Grapevine Trunk Diseases in Greece: Disease Incidence and Fungi Involved in Discrete Geographical Zones and Varieties.

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.

Disruption and Overexpression of the Gene Encoding ACC (1-Aminocyclopropane-1-Carboxylic Acid) Deaminase in Soil-Borne Fungal Pathogen Verticillium dahliae Revealed the Role of ACC as a Potential Regulator of Virulence and Plant Defense.

It has been suggested that some microorganisms, including plant growth-promoting rhizobacteria, manipulate the level of ethylene in plants by degrading 1-aminocyclopropane-1-carboxylic acid (ACC), an ethylene precursor, into α-ketobutyrate and ammonia, using ACC deaminase (ACCd). Here, we investigated whether ACCd of Verticillium dahliae, a soil-borne fungal pathogen of many important crops, is involved in causing vascular wilt disease. Overexpression of the V. dahliae gene encoding this enzyme, labeled as ACCd, significantly increased virulence in both tomato and eggplant, while disruption of ACCd reduced virulence. Both types of mutant produced more ethylene than a wild-type (70V-WT) strain, although they significantly differed in ACC content. Overexpression strains lowered ACC levels in the roots of infected plants, while the amount of ACC in the roots of plants infected with deletion mutants increased. To test the hypothesis that ACC acts as a signal for controlling defense, roots of WT and Never-ripe (Nr) tomato plants were treated with ACC before V. dahliae inoculation. Plants pretreated with ACC displayed less severe symptoms than untreated controls. Collectively, our results suggest a novel role of ACC as a regulator of both plant defense and pathogen virulence.

Successful Agrobacterium mediated transformation of Thielaviopsis basicola by optimizing multiple conditions.

Thielaviopsis basicola is a hemibiotrophic root pathogen causing black root rot in a wide range of economically important crops. Our initial attempts to transform T. basicola using standard Agrobacterium tumefaciens-mediated transformation (ATMT) protocols were unsuccessful. Successful transformation required the addition of V8 juice (to induce germination of T. basicola chlamydospores) and higher concentrations of acetosyringone in the co-cultivation medium, and of chlamydospores/endoconidia, A. tumefaciens cells during co-cultivation. With these modifications, two T. basicola strains were successfully transformed with the green (egfp) or red (AsRed) fluorescent protein genes. Chlamydospores/endoconidia transformed with the egfp gene exhibited strong green fluorescence, but their fluorescence became weaker as the germ tubes emerged. Transformants harbouring the AsRed gene displayed strong red fluorescence in both chlamydospores/endoconidia and germ tubes. Fluorescent microscopic observations of an AsRed-labelled strain colonizing roots of transgenic Nicotiana benthamiana plants, which express the actin filaments labelled with EGFP, at 24 hours post inoculation showed varying levels of fungal germination and penetration. At this stage, the infection appeared to be biotrophic with the EGFP-labelled host actin filaments not being visibly degraded, even in host root cells in close contact with the hyphae. This is the first report of ATMT of T. basicola, and the use of an AsRed-labelled strain to directly observe the root infection process.

The G protein ß subunit controls virulence and multiple growth- and development-related traits in Verticillium dahliae.

To gain insight into the role of G protein-mediated signaling in virulence and development of the soilborne, wilt causing fungus Verticillium dahliae, the G protein ß subunit gene (named as VGB) was disrupted in tomato race 1 strain of V. dahliae. A resulting mutant strain, 70ΔGb15, displayed drastic reduction in virulence, increased microsclerotia formation and conidiation, and decreased ethylene production compared to the corresponding wild type (wt) strain 70wt-r1. Moreover, 70ΔGb15 exhibited an elongated rather than radial growth pattern on agar media. A transformant of 70ΔGb15 (named as 70ΔGbPKAC1) that carries an extra copy of VdPKAC1, a V. dahliae gene encoding the catalytic subunit of the cAMP-dependent protein kinase A, exhibited wt growth pattern and conidiation, was unable to form microsclerotia, produced high amounts of ethylene, and exhibited virulence between that of 70ΔGb15 and 70wt-r1 on tomato plants. Phenotypical changes observed in 70ΔGb15 and 70ΔGbPKAC1 correlated with transcriptional changes in several genes involved in signaling (MAP kinase VMK1) and development (hydrophobin VDH1 and ACC synthase ACS1) of V. dahliae. Results from the present work suggest a linkage between VGB and VdPKAC1 signaling pathways in regulating virulence, hormone production and development in V. dahliae.

VdSNF1, the sucrose nonfermenting protein kinase gene of Verticillium dahliae, is required for virulence and expression of genes involved in cell-wall degradation.

Verticillium dahliae is a soilborne fungus causing vascular wilt in a diverse array of plant species. Its virulence has been attributed, among other factors, to the activity of hydrolytic cell wall-degrading enzymes (CWDE). The sucrose nonfermenting 1 gene (VdSNF1), which regulates catabolic repression, was disrupted in V. dahliae tomato race 1. Expression of CWDE in the resulting mutants was not induced in inductive medium and in simulated xylem fluid medium. Growth of the mutants was significantly reduced when grown with pectin or galactose as a carbon source whereas, with glucose, sucrose, and xylose, they grew similarly to wild-type and ectopic transformants. The mutants were severely impaired in virulence on tomato and eggplant (final disease severity reduced by an average of 87%). Microscopic observation of the infection behavior of a green fluorescent protein (gfp)-labeled VdSNF1 mutant (70ΔSF-gfp1) showed that it was defective in initial colonization of roots. Cross sections of tomato stem at the cotyledonary level showed that 70ΔSF-gfp1 colonized xylem vessels considerably less than the wild-type strain. The wild-type strain heavily colonized xylem vessels and adjacent parenchyma cells. Quantification of fungal biomass in plant tissues further confirmed reduced colonization of roots, stems, and cotyledons by 70ΔSF-gfp1 relative to that by the wild-type strain.