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Verticillium dahliae is a xylem‐invading fungal pathogen that causes vascular wilt in a wide range of angiosperms. The pathogen uses a variety of virulence factors to invade and colonize its hosts. Here, we report that VdNEP, an NLP (Necrosis and ethylene inducing peptide 1‐Like Protein), functions as one such factor in multiple hosts. Eggplant leaves treated with VdNEP developed necrotic symptoms. Overexpression of VdNEP by incorporating extra copies of the VdNEP gene increased virulence to cotton, eggplant and tomato plants, suggesting its role as a virulence factor in diverse plants. Increased expression of VdNEP among the transformants did not correlate with the number of VdNEP inserts, suggesting that its expression was affected by the genomic context of the insertion sites. Interestingly, a transformant derived from a defoliating strain with high VdNEP transcript levels caused disease symptoms in tomato plants, whereas the corresponding wild‐type strain did not cause visible symptoms. The amount of V. dahliae DNA in plants infected with this VdNEP‐overexpressing transformant was 22 times higher than that in plants infected with the wild‐type isolate, further supporting the critical role of VdNEP in infection. A VdNEP‐EGFP fusion was constructed to follow its localization in fungal cells and during infection. [ABSTRACT FROM AUTHOR]

A comprehensive study was conducted on the prevalence of grapevine trunk diseases in Greece, focusing specifically on contamination of grapevine propagation material by Phaeomoniella chlamydospora and black foot disease‐related species. Additionally, detection of esca pathogen Fomitiporia mediterranea, causing white rot in grapevine and other woody hosts, was assessed using a new PCR‐based assay that distinguishes F. mediterranea from F. punctata. Development of a nested PCR assay, combined with a cost‐effective DNA extraction protocol, revealed a high percentage of infection by P. chlamydospora (51%) and Ilyonectria species associated with black foot disease (28%) in different types of grapevine propagation material (dormant cuttings, field‐rooted benchgrafts and green‐growing plants). Interestingly, black foot disease‐related pathogens were more prevalent in nursery plants grown in the field for 6 months (57%) than in other types of propagation material and compared with P. chlamydospora (43%), indicating increased infection of propagation material during growth in the nursery field. The cost‐effective molecular method developed in this study could be used in mass inspections of propagation material for phytosanitary purposes. Finally, using primers specific for F. mediterranea and F. puncata developed in this study, combined with the universal ITS4 primer, a collection of Fomitiporia isolates from mature grapevine and other woody hosts from Greece and Italy (southern Europe) were characterized as F. mediterranea, whereas German and Swedish isolates from forest plants (central Europe) were classified as F. punctata. The developed primers can discriminate between the two Fomitiporia species, which are indistinguishable based on culture and morphological characteristics alone. [ABSTRACT FROM AUTHOR]

A three-year survey was conducted to estimate the incidence of grapevine trunk diseases (GTDs) in Greece and identify fungi associated with the disease complex. In total, 310 vineyards in different geographical regions in northern, central, and southern Greece were surveyed, and 533 fungal strains were isolated from diseased vines. Morphological, physiological and molecular (5.8S rRNA gene-ITS sequencing) analyses revealed that isolates belonged to 35 distinct fungal genera, including well-known (e.g., Botryosphaeria sp., Diaporthe spp., Eutypa sp., Diplodia sp., Fomitiporia sp., Phaeoacremonium spp., Phaeomoniella sp.) and lesser-known (e.g., Neosetophoma sp., Seimatosporium sp., Didymosphaeria sp., Kalmusia sp.) grapevine wood inhabitants. The GTDs-inducing population structure differed significantly among the discrete geographical zones. Phaeomoniella chlamydospora (26.62%, n = 70), Diaporthe spp. (18.25%, n = 48) and F. mediterranea (10.27%, n = 27) were the most prevalent in Heraklion, whereas D. seriata, Alternaria spp., P. chlamydospora and Fusarium spp. were predominant in Nemea (central Greece). In Amyntaio and Kavala (northern Greece), D. seriata was the most frequently isolated species (>50% frequency). Multi-genes (rDNA-ITS, LSU, tef1-α, tub2, act) sequencing of selected isolates, followed by pathogenicity tests, revealed that Neosetophoma italica, Seimatosporium vitis, Didymosphaeria variabile and Kalmusia variispora caused wood infection, with the former being the most virulent. To the best of our knowledge, this is the first report of N. italica associated with GTDs worldwide. This is also the first record of K. variispora, S. vitis and D. variabile associated with wood infection of grapevine in Greece. The potential associations of disease indices with vine age, cultivar, GTD-associated population structure and the prevailing meteorological conditions in different viticultural zones in Greece are presented and discussed. [ABSTRACT FROM AUTHOR]

The β-1,6-endoglucanase gene (vegB) of Verticillium dahliae was isolated using a genome walking technique. Nucleotide and deduced amino acid sequences of the gene showed high identity with the PAN1 sequence deposited at the Verticillium genome database (Broad Institute), but significant differences in intron numbers and sites of insertion. Detailed in silico analysis, accompanied by sequencing of both genomic and cDNA, as well as RT-PCR experiments, provided the correct size of the gene and the exact number, length and positions of introns. The putative protein of this gene was compared with corresponding β-1,6-endoglucanases from other fungi, and sequences were used to construct a phylogenetic tree. A clear differentiation between enzymes derived from plant pathogenic and mycoparasitic fungi was observed, fully supported by bootstrap data. An internal fragment (1.2kb) of vegB was used to disrupt the wild-type gene of a V. dahliae tomato race 2 strain, and the mutant strain, vegB-, was tested for pathogenicity on tomato plants. Results showed a small but constant reduction in disease symptoms only on eggplants for the vegB- strain in comparison with the wild type. Growth on minimal medium supplemented with different carbon sources showed reduced ability of the mutant to breakdown cellulose, whereas growth on glucose, pectin and sucrose was similar to the wild type. [ABSTRACT FROM AUTHOR]