R. Bandinelli2, B. Gini3 and E. Triolo1. 1Dipartimento di Coltivazione e Difesa delle Specie Legnose “G. Scaramuzzi”, Uni- Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 versità degli Studi, Via del Borghetto 80, 56124 Pisa, Italy. 2Dipartimento Ortoflorofrutticoltura, Università degli Studi di Firenze, Viale delle Idee 30, 50019 Sesto Fiorentino (FI), Italy. 3Vivai New Plants, Via Togliatti 41, 56040 Cenaia (PI), Italy. E-mail: etriolo@agr.unipi.it Grapevine plants marked by Radio frequency identification (RFID) system were monitored to determine whether the system interfered with the growth and health status of the vines. TAGs were inserted inside the rootstock pith in 2007, three cm below the grafting point, following two different procedures: (A) direct drilling of the pith from the upper end of the rootstock; (B) making a “U” cut laterally on the rootstock with a specially designed machine that involved tissues from bark to pith. After placing the microchip inside the pith, grafting was made. In 2008 and 2009, growth was monitored and histological observations were made on freshly cut trunk sections (20 µm thick) in proximity of the microchip. Based on growth data, the vines subjected to procedure A did not show any modification in the tissues compared with the control. Indeed, the insertion method did not cause additional wounds to the rootstock as only a small portion of pith is removed to accommodate the microchip. Conversely, procedure B was more invasive due to the “U” wound, causing partial loss of functionality in terms of open vessels. Image analysis of transections revealed that the damaged area did not exceed 27% and 19% of the total vascular area in 2008 and 2009, respectively. The extent of histological damage was limited to the microchip position: no histological alteration was noted above and below the microchip, and no growth decrement was recorded in the marked plants, compared with control. CONTROL OF VIRUSES IN SOIL BY STEAMING AND EXOTHERMIC REACTION. A. Luvisi, A. Colosimo, A. Mater- azzi and E. Triolo. Dipartimento di Coltivazione e Difesa delle Specie Legnose “G. Scaramuzzi”, Università degli Studi, Via del Borghetto 80, 56124 Pisa, Italy. E-mail: etriolo@agr.unipi.it Experimental trials were carried out in open-field conditions to assess the effectiveness of steam and chemical exothermic reaction (potassium hydroxide) against three widespread plant viruses: Tobacco mosaic virus (TMV), Potato virus Y (PVY) and Cucumber mosaic virus (CMV). In addition to crop residues, native flora is known as an important virus reservoir, thus a pre-planting treatment by steam/potassium hydroxide could be useful not only for fungal, bacterial and weed control, but also against viruses in the soil. Treatments were carried out with a self-propelled soilsteaming machine designed for steam release after incorporation into the soil of a substance that causes an exothermic reaction characterized by a thermal peak, capable of providing a satisfactory control of various soil-borne pathogens up to a 20 cm depth. Purified preparations of the three viruses were thoroughly mixed with air–dried soil samples taken from each experimental plot. The resulting mixtures were placed in nylon screen bags. Three bags per treatment were buried in each plot at depths of 14-16 cm 24 h before treatments with steam or steam/KOH. Significantly lower amounts of each virus were recovered from treated than untreated soil, in particular from soil samples that had been exposed to steam/KOH. Following mechanical inoculation of tobacco plants, no PVY and CMV infectivity was recovered from soil samples exposed to steam/KOH, whereas a significant reduction of infectivity was observed for TMV. These results show that this method can be useful for wide-spectrum control of soil-borne pathogens. S4.71 GENETIC VARIABILITY OF A POPULATION OF PHYTOPHTHORA NICOTIANAE ISOLATES FROM VARIOUS HOSTS. M.A. Mammella1, L. Schena1, A. Pane2, S.O. Cacciola3 and G. Magnano di San Lio1. 1Dipartimento di Gestione dei Sistemi Agrari e Forestali, Università Mediterranea, Località Feo di Vito, 89122 Reggio Calabria, Italy. 2Dipartimento di Scienze e Tecnologie Fitosanitarie, Università degli Studi, Via S. Sofia 100, 95123 Catania, Italy. 3Dipartimento di Chimica biologica, Chimica medica e Biologia molecolare, Università degli Studi, Viale Andrea 6, 95125 Catania, Italy. E-mail: gmagnano@unirc.it Phytophthora nicotianae is a polyphagous pathogen with more than 1000 hosts. The genetic variability of a population of P. nicotianae isolates of A1 and A2 mating types from diverse host plants and geographic origins was determined analysing two intergenic regions of the mitochondrial DNA. Two variable intergenic regions flanked by genes Trny and Rns (Trny/Rns) and genes Trnw and Cox2 (Trnw/Cox2) were identified by comparing the whole mitochondrial genome of P. infestans, P. ramorum and P. sojae and were amplified and sequenced using primers designed on flanking conserved genes. Amplified regions showed a very variable length comprised between 429 and 927 bp (Trny/Rns) and between 323 and 600 bp (Trnw/Cox2). All sequences were aligned using Clustal X and the phylogenetic analysis was performed with the Neighbor-Joining method. The analyses of the Trny/Rns region enabled the identification of 16 clusters (threshold limit 0.001). No correlation was found between the grouping of isolates and their mating type, growth rate, colony morphology, place and host of origin, with the only exception of isolates from citrus. All of them were in fact of the A1 mating type and almost all were comprised in the same cluster, irrespective of their geographic origin. Similar results were also obtained with the Trnw/Cox2 region although this latter region enabled a less accurate separation of the isolates. Mitochondrial intergenic regions are suitable for studying intraspecific genetic variability in P. nicotianae. INFLUENCE OF LOW OXYGEN LEVELS ON PENICILLIUM EXPANSUM DEVELOPMENT. M. Mari, R. Gregori, I. Donati and P. Bertolini. CRIOF, Dipartimento di Protezione e Valorizzazione Agro-Alimentare, Università degli Studi, Viale G. Fanin 46, 40127 Bologna, Italy. Email: marta.mari@unibo.it Blue mould caused by Penicillium expansum is one of the main postharvest diseases of pome fruits. In the past it has been controlled by postharvest chemical treatments, mainly thiabendazole, but the development of fungal strains resistant to this fungicide has reduced the effectiveness of treatments. To overcome this problem and to meet the requirements for fungicide-free products, various non chemical approaches have been investigated. Physical treatments, including storage atmosphere manipulation, have been applied to control postharvest fungal decay. The present study aimed at determining the influence of oxygen (O2) levels on the growth of P. expansum (P37 strain) in vitro and in vivo. Patulin production was also tested. In in vitro trials, 100 µl of conidial suspension (103 conidia per ml) were spread on PDA dishes, stored for 30 days at 0°C with 0.7, 1.5, 3, 6 and 21% O2. Carbon dioxide concentration was 1%. In in vivo trials, fruits were wounded and inoculated with 20 µl of a conidial suspension (103 conidia per ml) of P. expansum and stored with the same oxygen concentrations as above for five months at 0°C plus 3 days at 20°C. No significant differences were observed on P. expansum growth in vitro among different oxygen concentrations. The percentage of infected fruits was significantly higher at low oxygen concentrations (0.7, 1.5, 3%) than at 6 or 21%. Decay le- S4.72 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 sions on apple exposed to 6 and 21% O2 were significantly larger than those of apples stored at 0.7, 1.5 and 3% O2. PROTEOME-LEVEL DIFFERENCES IN THE INTERACTIONS BETWEEN BRASSICA NAPUS cv. SURPASS 400 AND VIRULENT OR AVIRULENT STRAINS OF LEPTOSPHAERIA MACULANS. R. Marra1, H. Li2, M.J. Barbetti3, K. Sivasitham- param2, F. Vinale1, S. Woo1, M. Ruocco1, S. Lanzuise1, P. Cavallo4 and M. Lorito1. 1Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università degli Studi di Napoli Federico II, Via Università 100, 80055 Portici (NA), Italy. 2School of Earth and Geographical Sciences, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley, WA 6009, Australia. 3School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley, WA 6009, Australia. 4Dipartimento di Scienze dell’Educazione, Università degli Studi di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy. E-mail: matteo.lorito@unina.it Leptosphaeria maculans causes blackleg disease on a wide range of oilseed Brassica species determining severe yield losses worldwide. The spring-type B. napus cultivar “Surpass 400” is a highly resistant cultivar to L. maculans, but examples of resistance breakdown have been reported. Many of the molecular factors involved in cv. Surpass 400 defence response against L. maculans, particularly in the case of resistance-breaking isolates, are yet unknown. In this work, we investigated, at the proteome level, the factors differentially produced by cv. Surpass 400 during compatible or incompatible interactions with L. maculans strains. We used 2D gel electrophoresis and in silico processing of the data to separate and analyse the differential proteins accumulated in this cultivar during the interaction with either a non resistance-breaking (UWA P11) or a resistance-breaking (UWA 192) isolate of L. maculans. Protein spots were identified by homology using either MALDI/TOF mass spectrometry (MS) or data from the literature. We found numerous differential spots involved in metabolic pathways or stress-related responses, which showed a marked increase of their relative intensity in the disease-determining combination (cv. Surpass 400 UWA 192), but not in the HR-inducing interaction (cv. Surpass 400 - UWA P11), as compared with the un-inoculated control. Our data indicate that B. napus susceptibility or resistance to a specific L. maculans isolate involves both a mechanism of plant defence suppression by the pathogen and an extensive modification of basic pathways, such as those that regulate nutrient metabolism, CO2 fixation, and antioxidative responses. PRELIMINARY INVESTIGATIONS ON ITALIAN STRAINS OF ALTERNARIA CAUSING A NEW APPLE DISEASE. K. Marschall1, E. Pattori2, E. Ortalda2, F. Rotondo3, M. Collina3, A. Brunelli3 and V. Rossi2. 1Centro per la Sperimentazione Agraria e Forestale di Laimburg, Vadena Laimburg 6, 39040 Ora, Italy. 2Istituto di Entomologia e Patologia Vegetale, Università Cattolica del Sacro Cuore, Via Emilia Parmense 84, 29100 Piacenza, Italy. 3Dipartimento di Protezione e Valorizzazione Agroalimentare, Università degli Studi, Viale G. Fanin 46, 40127 Bologna, Italy. E-mail: mcollina@agrsci.unibo.it A new apple tree disease, probably caused by a new pathotype of Alternaria alternata, was observed for the first time in Alto Adige (North-East Italy), in the year 2000. In the following years the disease spread southwards in Trentino, Veneto, Piedmont and, perhaps, Emilia-Romagna. The disease usually appears on the leaves in late spring as little, irregular to round, brown-reddish or black spots, sometimes surrounded by a reddish halo. Spots increase in number during the season and coalesce to form wide necrotic areas. Affected leaves can fall prematurely. Brown-blackish spots, 0.5 to 2 mm in diameter, appear also on the fruits. Incidence of affected fruits varies over the year and generally increases in the pre-harvest period. Preliminary investigations showed that the Italian strains of Alternaria from both leaves and fruits are probably able to cause the disease by producing host specific toxins, but these strains do not belong to the pathotype mali of A. alternata present in other apple-growing areas of the world. A bioassay was developed for detecting the Alternaria strains pathogenic to apple. Different inoculation methods were tested on wounded or unwounded leaves and fruits. A ring-test was carried out using five fungal isolates to evaluate repeatability and reproducibility of the bioassay. Necrotic spots consistently appeared only on wounded tissues of inoculated organs after 24-48 h of incubation. Availability of a robust bioassay is the first step for studying population dynamics of the apple-pathogenic strains, characterizing strains at the morphological, ecological, biochemical, and molecular levels, and investigating host–pathogen interactions. EFFICACY OF COMPOSTS ACTIVATED WITH THREE ISOLATES OF STREPTOMYCES spp. IN THE CONTROL OF TOMATO CORKY ROOT. A.D. Marsico1, A.M. Ligorio2, M. D’Amico1, M. Amenduni1 and M. Cirulli1. 1Dipartimento di Biologia e Patologia vegetale, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. 2Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. E-mail: amenduni@agr.uniba.it The efficacy of three commercial composts (obtained from distillery residues, cattle manure and green waste) and a commercial organic amendment (obtained from slaughterhouse residues) was evaluated for the control of tomato corky root, caused by Pyrenochaeta lycopersici, in a pot- experiment. The amendments were mixed at a rate of 10%, with soil, which was then artificially infested with the pathogen. The distillery residue compost significantly reduced corky root severity more than the other two composts, while the slaughterhouse residue amendment was phytotoxic. In addition, the distillery residue compost caused an increase in the soil populations of Bacillus spp., fluorescent pseudomonads and actinomycetes. Ability of three antagonistic streptomycete isolates to colonize the amendments tested was evaluated in vitro. The highest colonization level was observed in the distillery residue compost, especially when pasteurized. The distillery residue and the green waste composts, alone or in combination with three streptomycete isolates, and with or without pasteurization, were evaluated for the efficacy in controlling tomato corky root. Disease severity was assessed 90 days after transplanting. Activation of plant defense processes was periodically evaluated by quantitative analyses of plant enzyme systems such as chitinase, β-1,3-glucanase, peroxidase and phenylalanineammonia-lyase, and compared with those induced by the synthetic resistance activator acibenzolar-S-methyl. ARTICHOKE ITALIAN LATENT VIRUS AND RNA SILENCING: IMPLICATIONS IN SANITATION SCHEMES OF NEPOVIRUS-INFECTED PLANTS. T. Mascia1, M.I. Prigigallo2 and D. Gallitelli1,2. 1Istituto di Virologia Vegetale del CNR, Unità Organizzativa di Bari, Via Amendola 165/A, 70126 Bari, Italy. 2Dipartimento di Biologia e Patologia Vegetale, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. E-mail tizianamascia@ email.it Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 Artichoke Italian virus (AILV) is a nepovirus infecting globe artichoke and other plants in the family Compositae. Recent surveys in Apulia (Southern Italy) have shown that AILV infections are very frequent and detrimental to globe artichoke verieties of the reflowering type highlighting the importance of establishing new fields with virus-free propagation material. A sanitation protocol based on meristem tip culture (MTC) proved ineffective for AILV elimination. By contrast, MTC combined with either in vitro or in vivo thermopterapy eradicated the virus, so that virusfree plants were obtained. Recent insights propose that RNA silencing could be involved in the degradation of viral RNAs before entering meristems, a mechanism that is enhanced by thermoterapy so that the combined action should produce virus-free plants. AILV is seed-transmitted thus, it must be able to overcome the meristem exclusion mechanism. Most plant viruses code for genes that counteract host-mediated RNA silencing but no silencing suppressors have been identified in nepoviruses. After an initial phase of symptom appearance, plants infected by nepoviruses usually undergo a phase of recovery in which symptoms disappear. The recovery phenotype was attributed to the inability of nepoviruses to contrast silencing but it is still a matter of discussion whether the recovery phenotype could be associated with a strong reduction of virus titre in recovered tissues or not. As a preliminary approach to understand why AILV escaped sanitation by MTC, we have addressed some of these points in AILV infections in tobacco. COMPETITION FOR CROP RESIDUES AS A TOOL TO REDUCE FUSARIUM HEAD BLIGHT. F. Matarese. Dipartimento di Coltivazione e Difesa delle Specie Legnose “G. Scaramuzzi”, Sezione Patologia Vegetale, Università degli Studi, Via del Borghetto 80, 56124 Pisa, Italy. E-mail: fmatarese@agr.unipi.it Biological control of Fusarium graminearum and F. culmorum seems to be a considerably promising strategy to control Fusarium head blight (FHB) of wheat. This disease causes extensive damage by reducing yield and grain quality due to the presence of damaged kernels and of mycotoxins such as deoxynivalenol (DON). The aim of this work was to select fungal antagonists able to compete with FHB causal agents in crop residues. Potential antagonists were screened on the basis of their growth in presence of DON in multi-wells microplates. Ten of 100 Trichoderma spp. and 2 of 40 Pythium spp. strains showed a statistically comparable growth rate in the presence/absence of DON. Their cultural filtrates were submitted to HPLC analysis in order to investigate the fate of the mycotoxin. The 10 Trichoderma spp. isolates were also used in an in vitro test to investigate their antagonistic activity against a Fusarium culmorum and a F. graminearum mycotoxigenic strains. Only three Trichoderma strains, T. atroviride 6317, 6085 and T. velutinum 4837 seemed to be effective, showing antibiosis and mycoparasitism. Finally, T. velutinum 4837 was used in a preliminary competition test on natural substrates in order to evaluate the ability to prevent DON production by F. graminearum. The positive results obtained can be considered as a promising starting point for further investigations. PRESENCE AND DISTRIBUTION OF OLIVE-INFECTING VIRUSES IN TUSCANY AND LIGURIA. A. Materazzi, H. Bouyahia and E. Triolo. Dipartimento di Coltivazione e Difesa delle Specie Legnose “G. Scaramuzzi”, Sezione di Patologia Vegetale, Università degli Studi, Via del Borghetto 80, 56124 Pisa, Italy. E-mail: amatazzi@agr.unipi.it S4.73 In the framework of the national project “OLVIVA” addressing the conservation and multiplication of Italian olive (Olea europaea L.) varieties through the production of healthy “primary sources”, we have assayed 89 mother plants of 20 Tuscan and 8 Ligurian varieties from the respective regional varietal collection. In accordance with the project guidelines and respecting previously established protocols, all trees were sampled and tested twice (spring and autumn) in 2007-2009. Samples consisting of ten shoots were collected from the quadrant of the canopy. Phloem tissue recovered by scraping was powdered in liquid nitrogen and total RNA was extracted. One-step RT-PCR was performed for detection of nine olive-infecting viruses. i.e. Arabis mosaic virus (ArMV), Cherry leafroll virus (CLRV), Cucumber mosaic virus (CMV), Olive latent virus 1 (OLV-1), Olive latent virus 2 (OLV-2), Olive latent ringspot virus (OLRSV), Olive leaf yellowing-associated virus (OLYaV), Strawberry latent ringspot virus (SLRSV) and Tobacco necrosis virus (TNV). Molecular analysis revealed that 9 out of 89 (10.1%) samples were infected. Of the nine viruses looked for only OLV-1, OLYaV and SLRSV were detected with and incidecne of 6.7%, 2.2% and 1.1%, respectively. In particular, OLV-1 was detected exclusively in Liguria in six mother plants belonging to three different varieties (Razzola, Taggiasca and Merlina). The remaining three positive samples were found in the Tuscan varietal collection where two mother plants of cv. San Francesco were infected by OLYaV and one mother plant of cv. Leccio del Corno with SLRSV. The results indicate a satisfactory sanitary status of olive germplasm in Tuscany and Liguria enabling the constitution of at least two “primary sources” from all tested varieties. Work supported by the Project “OLVIVA-Qualificazione del vivaismo olivicolo”. GENETIC VARIATION AND MATING TYPES OF PODOSPHAERA XANTHII IN APULIA. M. Miazzi, C. Laguardia and F. Faretra. Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. E-mail: faretra@agr.uniba.it Seventy-eight isolates of Podosphaera xanthii, one of the causal agents of cucurbit powdery mildew, were collected in Apulia (Southern Italy) from naturally infected leaves from 38 fields in different locations, hosting different cucurbit species. Isolates were crossed by pairing in dual cultures with each of two reference strains of known and opposite mating types and fertility was judged based on the appearance of cleistothecia and ascospores. Genetic variation was explored by random amplified polymorphic DNA (RAPD) analysis using 18 primers, which provided reproducible electrophoretic patterns. All the isolates belonged to a single mating type, which explains why cleistothecia of P. xanthii have never been found in Apulia. RAPD analysis yielded a total of 236 amplicons, 216 of which were polymorphic markers. A high degree of variation was observed since each isolate displayed a unique aplotype. Cluster analysis of RAPD data did not discriminate isolates on the ground of host plants, mating types, physiological races, and locations. This finding suggest the existence of an efficient source of variation in fungal populations, but the occurrence of sexual recombination is prevented by the apparent absence of one of the two mating types. Further investigations are needed to confirm these data. Furthermore, efficient measures should be taken to prevent the introduction of the P. xanthii mating type not yet present in the region. S4.74 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 PHYSIOLOGICAL RACES OF THE CUCURBIT POWDERY MILDEW AGENT IN APULIA. M. Miazzi, C. Laguardia and F. Faretra. Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. E-mail: faretra@agr.uniba.it The development of cultivars displaying durable resistance to powdery mildew is one of the major aims of cucurbit breeding programmes. For a reliable assessment of the resistance of different plant genotypes, a comprehensive knowledge on the virulence of the pathogen is required. Several physiological races of Podosphaera xanthii, the obligate biotrophic fungus agent of powdery mildew of cucurbits are known. For monitoring their distribution in Apulia (Southern Italy), 84 monoconidial isolates coming from 38 fields from different locations were obtained and maintained over a period of 3 years on cotyledons of the highly susceptible zucchini cv. Diamant 1. Physiological races were sorted out with an assay based on a set of differential hosts made up of 8 melon genotypes. Results showed that, in addition to race 2 (17% isolates), already reported in the area, races 0 (1% isolates), 1 (20%), 3 (15%), 4 (4%), and 5 (25%) were also found. About 18% of the isolates did not belong to any race, which suggests the existence of still unknown physiological races of the fungus. Differences in the distribution of races were found depending on the location of fields. The detection frequency of different races varied also with host species: race 1 was prevalent (46% of isolates) on zucchini and race 5 (44%) on Cucumis melo. Race 3 was found only on C. melo, with some differences among plant genotypes, i.e. 16% on “winter melon”, 24% on “carosello” and 9% on “barattiere”. EPIDEMIOLOGICAL STUDY OF LASIODIPLODIA THEOBROMAE IN A SICILIAN VINEYARD. V. Mondello, G. Conigliaro, A. Alfonzo, L. Torta and S. Burruano. Dipartimento Dipartimento di Scienze Entomologiche, Fitopatologiche, Microbiologiche, Agrarie e Zootecniche, Sezione di Patologia Vegetale e Microbiologia Agraria, Università degli Studi, Viale delle Scienze, 90128 Palermo, Italy. E-mail: ezio_mondello@inwind.it During a study on Esca disease in Sicily an unusual decline was observed in a 14-year-old vineyard of cv. Insolia, located in Marsala (Western Sicily). The affected plants showed retarded growth, dieback of some spurs, and brown arc-shaped necrosis in cross-sectioned woody cylinder. Isolation and pathogenicity tests showed that the ascomycete Lasiodiplodia theobromae (Pat.) Griffon et Maubl. was the incitant of the disease. For a better evaluation of the occurrence and importance of the decline and associated symptoms an epidemiological study was carried out in 20072008 in the same vineyard. The incidence and severity of decline were evaluated on 550 vines initially twice a month, starting at sproutin time (April) until fruit-setting (June), then once a month, until October (post-harvest). In both years the decline reached a peak during pre-flowering, gradually decreasing towards harvest. Several affected plants, showing severe symptoms in 2007, did not sprout in 2008 and that the number of symptomatic plants increased by 1.7 % in 2008. SOIL MICROBIAL COMMUNITY AND COMPOST AMENDMENT IN AN ORGANIC SYSTEM: PRELIMINARY DATA. M. Montanari1, G. Innocenti1, C. Ciavatta2 and S. Scagliarini3. 1Dipartimento Protezione Valorizzazione Agroalimentare, Università degli Studi, Viale Fanin 46, 40127 Bologna, Italy. 2Dipartimento di Scienze e Tecnologie Agroambientali, Università degli Studi, Viale Fanin 40, 40127 Bologna, Italy. 3Centro Agricoltura e Ambiente Giorgio Nicoli, Via Argini Nord, 3351 Crevalcore (BO), Italy. Email: gloria.innocenti@unibo.it We studied the effect of a commercial compost amendment at the rate of 8 and 16 t/ha/year, on: (i) the number of cultivable fungi and bacteria in the soil, of fluorescent pseudomonads and actinomycetes in the rhizosphere of root fragments, by plate count on selective media; (ii) total soil activity expressed as concentration of fluorescin diacetate (FDA); (iii) soil health evaluated as suppressiveness using the pathosystem Pythium spp.-Beta vulgaris. Control treatments were two, i.e. a nitrogen organic treatment, and an unamended control. The experimental site was an organic pear orchard near Bologna (Northern Italy). Each plot was 232 m2 with 29 plants, and each treatment was replicated four times in a randomised block design. Soil samples were collected in the early autumn from 2006 to 2008 to a depth of 30 cm. The compost amendment at the highest rate significantly increased the total soil activity. Compost is a source of nutrients for soil micro-organisms thus, as a consequence, their activity is enhanced. Soil health was also improved by compost treatment. It has been reported that soil suppressiveness against plant pathogens such as Pythium and Phytophtora is positively related with microbial activity. However, the suppressiveness of compost is a complex phenomenon, not related only to microbial activity. The association of a species of Penicillium with compost treatments was observed in 2007 and 2008, and could constitute an indication of compost effect on soil microbiota. This aspect is now under investigation. IDENTIFICATION AND ANALYSIS OF CIS-ACTING REGULATORY MOTIFS ON THE PROMOTER OF FUM1, A KEY FUMONISIN BIOSYNTHETIC GENE OF FUSARIUM VERTICILLIOIDES. V. Montis1, I. Visentin1, D. Valentino1, G. Tamiet- ti1 and F. Cardinale2. 1Dipartimento di Valorizzazione e Protezione delle Risorse Agroforestali, Sezione di Patologia Vegetale, Università degli Studi di Torino, Via L. da Vinci 44, 10095 Grugliasco (TO), Italy. 2Dipartimento di Colture Arboree, Sezione di Fisiologia Vegetale, Università degli Studi di Torino, Via L. da Vinci 44, 10095 Grugliasco (TO), Italy. E-mail: francesca.cardinale@unito.it Fumonisins are mycotoxins produced mainly by Gibberella moniliformis [anamorph Fusarium verticillioides (Fv)], that contaminate maize and maize-based products and cause great concern for human and animal health. Several biosynthetic (FUM) genes are known; among them, FUM1 encodes a key polyketide synthase indispensable for fumonisin synthesis, and whose transcripts are always detectable when these metabolites are produced. To study the genetic and environmental factors linked to fumonisin production, we performed bioinformatic analyses on the putative promoter sequences of all known FUM genes, which are known to be co-regulated at the transcriptional level. Results showed the presence of a 6bp sequence statistically over-represented in the promoters of the genes in the FUM cluster compared to the rest of the genome. This motif could cis-act on the regulation of the FUM genes and notably of FUM1, since it is repeated twice in its putative promoter sequence (pFUM1). To validate the in silico prediction, a wild-type pFUM1 sequence from a toxigenic Fv strain and a synthetic version where this motif is mutated (mut-pFUM1) were obtained. Both pFUM1 and mutpFUM1 were used to drive the expression of GFP in transgenic Fv strains. Transcript quantification of the endogenous FUM1 and of GFP by RT-qPCR in both Fv transformants is under way, and will hopefully confirm the importance of the identified motif for wild-type expression of FUM1 under various conditions. Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 MOLECULAR VARIABILITY AND SEED TRANSMISSION OF GRAPEVINE RUPESTRIS STEM PITTING-ASSOCIATED VIRUS ISOLATES FROM SOUTHERN ITALY. M. Morelli1, A. Minafra2 and D. Boscia2. 1Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. 2Istituto di Virologia Vegetale del CNR, Unità Organizzativa di Bari, Via Amendola 165/A, 70126 Bari, Italy. E-mail: a.minafra@ba.ivv.cnr.it Thirty-four Vitis vinifera accessions from southern Italy were tested for the presence of Grapevine rupestris stem pitting-associated virus (GRSPaV), by Western blotting and RT-PCR (primer RSP48/RSP49). Due to the positive response of all samples, subsequent RT-PCR tests were done using three degenerate primer pairs, (GI, GII and GIII), designed in the CP region, for detecting putative molecular variants of GRSPaV. A very high percentange (94%) of the tested accessions were positive to GI used singularly (73%) or together with GII (18%), GIII (12%) or both (12%), whereas only a single infection was detected with GIII and none with GII. This suggests that at least three distinct molecular variants were found, of which GI is the most widespresd, although mixed infections are also common. Fifteen PCR amplicons (RSP48/RSP49 fragments; 327bp) were sequenced and submitted to computer-assisted analysis. The sequences shared a nucleotide identity ranging from 90% to 100%. In phylogenetic trees constructed with our and GenBank sequences previously reported as “type strains”, our isolates clustered in three different groups. In particualr, four isolates grouped with GRSPaV (AF026278), one with GRSPaV-BS (AY881627) and ten with GRSPaV-SG1(AY881626). Preliminary RT-PCR assays on GRSPaV transmission through pollen and seeds detected the virus in pollen collected from infected 110R rootstocks, but showed that it seems to be present on the outer surface rather than inside pollen grains. FIRST REPORT OF AMERICAN PLUM LINE PATTERN VIRUS IN FLOWERING CHERRY IN ITALY. A. Myrta1, J. Sanchez- Navarro2, O. Potere3, D. Boscia4 and V. Pallás2. 1Certis Europe B.V., Via Guaragna 3, 21047 Saronno (VA), Italy. 2Instituto de Biologia Molecular y Celular de Plantas, Universidad Politecnica, CSIC, Avenida de los Naranjos s/n 46022 Valencia, Spain. 3Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. 4Istituto di Virologia Vegetale del CNR, Unità Organizzativa di Bari, Via Amendola 165/A, 70126 Bari, Italy. E-mail: d.boscia@ba.ivv.cnr.it American plum line pattern virus (APLPV), a member of the genus Ilarvirus thought for long time to occur only in North America, was recorded during the last few years in isolated trees/foci from several Mediterranean countries in Japanese plum and sweet cherry. Line pattern symptoms were observed during spring 2009 in some flowering cherries (P. serrulata) in Northern Italy (Piedmont and Lombardia). A total of 22 trees (symptomatic and symptomless) were sampled randomly from seven locations: Verbania, Saronno, Rovellasca, Rovello Porro, Cantù, Bregnano and Solaro. All sampled trees were ELISA-tested for the following viruses: Plum pox virus (PPV), Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Apple mosaic virus (ApMV), Apple chlorotic leafspot virus (ACLSV) and APLPV. The same samples were tested by molecular hybridization (poly and virus-specific probes) and by RT-PCR (simultaneous and virus-specific) for the same viruses and for Apricot latent virus (ApLV), Plum bark necrosis stem pitting-associated virus (PBNSPaV), Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd). Two samples were positive for APLPV and four for S4.75 PDV, including one mixed infections. APLPV-positive samples were found in Verbania. Sequence analysis of a PCR amplicon confirmed the presence of this virus. To our knowledge, this is the first report of APLPV in flowering cherry in Italy. However, more extensive studies are needed to check the virus status of ornamental nurseries, to evaluate virus incidence in the territory, and the potential risks of transmission to cultivated stone fruits. ANTIMICROBIAL ACTIVITY OF PEACH AND GRAPE DEFENSINS DIFFEREN-TIALLY EXPRESSED DURING FRUIT RIPENING. V. Nanni1, E. Baraldi1, M. Bellucci2 and P. Bertolini1. 1CRIOF, Dipartimento di Protezione e Valorizzazione Agroalimentare, Laboratorio di Biotecnologie, Università degli Studi, Viale Fanin 46, 40127 Bologna, Italy. 2Dipartimento di Scienze e Tecnologie Agroambientali, Laboratorio di Chimica Bioinorganica, Università degli Studi, Viale Fanin 46, 40127 Bologna, Italy. Email: elena.baraldi@unibo.it Plant defensins are a large family of small, cationic, cysteinerich peptides playing a crucial role in plant innate immunity. Because of their known antimicrobial activity against important plant pathogens, plant defensins are very interesting targets for agrobiotechnology applications. In this study we show that defensin genes from peach (Prunus persica) and grape (Vitis vinifera) are highly expressed at different stages of fruit ripening. In peach fruits, the defensin transcript level drastically decreases from the early (S1 and S2) to the late (S3, S4) stages of ripening, whereas, in grape, defensin expression increases at veraison and remains stable afterwards. Two recombinant defensins were expressed in E. coli and purified to homogeneity. The recombinant proteins were tested for their antimicrobial activity against important fungal pathogens and human and plant bacterial pathogens. Both specifically inhibited the germination of Penicillium expansum and Botrytis cinerea while peach defensin inhibited also the germination of Monilia laxa. Furthermore, in accordance with other plant defensins, both defensins did not show antibacterial activity against plant and human pathogens. IDENTIFICATION AND CHARACTERIZATION OF POTATO SPINDLE TUBER VIROID INFECTING TOMATO IN ITALY. B. Navarro1, M.R. Silletti2, V.N. Trisciuzzi2 and F. Di Serio1. 1Istituto di Virologia Vegetale del CNR, Unità Organizzativa di Bari, Via Amendola 165/A, 70126 Bari, Italy. 2Centro di Ricerca e Sperimentazione in Agricoltura Basile Caramia, Via Cisternino 281, 70010 Locorotondo (BA), Italy. E-mail: f.diserio@ba.ivv.cnr.it Potato spindle tuber viroid (PSTVd), a quarantine plant pathogen in Europe, has been detected in tomato plants in northern Italy. Infected plants, showing shortened internodes, deformed and necrotic leaves, and abnormal fruit maturation, were found close to symptomless Solanum jasminoides plants also infected by PSTVd. Molecular characterization of field isolates, biological assays with viroidal cDNA infectious clones and sequence analyses of the resulting progenies support the possibility of PSTVd transmission to tomato plants from the neighbouring infected S. jasminoides plants. This is the first report of tomato plants infected by PSTVd in Italy and the first well-supported evidence on the risk of PSTVd epidemic spread from symptomless ornamental Solanaceae to susceptible horticultural crops when prophylactic measures are not enforced. These results also suggest that occasional PSTVd outbreaks in tomato crops reported in several European countries in the last two decades could have a similar origin. S4.76 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 INFLUENCE OF FRUIT AROMA COMPOUNDS ON PENICILLIUM EXPANSUM DEVELOPMENT. F. Neri, F. Veronesi genic ability of AG-2-1/Nt is not restricted to tobacco, and that future investigations may disclose an even wider host range. and M. Mari. CRIOF, Dipartimento di Protezione e Valorizzazione Agroalimentare, Università degli Studi di Bologna, Via Gandolfi 19, 40057 Cadriano (BO), Italy. E-mail: fiorella.neri@unibo.it Twelve synthetic aroma compounds of different fruits, chosen among typical (pome fruits) or less common (stone fruits, soft fruits and kiwifruits) hosts of Penicillium expansum, were individually tested in vitro for their influence on the pathogen’s conidial germination and mycelial growth. Low concentrations (0.62 µl/l) of 2-methyl butyl acetate, ethyl 2-methyl butyrate, linalool and γ−decalactone, close to their natural amount in pome fruits or stone fruits, and concentrations ranging from 0.62 to 984.3 µl/l of β-ionone, occurring in stone fruits and soft fruits, stimulated conidial germination of P. expansum. Vice versa, high concentrations of most of the tested compounds showed an inhibitory effect on both conidial germination and mycelial growth. Linalool (occurring in stone fruits and strawberries) and γ−decalactone (typical of stone fruits), showed a fungicidal inhibition at 492.13 µl/l and 984.25 µl/l, respectively, while hexyl acetate, ethyl 2-methyl butyrate, 2-methyl butyl acetate, butyl acetate, ethyl butyrate and methyl butyrate, typical of pome fruits and/or strawberries and kiwifruits, showed only a fungistatic effect at higher concentrations (minimum inhibitory concentrations ranging from 1107.28 to 3075.79 µl/l). Hexyl butyrate and β-ionone did not completely inhibited conidial germination or mycelial growth also at 6151.37 µl/l. No effect on P. expansum was exhibited by γ-decalatactone. These results provide evidence that fruit aroma compounds can influence P. expansum development both with stimulatory and inhibitory effect, in relation to their concentration. A role of some tested compounds in host recognition could be hypothesized, however further investigations are needed. OCCURRENCE OF RHIZOCTONIA SOLANI AG-2-1/Nt. R. Nicoletti, E. Lahoz, A. Carella and R. Caiazzo. CRA, Unità di Ricerca per le Colture Alternative al Tabacco, Via Vitiello 108, 84018 Scafati (SA), Italy. E-mail: rosario.nicoletti@entecra.it Among fungal plant pathogens Rhizoctonia solani (teleomorph Thanatephorus cucumeris) stands out for being a collective species composed by a number of infraspecific entities defined as anastomosis groups (AGs). Actually, as hyphal anastomoses are essential for nuclear exchange and heterokaryon formation, AGs represent genetically isolated and phylogenetically diverging entities that can be considered as true biological species. Moreover, the existence of subgroups and subsets of isolates displaying a lower anastomosis affinity with tester strains has been pointed out in several AGs. Within AG-2, five subgroups and a number of subsets have been described, including the quite puzzling AG2-1/Nt that was first identified in isolates recovered from tobacco in Italy and France displaying quite a reduced anastomosis affinity with AG-2-1 tester strains. Despite a high DNA-sequence homology, the peculiarity of subset Nt has been outlined by means of biochemical and molecular techniques, such as polygalacturonase isozymes, composition of whole-cell fatty acids, and RFLPs of rDNA-ITS. The availability in GeneBank of ITS sequences has recently increased the possibility to identify isolates belonging to this grouping, and investigations carried out throughout Europe have evidenced their widespread distribution. Particularly, besides the reported occurrence in tobacco, Nt isolates have been found in pepper, bean, periwinkle and sugar beet in Italy, sugar beet in Spain, cauliflower in Belgium, potato in Great Britain, and again tobacco in Turkey. Therefore it appears that the patho- A PHYSIOLOGICAL AND MOLECULAR APPROACH FOR STUDYING THE INTERACTION AMONG DON-PRODUCER FUSARIUM GRAMINEARUM ISOLATES AND TOLERANT OR SUSCEPTIBLE VARIETIES OF TRITICUM AESTIVUM. C. Nobili1, A. Ricelli2, M. Reverberi3, S. Gatta3, V. Scala4, G. Aureli4, A.A. Fabbri3 and C. Fanelli3. 1Sezione Genetica e Genomica Vegetale, Dipartimento di Biotecnologie, Agroindustria e Protezione della Salute, ENEA, Via Anguillarese 301, 00123 S.M. di Galeria (Roma) S.P. 026, Italy. 2Istituto di Chimica Biomolecolare del CNR, Piazzale Aldo Moro 5, 00185 Roma, Italy. 3Dipartimento di Biologia Vegetale, Università “Sapienza”, Largo Cristina di Svezia 24, 00165 Roma, Italy. 4CRA, Unità di Ricerca della Valorizzazione Qualitativa dei Cereali, Via Cassia 166, 00191 Roma, Italy. E-mail: chiara.nobili@enea.it Fusarium graminearum can cause Fusarium head blight (FHB) of wheat, leading to a severe reduction of grain yield and quality. The virulence factor involved in FHB disease is the production of toxins, predominantly deoxynivalenol (DON) which inhibits the activity of some plant defence proteins and induces a resistant response in tolerant varieties such as hydrogen peroxide production and the onset of programmed cell death. One of the biochemical mechanisms of resistance to DON is the plant ability to convert DON in a less toxic glucosylated form, via a reaction carried out by glucosyl-transferase. In this study, we analysed by a SYBR green RT-PCR approach the expression of different genes, whose products are involved in the interaction of tolerant or susceptible early flowering-soft wheat varieties with both a DON or a DON-zearalenone (ZEA) producer strains of F. graminearum. The fungal genes analysed encode proteins putatively involved in the onset of disease progression such as an exopolygalacturonase (ePG1) related to plant cell wall degradation, an orthologue of yAP-1, that is a yeast transcription factor active in the cell defence against oxidative stress, and Tri6, one of the thricotecenes biosynthesis regulator. In order to correlate the tolerant and susceptible phenotype of the two Triticum aestivum varieties with some of plant defence-related activities, the analysis of expression of three genes which encode glucosyl transferases and the pathogenesis-related protein PR1 have been carried out. Furthermore, we have analyzed, by LC-MS, DON and derivatives extracted from infected wheat. The aim of this work was to find a correlation between the expression of some F. graminearum genes involved in the onset of the FHB disease with the different ability of tolerant and susceptible varieties of T. aestivum to achieve DON inactivation. ACTIVATION OF DEFENSE RESPONSES IN TOMATO PLANTS INFECTED WITH NECROGENIC AND NON NECROGENIC STRAINS OF CUCUMBER MOSAIC VIRUS. C. Paciolla1, S. De Leonardis1, T. Mascia2 and F. Cillo2. 1Dipartimento di Biologia e Patologia Vegetale, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. 2Istituto di Virologia Vegetale del CNR, Unità Organizzativa di Bari, Via Amendola 165/A, 70126 Bari, Italy. E-mail: f.cillo@ba.ivv.cnr.it The increase in reactive oxygen species (ROS) has been found in plant cell in response to infection of various pathogens, including viruses. In the cell, the ROS level is controlled by enzymatic and not-enzymatic antioxidant systems. Among them, the ascorbate-glutathione cycle and other defence agents such as the en- Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 zymes catalase, superoxide dismutase and peroxidase prevent the accumulation of toxic level of H2O2 and other ROS. The objective of this study was to determine the changes of these soluble antioxidants in tomato plants infected with different strains of Cucumber mosaic virus (CMV). In particular, the antioxidant enzymes were examined in Solanum lycopersicum cv. UC82 plants inoculated with the aggressive strain CMV-Fny and with the same strain co-inoculated with a necrogenic variant of satellite RNA (CMV-Fny/77-satRNA), a combination inducing systemic necrosis and plant death. Analyses performed included both the transcriptional profiling and the determination of the enzymatic activity of such antioxidant components. In addition, gene expression analysis was carried out on genes involved in ethylene biosynthesis and signaling, in general defence responses and in programmed cell death (PCD) processes. Results showed that some of the antioxidative systems and components involved in PCD analyzed were commonly activated by both viral strains despite the different disease phenotype induced. Peculiar responses to either specific CMV inoculum were also detected, that could have some role in the determination of the observed disease phenotypes. CHANGES OF ANTIOXIDANT ENZYMES IN FUSARIUM VERTICILLIOIDES-MAIZE PATHOSYSTEM. C. Paciolla1, S. De Leonardis1, G. Mulè2, V. Ricci2, A. Logrieco2 and A. Marocco3. 1Dipartimento di Biologia e Patologia Vegetale, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. 2Istituto di Scienze delle Produzioni Alimentari del CNR, Via Amendola 122/0, 70126 Bari, Italy. 3Istituto di Agronomia Generale e Coltivazioni Erbacee, Università Cattolica del Sacro Cuore, Via E. Parmense 84, 29100 Piacenza, Italy. E-mail: paciolla@botanica.uniba.it Fusarium verticillioides is a fungus of particular importance for its widespread occurrence in cereals, especially in maize. F. verticillioides causes ear and stalk rot of maize, frequently colonizes maize tissue sometimes without causing disease symptoms and produces fumonisins. It is known that an early plant response to pathogen infection is an oxidative burst characterized by a huge production of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide anion that are potentially toxic for the cells. The ROS level in the cells is under the control of antioxidant defences, such as detoxifying enzymes and low-molecular weight antioxidants. Our aim was to study the trend of some detoxifying enzymes such as superoxide dismutase, peroxidase, catalase and ascorbate peroxidase during the defensive strategy activated by resistant or susceptible plants at five days post treatment with F. verticillioides. Superoxide dismutase and ascorbate peroxidase, the latter considered the key enzyme in cellular H2O2 removal, showed higher activity in resistant than in susceptible caryopses, highlighting their involvement in counteracting the pathogen colonization and mycotoxin accumulation. On the contrary, the decrease of catalase and total peroxidase activity underlined a lower cellular H2O2-detoxifying capacity than other enzymes. This study contributes to better understanding of the molecular and biochemical mechanisms that take part in the activation of the defence responses during biotic stress and that can be useful for fast plant resistance selection programmes. FIRST OUTBREAKS OF THE MARCUS STRAIN OF PLUM POX VIRUS IN APULIA. F. Palmisano1, A. Minafra1, M. Digia- ro2, A. Percoco3, T. Elbeaino2 and D. Boscia1. 1Istituto di Virologia Vegetale del CNR, Unità Organizzativa di Bari, Via Amendola 165/A, 70126 Bari, Italy. 2Istituto Agronomico Mediterraneo, Via S4.77 Ceglie 9, 70010 Valenzano (BA), Italy. 3Osservatorio Fitosanitario Regionale, Lungomare Nazario Sauro 45-47, 70121 Bari, Italy. Email: d.boscia@ba.ivv.cnr.it Plum pox virus (PPV), the causal agent of Sharka, was first detected in Apulia in 1988 in a young apricot orchard of cv. Tyrinthos. Since then, few outbreaks, all of them subjected to eradication, were identified in apricot and plum orchards. In all cases, the eliciting strain was PPV-D, except for a single record of PPV-Rec. Two new PPV foci were recently found in apricot (cv. Ninfa, 2008) and peach (cv. Big Top, 2009) orchards at Cerignola (Northern Apulia, Italy) established with propagative materials from nurseries of northern Italy. Preliminary serological typing carried out by DASI-ELISA with strain-specific monoclonal antibodies were in both cases positive for PPV-M (Mab.AL). To exclude the presence of recombinant (M-D) isolates, molecular characterization was done by RT-PCR through: (i) amplification of the N terminal region of the coat protein (CP) gene using the P3MP4b set of primers; (ii) amplification of the P3-6K1 region using the PP3-PCI set of primers; (iii) sequencing of both PCR products; (iv) multiple sequence alignments and phylogenetic analysis. Molecular typing confirmed the results of serology since both isolates proved to belong to the M strain, while there was no evidence of the presence of recombinant PPV. This represents the first detection of Sharka in peach in Apulia, and the first record of PPV-M. This finding highlights once again the need to enforce strict and effective controls of the health status of propagation material, especially when it comes from highly contaminated areas, and the need to protect PPV-free areas with appropriate phytosanitary measures (e.g., establishment of “protected zones”). DENDRIMERS AS ACTIVITY ENHANCERS IN PLANT CHEMOTHERAPY. A. Panattoni1, A. Luvisi1, F. D’Andrea2, F. Giorgelli2, G. Catelani2 and E. Triolo1. 1Dipartimento di Coltivazione e Difesa delle Specie Legnose “G. Scaramuzzi”, Università degli Studi, Via del Borghetto 80, 56124 Pisa, Italy. 2Dipartimento di Scienze Farmaceutiche, Università degli Sudi, Via Bonanno Pisano 6, 56126 Pisa, Italy. E-mail: apanatto@agr.unipi.it Dendrimers are macromolecules recently developed for medical use, which enhance the activity of small chemicals. In fact, one of main limits of chemicals used in cancer or antiviral therapy relies in the low efficacy of receptor/ligand binding, thus an useful tool could be the building of high molecular weight clusters, characterized by functional chemicals linked to an inert scaffold. These dendrimeric structures show branches that are able to bind many receptor sites, increasing binding stability and local concentration. This feature could be useful for antiviral therapy in plants, considering that a few chemicals are available, especially in cases of limited efficacy against plant viruses. Among available scaffolds usable as dendrimer core, polyamidoamine (PAMAM) is simple to use, versatile and biologically compatible, so that a protocol for binding 2 or 4 molecules of mycophenolic acid (MPA) was developed. This chemical eradicated Cucumber mosaic virus (CMV), but limited antiviral effects was shown against other woody plant viruses. Thus, MPA efficacy could be theoretically increased or extended as MPA-dendrimers. A preliminary study of these novel drugs in in vitro experimental system (Nicotiana tabacum L. cv. Xanthi/CMV) was carried out, with the aim of investigating drugs toxicity, optimal dosage and potential antiviral efficacy. Results considering the lack of toxicity up to 0.30 mM application are encouraging, and these novel drugs seem to be strongly effective against virus replication, suggesting this approach as potentially useful for enhancing antiviral chemicals. S4.78 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 SUPPRESSION OF TELLURIC PLANT DISEASES BY APPLICATION OF THERMOPHILIC BACTERIAL STRAINS ISOLATED FROM COMPOST AND COMPOST-AMENDED SOILS. C. Pane, D. Villecco and M. Zaccardelli. CRA, Azienda these species but one are native to Australia, suggesting that P. niederhauserii has been introduced only recently in nurseries of ornamental plants, probably from Australia. Agraria di Battipaglia, S.S. 18 n. 204, 84091 Battipaglia (SA), Italy. E-mail: catello.pane@libero.it A great diversity of microorganisms, mainly bacteria, able to suppress pathogens, naturally colonize compost, especially in the thermophylic phase of the composting process. The activity of microflora was described as the principal responsible of soilborne disease suppression in compost- amended soils. More than 200 thermophylic bacterial strains, selected as potential biocontrol agents (BCAs) by preliminary plate challenge experiments, were isolated from municipal waste compost (MWC) and from MWC-amended soils. In in vitro assays, the best 24 suppressive bacterial isolates of this wide collection, resulted effective antagonists against Rhizoctonia solani, Fusarium solani, Fusarium oxysporum f.sp. lycopersici, Sclerotinia minor and Pyrenochaeta lycopersici. This group of isolates was separated in seven major genetically distinct subgroups by polymorphic analyses with coliphage M13 sequenced-based PCR fingerprints. One member for each subgroups was chosen, on the basis of the in vitro performances, for further bioassay with R. solani, F. solani and S. minor on Lepidium sativum. Interestingly, these experiments showed that the selected microorganisms reduced the severity of seedling damping-off. Moreover, no phytotoxic effects due to bacterial inocula were observed on the plants. These results confirm that compost and amended-soils can be potential sources of beneficial microorganisms useful for crop protection. In particular, the isolates selected appeared to be promising for their successful use in the control of soil-borne pathogens. PHYOTPHTHORA NIEDERHAUSERII, A NEW SPECIES RECOVERED FROM ORNAMENTAL PLANTS IN ITALY. A. Pane1, S.O. Cacciola2, P. Martini3, C. Rizza2 and F. Raudino1. di Scienze e Tecnologie Fitosanitarie, Università degli Studi, Via S. Sofia 100, 95123 Catania, Italy. 2Dipartimento di Chimica biologica, Chimica medica e Biologia molecolare, Università degli Studi, Viale A. Doria 6, 95125 Catania, Italy. 3Istituto Regionale per la Floricoltura, Via G. Carducci 12, 18038 Sanremo, Italy. Email: apane@unict.it 1Dipartimento Phytophthora taxon niederhauserii Z.G. Abad and J.A. Abad is a species in the ITS-clade 7 that has been identified on a molecular basis but has not been formally described. First records of this new species date back to 2001. Since then P. niederhauserii has been reported on numerous host plants from the United States and Europe. In Australia it was isolated from native plant species in forest ecosystems. In a survey of ornamental plant nurseries in Italy, P. niederhauserii was found associated with root and basal stem rot of banksia (Banksia speciosa R. Br., B. baxteri R. Br.and B. prionotes Lindl.), bottlebrush [Callistemon citrinus (Curtis.) Skeels.] and rock rose (Cistus salvifolius L.). It was isolated from infected tissues with selective media and identified by amplification of the internal transcribed spacer (ITS) region of the rDNA gene. Sequences were compared with those of existing species and undescribed taxa deposited in GenBank. In pathogenicity tests, five italian isolates, IMI 393960 and 466/03 from banksia, IMI 391712 and IMI 391713 from rock rose, and IMI 391708 from bottlebrush induced symptoms of root and collar rot on their respective hosts with final collapse of the entire plant, thus fulfilling Koch’s postulates. Although P. niederhauserii is potentially polyphagous, at present its host range in Italy is restricted to few plant species that are not taxonomically related. All COMPOST AMENDMENTS ENHANCE SUPPRESSIVENESS TO PYTHIUM ULTIMUM, RHIZOCTONIA SOLANI AND SCLEROTINIA MINOR. C. Pane1,2, G. Bonanomi1, D. Sme- jkalovà3, A. Piccolo3 and F. Scala1. 1Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Universita` di Napoli Federico II, Via Università 100, 80055 Portici (NA), Italy. 2CRA, Centro di Ricerche per l’Orticoltura, Azienda Agraria di Battipaglia, S.S. 18 n. 204, 84091 Battipaglia (SA), Italy. 3Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali, Università di Napoli Federico II, Via Università 100, 80055 Portici (NA), Italy. E-mail: catello.pane@libero.it Peat is the most common organic material used for the preparation of potting mix because of the homogeneous and favourable agronomic characteristics. However, potting mixes are poorly suppressive against soilborne pathogens, so that fungicides are used routinely to manage damping-off diseases. In the present study we investigated the suppressive capability of five compost-peat potting mixes. The suppressive effect was evaluated on Pythium ultimum-, Rhizoctonia solani- and Sclerotinia minor-Lepidium sativum pathosystems. Organic media were chemically and biologically characterized, and the measured parameters were correlated with disease suppression. A compost derived from animal manure showed the highest disease suppression. Sterilization decreased or eliminated suppressivity of all mixes. It has been hypothesized that compost microflora suppresses the pathogens through competition for organic carbon resources and, in specific cases, by production of cell wall degrading enzymes. The key role of compost microflora in disease control was shown because the microbial functional diversity, assessed with the BiologTM method, and several enzymatic activities (hydrolysis of fluorescein diacetate, chitobiosidase, glucanase, N-acetyl-glucosaminidase and chitinase) were positively correlated with disease suppression. Moreover, 13C NMR spectroscopy showed that availability of organic carbon resources, well as the hydrophobicity of organic C compounds are significantly correlated with disease suppression. These results suggest that the carbohydrate content and their physical accessibility are key parameters to predict potting mix suppressiveness. Our results show that the addition of composts to peat could be useful for the control of soilborne pathogens. POSTHARVEST BIOLOGICAL CONTROL OF APPLE BLUE MOLD BY COMBINED APPLICATION OF COMPATIBLE BIOCONTROL AGENTS. S. Panebianco, G. Scuderi, C. Plata- nia and G. Cirvilleri. Dipartimento di Scienze e Tecnologie Fitosanitarie, Università di degli Studi, Via S. Sofia 100, 95123 Catania, Italy. E-mail: gcirvil@unict.it The effectiveness of 10 Pseudomonas syringae strains, applied alone or in combination with Trichoderma atroviride strain P1, to control blue mould of apple fruits was evaluated. In vitro assays showed that all bacterial strains inhibited, to a different extent, the growth of the pathogen. Spore germination in PDB was also largely inhibited by P. syringae cell suspensions as well as by culture filtrates. High levels of inhibition of Penicillium expansum growth were observed in presence of T. atroviride strain P1. Application of mixtures of P. syringae strains and T. atroviride P1 with the pathogen in co-inoculation experiments resulted in a Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 greater control of the pathogen development compared with the application of bacterial strains alone. In vivo antagonistic activity of the strains was evaluated on apples cv. Golden delicious. The incidence of apple decay and the diameter of lesions were greatly reduced when the biocontrol agents were applied to wounds 24 h and 72 h before challenging with P. expansum. The combination of P. syringae PVCT 48SR2, PVCTB728a and PVCT335 with T. atroviride P1 had a higher biocontrol activity than that of the single microbes used alone. These results clearly indicate that Pseudomonas spp. and Trichoderma spp. strains could be considered as interesting biocontrol agents for apple blue mould. Moreover, a more effective disease control could be afforded by the combined action of the two agents. Further trials are needed to develop strategies for compatible biocontrol agents implementation and management at the practical level. A NEW POTYVIRUS FOUND IN SILENE ALBA. G. Parrella1, A. De Stradis2, A. Cirillo3, N. Acanfora1 and A. Ragozzino3. 1Istituto per la Protezione delle Piante del CNR, Unità Organizzativa di Napoli, Via Università 13, 80055 Portici (NA), Italy. 2Istituto di Virologia Vegetale del CNR, Unità Organizzativa di Bari, Via Amendola 165/A, 70126 Bari, Italy. 3Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università di Napoli Federico II, Via Università 100, 80055 Portici (NA), Italy. E-mail: parrella@ipp.cnr.it During March 2008 and again during April 2009, several plants of Silene alba L. (family Caryophyllaceae) with leaf symptoms consisting in vein clearing, light chlorotic flecks, mottling and malformations were observed in the archeological area of Cuma (Southern Italy). Leaf dips showed the presence of filamentous virus particles 750-800 nm long. Cytoplasmic cylindrical inclusions seen in ultrathin sections of leaves were consistent with a potyviral infection. Attempts to isolate the virus on erbaceous plants of the families Solanaceae, Chenopodiaceae and Cucurbitaceae were unsuccessful and no latent infection was detected in inoculated hosts by dot blot assays with a specific riboprobe. By converse, the virus was readily transmitted mechanically to Silene plantlets. Reverse-transcription RT-PCR using degenerate potyvirus primers, conducted on nucleic acids extracted from infected plants produced an amplification product ca. 1.7-kb in size. This amplicon contained the 3’ end of the NIb region, the coat protein, and the complete 3’-UTR of a potyvirus genome. The putative coat protein (CP) showed the highest percentage of identity with the corresponding CP of the potyvirus Carnation vein mottle virus (accession No. AB017630) both at the nucleotide (71%) and amino acid (59%) level. According to the current criteria for potyvirus classification the virus found in S. alba should be considered a new member of the Potyvirus genus. S4.79 low leaf mosaic. TEC-1 was characterized based on biological reactions and partial nucleotide sequence analysis. Following mechanical inoculation it elicited necrotic local lesions following by systemic necrosis in Solanum lycopersicum, chlorotic spot and systemic mild mottling in Chenopodium amaranticolor, small local chlorotic spots in Cucumis sativus, local necrotic etching and systemic slight vein clearing and mottling in Nicotiana tabacum cvs. Samsun and White Burley. When the cDNA product of TEC-1 coat protein (CP) gene, obtained directly by RT-PCR from a Cape honeysuckle plant, was digested with BamHI, the fragment pattern was identical to that obtained from previously described AMV subgroup I isolates. Nevertheless, phylogenetic analysis revealed that TEC-1 clustered separately from subgroups I and II. The nucleotide and amino acid sequences of TEC-1 CP were 9495% and 92-96% identical to subgroup I and II isolates, whereas the identities among isolates belonging to subgroup I were 9699% at the nucleotide and 94-99% at the amino acid level and 97-99% at the nucleotide and 96-100% at the amino acid level among subgroup II isolates. Overall, the results of this study suggested that the isolate TEC-1 should be tentatively considered a member of a new AMV subgroup. Genome sequencing of TEC-1 is under way to complete its molecular characterization. EVALUATION OF ELICITORS FOR THE CONTROL OF POSTHARVEST DECAY OF CITRUS FRUIT. C. Platania, S. Panebianco, G. Scuderi and G. Cirvilleri. Dipartimento di Scienze e Tecnologie Fitosanitarie, Università degli Studi, Via S. Sofia 100, 95123 Catania, Italy. E-mail: gcirvil@unict.it CHARACTERIZATION OF AN ALFALFA MOSAIC VIRUS ISOLATE FROM CAPE HONEYSUCKLE IN SPAIN: A NEW SUBGROUP OF THE VIRUS? G. Parrella1, N. Acanfora1, A.F. Use of elicitors for the control of postharvest diseases received increasing attention over recent years. In the present work, acibenzolar-S-methyl (ASM), b-aminobutyric acid (BABA) and chitosan, known for their ability to induce resistance in the host, were also evaluated for their direct antifungal activity against postharvest citrus moulds. Orange, grapefruit and lemon fruits were wound-inoculated with ASM and chitosan (from 0.02 to 0.5%) and with BABA (from 1 to 1000 mM), then challenged with Penicillium digitatum 24 h post treatment. Disease incidence and severity were significantly reduced by chitosan at 0.05 and 0.1%, whereas ASM did not show any effectiveness in reducing disease incidence. On the contrary, BABA inhibited the development of green mould at all concentrations, although it was more effective at 10 mM. Decay control was higher on grapefruit than on orange and lemon fruits. Even if the pathogen was inoculated at a distance of 1 cm from the BABA-treated site, the level of decay on treated grapefruit and lemon fruits was significantly lower than in the water-treated control. In vitro tests on PDA and on orange peel extract indicated that BABA inhibited mycelial growth and conidial germination of P. digitatum, P. italicum and Geotricum candidum at concentration lower than 10 mM, whereas ASM was weakly effective only at 0.5 and 1%, and chitosan was always effective at all tested concentrations. The results suggest that, in addition to inducing pathogen resistance, BABA also inhibits postharvest citrus pathogens directly, suggesting future investigations on the use of elicitors in postharvest disease control. Orílio2 and J. Navas-Castillo2. 1Istituto per la Protezione delle Piante del CNR, Via Università 13, 80055 Portici (NA), Italy. 2Estación Experimental “La Mayora”, Consejo Superior de Investigaciones Científicas, 29760 Algarrobo-Costa, Málaga, Spain. Email: parrella@ipp.cnr.it ACTIVITY OF ENDOPHYTIC ALTERNARIA spp. STRAINS IN THE CONTROL OF PLASMOPARA VITICOLA. R. Polizzotto1, An isolate of Alfalfa mosaic virus (AMV), named TEC-1, was obtained from a Cape honeysuckle plant [Tecomaria capensis (Thunb.) Spach, syn. Bignonia capensis Thunb.] growing in a private garden in Almeria (Southern Spain) and showing bright yel- S. D’Agostin2, S. Grisan1, G. Assante3, I. Pertot2, B. Andersen4 and R. Musetti1. 1Dipartimento di Biologia e Protezione delle Piante, Università degli Studi, Via delle Scienze 208, 33100 Udine, Italy. 2IASMA Research and Innovation Center, Fondazione Edmund Mach, Genetics and Crop Biology Area, Via Mach 1, 38010 S4.80 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 S. Michele all’Adige (TN), Italy. 3Istituto di Patologia Vegetale, Università degli Studi, Via Celoria 2, 20133 Milano, Italy. 4Department of Systems Biology, Center for Microbial Biotechnology, Technical University of Denmark, Søltofts Plads, 2800 Kgs. Lyngby, Denmark. E-mail: rachele.polizzotto@uniud.it Endophytic Alternaria spp. are able to synthesize bioactive compounds involved in plant defence mechanisms. In the case of grapevine downy mildew, it has been reported that four endophytic isolates belonging to Alternaria NEES, recovered from grapevines in Tuscany, and their secondary metabolites, inhibited Plasmopara viticola sporulation on grapevine leaves in moisture chambers as well as on plants maintained in the greenhouse. To better understand grapevine-Alternaria - P. viticola relationships, 14 endophytic Alternaria strains had been isolated from different grapevine cultivars in Friuli Venezia Giulia (Northern Italy) and characterized by molecular and biochemical methods, showing that differences among strains occur. Aim of this work was to compare the effectiveness of seven endophytic Alternaria isolates, chosen as representative of the 14 ones, and of two other Alternaria strains, belonging to A. tenuissima and A. alternata species, against P. viticola in greenhouse-grown grapevines. Eight grapevines were treated with each Alternaria culture broth, half of which were inoculated before P. viticola infection and half after. Untreated grapevines and copper-treated vined were included as controls. The inhibition of P. viticola was evaluated assessing the colonization and sporulation level on the leaves by observing symptom severity and pathogen DNA quantification by qRT-PCR. All the Alternaria strains inhibited P. viticola. The majority of them (including A. tenuissima and A. alternata) showed activity in pre-infection treatment and very few both in pre- and in post-infection. Symptom severity evaluations were confirmed by data obtained by qRT-PCR. Mechanism(s) involved in antagonistic activity against P. viticola will be discussed. LONG-TERM OBSERVATIONS ON THE SPATIAL DISTRIBUTION OF ESCA DISEASE IN VINEYARDS. S. Pollastro1, C. Dongiovanni2, W. Habib1 and F. Faretra1. 1Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. 2Centro di Ricerca e Sperimentazione in Agricoltura “Basile Caramia”, Via Cisternino 281, 70010 Locorotondo (BA), Italy. E-mail: faretra@agr.uniba.it In last decades, severity of esca disease has increased wherever grapevine is grown and nowadays the disease is common even in young vineyards. Many aspects of the disease, including the way of its spreading in the vineyards remain to be clarified. This study reports the spatial distribution of esca-diseased vines in Apulian (Southern Italy) vineyards. Field surveys were carried out from 1997 to 2008, in 11 vineyards, representative of different farming conditions. During the period July-October of each year, a total of 16,554 vines were singularly inspected for the presence of symptoms on leaves and bunches or for apoplexy. Detailed bi-dimensional maps of monitored vineyards were prepared each year and for the whole duration of monitoring. A broad variability in the expression of symptoms in different years was observed. Symptoms were more frequent on leaves (average value: 2.6%) than bunches (average value: 0.2%), or on both organs (average value: 0.4%). Where detected, apoplexy was observed on a maximum of 1.5% of the vines (average value: 0.2%). Annual and cumulative maps were submitted to spatial analysis using three different methodologies: spatial autocorrelation by a causality test, spatial analysis by distance indices, and semi-variogram. All the methods yielded similar results. Usually, symptomatic vines showed a trend to aggregation in vineyards except in a few cases in which they were distributed at random. A regular distribution of symptomatic vines were never observed. These findings suggest a role of soil-borne fungal inoculum and/or that the disease can be transmitted from an affected vine to its neighbours by natural or man-mediated ways. EVIDENCE OF MATING IN NATURE BETWEEN INDIVIDUALS OF ERYSIPHE NECATOR FROM DIFFERENT GENETIC GROUPS. I. Portillo1, A. Brunelli1, M.G. Milgroom2 and P. Corte- si3. 1Dipartimento di Protezione e Valorizzazione Agro-Alimentare, Università degli Studi, Viale Fanin 46, 40100 Bologna, Italy. 2Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, 14853 NY, USA. 3Università degli Studi, Dipartimento di Protezione dei Sistemi Agroalimentare e Urbano e Valorizzazione delle Biodiversità, Via Celoria 2, 20133 Milano, Italy. Email: ivanportillo785@hotmail.com Erysiphe necator (formerly Uncinula necator) is a biotrophic ascomycete that causes powdery mildew on grapevine. For this pathogen, two sympatric populations (groups A and B) have been described in Europe and Australia. The two genetic groups differ in multiple genetic loci and previous studies reported a lack of interfertility. Twenty flagshoots were sampled in an untreated vineyard in Emilia-Romagna (northern Italy) early in the growing season, and one single-conidial isolate was obtained from each. Additional single-conidial isolates were obtained from infected leaves and berries later in the epidemics. Cleistothecia were collected in the same vineyard from leaves before senescence, and from bark, before budbreak in the following season. DNA was purified from 60 single-conidial isolates and from 250 cleistothecia and used as template for PCR to amplify β-tubulin and IGS sequences. Amplified DNA from these two loci was digested with AciI and XhoI, respectively, to show single-nucleotide polymorphisms specific for the two genetic groups. Individuals of genetic groups A and B coexisted in the vineyard throughout the season, with no change in frequency. DNA amplified from cleistothecia showed the presence of markers diagnostic for both groups A and B and a mixture of the two. Mixtures of A and B were found in 12% of cleistothecia collected in autumn and in 36% of cleistothecia collected in spring from the bark. These results indicate that these two groups mated and produced ascospores in nature, although we found no evidence of recombination between A and B in field isolates. BIOLOGICAL EFFECTIVITY OF PLANT ESSENTIAL OILS AGAINST THE CAUSAL AGENTS OF SIGNIFICANT BACTERIAL DISEASES OF STONE FRUITS AND WALNUTS. D. Pou- vová1, B. Kokošková2, R. Pavela3 and P. Ryšánek1. 1Faculty of Agrobiology, Food and Natural Resources, Czech Univerzity of Agriculture, 165 21 Prague 6, Czech Republic. 2Department of Bacteriology, Plant Medicine Division, Research Institute of Crop Production, 161 06 Prague 6, Czech Republic. 3Department of Entomology, Plant Medicine Division, Research Institute of Crop Production, 161 06 Prague 6, Czech Republic. E-mail: dagmarpouvova@seznam.cz Stone fruits suffer from various bacterial diseases. They can be often infected by plant pathogenic bacteria such as Pseudomonas syringae pv. syringae (Pss) and Pseudomonas syringae pv. morsprunorum (Psm), which cause bacterial canker, and also with Xanthomonas arboricola pv. pruni (Xap) causing bacterial spot and Agrobacterium tumefaciens (At), the causal agent of crown gall. Walnuts are often infected with Xanthomonas arboricola pv. jug- Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 landis (Xaj), which causes bacterial bligth. For chemical control, copper-containing preparations are usually used, but they are not sufficiently effective. Antibiotics such as streptomycin are more effective than copper compounds, but they are banned from use for plant protection in majority of EU countries. In our tests, thirtyfour essential oils obtained from different aromatic plants were tested as potential inhibitors of plant pathogenic bacteria. Essential oil as a crude extract was dropped on the surface of agar contaminated by the target bacterium in Petri dish. Streptomycin was used as control. Plant essential oils from Origanum compactum, O. vulgare, Ocimum basilicum and Thuja occidentalis were the most effective against At. Plant essential oils from Origanum compactum, O. vulgare, Thymus vulgaris and Eugenia caryophyllata were the most effective against Pss. Essences from Eugenia caryophyllata, Origanum vulgare, O. compactum, Citrus aurantifolia, and Abies siberica were the most effective against Psm. Oils from Thymus vulgaris, Tsuga canadensis, Origanum compactum and O. vulgare were most effective against Xap and Xaj. Work supported by MZe _R 0002700604 and by the Ministry of Education, project No. MSM6046070901. THE EFFECT OF DIFFERENT INFECTION PATHWAYS ON MAIZE KERNEL INFECTION BY FUSARIUM AND FUMONISIN CONTAMINATION. S. Povero1, I. Visentin1, D. Valentino1, F. Vanara2, M. Blandino2 and G. Tamietti1. 1Dipartimento di Protezione e Valorizzazione Agroalimentare, Sezione di Patologia Vegetale, Università degli Studi di Torino, Via L. da Vinci 44, 10095 Grugliasco (TO), Italy. 2Dipartimento di Agronomia, Selvicoltura e Gestione del Territorio, Università degli Studi di Torino, Via L. da Vinci 44, 10095 Grugliasco (TO), Italy. E-mail: giacomo.tamietti@unito.it Fusarium verticillioides is commonly reported as the causal agent of maize pink ear rot. It produces fumonisins, compounds contaminating the kernels, toxic to animals and humans. It can infect maize kernels through silks, lesions caused by insects in the ears, and via internal, systemic infection of the corn plant. The objective of this study was to evaluate the relative importance of such pathways for maize kernel infection by F. verticillioides and fumonisin contamination. In 2007-08 in Northwest Italy, in a full factorial field experiment, the effects of seed-inoculation, fungicide protection of the silks at flowering, and physical ear protection against the European corn borer (ECB) were compared. The experiments were a repeated measure design with eight replicate blocks. Fusarium species were isolated from maize plants and seeds at different phenological stages, up to grain physiological maturity. Fumonisin contamination was quantified by HPLC. The infection rate of epicotyl and crown increased following F. verticillioides seed inoculation (+55%). Stem colonization decreased dramatically after the 2nd node, to reach 0-3% at the 7th node. Maize silks were Fusarium-free until they turned brown. The kernels were healthy during growth; infections started at the milk stage and increased during ripening. Seed inoculation and fungicide application at flowering did not show any significant effect, whereas the physical protection of the hears against ECB injuries reduced F. verticillioides infection by 49% and fumonisins by 56%. These results emphasize the epidemiological role of both F. verticillioides air-borne inoculum and ECB larvae in maize Fusarium pink ear rot. FUSARIUM SPECIES INFECTING WINTER WHEAT HEADS IN PIEDMONT, AND THE ROLE OF SOME AGRO-TECH- S4.81 NIQUES ON GRAIN MYCOTOXIN CONTAMINATION. S. Povero1, I. Visentin1, D. Valentino1, F. Vanara2, M. Blandino2 and G. Tamietti1. 1Dipartimento di Protezione e Valorizzazione Agroalimentare, Sezione di Patologia Vegetale, Università degli Studi di Torino, Via L. da Vinci 44, 10095 Grugliasco (TO), Italy. 2Dipartimento di Agronomia, Selvicoltura e Gestione del Territorio, Università degli Studi di Torino, Via L. da Vinci 44, 10095 Grugliasco (TO), Italy. E-mail: giacomo.tamietti@unito.it The contamination of winter wheat grains by Fusarium mycotoxins is threatening the quality of the wheat produced in NorthWest Italy, mainly in the years with rainy spring. In 2007-2008 in a typical cereal area of Piedmont, we investigated both the Fusarium species involved in the disease and the effects of different crop managements on Fusarium head blight (FHB) and mycotoxin contamination of grains. Fusarium species were isolated by plating 100 disinfected wheat grains per treatment on Komada’s Fusarium selective medium, and classified based on their morphological traits and by PCR analysis. Deoxynivalenol (DON) was determined by ELISA. Fusarium grain infections ranged from 0 to 3.8% in 2007 and from 32 to 92% in 2008 depending on the wheat cultivar, soil cultivation and fungicide treatments, and were caused mainly by F. graminearum (97.05%), F. avenaceum (1.55%), F. poae (0.54%). DON contamination ranged rom 75 to140 ppb in 2007 and from 8,560 to 17,429 ppb in 2008. Direct sowing significantly promoted the disease in comparison with soil ploughing (76,7 and 52.2 % of infected grains, respectively) and significant positive interactions were observed on yield weight, TEST WEIGHT, and reduction of DON contamination, between soil ploughing, reaction of wheat cultivar to Fusarium infections, and fungicide treatments. Among the tested fungicides, Metconazole was the most effective in reducing FHB and DON, by about 51%. DON contamination was directly correlated with the percentage of infected grains (R2 = 0.7845). PRESENCE OF DEOXYNIVALENOL AND NIVALENOL CHEMOTYPES OF FUSARIUM GRAMINEARUM ISOLATED FROM DURUM WHEAT IN ITALY. A. Prodi1, P. Nipoti1, E. Bertacchini1, S. Tonti1,2, I. Alberti2, M. Dal Prà2, M. Montanari3, D. Pancaldi4, L. Covarelli5, V.A. Infantino6, A. Santori6 and V. Balmas7. 1Dipartimento di Scienze e Tecnologie Agroambientali, Sezione di Patologia Vegetale, Università degli Studi, Viale Fanin 40, 40127 Bologna, Italy. 2Ente Nazionale Sementi Elette, Via Ca’ Nova Zampieri 37, 37057 S. Giovanni Lupatoto (VR), Italy. 3Ente Nazionale Sementi Elette, Via Sicilia 2, 40060 Osteria Grande (BO), Italy. 4Dipartimento di Protezione e Valorizzazione Agroalimentare, Università degli Studi, Viale Fanin 46, 40127 Bologna, Italy. 5Dipartimento di Scienze Agrarie e Ambientali, Università degli Studi, Borgo XX Giugno 74, 06121 Perugia, Italy. 6CRA, Centro di Ricerca per la Patologia Vegetale, Via C.G. Bertero 22, 00156 Roma, Italy. 7Dipartimento di Protezione delle Piante, Università degli Studi, Viale E. De Nicola 9, 07100 Sassari, Italy. Email: antonio.prodi@unibo.it Durum wheat production in Italy is of great economical importance. Fusarium graminearum is the main Fusarium head blight (FHB) causal agent in Italian wheat crops, reducing both grain quality and yield. This fungus produces trichothecene mycotoxins such as deoxynivalenol (DON) and nivalenol (NIV) that are toxic to humans and animals. A population of F. graminearum sensu stricto strains was collected from symptomatic durum wheat heads and grains from several naturally infected fields in different Italian regions such as Emilia Romagna, Latium, Marche, Piedmont, Sardinia, Umbria and Veneto. A multiplex PCR in the Tri12 region of the trichothecene gene cluster was S4.82 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 used to assign each strain to one of the trichothecene chemotype profiles: NIV, 3-acetyldeoxynivalenol (3ADON) and 15acetyldeoxynivalenol (15ADON). In order to evaluate possible mycotoxin contamination risks it is very important to know which chemotype is the prevalent in a F. graminearum population. Our research showed that the15ADON chemotype was the most frequent. This work, conducted for the first time in Italy, allowed us to better understand the distribution of F. graminearum chemotypes. PRELIMINARY INVESTIGATIONS ON THE PRESENCE OF VIRUSES IN OLEA EUROPEA IN SARDINIA. V. Prota and R. Garau. Dipartimento di Protezione delle Piante, Sezione di Patologia Vegetale, Università degli Studi, Via E. De Nicola 1, 07100 Sassari, Italy. E-mail: garau@uniss.it Olive trees, Mediterranean species with a rich and varied germplasm, are host of many viruses, most of which have been described and characterized. This study was carried out in 20072008 in Sardinia to obtain information on the sanitary status of 122 olive plants of five cultivars (Bosana, Semidana, Tonda di Cagliari, Nera di Gonnos and Nera di Villacidro) with good pomological characters, in the framework of a programme for the improvement of the health conditions of Sardinian olive germplasm. Tests were performed from cortical scrapings of young twigs colelcted from the qudrant of each tree, according to a protocol set up by the ‘OLVIVA’ project (extraction of nucleic acid with “RNeasy Plant Mini kit – Qiagen”, to serve as template for one-step RT-PCR) for the identification of. Arabis mosaic virus (ArMV), Cherry leafroll virus (CLRV), Cucumber mosaic virus (CMV), Olive latent virus 1 (OLV-1), Olive latent virus 2 (OLV-2), Olive leaf yellowing-associated virus (OLYaV), Strawberry latent ringspot virus (SLRSV) and Tobacco necrosis virus (TNV) using virus-specific primers. All samples were free from ArMV, CLSV, SLRV, CMV and TNV. Positive results were obtained for OLYaV, which was detected in 56% of the samples, while OLV-2 e OLV-1 were identified in 18% and 14,5% of the trees, respectively. The highest infection rates were found in cv. Tonda di Cagliari for OLYaV and OLV-1 (83.3% and 26.3% infection, respectively), and cv. Semidana for OLV-2 (36% infection). All varieties showed mixed infections of the three above-mentioned viruses, with the exception of Nera di Villacidro which was infected only by OLYaV and OLV-2. These results, which represent new records for Sardinia, open up promising new prospects for the implementation of sanitary and clonal selection programmes. Research carried out in the framework of the inter-regional project OLVIVA “Qualificazione del vivaismo olivicolo” (DM 25279 of 23.12.03) FIRST SELECTION OF VIRUS-FREE AND VIRUS-TESTED CLONES IN VARIETIES OF OLEA EUROPEA IN SARDINIA. V. Prota1, P. Sedda2 and R. Garau1. 1Dipartimento di Protezione delle Piante, Sezione di Patologia Vegetale, Università degli Studi, Via E. De Nicola 1, 07100 Sassari, Italy. 2Agris Sardegna Innovazione e Ricerca, Loalità Bonassai, S.S 291 Sassari-Fertilia, Italy. E-mail: garau@uniss.it Sanitary selection is one of the most significant aspects in of the process aimed at improving the quality and quantity of fruit crops. As to olive, a protocol enforced in Italy since June 2007 defines propagative material and nursery productions of olive to be virus-free (VF) if it does not contain Arabis mosaic virus (Ar- MV), Cherry leafroll virus (CLRV), Olive latent virus 1 (OLV-1), Olive leaf yellowing-associated virus (OLYaV), Strawberry latent ringspot virus (SLRSV), Olive latent virus 2 (OLV-2), Cucumber mosaic virus (CMV) and Tobacco necrosis virus (TNV) and virustested (VT) if it is free rom the first five virus species. The present work, carried out in 2007-2008 within the OLVIVA project, aimed at identifying accessions elegible for certification by testing 122 plants of cvs Bosana, Semidana, Tonda di Cagliari, Nera di Gonnos and Nera di Villacidro. These plants were grown in a collection field at Villasor (Southern Sardinia), belonging to Agris-Sardegna. Assays to determine the presence of the abovementioned viruses were performed by RT-PCR with virus-specific primers on total RNAs extracted from cortical scrapings of young twigs, using “RNeasy Plant Mini kit – Qiagen”. The results were extremely interesting for 20 virus-free and 5 virus-tested clones were identified in the assayed cultivars (20% of the total). In particular, 2 VF and 2 VT clones were found in cv. Tonda di Cagliari, 6 VF clones in cv. Bosana, 6 VF and 3 VT clones in cv. Semidana, 1 VF clone in cv. Nera di Gonnos and 5 VF clones in cv. Nera di Villacidro. These results may be indicative of a relatively satisfactory sanitary status of Sardinian olive industry, which opens to the hope that similar conditions may exist in other cultivars for olive oil production of interest for this region. Research carried out in the framework of the inter-regional project OLVIVA “Qualificazione del vivaismo olivicolo” (DM 25279 of 23.12.03). EFFECTS OF ELEVATED CO2 AND TEMPERATURE ON INFECTION OF GRAPEVINE BY POWDERY MILDEW UNDER CONTROLLED ENVIRONMENT. M. Pugliese1,2, M.L. Gullino1 and A. Garibaldi1. 1Centro di Competenza per l’Innovazione in Campo Agro-ambientale, Università degli Studi di Torino, Via L. da Vinci 44, 10095 Grugliasco (TO), Italy. 2Dipartimento di Valorizzazione e Protezione delle Risorse Agroforestali, Sezione di Patologia Vegetale, Università degli Studi di Torino, Via L. da Vinci 44, 10095 Grugliasco (TO), Italy. E-mail: massimo.pugliese@unito.it Plant responses to elevated CO2 and temperature have been much studied in recent years, but the effects of climate change on pathological responses are still largerly unknown. The grapevinepowdery mildew pathosystem was chosen as a model to assess the potential impact of increased CO2 and temperature on disease incidence and severity. Potted grapevine plants of cvs Moscato and Barbera, were grown in phytotrons under four different simulated climatic conditions: standard CO2 concentration for the area (450 ppm) with standard (ranging from 18 to 26°C) or elevated temperature (4°C higher than standard) and elevated CO2 (800 ppm) with standard or elevated temperature. Disease index and physiological paramenters (chlorophyll content, fluorescence, assimilation rate) were assessed. Results showed an increase of the chlorophyll content with higher temperatures and CO2 concentration, to which a higher fluorescence index corresponded. Disease incidence varied with the cultivar, but differences were not statistically significant. In conclusion, an increase in CO2 did not affect powdery mildew incidence, probably due to the increased photosynthetic activity of the vines under such conditions. COMPOST AS A SOURCE OF ANTAGONISTS AGAINST SOIL-BORNE PATHOGENS. M. Pugliese1,2, M.L. Gullino1 and A. Garibaldi1. 1Centro di Competenza per l’Innovazione in Campo Agro-ambientale, Università degli Studi di Torino, Via L. da Vinci 44, 10095 Grugliasco (TO), Italy. 2Dipartimento di Valorizzazione Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 e Protezione delle Risorse Agroforestali, Sezione di Patologia Vegetale, Università degli Studi di Torino, Via L. da Vinci 44, 10095 Grugliasco (TO), Italy. E-mail: massimo.pugliese@unito.it Suppression of soil-borne plant diseases with composts has widely been studied. The objective of the present work was to isolate microorganisms from a compost and to test them for their activity against soil-borne pathogens. Twenty eight microorganisms were isolated from a compost originated from food and garden wastes and were tested under laboratory conditions on tomato seedlings growing on perlite medium in Petri plates infected with Fusarium oxysporum f. sp. radicis-lycopersici. Microorganisms that controlled tomato wilt were tested under greenhouse condition on three pathosystems: Fusarium oxysporum f. sp. basilici/basil, Phytophthora nicotianae/tomato and Rhizoctonia solani/bean. In general, Fusarium spp. afforded a fair control of Fusarium wilts, whereas only bacteria were able to control P. nicotianae on tomato. None of the microorganisms tested was able to control all the soil-borne pathogens. The selection of antagonists from compost is a promising strategy for the development of new biological control agents against soil-borne pathogens. POLYPHASIC IDENTIFICATION OF XANTHOMONADS CAUSING BACTERIAL LEAF SPOT ON SENISE IGP PEPPERS. G. Puopolo1, L. Cozzolino1, A. Raio2, C. Nigro3 and A. Zoina1. 1Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università degli Studi di Napoli Federico II, Via Università 100, 80055 Portici (NA), Italy. 2Istituto Protezione delle Piante del CNR, Via Madonna del Piano 10, 50019 Sesto Fiorentino (FI), Italy. 3Azienda Agricola Sperimentale Dimostrativa “Bosco Galdo”ALSIA, Via Grumentina 118, 85050 Villa d’Agri (PZ), Italy. Email: zoina@unina.it Members of the species Xanthomonas euvesicatoria, X. gardneri and X. vesicatoria are responsible for pepper bacterial leaf spot, a disease causing severe losses to pepper production worldwide. Xanthomonas-like isolates recovered from diseased leaves and fruits of peppers grown in the Senise area (Basilicata, Southern Italy) were identified using a polyphasic approach based on physiological and molecular methods. The molecular identification relied on sequencing of the DNA fragment (ITS region) placed between genes coding for 16S rRNA and 23S rRNA while the Biolog test was used to determine the physiological profile of bacterial isolates. A new database (named Xantho1) containing the physiological profiles of the type strains of the causal agents of pepper bacterial leaf spot was created by our group since Biolog database does not comprise any of the Xanthomonas spp. associated to this disease. The totality of bacterial isolates analyzed during this work was assigned to X. euvesicatoria on the basis of phylogenetic analyses of the ITS region. At the same time, the of Xantho1 database pointed out that 60% of the bacterial isolates associated with diseased peppers grown in the Senise area had a high physiological similarity with the type strain of X. euvesicatoria. The combination of the phylogenetic analyses of ITS region and a new improved Xantho1 database could be useful in the future for an accurate polyphasic identification of xanthomonads associated with pepper bacterial leaf spot. FIRST REPORT OF CHALARA ELEGANS ON SQUASH IN ITALY. M. Quaglia1, V.M. Stravato2, G. Carannante2 and C. Cap- pelli1. 1Dipartimento di Scienze Agrarie e Ambientali, Università degli Studi, Borgo XX Giugno 74, 06121 Perugia, Italy. 2Genista s.r.l., S.S. Flacca, 04022 Fondi (LT), Italy. E-mail: mara.quaglia@unipg.it S4.83 In February and March 2007 and 2008 unusual symptoms of stunting associated with severe leaf chlorosis were observed in some squash (Cucurbita pepo L.) crops near Sabaudia (Latium, Southern Italy). Roots of diseased plants showed a reduced growth, brown root rot and premature death. Microscopic observations symptomatic roots showed the presence of Chalara elegans Nag Rai & Kendrik. The same fungus was consistently isolated on potato dextrose agar (PDA) from diseased roots. To verify the pathogenicity of two fungal isolates, disks of 8-day-old colonies, 5 mm in diameter, were placed on the collar of 15 seedlings in which small lesions were made with a sterile needle. Control plants were treated with sterile PDA. The seedlings were grown in a greenhouse at 20±2°C with 14 h photoperiod. After four weeks only the inoculated plants showed the same symptoms observed in the field. C. elegans was successfully reisolated from the inoculated plants, proving that this fungus is the causal agent of black root rot of squash. C. elegans has been recorded in Italy from a wide range of important hosts, in both protected and open field crops. The occurrence of this new disease could be due to the inoculum persisting in soils where other susceptible species were previously grown in which chlamidospores can survive for more than 5 years. To our knowledge this is the first report of C. elegans on squash in Italy. IDENTIFICATION OF AMINO ACID RESIDUES OF FUSARIUM VERTICILLIOIDES ENDO-POLYGALACTURONASE REQUIRED TO ESCAPE THE INHIBITION BY HOST PLANT PGIP. A. Raiola, C. Castiglioni, A. Nesler, I. Elmaghraby and F. Favaron. Dipartimento Territorio e Sistemi Agro-Forestali, Università di Padova, Viale dell’Università 16, 35020 Legnaro (PD), Italy. E-mail: alessandro.raiola@unipd.it Endopolygalacturonases (Endo-PGs) play an important role in fungal infection since they depolymerize the pectin component of plant cell wall. Most plant have evolved defence mechanisms to contrast the action of endo-PGs. A direct mechanism is the interaction with plant cell wall polygalacturonase-inhibiting proteins (PGIPs) that block polygalacturonase activity. However, fungal pathogens can elude the inhibition by plant PGIP, producing PGs refractory to inhibition. Fusarium verticillioides endo-PG is unaffected by host PGIPs (asparagus and leek) as well as by Phaseolus vulgaris PGIP, that is the most efficient PGIP so far characterized. Recently, we have identified a specific PG amino acid residue responsible for the lack of recognition by bean PGIP. Now we have mutated few amino acids of the F. verticillioides endo-PG which allow the recognition by host PGIP. This finding highlights the structural features of the enzyme to escape PGIP inhibition. Moreover, this modified PG could be used to replace native F. verticillioides PG in order to better clarify the role of constitutive PGIP in plant defense. EFFECT OF GREEN MANURE ON CORKY ROOT OF TOMATO AND VERTICILLIUM WILT OF EGGPLANT. G.E. Rescigno, M. D’Amico, M. Amenduni and M. Cirulli. Dipartimento di Biologia e Patologia Vegetale, Università degli Studi, Via G. Amendola 165/A, 70126 Bari, Italy. E-mail: amenduni@agr.uniba.it In the present study, the efficacy of green manuring with Helianthus annuum, Sorghum bicolor and Trifolium subterraneum in the control of tomato corky root, caused by Pyrenochaeta lycopersici, and Verticillium wilt of eggplant, caused by Verticillium dahliae, was evaluated in fields naturally infested by these pathogens. Crops used for green manure were grown according S4.84 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 to ordinary agricultural practices, and soil incorporation of plant debris was made at the flowering stage by rotovation. Planting of tomato and eggplant was carried out in the following year. A randomized block design with three replications for T. subterraneum, or four replications for H. annuum and S. bicolor, was used. Green manure with all the three crops reduced corky root severity up to 36%, but did not provide any control of Verticillium wilt. On the other hand, green manure treatments did not affect yield of tomato, while green manure with T. subterraneum significantly increased eggplant yield by 30%. The present paper reports the results of the first experiments, which are being replicated. ORGANIC SEEDS, AN AREA THAT REQUIRES RESEARCH AND DEVELOPMENT. L. Riccioni1, M. Zaccardelli2, M. Pasquini3, C. Micheloni4 and M. Malinconico5. 1CRA, Centro di Ricerca per la Patologia Vegetale, Via C.G. Bertero 22, 00156 Roma, Italy. 2CRA, Unità di Ricerca per la Valorizzazione Qualitativa dei Cereali, Via Cassia 176, 00191 Roma, Italy. 3CRA, Centro di Ricerca per l’Orticoltura, Via dei Cavalleggeri 25, 84098 Pontecagnano (SA), Italy. 4Istituto di Chimica e Tecnologia dei Polimeri del CNR, Via Campi Flegrei 34, 80078 Pozzuoli (NA), Italy. 5Associazione Italiana Agricoltura Biologica, Via Piave 14, 00187 Roma, Italy. E-mail: luca.riccioni@entecra.it For obtaining organic productions, one of the requirements to be followed is the use of seeds from mother plants grown using organic methods (Reg. EC No 2092/91). In the early stages, given the total absence of organic seeds on the market, the EU had to provide mechanisms for exemption (Reg. EC No 1452/2003) to use conventional non chemically treated seeds. Data, available on the ENSE website, demonstrate the unavailability of organically produced seeds, and the use of massive amounts of untreated conventional seed. To encourage the development of organic seed production, the Italian Ministero delle Politiche Agricole, Alimentari e Forestali has therefore launched the project Pro.vi.se.bio, in which the Subproject “Organic seeds” aims at the “identification of the best agronomic practices to prevent infection to seeds and limit the use of treatments for the defense in the growing stages after sowing, and to the identification of new natural compounds for organic seed treatment”. Three Institutions of CRA (CRA-PAV, CRA-QCE and CRA-ORT), the ICTPCNR and AIAB participate in this Subproject. Technical choices will be investigated as the most suitable to contain the contamination/infection by: (i) Fusarium spp. to seeds of durum and bread wheat; (ii) Colletotrichum lindemuthianum to bean seeds; (iii) Ascochyta rabiei to chickpea seeds; (iv) Alternaria spp. to carrot seeds. Economically sustainable strategies for organic seed treatment will also be developed for the control of seed-borne fungi, without affecting negatively other agronomic parameters, and for protecting seedlings from attacks by pathogenic soil fungi. Preliminary results obtained during the first phase of the Subproject are reported. SURVEY OF FUNGI ASSOCIATED WITH ACTINIDIA CHINENSIS PRUNING WOUNDS. L. Riccioni and L. Orzali. CRA, swellings and bark cracking. Streaky brown lesions are observed in the wood underneath. The lesions cause localized blockage of the xylem that results in the death of single canes or entire leaders when the blockage is complete, associated with a significant yield losses. Lesions are associated with unprotected pruning wounds which may constitute the main point of entry for infections. For this reason an investigation on the fungal species present in pruning wounds both treated and not with various fungicides was carried out in two orchards of Latina (Latium) over three years (2007-2009). Penetration of the fungi into the wood was assessed after 6 to 8 months by plating pieces of wood tissue on agar medium. Isolations from these pruning cut samples allowed to draw up a list of the most common fungal species associated with pruning wounds: Cadophora luteo-olivacea was the most frequently isolated species, followed by Paraconiothyrium spp. in one orchard and Acremonium strictum in the other. Occasionally, Pheoacremonium spp., Phoma spp. and Diaporte/Phomopsis spp. were also isolated. There were no significant differences between treated and non-treated samples mainly with regard to C. luteo-olivacea. GENE EXPRESSION ANALYSIS IN SOUR ORANGE SEEDLINGS INOCULATED WITH CITRUS TRISTEZA VIRUS. S. Rizza1, G. Nobile1,2, V. De Luca3, A. Catara1,2, C. Ca- passo3 and V. Carginale3. 1Dipartimento di Scienze e Tecnologie Fitosanitarie, Università degli Studi, Via S. Sofia 100, 95123 Catania, Italy. 2Laboratorio di Diagnosi e Biotecnologie Fitosanitarie, Parco Scientifico e Tecnologico della Sicilia, Blocco Palma I Z.I., 95030 Catania, Italy. 3Istituto di Biochimica delle Proteine del CNR, Via P. Castellino 111, 80131 Napoli, Italy. E-mail: srizza@unict.it Twenty three genes, identified in Etrog citron in response to Citrus dwarfing viroid (CDVd) infection, were used as probes to evaluate their expression level in sour orange seedlings inoculated with a severe isolate of Citrus tristeza virus (CTV) from Sicily (CTV S29). Northern blot analysis of leaf extracts revealed that ten genes exhibited the same pattern of expression (up regulation) following both CDVd and CTV infection, suggesting that they are involved in general response to stress factors. On the contrary, the remaining genes showed different patterns of expression, indicative of a pathogen-specific regulation. Particularly interesting appears the fact that a gene encoding a gene silencing suppressor [regulator of gene silencing-calmodulin-like protein (rgs-CaM)] was induced in both infections. A messenger RNA differential display RT-PCR analysis (DDRT-PCR) of sour orange seedlings inoculated with the viral isolate CTV S29 in comparison with healthy controls, was also carried out for identifying further differentially expressed genes Four new transcripts were identified in the first fingerprints obtained. Three of them are positive regulated in response to CTV infection, two show high sequence similarity with two different EST of Citrus sinensis, whose biological function is unknown, the third one shows sequence similarity with a retrotransposon of Arabidopsis thaliana, whose role under conditions of biotic and abiotic stress is largely reported in literature. The last transcript, which is negatively regulated, encodes a protein belonging to the family of cytochrome P450. Centro di Ricerca per la Patologia Vegetale, Via C.G. Bertero 22, 00156 Roma, Italy. E-mail: luca.riccioni@entecra.it A new disease of yellow-fleshed kiwifruit vines (Actinidia chinensis) ‘Hort16A’ known as “leader die-back”, is spreading in some orchards of Latium and Emilia Romagna (Central and Northern Italy). The symptoms of leader die-back are typically MOLECULAR MONITORING OF PHYTOPHTHORA POPULATIONS IN FOREST AND NATURAL ECOSYSTEMS IN SICILY. C. Rizza1, S. Scibetta2, G. Magnano di San Lio3 and S.O. Cacciola2. 1Dipartimento di Scienze Entomologiche, Fitopato- Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 logiche, Microbiologiche Agrarie e Zootecniche, Università degli Studi, Viale delle Scienze 2, 90128 Palermo, Italy. 2Dipartimento di Chimica Biologica, Chimica Medica e Biologia Molecolare, Università degli Studi, Viale Andrea Doria, 6, 95125 Catania, Italy. 3Dipartimento di Gestione dei Sistemi Agrari e Forestali, Università Mediterranea, Località Feo di Vito, 89122 Reggio Calabria, Italy. E-mail: olgacacciola@unict.it Phytophthora population was monitored in soil and water samples from mixed oak and beech stands with symptoms of decline in the Regional Park of Madonie (Sicily) using a nested-PCR amplification of the ITS1 region with genus-specific primers. DNA was extracted directly from soil samples while DNA from water samples was concentraded by filtration on paper filters and extracted from them. The amplified product (ca 260 bp) was cloned and phylogenetic tools were used for identifying the clones with the related species or phylotypes of Phytophthora. Phytophthora DNA was detected in 44.8% of soil samples and in all water samples. Nine Phytophthora taxa were identified, including seven known species, P. cambivora, P. citricola, P. gonapodyides, P. inundata, P. psychrophila, P. quercina and P. syringae, and two new phylotypes (Blast identity with known taxa <97%) that were provisionally named P. europea-like and P. porrilike, respectively, because of the phylogenetic relationship with these two known species. P. psychrophila was the most frequent species in soil samples (eight out of 29 analysed samples), followed by P. quercina (seven samples), P. syringae (one sample), P. citricola (one sample) and P. europea-like (one sample). P. gonapodyides was the most frequent species in water samples (five out of six analysed samples), followed by P. syringae (four samples), P. cambivora (two samples), P. psychrophila (two samples), P. inundata (one sample) and P. porri-like (one sample). This molecular method is a powerful tool for detecting new threatening species of Phytophthora that are not traceable by traditional isolation methods. POTENTIAL OF BIOFUNGICIDES FOR THE CONTROL OF STEMPHYLIUM VESICARIUM ON PEAR. R. Roberti, A. Veronesi, G. Alberoni and A. Brunelli. Dipartimento di Protezione e Valorizzazione Agroalimentare, Università degli Studi, Viale Fanin 46, 40127 Bologna, Italy. E-mail: roberta.roberti@unibo.it The control of brown spot of pear caused by Stemphylium vesicarium (Pleospora allii), is mainly based on repeated chemical treatments. However, new perspectives are offered by the integration with biological products based on Trichoderma, which were shown to reduce the pathogen’s inoculum on the orchard floor. The aim of this study was to test the competitive interaction and the mechanisms of action of several biocontrol agents against S. vesicarium. We considered the biofungicides Rootshield (Trichoderma harzianum T22), Remedier (T. asperellum ICC012 and T. gamsii ICC080), Trichover (T. harzianum ITEM 908), Mycostop (Streptomyces griseoviridis K61) and Serenade (Bacillus subtilis QST713). Each microorganism was tested for substrate colonization and inhibitory effect against S. vesicarium. T. harzianum T22 and S. griseoviridis K61 were the most active in inhibiting the pathogen mycelial growth, while T. harzianum ITEM908 showed the highest substrate colonization. Trichoderma strains exhibited parasitic action of pathogen hyphae, visible as coiling, lysis and collapse. All microorganisms produced chitinolytic enzymes. B. subtilis QST713 produced the highest level of glucosaminidase, S. griseoviridis K61 the highest level of chitobiosidase and T. asperellum ICC012 the highest level of endochitinase. Non volatile metabolites produced by T. harzianum T22, S. griseoviridis K61 S4.85 and B. subtilis QST713 on PDA were the most active in reducing the pathogen’s mycelial growth. Metabolites caused also morphological alteration of the hyphae. Metabolites from liquid culture of the microorganisms, did not reduce conidial germination, but reduced the number of germ tubes/conidia (B. subtilis QST713) and their elongation (T. asperellum ICC012 and T. gamsii ICC080). WOOD-ROTTING FUNGI ASSOCIATED WITH CITRUS ORCHARDS IN SOUTHERN ITALY. A. Roccotelli1, L. Schena1, A.M. Ligorio2, S.O. Cacciola3 and A. Ippolito2. 1Dipartimento di Gestione dei Sistemi Agrari e Forestali, Università Mediterranea, Località Feo di Vito, 89122 Reggio Calabria, Italy. 2Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. 3Dipartimento di Chimica biologica, Chimica medica e Biologia molecolare, Università degli Studi, Viale Andrea 6, 95125 Catania, Italy. E-mail: lschena@unirc.it The distribution of wood-rotting fungi infecting citrus trees was investigated in Southern Italy. Isolations were carried out on PDA from decaying wood of clementine trees, showing leaf dropping, chlorosis and twig dieback and from apparently healthy sweet orange trees. Cross-sections of clementine trunks and branches revealed symptoms resembling those of esca disease of grapevines consisting of a light-coloured rot surrounded by brown and hard necrotic wood. Dark-coloured rots were observed in orange trees. Fungal isolates were grouped according to their morphology and identified by the analysis of the internal transcribed spacer regions of rDNA. A single fungal species was recovered from each sample. Fomitiporia mediterranea, the most common species in clementine trees, was recovered occasionally from 15- to 20-year-old trees, but was common in older trees. It was isolated also from the wood of both ‘Tarocco’ orange and declining olive trees grown as windbreaks in orange groves. Two different ITS genotypes closely related to Phellinus spp. were also associated with “white rot” symptoms and occasionally isolated from ‘Fedele’ clementine. On ‘Valencia late’ sweet orange another fungus, still under investigation, was frequently associated with “brown rot” symptoms. RESISTANCE OF BOTRYOTINIA FUCKELIANA TO ANILINOPYRIMIDINE AND PHENYLPYRROLE FUNGICIDES IN SOUTHERN ITALY. C. Rotolo, R.M. De Miccolis Angelini, S. Pollastro, A. Santomauro and F. Faretra. Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. E-mail: faretra@agr.uniba.it Botryotinia fuckeliana (de Bary) Whetz. (Botrytis cinerea Pers.) is a pathogen at high risk of acquired resistance to fungicides. The effectiveness of chemical control can be compromised especially when intensive spray schedules are used. Anilinopyrimidine fungicides (APs), pyrimethanil, mepanipyrim and cyprodinil (used in mixture with phenylpyrrole fludioxonil), are fungicides highly effective against the pathogen and are commonly used on several crops including grapevine. In 2008, a monitoring of sensitivity to APs and phenylpyrroles in B. fuckeliana populations was carried out in 18 vineyards with different spray histories. Isolates resistant to pyrimethanil were detected with a high frequency (52-91% of conidia) in vineyards treated intensively (4-7 sprays/season) or exclusively with APs. The frequency of resistant conidia was lower (up to 23%) when 1-3 sprays per season S4.86 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 were applied. Fungal isolates showing a slightly decreased sensitivity to fludioxonil were detected with a frequency up to 50%. Response to pyrimethanil and fludioxonil was evaluated through colony growth tests. AP sensitive wild-type isolates showed EC50 < 0.1 µg/ml and MIC = 1 µg/ml. At least three levels of resistance to APs were found: low (EC50 = 0.3-1 µg/ml; MIC = 3-10 µg/ml), moderate (EC50 = 1-10 µg/ml; MIC ≥ 100 µg/ml) and high (EC50 = 30-100 µg/ml; MIC > 100 µg/ml). All fludioxonil-resistant isolates showed EC50 values (0.1-1 µg/ml) and MIC (1-3 µg/ml) significantly higher than sensitive wild-type isolates (EC50 = 0.020.03 µg/ml; MIC = 0.2 µg/ml) and were also resistant to APs. FIRST REPORT OF BOTRYOTINIA FUCKELIANA MUTANTS RESISTANT TO QOI FUNGICIDES IN SOUTHERN ITALIAN VINEYARDS AND THEIR GENETIC CHARACTERIZATION. C. Rotolo, R.M. De Miccolis Angelini, S. Pollastro, and F. Faretra. Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. E-mail: faretra@agr.uniba.it QoI fungicides, inhibitors of mitochondrial respiration, are considered at high risk of acquired resistance. In several phytopathogenic fungi, resistance is caused by mutations (more often G143A) in the mitochondrial cytochrome b gene (cytb). Resistance to QoIs was monitored in field populations of Botryotinia fuckeliana (de Bary) Whetz. (Botrytis cinerea Pers.). Conidia, collected from naturally infected berries sampled in 18 Apulian (southern Italy) vineyards, were plated on malt extract agar amended with 1 mg/l trifloxystrobin and 2 mM salicylhydroxamic acid (SHAM), a specific inhibitor of alternative oxidase. QoIresistant mutants were recovered in 11 vineyards with a frequency ranging from 1.5×10-4 to 2.8×10-2. Sensitivity to trifloxystrobin was evaluated in colony growth test. All mutants showed high level of resistance (EC50 and MIC > 100 µg/ml) as compared with sensitive wild-type strains (EC50 = 0.03-0.1; MIC = 1 µg/ml). Cleaved amplified polymorphic sequences (CAPS) analysis using the restriction enzyme AluI showed that all the tested QoI-resistant mutants carried the G143A mutation in the cytb gene. Analysis of meiotic progenies of appropriate crosses with sensitive reference strains proved maternal (sclerotial) inheritance of QoI-resistance, confirming that it was caused by cytoplasmic genetic determinants. None of the mutants resistant to QoIs showed intronlike sequence (with high homology with conserved regions of group I introns) flanking downstream the codon 143 of the cytb gene. A so located intron, previously detected by PCR in around 15% wild-type sensitive B. fuckeliana isolates, is supposed to prevent the occurrence of the G143A mutation and, consequently, the acquisition of QoI resistance. TIME AND SPACE MONITORING OF PHOMA TRACHEIPHILA PROPAGULES IN LEMON ORCHARDS. M. Rus- so, P. Bella, V. Catara and A. Catara. Dipartimento di Scienze e Tecnologie Fitosanitarie, Via S. Sofia 100, 95123 Catania, Italy. Email: vcatara@unict.it Citrus ‘mal secco’, caused by Phoma tracheiphila, is the main limiting factor of lemon crops. Investigations in various citrusgrowing areas and the evermore-frequent epidemics suggested that the soil plays an important role as reservoir of inoculum. Reducing the amount of pathogen in the soil has never been seriously considered by researchers or by farmers, possibly due to the high cost of manual labour needed to remove and burn all the infected material. An original quantitative real-time PCR protocol, previously devised and validated to evaluate P. tracheiphila propagules in artificially inoculated soil, was used to monitor the presence of the fungus in naturally infested citrus soil. A standard curve was generated by plotting the threshold cycle values against the log of the amount of P. tracheiphila DNA and the amount of fungal DNA for unknown samples inferred from the regression line. Seventy-seven out of 98 soil samples collected from four lemon orchards showing severe symptoms of mal secco (leaf and shoot chlorosis, leaf dropping, dieback of twigs and branches) revealed a variable level of P. tracheiphila DNA concentration, not related to the severity of disease. The highest amount of propagules was detected in samples taken in March and April (0.30-0.43 ng DNA/g of soil), whereas it become nearly undetectable in July and August (0.05-0.06 ng DNA/g of soil). DNA content raised in September-November (0.10-0.24 ng DNA/g of soil). Data confirm a relation bertween temperature, rain and relative humidity and survival/accumulation of P. tracheiphila in the soil. TRANSGENIC ENDOCHITINASE TROYER CITRANGE SHOWS HIGHER CHITINASE AND GLUCANASE ACTIVITY AND REDUCED COLONIZATION BY FUNGI. M. Russo1, S. La Malfa2, C. Abbate3, A. Gentile2, G. Cirvilleri1 and A. Catara1. di Scienze e Tecnologie Fitosanitarie, Università degli Studi, Via S. Sofia 100, 95100 Catania, Italy. 2Dipartimento di OrtoFloroArboricoltura e Tecnologie Agroalimentari, Università degli Studi, Via Valdisavoia 5, 95123 Catania, Italy. 3Dipartimento di Scienze Agronomiche, Agrochimiche e delle Produzioni Animali, Università degli Studi, Via S. Sofia 98, 95100 Catania, Italy. Email: russomar@unict.it 1Dipartimento The introduction of exogenous genes has open new perspectives for disease control, as suggested by the results obtained after the introduction of endochitinase in different species. Citranges (Poncirus trifoliata x Citrus sinensis) are widely used as citrus rootstocks in countries were CTV is spread because of their tolerance to Citrus tristeza virus (CTV). Unfortunately, they are susceptible to some diseases, such as Fusarium spp.-induced dry root rot, a limiting factor in nursery and field crops. In the attempt to overcome this problem citrange the endochitinase gene of Trichoderma harzianum (chit42) was introduced into Troyer through Agrobacterium-mediated transformation of stem internode segments. Regenerated transgenic plants (GM) were tested in comparison to wild type (WT) for their chitinase and glucanase activities, for resistance to two specific citrus pathogens. The root-associated microbial population was also evaluated. The level of both enzymes in root apex was higher in transgenic plants, i.e up 2.2 fold for chitinase and up to 1.7 fold for glucanase. WT and GM rooted shoots growing under controlled environments were leaf inoculated with a conidial suspension of Phoma tracheiphila and the roots were inoculated with Fusarium solani A1. GM shoots inoculated with P. tracheiphila showed a reduction of infected sites and tissue colonization around the inoculation point, whereas those inoculated with F. solani did not differ virtually from WT. Interestingly, when GM shoots were immersed in crude filtrates of P. tracheiphila they proved to be more tolerant than WT to wilting. Finally, root-associated microbial population was almost equivalent in GM and WT plants. OCCURRENCE OF MYCOTOXIGENIC FUSARIUM SPECIES AND MYCOTOXINS IN MAIZE GRAIN IN UMBRIA. S. Salvi, M. De Berardinis, G. Beccari and L. Covarelli. Dipartimento di Scienze Agrarie e Ambientali, Università degli Studi, Borgo XX Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 Giugno 74, 06121 Perugia, Italy. E-mail: lorenzo.covarelli@ unipg.it Surveys were carried out on on 47 and 36 maize grain samples collected in different locations of Umbria (Central Italy) in 2006 and 2007, respectively. After a preliminary visual observation of rotten kernels, grain samples were subjected to pathogen isolation by the deep-freezing blotter method, DNA extraction for fungal detection by PCR assays and mycotoxin extraction for determining the content of fumonisins, deoxynivalenol and aflatoxins by commercial ELISA kits. Fusarium isolates were identified both by morphological observations and PCR. The incidence of Fusarium infections ranged, in average, from 66% in 2006 to 79% in 2007. Both traditional and molecular methods showed that F. verticillioides was the main pathogen in maize kernels, followed by the sporadic presence of F. proliferatum and F. subglutinans, whereas F. graminearum, F. culmorum and F. sporotrichioides were detected only in 2006. Maize grain was characterized by the constant presence of mycotoxins, fumonisins in particular, which in a few cases exceeded the EU legal limits. This study represents the first accurate survey carried out in Umbria for the identification of mycotoxigenic Fusarium species and the presence of mycotoxin contamination in maize grain. Our results indicate that the mycotoxin problem in maize in this Italian region is generally minor, however the adoption of appropriate prevention control strategies and a continuous monitoring are necessary for not exceeding the law limits. The different F. verticillioides isolates obtained are currently being evaluated for their molecular, pathogenic and mycotoxigenic characteristics. EFFECTS OF PHENOLIC COMPOUNDS ON THE BIOCONTROL ACTIVITY OF YEAST ANTAGONISTS AGAINST PENICILLIUM EXPANSUM ON APPLES. S.M. Sanzani1, R. Castoria2, L. Schena3, A. De Girolamo4, F. Nigro1, M. Solfrizzo4 and A. Ippolito1. 1Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. 2Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Università degli Studi del Molise, Via De Sanctis, 86100 Campobasso, Italy. 3Dipartimento Gestione dei Sistemi Agrari e Forestali, Università Mediterranea, Località Feo di Vito, 89124 Reggio Calabria, Italy. 4Istituto di Scienze delle Produzioni Alimentai del CNR, via G. Amendola 122/O, 70126 Bari, Italy. Email: ippolito@agr.uniba.it; simona.sanzani@agr.uniba.it Blue mould caused by Penicillium expansum Link is a major postharvest rot of pome fruits. This disease causes economic losses not only to fresh fruits but also to the fruit-processing industry since some strains of the pathogen produce patulin, a mycotoxin which is transferred into fruit juices. So far, control strategies have mainly relied on synthetic fungicides. However, concerns about the environment and human health, as well as the onset of resistant pathogen’s strains, have fostered the search for more eco-friendly measures. Although the use of microbial antagonists is considered as one of the most desirable alternatives, it faces some disadvantages such as the inconsistency of results. The aim of this study was to enhance the efficacy of selected antagonist yeasts against blue mould of apples by combining them with three phenolic compounds (quercetin, umbelliferone and ferulic acid), that in previous studies proved to be effective in controlling the disease and/or patulin accumulation. Trials were carried out to determine the compatibility of phenolics with the yeasts Rhodosporidium kratochvilovae (LS11) and Cryptococcus laurentii (LS28). Strain LS11 was compatible with phenolic concentrations not exceeding 1 µg/ml, but combined applications did not provide any improvement in efficacy. By contrast, strain LS28 was S4.87 compatible with the tested substances up to 10 µg/ml and its efficacy was enhanced when the antagonist was combined with quercetin. In particular, a synergic controlling effect on disease severity (38%) and patulin accumulation (88%) was recorded. Further studies are in progress. IMPROVEMENT OF CITRUS LEAF BLOTCH VIRUS DETECTION USING CONVENTIONAL AND REAL-TIME RT-PCR. M. Saponari1, G. Loconsole2 and V. Savino1,2. 1Istituto di Virologia Vegetale del CNR, Unità Organizzativa di Bari, Via Amendola 165/A, 70126 Bari, Italy. 2Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. E-mail: m.saponari@ba.ivv.cnr.it Citrus leaf blotch virus (CLBV) was first reported from Italy in 2006 associated with bud union disorder of ‘Nagami’ kumquat scions on citrange Troyer. In 2008, surveys were conducted to assess the presence of the virus in the foundation block of certified trees, as well as in commercial citrus orchards of Apulia (Southern Italy). RT-PCR assays were performed using the standard protocol developed by Galipienso et al. (Eur. J. Plant Pathol. 110: 175-181, 2004). Although, most of the samples tested negative for CLBV, a few yielded faint DNA bands. Due to these inconsistent results, two new primer sets for conventional and real-time RTPCR were developed. Both primer sets were selected in the 3’ portion of ORF1 of the viral genome. Using one-step RT-PCR the primers CLBV-1/CLBV-2 (5’-TTTTGGTACAGTTCGCTCCA3’ and 5’ATCAGATGGACCTC GAAGGA-3’) amplified the expected product of 292 bp only from CLBV-infected controls, while none of 150 samples tested proved to be infected. A TaqMan based real-time RT-PCR assay was also developed the results of which agreed with those of conventional one-step RT-PCR. Ten-fold serial dilutions of total RNA extracted from a CLBV-infected kumquat were used to assess the sensitivity of the newly developed assays. Target RNA was detected up to a dilution of 10-5 and 10-6 using the one-step RT-PCR and the real-time RTPCR assays, respectively. When compared with the protocol previously available, the present assays proved to be up to 100 times more sensitive. However, none of the commercial citrus varieties tested in Apulia was found to be infected by CLBV. LOW GENETIC COMPLEXITY IN THE CITRUS TRISTEZA VIRUS POPULATION SPREADING IN APULIA. M. Saponari1, R. Abou Kubaa2, G. Loconsole2, A. Percoco3 and V. Savino1,2. 1Istituto di Virologia Vegetale del CNR, Unità Organizzativa di Bari, Via Amendola, 165/A, 70126 Bari, Italy. 2Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola, 165/A, 70126 Bari, Italy. 3Servizio Fitosanitario Regionale, Regione Puglia, Via Mele 2, 70121 Bari, Italy. Email: m.saponari@ba.ivv.cnr.it Since 2001, recurrent outbreaks of tristeza, caused by Citrus tristeza virus (CTV) have occurred in the main citrus growing areas of Apulia (Southern Italy). To restrain the spread of the disease, a control program has been implemented which includes surveys, eradication of infected trees and characterization of CTV strains associated with field infections. Between 2007 and 2008, CTV isolates from 200 trees were collected from 13 different CTV-infected groves and local nurseries. Single strand conformation polymorphism (SSCP) analysis, PCR with multiple molecular markers (MMM), real-time qRT-PCR using strain-specific TaqMan probes, restriction fragment length polymorphism (RFLP) and sequence analysis were used to assess the genetic di- S4.88 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 versity of these isolates. Based on field symptomatology and on the result of molecular analyses, 10 isolates were selected and graft-inoculated to a standard set of indicators, for determining their virulence. SSCP patterns of the coat protein (CP) amplicon showed a consistent simple pattern, suggesting that the tested isolates are likely to be all the same. MMM, RFLP and qRT-PCR assays indicated that the isolates are genetically closely related. Furthermore, sequence analysis of the CP gene showed that all isolates grouped in the same phylogenetic cluster, along with the typical mild isolates from Florida (T30) and Spain (T385). Preliminary results from indexing support the notion that all isolates are of the mild type. So far, no stem pitting has been observed in sweet orange or grapefruit seedlings. Work supported in part by the Regione Puglia, Servizio Fitosanitario Regionale. PHENOTYPIC AND GENOTYPIC DIVERSITY OF SEVERAL ITALIAN ISOLATES OF PHYTOPHTHORA INFESTANS. F. Savazzini, P.L. Burzi, S. Galletti and C. Cerato. CRA, Centro di Ricerca per le Colture Industriali, Via di Corticella 133, 40128 Bologna, Italy. E-mail: stefania.galletti@entecra.it.it Phytophthora infestans (Mont.) de Bary is the causal agent of potato late-blight, which devastated the British Isles potato crops in the mid 1800s. Since then, this disease has been constantly monitored. Before the 1970s, the European populations of the pathogen appeared homogeneous, but, in the past 30 years, the changes occurred whitin them were associated with an increased virulence. The monitoring of these “new populations” as well as the understanding of the population dynamics and evolution are crucial for the management of potato crops and breeding. While the genotypic and phenotypic diversity of P. infestans populations have been deeply examined in Europe and the Americas, the characterization of Italian populations is still scarce and a few studies are available. These latter are mainly based on phenotypic traits obtained by in planta virulence tests. This study aimed at investigating the genetic structure of the population of this important pathogen. Several isolates from infected potatoes were collected in the last years and the phenotypic diversity was investigated by mating type and metalaxyl sensitivity tests. Moreover, the research was addressed at completing the analysis of the population diversity, using several genotypic markers such as mitochondrial DNA haplotype, simple sequence repeats (SSRs) and the sequence of the rDNA internal trascribed spacers regions (ITS). Finally, the comparison of the Italian isolates with the European populations would permit to understand the extant relationships and offer a first overview of the genetic and phenotypic structure of the Italian population of P. infestans. HORIZONTAL TRANSFER OF THE OPHIOSTOMA GENE ENCODING CERATO-ULMIN INTO UNRELATED SPECIES OF THE GENUS GEOSMITHIA. A. Scala1, C. Comparini1, L. Carresi1, P. Bettini2, A. Santini3, A.L. Pepoli3, G. Cappugi4, L. Pazzagli4, F. Martellini4 and F. Scala5. 1Dipartimento di Biotecnologie Agrarie, Laboratorio di Patologia Vegetale Molecolare, Università degli Studi di Firenze, Via della Lastruccia 12, 50019 Sesto Fiorentino (FI), Italy. 2Dipartimento di Biologia Evoluzionistica “Leo Pardi”, Via Romana 17, 50125 Firenze, Italy. 3Istituto per la Protezione delle Piante del CNR, Via Madonna del Piano 10, 50019 Sesto Fiorentino (FI), Italy. 4Dipartimento di Scienze Biochimiche, Università degli Studi, Viale Morgagni 50, 50134 Firenze, Italy. 5Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Sezione di Patologia Vegetale, Università di Napoli Federico II, Via Università 100, 80055 Portici (NA), Italy. E-mail: aniello.scala@unifi.it Horizontal gene transfer (HGT) is a relevant evolutionary mechanism by which plant pathogens have emerged in agroecosystems over different time scales. In the prokaryotes HGT is generally considered a major factor for the evolution of genomes, whereas in multicellular eucaryotes HGT is assumed to play a minor role. In fungi, HGT has been invoked to justify unusual features of genetic elements such as single genes or gene clusters. Among the few examples of horizontally transferred genes, we can mention those for the biosynthesis of host-selective toxins, clustered in Alternaria alternata, Cochliobolus carbonum and C. heterostrophus, the pea pathogenicity (PEP) cluster in Nectria haematococca and the toxA gene from Phaeosphaeria nodorum into Pyrenophora tritici-repens. In the present work we demonstrated that strains belonging to Geosmithia pallida and G. langdonii possess and express the cu gene coding the cerato-ulmin (CU) hydrophobin. CU is produced by various Ophiostoma species (a taxon distant from the genus Geosmithia), and to give the Ophiostomas causing Dutch elm disease key advantages in parasitic fitness and virulence. We are working to understand how and why a portion of the genome of Ophiostoma novo-ulmi, including the cu gene, has been transferred in Geosmithia strains, and what advantages, in terms of fungal fitness, there are for the Geosmithias by acquiring this gene. Moreover, we are characterizing the CUs produced by the Geoesmithias and investigating how they behave towards the elms. THE CERATO-PLATANINS: NON-CATALYTIC PROTEINS WITH FUNCTIONS IN THE RELATIONAL LIFE OF FUNGI. A. Scala1, I. Baccelli2, R. Bernardi2, G. Cappugi3, L. Carresi1, D.O. Cicero4, C. Comparini1, F. Martellini3, L. Pazzagli3, T.A. Pertinhez5 and A. Spisni5. 1Dipartimento di Biotecnologie Agrarie, Laboratorio di Patologia Vegetale Molecolare, Università degli Studi di Firenze, Via della Lastruccia 12, 50019 Sesto Fiorentino (FI), Italy. 2Dipartimento di Biologia delle Piante Agrarie, Sezione di Genetica, Università degli Studi, Via Matteotti 1/B, 56124 Pisa, Italy. 3Dipartimento di Scienze Biochimiche, Università degli Studi, Viale Morgagni 50, 50134 Firenze, Italy. 4Dipartimento di Scienze e Tecnologie Chimiche, Università “Tor Vergata”, Via della Ricerca Scientifica 1, 00133 Roma, Italy. 5Dipartimento di Medicina Sperimentale, Università degli Studi, Via Volturno 39, 43100 Parma, Italy. E-mail: aniello.scala@unifi.it Plant pathogenic fungi produce a lot of proteins without catalytic activity, which are involved in various aspects of parasitism and in the development of disease, such as the dissemination of fungi by vectors, the attachment of the fungus to the surface of plant organs, the expression of symptoms, and the elicitation of defense responses. Currently, seven non-catalytic fungal protein families have been identified, including the cerato-platanin family (PF07249), the class I hydrophobins (PF01185), the class II hydrophobins (PF06766), the elicitins (PF00964), the PcF family (PF09461), and the NIP-1 protein family (PF08995); the CFEM family (PF05730) contains a cysteine-rich domain present into some other fungal proteins for which a role in pathogenesis has been proposed. Since different families may contain proteins with similar functions in the plant pathogenesis, and a single family contains proteins with different functions, we aimed to investigate why this happens. For this purpose we developed a study model represented by two proteins belonging to the cerato-platanin family: cerato-platanin (CP), the founder protein of the family produced by Ceratocystis platani, and cerato-populin Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 (Pop1) produced by C. populicola. Both CP and Pop1 have been purified, cloned in Pichia pastoris and characterized biochemically and functionally. They are well-structured α/β proteins (but with different percentages of the α-helix), and have only the 70.1% of the amino acids identical or highly conserved. Moreover, they behave as PAMPs, since it stimulated plants to activate defense responses able to reduce consistently the fungal growth. Their capability to elicit differently defense events is under study. PHYTOPHTHORA spp. IN FOREST NURSERIES IN SARDINIA. B. Scanu1, B.T. Linaldeddu1, A. Schiaffino1, J.F. Webber2 and A. Franceschini1. 1Dipartimento di Protezione delle Piante, Sezione di Patologia vegetale, Università degli Studi, Via E. De Nicola 9, 07100 Sassari, Italy. 2Forest Research, Alice Holt Lodge, Farnham, Surrey GU10 4LH, UK. E-mail: bscanu@uniss.it In summer 2008 several cases of decline and mortality were observed in two forest nurseries located in central Sardinia (Italy). Seedlings and potted saplings of various Mediterranean shrubs and trees (Arbutus unedo, Castanea sativa, Juglans regia, Myrtus communis, Pistacia lentiscus, Quercus ilex and Taxus baccata) showed symptoms of dieback, leaf withering, collar and root rot. Since a Phytophthora was suspected to be the causal agent, isolations from soil and roots were made by baiting with apples and rhododendron leaves, plating onto the selective medium synthetic mucor agar (SMA) and transferring to carrot agar (CA). The isolates obtained were identified on the basis of morphological and growth features. Identification was confirmed by sequencing the ITS region of rDNA and comparing the sequences with those of the Phytophthora species in NCBI database. Two heterothallic species, Phytophthora cinnamomi and P. nicotianae, were the most commonly isolated, but other Phytophthora spp. were also detected and are still under investigation. The virulence of all Phytophthora species obtained in this study was confirmed by apple bioassay, and that of P. cinnamomi also by pathogenicity tests on 3-month-old Q. ilex seedlings. The latter Phytophthora species is a dangerous and polyphagous pathogen that could constitute a serious risk for the health of Sardinian forests if disseminated by infected nursery plants. VERSICOLOR EXTRACELLULAR GLUCAN OLIGOMERS ARE ABLE TO INHIBIT AFLATOXINS BIOSYNTHESIS IN ASPERGILLUS FLAVUS. M. Scarpari1, M. TRAMETES Reverberi1, S. Zjalic1 P. Cescutti,2 R. Rizzo2, A. Ricelli3, A.A. Fabbri1 and C. Fanelli1. 1Dipartimento Biologia Vegetale, Università “Sapienza”, Largo Cristina di Svezia 24, 00165 Roma, Italy. 2Dipartimento Scienze della Vita, Università degli Studi, Via Giorgieri 1, 34127 Trieste, Italy. 3Istituto di Chimica Biomolecolare del CNR, Piazzale Aldo Moro 5, 00185 Roma, Italy. E-mail: marzia.scarpari@uniroma1.it Aflatoxins are toxic secondary metabolites produced by Aspergillus flavus. It has been largely shown that the synthesis of aflatoxins is closely related with the oxidative stress inside and outside the cell. Trametes versicolor is a basidiomycete known for its therapeutic effects mainly due to production of several glycoproteins and exoglucans which display anti-tumoral actions and anti-oxidative effects. The addition of lyophilised filtrate (LF) of T. versicolor, in a concentration range of 0.01-2%, in the the culture medium of A. flavus strongly inhibits the biosynthesis of aflatoxin and reduces mycelium growth. If LF is previously dialysed (membrane cutoff 1000) it induces a lowering (~55-60%) of toxin inhibition ability and a similar decrease is observed when S4.89 LF is digested with trypsin or with glucanases. GC-MS and 1HNMR analysis show the presence of highly branched polysaccharides (α and _-glucans) with mol. wt between 20 and 6 kDa. These glucans are probably digested by T. versicolor glucanase to biologically active smaller fragments. The presence is therefore assumed of enzymatic and oligosaccharidic components, which act in a complementary way to inhibit aflatoxins. How can extracellular glucan oligomers of T. versicolor inhibit aflatoxin biosynthesis in A. flavus? A putative receptor for glucans was identified in A. flavus genome which is similar to a plant receptor related to the onset of plant defence responses. It can be hypothesised that these fungal oligomers can act as non self signals able to modulate secondary metabolism in mycotoxigenic fungi. GENE SILENCING TARGETED AT THE TRICHOTHECENE BIOSYNTHESIS GENE TRI6 IN FUSARIUM CULMORUM AND ITS EFFECT ON PATHOGENICITY TOWARDS DURUM WHEAT. B. Scherm1, M. Orrù1, V. Balmas1, F. Spanu1, E. Azara2, G. Delogu2, T.M. Hammond3, N.P. Keller4 and Q. Migheli1. 1Dipartimento di Protezione delle Piante, Unità di Ricerca Istituto Nazionale di Biostrutture e Biosistemi, Università degli Studi, Via E. De Nicola 9, 07100 Sassari, Italy. 2Istituto di Chimica Biomolecolare del CNR, Traversa La Crucca 3, 07100 Sassari-Baldinca, Italy. 3Division of Biological Sciences, University of Missouri, 105 Tucker Hall, Columbia, MO 65211, USA. 4Department of Plant Pathology, University of Wisconsin, 1630 Linden Drive, Madison, WI 53706-1598, USA. E-mail: qmigheli@uniss.it The efficacy of an RNA silencing construct in suppressing mycotoxin production was tested in the plant pathogenic fungus Fusarium culmorum, incitant of crown and foot rot of wheat. Transformation of a highly virulent wild type strain of F. culmorum and its nitrate reductase deficient mutant with inverted repeat transgenes (IRT) containing sequences corresponding to the trichothecene biosynthesis gene tri6 was obtained using the hygromycin B resistance gene hph as selectable marker. The pattern of integration indicates that a high proportion of cotransformants underwent homologous recombination events with partial deletion of the endogenous tri6 gene. The analysis of cotransformants with a single site integration showed that tri6-specific IRT did not alter physiological characteristics, such as spore production, pigmentation, and growth rate on solid media. Northern blot analysis of selected cotransformants after 7-13 days in culture revealed an intense tri6-specific smear, indicating the presence of silenced RNAs. Gene expression profiles of tri5, encoding a trichodiene synthase regulated by the tri6 protein, were determined by realtime RT-PCR analysis, confirming that in most cotransformants the tri5 gene was not expressed. Accordingly, the production of deoxynivalenol (DON) and its derivatives in vitro, detected by LC-MS upon extraction of the mycelia and the culture filtrates, revealed that silenced mutants failed to produce trichothecenes. Pathogenicity assays were carried out to evaluate whether a reduced DON production by the tri6-IRT strains correlated with a loss of virulence, and showed significantly decreased disease indices (20-50%) in most of the tested strains. PRELIMINARY STUDY ON THE GENETIC VARIABILITY OF TUBER BORCHII. A. Schiaffino1 and F. Marras1,2. 1Diparti- mento di Protezione delle Piante, Sezione di Patologia Vegetale, Università degli Studi, Via E. De Nicola 9, 07100 Sassari, Italy. 2Istituto per lo Studio degli Ecosistemi del CNR, Traversa La Crucca 3, Regione Baldinca, 07040 Li Punti (SS), Italy. E-mail: fmarras@uniss.it S4.90 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 Tuber borchii Vittad is a very common truffle in Sardinia and all the other Italian regions. It is an ubiquitous species, for it can be found either in the inland hilly areas and the littoral zones, especially when covered by coastal pine forests. It can also be found in a wide range of soils from clayey to loose and sandy type, with sub-alkaline or sub-acid reaction, or even as acid as in the cork oak stands of Gallura. In this survey the genetic variability of T. borchii was analysed by sequencing the ITS1-5.8S-ITS2 region and the genes coding for β-tubulin and elongation factor 1α (EF-1α), in fungal samples collected in Sardinia and Apulia (Southern Italy). Comparison of the sequences disclosed that there are two different groups of “whitish truffle”, A and B, with a B1 subgroup. The results of this preliminary study confirmed the great genetic variability of T. borchii and underlined the presence in this species of two or three groups of different genetic sequences which could indicate the existence of as many populations comparable to sub-specific taxa. STUDY OF RELATIVE EXPRESSION OF LIPODEPSIPEPTIDES SYNTHETASE AND DEFENCE-RELATED GENES IN ORANGE FRUIT IN RESPONSE TO ANTAGONISTPATHOGEN INTERACTION. G. Scuderi, S. Panebianco and G. Cirvilleri. Dipartimento di Scienze e Tecnologie Fitosanitarie, Università degli Studi, Via S. Sofia 100, 95123 Catania, Italy. Email: gcirvil@unict.it The production of syringomycin and syringopeptin toxins is a distinctive feature of Pseudomonas syringae pv. syringae and has been correlated with post-harvest disease control. Furthermore, biocontrol agents can induce defence mechanisms of resistance to disease in citrus such as production of reactive oxygen species, systemic acquired resistance and induced systemic resistance. Aim of this research was to evaluate the expression of syringomycin (syrB1) and syringopeptin (sypA) synthetase genes from two P. syringae biocontrol strains in orange fruit in the presence/absence of Penicillium digitatum. Quantitative reverse transcriptase PCR (qRT-PCR), used to determine the level of each transcriptional-product, indicated that expression of syrB1 and sypA genes was induced in orange peel tissue, and that the presence of P. digitatum increased syrB1 gene expression 24 and 72 h after orange inoculation. Similarly, the expression level of genes that encode chitinase (CHI), gluthatione peroxidase (GPX), allene oxide synthase (AOS), glucanase (GNSL) and phenylalanine-ammonia lyase (PAL) in inoculated orange tissues was measured by qRT-PCR during the first 72 h after inoculation. Expression of defence-related genes showed that antagonistic P. syringae induces the transcription of chitinase and that presence of P. digitatum strongly enhances the expression of chitinase, allene oxidase synthase and gluthatione peroxidase in orange tissues. Our results suggest that in the interaction among antagonist, pathogen and orange tissue, the expression of both syringomycin/syringopeptin synthetase and defence-related genes could play an important role in the biocontrol activity of P. syringae strains. MOLECULAR CHARACTERIZATION OF COLLETOTRICHUM ISOLATES FROM OLIVE IN AUSTRALIA. V. Sergeeva1, L. Schena2, M.A. Mammella2, R. Faedda3 and S.O. Cacciola4. 1Centre for Plant and Food Science, University of Western Sydney, Locked Bag1797, South Penrith, DC, NSW, Australia. 2Dipartimento di Gestione dei Sistemi Agrari e Forestali, Università Mediterranea, Località Feo di Vito, 89122 Reggio Calabria, Italy. 3Dipartimento di Scienze e Tecnologie Fitosanitarie, Università degli Studi, Via S. Sofia 100, 95123 Catania, Italy. 4Diparti- mento di Chimica Biologica, Chimica Medica e Biologia Molecolare, Università degli Studi, Viale Andrea 6, 95125 Catania, Italy. E-mail: olgacacciola@unict.it Olive isolates of Colletotrichum from diverse olive-growing areas of Australia were characterized by RAPD-PCR with decamer primers and by amplifying and sequencing two genomic regions of approximately 600 and 1000 bp from the β-tubulin genes 1 and 2, respectively. Both β-tubulin genes were amplified and sequenced using degenerate primers designed by aligning and comparing more that 1000 available sequences. Other olive isolates of Colletotrichum from Italy and Portugal as well as isolates of various geographic origins from citrus, camellia, basil, strawberry, azalea, cherry and oleander, an Italian C. musae isolate from banana and almond isolates from Australia were included in this study. Published sequences of Glomerella acutata, C. gloeosporioides and C. graminicola (accessions Nos. AB273716, U14138 and M34492, respectively) were used as references while sequences of Verticillium dahliae var. longisporum, V. dahliae, Fusarium lunatum and Fusarium spp. (accessions Nos. DQ166861, DQ166894, EU926357 and EU926418, respectively) were included as outgroup sequences. Grouping of isolates based on RAPDPCR was consistent with the groups separated by the β-tubulin genes sequencing. Australian isolates from olives infected by antrachnose were identical or very close to G. acutata, teleomorph of C. acutatum. They were tolerant to benomyl (MIC > 102 µg/ml) and clustered with almond and oleander isolates from Italy but formed a C. acutatum subgroup distinct from both strawberry and olive isolates from Portugal. These results indicate that the Colletotrichum population associated with olive antrachnose in Australia is distinct from Colletotrichum populations causing the same disease in Italy and Portugal. SURVEY OF POTATO VIRUS Y IN POTATO SEED AND CONSUMER TUBERS IN CENTRAL AND SOUTHERN ITALY. L. Sigillo1, V. Senape1, V Lumia2, G. Serratore1 and L. Tomassoli2. Nazionale delle Sementi Elette, SS 18 km 77,700, 84091 Battipaglia (SA), Italy. 2CRA, Centro di Ricerca per la Patologia Vegetale, Via C.G. Bertero 22, 00156 Roma, Italy. E-mail: laura.tomassoli@entecra.it 1Ente In 2009 a survey was carried out to check the distribution of Potato virus Y (PVY) genotypes (N; O; NTN; N:O) in consumer and seed tubers in Central and Southern Italy. Twenty tuber stocks were analyzed from Latium, 3 from Campania, 20 from Apulia, 15 from Sicily and 87 seed stocks from Calabria. Virus detection was carried out by TAS-ELISA and the identification of PVY variants was made using multiplex PCR according to Lorenzen [Plant Dis., 90 (7), 935-940, 2006]. Based on TAS ELISA, PVY was detected in 44% of the samples from Latium, 26% from Apulia, 87% from Sicily and in the totality of Campanian stocks. Infection rate was 10 to 70%. Infected stocks from Calabria had an average infection rate lower than 10% with only 6% of them exceeding 40%. After multiplex PCR assays, all the isolates from Campania and Apulia were identified as PVY N:O and PVY-NTN, respectively. PVYNTN was identified in about 60% of infected samples from Latium and Sicily; PVY N:O was found in 13% and 40% of samples from Latium and Apulia, respectively. In Latium, molecular assays detected 22% mixed infections caused by N:O and NTN strains. No single infection of PVY-N or PVY-O was detected. In PVY-positive field samples, symptoms were rarely obvious, especially for PVY- NTN which is responsible for necrotic ringspots on tubers. To investigate PVY strains variability, some Italian isolates were sequenced in the VPG-NIa re- Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 gion and preliminary phylogenetic analysis confirmed their distribution in genotype clusters. EFFECTS OF CITRUS TRISTEZA VIRUS ON FRUIT QUALITY OF TAROCCO COMUNE SWEET ORANGE IN SICILY. G. Sorrentino1, S. Davino2, M. Guardo1, G. Giuffrida1, G. Iacono3, A. Caruso1 and M. Davino3. 1CRA, Istituto Sperimentale per l’Agrumicoltura, Corso Savoia 190, 95024 Acireale (CT), Italy. 2Dipartimento di Scienze Entomologiche, Fitopatologiche, Microbiologiche, Agrarie e Zootecniche Sezione di Patologia Vegetale e Microbiologia Agraria, Università degli Studi, Viale delle Scienze, 90128 Palermo, Italy. 3Dipartimento di Scienze e Tecnologie Fitosanitarie, Sezione di Patologia Vegetale, Università degli Studi, Via Santa Sofia 100, 95123 Catania, Italy. E-mail: davino@unipa.it Since 1999, many foci of Citrus tristeza virus (CTV) have been discovered in different areas of Eastern Sicily (Southern Italy), particularly in Catania and Syracuse provinces and in the mainland (Apulia and Calabria). Different CTV strains have been detected. A severe strain was detected in the Baè area in Catania province, where some parameters were evaluated to ascertain the effects of CTV infection on fruit quality. Plants at different infection stages were evaluated with an empirical scale: 0, no infection; 1, infected with no symptoms; 2, light symptoms; 3 severe symptoms. All trees monitored were about thirty-year-old cv. Tarocco ‘Comune’ sweet oranges grafted onto sour orange. Every year for four successive years, yield, average fruit weight, juice content, percentage of total soluble solids (TSS), of total acidity (TA), ripening ratio (TSS/TA), peel colour, pulp colour, juice colour and SPAD for leaf chlorophyll were evaluated. Data were subjected to simple variance analysis and the means were compared (Tukey). The results for group 1 regarding production, average fruit weight, SPAD for leaf chlorophyll and total soluble solids were significantly lower compared to uninfected plants. Consequently, the maturation ratio (S/A) was significantly lower than in uninfected plants. The negative effects of CTV infection on production parameters even in the initial stages of viral infedtion were confirmed by discriminatory analysis. MONILINIA FRUCTICOLA AMONG THE PATHOGENS OF STONE FRUITS IN NORTHERN ITALY. D. Spadaro1, C. Pellegrino2, A. Garibaldi2 and M.L. Gullino2. 1Dipartimento di Valorizzazione e Protezione delle Risorse Agroforestali, Sezione di Patologia Vegetale, Università degli Studi di Torino, Via L. da Vinci 44, 10095 Grugliasco (TO), Italy. 2Centro di Competenza per l’Innovazione in Campo Agro-ambientale, Università degli Studi di Torino, Via L. da Vinci 44, 10095 Grugliasco (TO), Italy. E-mail: davide.spadaro@unito.it Monitoring of postharvest rots of stone fruit was carried out in Northern Italy in 2008. Twelve cultivars of apricot, peach and nectarine were monitored in the orchard and during storage for eight weeks. Early-ripening cultivars showed higher susceptibility to rots than late-ripening cultivars. Peach varieties were more susceptible to postharvest storage than nectarines. Peaches and nectarines were harvested twice. Fruit of the second harvest resulted more severely affected. Most of the postharvest damages to peaches and nectarines were caused by Botrytis cinerea. Monilia laxa was the most frequent species isolated from apricots, peaches and nectarines. M. fructigena was present only on apricots. M. fructicola, a quarantine pathogen in Europe, was reported for the first time in Italy on stone fruits. In particular, it was detected on some varieties of nectarines starting 3 weeks after harvest. Prelim- S4.91 inary morphological identification of M. fructicola was confirmed by PCR with a common reverse primer and three species-specific forward primers obtained from a sequence-characterized amplified region, which yielded a 535 bp product diagnostic for M. fructicola. Two sequences obtained through the amplification of ribosomal region ITS1-5.8S-ITS2, showed 100% similarity with the same ribosomal sequence of M. fructicola. The pathogen was detected on some mummified fruit from the same orchards in early November 2008. Pathogenicity was confirmed by spraying conidia on nectarines. M. fructicola was reisolated from the inoculated fruit on PDA medium. PHYTOPLASMOSIS OF WEEDS IN SICILY. M. Tessitori1, C. Oliveri1, R. La Rosa1 and C. Marzachì2. 1Dipartimento di Scienze e Tecnologie Fitosanitarie, Università degli Studi, Via S. Sofia 100, 95123 Catania, Italy. 2Istituto di Virologia Vegetale del CNR, Strada delle Cacce 73, 10135 Torino, Italy. E-mail: mtessitori@unict.it Spontaneous and invasive plants can be infected by phytoplasmas and show specific symptoms. Moreover weeds often act as alternative hosts of phytoplasmas agents of important diseases of cultivated crops (e.g. grapevine yellows). A study of the role of weeds in the epidemiology of bois noir (BN) disease was made in an organic vineyard in Sicily. After extensive inventory of spontaneous plant species inside and at the border of the field in the different seasons, phytoplasma-specific symptoms were observed and potential vectors were sweep-collected on symptomatic weeds. Two important syndromes were observed on Cynodon dactylon and Conyza bonariensis. The first host showed yellow stripes and white leaf, while asthma weed (C. bonariensis) showed evident shortening of the internodes. Molecular assays and sequencing showed the presence of “Candidatus Phytoplasma cynodontis” in bermuda grass and of “Ca. P. solani” in C. bonariensis. This latter species can be defined as a new natural host of the BN phytoplasma. During a survey in an uncultivated field, sectorial symptoms of fasciation were observed on Euphorbia linifolia. Molecular analysis confirmed that this species was also affected by the same “Ca. P. cynodontis”. These new findings, and the observation of similar symptoms on Bermuda grass in several areas of the region, can also suggest the involvement of an yet unknown efficient vector of this disease in Sicily. RECURRENCE OF PARIETARIA MOTTLE VIRUS ON TOMATO IN LATIUM. A. Tiberini1,4, R. Parisella2, C. Boc- congelli3 and L. Tomassoli1. 1CRA, Centro di Ricerca di Patologia Vegetale, Via C.G. Bertero 22, 00156 Roma, Italy. 2De Ruiter Sementi snc, Via Mortacino, 04019 Terracina (LT), Italy. 3Monsanto Agricoltura Italia S.p.A., Strada Canneto di Rodi, 04100 Latina, Italy. 4Dipartimento di Gestione dei Sistemi Agrari e Forestali, Università Mediterranea, Località Feo di Vito, 89060 Reggio Calabria, Italy. E-mail: laura.tomassoli@entecra.it Parietaria mottle virus (PMoV), a member of genus Ilarvirus, family Bromoviridae, was originally isolated from a weed species, Parietaria officinalis in 1989. The virus has quasi isometric to ovoid particles 24-36 nm in diameter, a capsid protein (CP) of 24,3 kDa, and a tripartite genome composed of three singlestranded positive sense RNA molecules. No serological relationship with other member of genus Ilarvirus is known, thus PMoV was assigned to a separated subgroup within the genus. A variant of PMoV, named tomato strain (PMoV-T), was reported as the cause of a disease of tomato in France, Spain, Greece and Italy. Further, PMoV was reported infecting Capsicum annum. Since S4.92 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 1971, PMoV-T was observed sporadically in many Italian areas including Apulia, Basilicata, Campania, Latium, Liguria, Sardinia and Sicily. Yearly occurrence of PMoV-T in an erratic and scattered manner was reported form tomato in Latina province (Latium). Initially, infected plants develop a bright necrotic mosaic on the leaves that turns into necrosis, of laves, stem and, occasionally, apex. Fruits of affected plants display corky rings and brown patches, which develop into ridges with necrotic scars. Recently, samples were assayed in RT-PCR with two different sets of specific primers, for the putative movement protein (MP) PMoV 1F/R and coat protein (CP) PMoV 4F/R. Amplicons of expected length (712 bp and 663 bp respectively) were obtained and sequenced. Phylogenetic analysis confirmed that our isolates belong to PMoV-T cluster sharing 99% nucleotide sequence homology with Spanish isolates. 2009 in Apulia and Basilicata (Southern Italy). Five- to sevenyear-old stone fruit trees were sampled in the provinces of Taranto (Castellaneta, Lizzano, Massafra, Mottola, Palagiano), Bari (Acquaviva delle Fonti, Bitonto, Castellana Grotte, Molfetta, Sammichele di Bari, Terlizzi, Turi), Foggia (Trinitapoli) and in Basilicata (Policoro). Apple, pear and quince trees were sampled in several Apulian areas. Based on visual inspections conducted in spring, symptomless plants were sampled and ELISA-tested for the following viruses: Apple chlorotic leaf spot virus (ACLSV) (all trees), Prune dwarf virus (PDV), Prunus necrotic ring spot virus (PNRSV), and Plum pox virus (PPV) (stone fruits only), Apple stem grooving (ASGV) (pome fruits) and Apple mosaic virus (ApMV) (apple). None of the tested trees was infected by PPV, a quarantine pathogen, and by ASGV. The other viruses had a low incidecne, i.e. 3.6% for PDV and 1.6% for PNRSV and ACLSV. PHYTOPATHOLOGICAL SURVEYS ON ORGANIC SMALL GRAIN CEREALS. M. Tinelli, S. Salvi, G. Beccari and L. Co- INDUCED RESISTANCE IN ORANGE FRUIT TREATED WITH CHITOSAN. M.C. Trullo1, P. Merelo2, L. Schena3, F.R. varelli. Dipartimento di Scienze Agrarie e Ambientali, Università degli Studi, Borgo XX Giugno 74, 06121 Perugia, Italy. E-mail: lorenzo.covarelli@unipg.it Tadeo2, M. Talón2 and A. Ippolito1. 1Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. 2Instituto Valenciano de Investigaciones Agraria, Centro de Genómica, Moncada, Valencia, Spain. 3Dipartimento di Gestione dei Sistemi Agrari e Forestali, Università Mediterranea, Località Feo di Vito, 89122 Reggio Calabria, Italy. E-mail: ippolito@agr.uniba.it The purpose of the present study was to investigate phytosanitary status of the seed of organic small grain cereals (Panicum miliaceum, Triticum dicoccum, Hordeum vulgare, Secale cereale, Triticum aestivum, Zea mays, Avena sativa), harvested in Central Italy in 2008 and destined for human consumption. The occurrence of seed-transmitted and mycotoxigenic fungi was evaluated by pathogen isolation on artificial media, followed by morphological identification, and by PCR. The work was mainly focused on Fusarium species, which can currently be considered the most dangerous pathogens in cereals due to their capability to cause serious plant diseases and to produce mycotoxins in the grain. Moreover, in maize, spelt, millet and barley, the effects on the above mentioned parameters of technological operations (drying, decortication, pearling) were investigated. The presence of deoxynivalenol and fumonisins was assessed by immuno-enzymatic assays. Microdochium nivale and Fusarium spp. were the most frequently isolated pathogens from the examined seed samples, but also Alternaria spp. and Aspergillus spp. were regularly isolated. Pearling and decortication significantly decreased the isolation frequency of Fusarium, Aspergillus and Alternaria species, particularly in millet and barley whereas drying operations had a strong effect on reducing the presence of Fusarium verticillioides in maize grains. Seed infections by the different pathogens in the examined cereals, as detected by traditional and molecular methods, are discussed. Very low mycotoxin levels were found in the analyzed samples. INCIDENCE OF VIRUS INFECTIONS IN STONE AND POME FRUIT SELECTED FOR NURSERY INDUSTRY IN SOUTHERN ITALY. N. Trisciuzzi, M.R. Silletti and P. Pollastro. Centro di Ricerca e Sperimentazione in Agricoltura “Basile Caramia”, Via Cisternino 281, Locorotondo (BA), Italy. E-mail: info@crsa.it EU regulations for nursery plant production impose specific phytosanitary requirements to ensure high quality standards of the marketed productions and to contain the spread of harmful organisms. To fulfill these requirements, mother plants must be regularly inspected and laboratory tested to assess the absence of specific harmful pathogens such as viruses, viroids and fungi. In the framework of this program, a total of 1860 stone fruit trees and 215 apple, pear and quince trees were tested during 2004- Chitosan is widely recognised as an alternative and safe means to control postharvest diseases of fruit and vegetables. Its mode of action has not been completely elucidated, however it is known that it can act on host and pathogen inducing resistance. In the present study specific trials were conducted to evaluate the activation of resistance genes in citrus fruit treated with chitosan. Surface sterilised oranges were treated by applying chitosan or water in a wound and Penicillium digitatum in another wound made 48 h later and 0.5 cm apart. Notwithstanding the spatial separation between chitosan and pathogen, incidence and severity of decays were significantly reduced in chitosan- treated fruit, clearly indicating the activation of resistance mechanisms. To investigate the induction of resistance at a molecular level, gene expression profiles were analysed by using a 20K citrus cDNA microarray containing 22.176 citrus Expressed Sequence Tags (ESTs) in albedo and flavedo tissues. Chitosan effect was detected by hybridizing RNA from chitosan- and water-treated tissues. A total of 671 genes were statistically significant (P≤0.005) and among these 220 were differentially expressed (showing changes in gene expression equal or higher than ± 2 fold) in tissues treated with chitosan. According to the functional classification based on Gene Ontology analysis, many of the selected genes are involved in defence mechanism, protein metabolism, transport, and signal transduction. On the whole, data indicate that the induction of resistance plays an important role in determining the efficacy of chitosan against Penicillium rots in citrus fruit. INDUCED RESISTANCE IN TOMATO BY OLIGOSACCHARIDES AND GLUTATHIONE. A. Veronesi, R. Roberti, I. Por- tillo and A. Brunelli. Dipartimento di Protezione e Valorizzazione Agroalimentare, Università degli Studi, Viale Fanin 46, 40127 Bologna, Italy. E-mail: roberta.roberti@unibo.it The enhancement of the natural defense of plants against pathogens is an attractive perspective that would allow a reduction of pesticide pollution of the environment. Oligosaccharides and glutathione are components of various products with plant Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 biostimulant activity which are commercialized as fertilizers and are proposed as promoters of plant growth and inducers of defense mechanisms. The aim of this study was to evaluate the resistance induced in tomato by oligosaccharides and glutathione through the detection of foliar enzymatic activities correlated with biochemical plant defense. The basal part of tomatoes at the eight true leaf stage, was treated with oligosaccharides (1 mg/hl), glutathione (60.6 mg/hl), singly and in combination, and acibenzolar S-methyl (1.125 g/hl). Total proteins of the leaves removed from the upper part of each plant, were extracted two days after treatment and their peroxidase, chitinase and glucanase activities were determined. Glutathione, oligosaccharides and their mixture caused significant increases of all three enzymatic activities compared with the non treated control, however no synergistic effect of the mixture was observed. Peroxidase and glucosaminidase activities following treatment with glutathione, oligosaccharides or their mixture were lower than those stimulated by acibenzolar S-methyl. Based on these results, the application of products containing glutathione and oligosaccharides could represent a way to enhance tomato plant defense response to pathogen attacks. BENEFICIAL MICROBES USEFUL FOR BIOCONTROL OF PLANT PATHOGENS AND BIOREMEDIATION OF SITE CONTAMINATED BY METHYL TERT-BUTYL ETHER. F. Vinale1, V. Aloj1, O. Pepe2, S. Woo1, R. Marra1, M. Ruocco3, S. Lanzuise1, F. Scala1, A. Ritieni2, P. Cavallo4 and M. Lorito1. 1Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università degli Studi di Napoli Federico II, Via Università 100, 80055 Portici (NA), Italy. 2Dipartimento di Scienza degli Alimenti, Università degli Studi di Napoli Federico II, Via Università 130, 80055 Portici (NA), Italy. 3Istituto per la Protezione delle Piante del CNR, Via Università 133, 80055 Portici (NA), Italy. 4Dipartimento di Scienze dell’Educazione, Università degli Studi di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy. E-mail: matteo.lorito@unina.it Methyl tert-butyl ether (MtBE) is a gasoline component frequently used as an alternative to lead-based additives. The widespread use of this compound and its persistence in soil and water resulted in a widespread contamination. The use of microorganisms, plants and/or their metabolites to bioremediate polluted sites represents one of the most promising strategy to recover at least partially natural environment heavily altered by man-made contaminants. The aim of this work was to isolate and identify microorganisms able to degrade MtBE and to control simultaneously phytopathogenic agents. Sixteen different bacterial strains were isolated from diverse gasoline-contaminated soils by the enrichment culture technique, and identified by 16S rDNA analysis and phenotipe characterization. These isolates were screened for their ability to degrade MtBE and grow on minimal salt medium amended with this pollutant. Three of them, classified as Bacillus spp., Thiobacillus spp. and Mycobacterium spp., significantly reduced MtBE concentration compared to the untreated control after 7 days of incubation. Moreover, these 3 isolates significantly controlled in vitro and in vivo the growth of the fungal pathogens Pythium. ultimum, Botrytis cinerea and Alternaria alternata. The major secondary metabolites produced by these bacteria were isolated and characterized. Surfactant lipopeptides and small aromatic compounds with antibiotic activity were detected in bacterial culture filtrates. Our results support the use of naturally-occurring beneficial microbes as an effective and low cost strategy for combining biocontrol and bioremediation. S4.93 PUTATIVE MECHANISMS INVOLVED IN THE INTERACTION BETWEEN BACILLUS AMYLOLIQUEFACIENS STRAIN BO7 AND FUSARIUM OXYSPORUM. D. Vitullo1, A. Di Pietro2, F. De Curtis1, V. Lanzotti3 and G. Lima1. 1Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Università degli Studi del Molise, Via de Sanctis, 86100 Campobasso, Italy. 2Departamento de Genética, Universidad de Córdoba, Córdoba, Spain. 3Dipartimento di Scienze e Tecnologie Agro-alimentari Ambientali e Microbiologiche, Università degli Studi del Molise, Via de Sanctis, 86100 Campobasso, Italy. E-mail: domenico.vitullo@unimol.it Biological control of plant pathogens is a promising alternative to the extensive use of synthetic fungicides. Bacillus amyloliquefaciens, a gram positive bacterium, was recently reported as an effective biocontrol agent of soil-borne pathogens. However, its mechanisms of action have not been investigated in detail. The aim of this work was to elucidate the physical, biochemical and molecular mechanisms involved in the action of a novel strain (BO7) of B. amyloliquefaciens against the vascular wilt fungus Fusarium oxysporum used as a soil-borne pathogen model. Strain BO7 was selected from a suppressive soil and tested against a collection of genetically characterized mutants of F. oxysporum f. sp. lycopersici (Fol). Bacterial culture filtrates, fractionated according to molecular weight, were tested for in vitro activity against Fol wild type strain and Fol gene knockout mutants. Fol mutants with cell wall alterations were highly susceptible to low molecular weight fractions of the culture filtrates, suggesting that the antifungal effect of BO7 metabolites is enhanced by loss of cell wall integrity. HPLC and 1H-NMR analysis of the most active fractions indicated that key biomolecules in the antagonism of BO7 are lipopeptides. Light fluorescence microscope observation of Fol hyphae grown in the presence of BO7 cells showed irreversible modification and damage of the cell walls. The results of this study will be discussed in relation to the role of the different modes of action by which the novel biocontrol strain BO7 of B. amyloliquefaciens exerts its strong antifungal activity. COLOMBIAN DATURA VIRUS IN BRUGMASIA spp. IN ITALY. C. Vovlas1, A. De Stradis2 and G. Parrella3. 1Dipartimento di Protezione delle Piante e Microbiologia Applicata, Via Amendola 165/A, 70126 Bari, Italy. 2Istituto di Virologia Vegetale, CNR, Unità Organizzativa di Bari, Via Amendola 165/A, 70126 Bari, Italy. 3Istituto per la Protezione delle Piante del CNR, Via Università 13, 80055 Portici (NA), Italy. E-mail: parrella@ipp.cnr.it In recent years, symptoms of likely viral origin were observed in plants of Brugmasia spp. growing in private gardens and in one plant nursery near to Bari (Apulia, Southern Italy). Symptomatic plants showed small leaves with vein banding and chlorotic flecking. Leaf samples from symptomatic and symptomless Brugmasia were used for mechanical inoculation to herbaceous hosts and electron microscope observations (leaf dips, ISEM, and thin sectioning). The presence of pinwheels, tubes and scrolls in thin-sectioned cells and of filamentous virus particles ca. 850 nm long in leaf dips, suggested that symptomatic plants hosted a potyvirus. This virus was easly recovered by manual inoculation to different solanaceous species (Nicotiana rustica, N. tabacum, N. benthamiana, N. occidentalis, N. clevelandii and Solanum lycopersicum) which reacted with local and/or systemic symptoms. Sequence analysis of a genome fragment ca. 1.7 kb in size, amplified by RTPCR from total nucleic acids extracted fom infected plants showed 99% nucleotide identity with the corresponding region of Colombian datura virus (CDV) sequence (accession No. AB179622). In addition, virus particles were clearly decorated with an antiserum to CDV as observed in IEM assays, confirming S4.94 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 the identity of the virus. This represent the first report of CDV in Italy and confirms that this virus is emerging in different part of the world and might became a serious threat, expecially for solanaceous crops. CHARACTERIZATION OF TRICHODERMA SPECIES ASSOCIATED WITH THE PRODUCTION OF PLEUROTUS OSTREATUS IN ITALY. S.L. Woo1, C.P. Kubicek2, I.S. Druzhinina2, F. Vinale1, P. Cavallo3 and M. Lorito1. 1Dipartimento di Arboricoltura, Botaninca e Patologia Vegetale, Sezione di Patologia Vegetale, Università degli Studi di Napoli Fedrico II, Via Università 100, 80055 Portici (NA), Italy. 2Institute of Chemical Engineering, Vienna University of Technology, Getreidemarkt 9/1665, 1060 Vienna, Austria. 3Dipartimento di Scienze dell’Educazione, Università degli Studi di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy. E-mail: woo@unina.it Trichoderma spp. infestations of Pleurotus production are relatively recent to Italy in comparison with noted problems to Agaricus with T. aggressivum f. europaeum (Europe) and T. a. f. aggressivum (North America). Samples were collected from a compost producer and mushroom farms cultivating P. ostreatus, and pure fungal cultures were obtained. Total fungal counts were conducted in all composting stages. No fungi were detected after pasteurization or grain spawn inoculation, but fungi, including Trichoderma spp., were detected later during incubation and fruiting stages. Biological, phenotypic and genetic characteristics of all Trichoderma isolates were analyzed. Various growth conditions were tested in vitro on both pathogenic and commercial fungi. At 25°C there was optimal sporulation and mycelial growth of the 26 Trichoderma isolates; always more rapid in growth than the 11 Pleurotus varieties at all tested temperatures. Trichoderma grew over a wide pH range, but best at pH 5-7. Applications of different doses of chemical fungicides, a.i. procloraz and/or thiabendazole, controlled spore germination, mycelial and spore development of Trichoderma without negative effects to Pleurotus. Trichoderma isolates problematic to P. ostreatus differed in form and rate of mycelial growth, as well as in type and time to sporulation, from isolates used for biological control of plant pathogens and species pathogenic to A. bisporus. Analysis of internal transcribed sequences (ITS) of the Trichoderma isolates identified: 10 as the common species T. harzianum, 15 as T. pleuroticolum sp. nov. and 1 as T. pleurotum sp. nov., two new species to Italy. EFFECT OF FUNGAL AND BACTERIAL BASED BIOPRODUCTS FOR CONTROLLING VERTICILLIUM WILT OF OLIVE IN THE NURSERY. T. Yaseen1, A.M. Ismail1, A.M. D’Onghia1, A. Ippolito2 and F. Nigro2. 1Istituto Agronomico Mediterraneao, Via Ceglie 9, 70010 Valenzano (BA), Italy. 2Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. E-mail: nigrof@agr.uniba.it Verticillium wilt, caused by the soil-borne fungus Verticillium dahliae Kleb., is one of the most important disease of olive worldwide. The pathogen is a soil inhabiting fungus and inoculum consists of microsclerotia, resting structures that survive for many years. Chemical control of microsclerotia in the nursery is difficult, and the implementation of organic farming system also in olive nursery production urgently requires new and eco-compatible control methods. In this study fungal (Trichoderma harzianum Fv178, Clonostachys rosea Fv114) and bacterial (Bacillus licheniformis and B. subtilis) based bio-products were tested for their activity in controlling Verticillium wilt in nursery-produced olive planting stocks, under controlled conditions. The bio-products were added to the growing media (20% sand, 45% peat moss, 20% pumice, 15% perlite, macro and microelements) seven days before transplanting and one day after transplanting. Olive plantlets were inoculated by dipping the roots in a conidial suspension (4x106 CFU/ml) of the pathogen. After 15 months, the inoculum density of V. dahliae in the growing medium and the incidence of infected olive roots were assessed by Real-time PCR. Plant height, fresh weight, and root weight were also determined. Olive plantlets growing in media treated with the fungal bioproducts showed better vegetative parameters than those grown in media trated with bacterial byproducts, and were significantly higher than untreated controls. Both bio-products reduced the inoculum density of V. dahliae in the medium and the incidence of infected roots. ACTIVITY OF A MIXTURE OF PERACETIC ACID AND HYDROGEN PEROXIDE IN SUPPRESSING PHYTOPATHOGENIC FUNGI AND BACTERIA IN VITRO AND IN COLD STORAGE ROOM. K. Youssef1,2, C. Cariddi1, A. Myrta3 and A. Ippolito1. 1Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via G. Amendola 165/A, 70126, Bari, Italy. 2Plant Pathology Research Institute, Agricultural Research Center, Giza-Egypt. 3Certis Europe B.V., Via A. Guaragna 3, 21047 Saronno (VA), Italy. E-mail: ippolito@agr.uniba.it The use of synthetic fungicides for controlling diseases of fresh fruit and vegetables is restricted by the actual EU and national legislations due to the possible risks for human and environmental health. Therefore, the need for unconventional and preventative control means, that guarantee a more eco-compatible agriculture, is always increasing. The effect of Jet 5 (a mixture of peracetic acid 5% and hydrogen peroxide 20%) was tested in vitro against some important phytopathogenic fungi and bacteria and in a cold storage room for fresh fruit and vegetables. In particular, Jet 5 at concentrations of 0.25, 0.5, 0.75 and 1% was mixed with potato dextrose agar (PDA) medium and tested against Penicillium italicum, P. expansum, Monilinia laxa and Botrytis cinerea. The same compound at concentrations of 0.062, 0.125, 0.25, 0.5, and 1% mixed with sucrose nutrient agar (SNA) was tested against several phytopathogenic bacteria including Erwinia amylovora, Agrobacterium tumefaciens, Clavibacter michiganensis subsp. michiganensis, Pseudomonas spp., Xanthomonas campestris pv. vesicatoria and Pectobacterium carotovorum subsp. carotovorum. Jet 5 at 0.75% gave a complete growth inhibition of all tested fungi, except for B. cinerea, which was completely inhibited at 1%. The mixture at 0.125% gave a complete inhibition of all bacterial species, except for Pseudomonas viridiflava which was completely inhibited at 0.25%. In the application as aerosol for air sanitation in a cold storage room, Jet 5 applied at 0.6 ml/m3, reduced microbial air population of Penicillium spp. and Cladosporium spp. by 88.5%. and by 60.5% for yeasts. STUDIES ON MIXED APPLICATION OF SALTS AND WAX FOR CONTROLLING POSTHARVEST ROTS OF CITRUS FRUIT. K. Youssef1,2, A.M. Ligorio1, F. Nigro1 and A. Ippolito1. 1Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via G. Amendola 165/A, 70126, Bari, Italy. 2Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt. E-mail: ippolito@agr.uniba.it The appearance of pathogen strains resistant to fungicides Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 and the increasing concern about environment and public health have raised the demand for alternative means to control postharvest disease. Beside fungicide application, during postharvest handling citrus fruit are waxed to improve their overall appearance and to preserve the internal quality. The role of mixed application of wax and salts on the development of postharvest diseases was examined on two citrus varieties, ‘Comune’ clementine and ‘Tarocco’ orange. Common food additives, i.e. sodium carbonate and bicarbonate, potassium carbonate and bicarbonate, ammonium bicarbonate and potassium sorbate, at a concentration of 6% (w/v), in combination with a commercial wax, were evaluated for their activity against postharvest rots. Fruit were stored for one month at 4°C (‘Tarocco’ orange) or 6°C (‘Comune’ Clementine), followed by one week of shelf life at 20±2°C. Major rots were due to Penicillium digitatum and P. italicum, with an incidence of 11 and 4% for ‘Comune’ clementine and ‘Tarocco’ orange, respectively. The incidence of P. ulaiense infection was negligible and appeared at the end of storage. Rots caused by Botrytis cinerea and Alternaria spp. showed an incidence, on the whole, lower than 1% on ‘Tarocco’ orange and around 3% on ‘Comune’ Clementine. The incidence of post-harvest rots on fruit treated with wax was significantly higher as compared with fruit treated with water, whereas, in fruits treated with wax combined with different salts, rot incidence was significantly lower than the control, the combination wax/potassium sorbate being the most effective. POTENTIAL USE OF ANTAGONISTIC BACTERIA IN COMBINATION WITH BRASSICA CARINATA AND HELIANTUS ANNUS MEALS. M. Zaccardelli, D. Villecco and D. Ronga. CRA, Centro di Ricerca per l’Orticoltura, Azienda e laboratori di Battipaglia, Strada Statale 18 n° 204, 84091 Battipaglia (SA), Italy. E-mail: massimo.zaccardelli@entecra.it The development of the biodiesel industry for the production of bioenergy will produce an high quantity of meals, such as those from the biocide Brassica carinata and the no-biocide Heliantus annus. A recent work has shown that is possible to improve biological control by adding B. carinata meals to a Trichoderma strain no-sensitive to glucosinolates. To investigate if it is possible to improve the biological control of soil-borne pathogens, 22 bacterial strains, showing PGPR/ISR and/or antibiosis activities, were tested for their ability to grow on meals of the two biodiesel species. All the strains were able to grow on agar-plates or in liquid medium containing only sterilized meals of B. carinata or sunflower. In vitro tests were performed with activated B. carinata meals, that delivered isothiocyanates by hydroloysis of the glucosinolates, contained in the meals. Results showed that B. carinata meals were able to partially inhibit the growth of all the strains within 24 h, except for two PGPR/ISR strains of Pseudomonas fluorescens and P. putida, that were totally insensitive to isothiocyanates. After 48 h all strains grew. Biological control of different soil-borne pathogen/host plant systems are in progress using antagonistic bacteria insensitive or slowly sensitive to B. carinata meals. The combination of this biocide meal with the antagonistic bacteria reduces the risk of the growth of phytopathogenic soil-borne fungi on B. carinata meal, after hydrolysis of all glucosinolates. OCCURRENCE AND DIVERSITY OF VEGETATIVE COMPATIBILITY TYPES OF CRYPHONECTRIA PARASITICA IN SARDINIAN CHESTNUT STANDS. A. Zanda, B.T. Linaldeddu, B. Scanu and A. Franceschini. Dipartimento di Protezione delle S4.95 Piante, Sezione di Patologia Vegetale, Università degli Studi, Via E. De Nicola 9, 07100 Sassari, Italy. E-mail: ben@uniss.it The occurrence and diversity of vegetative compatibility (vc) types in populations of Cryphonectria parasitica, the causal agent of chestnut blight, were examined in five chestnut-growing areas of Central Sardinia (Italy). Four vc types were identified among 131 isolates obtained from 150 randomly selected cankers (30 for each area), by means of European tester isolates (EU). EU-12 and EU-2 groups were the dominant vc types, reaching a percentage of 48.9% and 46.6% respectively, followed by EU-14 (3.8%) and EU-1 (0.7%). EU-12 was found in all five populations investigated, whereas EU-2 was isolated from four populations. EU-1 and EU-14 were each found in only one population. The two dominant vc types found in Sardinian populations of C. parasitica have a different geographic distribution at the European level: EU-12 is more frequent in Southern Italy, the Balkans and in Greece, whereas EU-2 is dominant in Northern Italy and Western Europe. The diversity of vc types in all five populations studied was very low. Values of the Shannon diversity index, ranging from 0.49 to 0.78, were comparable with those reported for C. parasitica populations in other southern Italian regions. The low diversity in vc types, the natural occurrence of superficial non-lethal cankers, and the high isolation frequency of hypovirulent strains in all chestnut-growing areas examined, support the possibility of the successful application of biological control measures at a local level in Sardinian chestnut stands. CAULIFLOWER MOSAIC VIRUS TRANSPORT STRATEGY IS IMPOSED BY MOVEMENT PROTEIN PROPERTIES. S. Zicca1,2, J. A.Sánchez-Navarro3, V. Pallás3 and L. Stavolone1. 1Istituto di Virologia Vegetale del CNR, Unità Organizzativa di Bari, Via Amendola 165/A, 70126 Bari, Italy. 2Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Via Amendola 165/A, 70126 Bari, Italy. 3Instituto de Biologìa Molecular y Celular de Plantas, Universidad Politécnica de Valencia-CSIC, Avenida de los Naranjos sn, 46022 Valencia, Spain. E-mail: l.stavolone@ba.ivv.cnr.it Cell-to-cell movement of plant viruses occurs via plasmodesmata (PD) and is mediated by movement proteins (MPs). These are virus-encoded proteins that modify the size exclusion limit of PD allowing transport of viruses to adjacent cells as nucleoprotein-complex (NCP) or as tubule-guided whole particles. To explore how and why MPs specialized in one or the other strategy, we tested the exchange ability of several tubule forming MPs, encoded by DNA and RNA viruses, by means of an engineered Alfalfa mosaic virus (AMV) system. Previous results have shown that MPs of distinct RNA virus species can efficiently replace AMV MP and mediate cell-to-cell and long distance virus movement. When AMV MP was replaced with the MP of Cauliflower mosaic virus (CaMV), it mediated the movement of encapsidated AMV only. CaMV is a DNA virus moving as whole particles through tubules. However, when we tested the hypothesis that tubuleforming MPs could mediate movement of encapsidated virus only, we found that the replacement of AMV MP with that of two other RNA viruses, i.e. Grapevine fanleaf virus and Cowpea mosaic virus which also move via tubules did not impair AMV NCP transport. Evidence that the MP of Cestrum yellow leaf curling virus (CYLCV) another DNA virus, acted like that of CaMV, indicated that the critical factor for such behavior was the MP-encoding viral genome rather than the movement strategy. We discuss a possible explanation of such limitation, suggesting a forced evolution of DNA virus MPs to mediate genome entry into the non-cell-autonomous RNA-trafficking plasmodesmata pathway. S4.96 Journal of Plant Pathology (2009), 91 (4, Supplement), S4.45-S4.96 SANITARY SELECTION OF LOCAL GRAPEVINE VARIETIES IN ITALY. A. Zorloni, P.A. Bianco and G. Belli. Dipartimento di Produzione Vegetale, Sezione di Patologia Vegetale, Università degli Studi, Via Celoria 2, 20133 Milano, Italy. E-mail: piero.bianco@unimi.it Sanitary selection of grapevine is an important activity, which allows to prevent spreading of viral diseases and to produce better quality clonal material. Recently, a great importance has been given to local cultivars by vinegrowers and consumers. Results of sanitary selection of clones belonging to autochthonous varieties, selected during five years (2004-08) in different Italian regions, are reported. Woody cuttings collected in winter from 1165 candidate clones, i.e. 609 selected in Lombardia, 88 from Piedmont, 150 from Marche, 103 from Umbria, 114 from Campania, 101 from Sicity, were ELISA test- ed for the presence of Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Grapevine leafroll-associated virus 1 (GLRaV-1), 2 (GLRaV-2) and 3 (GLRaV-3), Grapevine virus A (GVA) and B (GVB). In all the areas surveyed was the incidence of GLRaV-1 and GLRaV-3, involved in grapevine leafroll, and of GVA, involved in Kober stem grooving, was very high. These results confirm the prevalence in our country of these viruses, transmitted by the same vector species. GVB, involved in corky bark, was found only in three samples from Northern Italy, and the presence of GFLV and ArMV, involved in degenerative diseases was limited to a few cases. The results obtained deserve some considerations: the importance of visual selection (selected clones were sampled without a careful symptom observation), and the need to continue sanitary selection, to avoid the propagation of infected materials and further spread of damaging viral diseases.