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Sixty-eight isolates of Phaeomoniella chlamydospora obtained from symptomatic esca and Petri disease grapevines, obtained mostly within the Portuguese Dão appellation, were investigated regarding their phenotypic and molecular diversity, in order to determine intraspecific variability and population structure. Phenotypic features such as texture, colour, growing margin zonation, hyphal morphology and diameter growth were evaluated. Molecular characterization was performed through the sequencing of the total ITS region and molecular analyses were used to infer phylogenetic relationships, using the Maximum Likelihood approach. Isolates were divided into two groups, by both phenotypic and molecular analysis, but no clear correspondence was found between the two approaches. Nevertheless, both phenotypic and molecular analysis revealed a strong homogeneity among all isolates, despite their geographical origin, year of isolation and scion/rootstock combination, therefore supporting the clonal reproduction strategy described for this species.

Phaeomoniella chlamydospora is an important tracheomycotic fungus involved in different syndromes of the esca disease complex affecting young and mature grapevine plants. Although grapevine planting material is frequently infected by this pathogen, no regular screening to detect the fungus is carried out, mainly due to the lack of fast, sensitive and affordable methods for large-scale application. A monoclonal antibody-based triple antibody sandwich (TAS) enzyme-linked immunosorbent assay (ELISA) was developed to specifically detect Pa. chlamydospora from grapevine wood tissues. This assay could be especially useful for large-scale application in nurseries, to ensure Pa. chlamydospora-free grapevine stocks, and thus contributing to the production of healthy propagation material.

Fungi of the Ilyonectria genus are the main causal agents of black foot disease of grapevine. These pathogens cause necrosis in the basal end of the rootstock, leading to the early decline and the death of vines in nurseries and young vineyards. In the present study a collection of isolates of the genus Ilyonectria obtained from a vineyard located in the Alentejo region, Portugal, was characterised. This vineyard was established with planting material originating from three different nurseries. To assess the inter- and intra-specific variability among isolates, morphological, cultural and biomolecular characteristics were evaluated. Morpho-cultural and molecular data (RAPD and ISSR markers and histone H3 nucleotide sequence) identified I. estremocensis, I. europaea, I. liriodendri, I. macrodidyma, I. torresensis, I. vitis and “Cylindrocarpon” pauciseptatum. Ilyonectria torresensis was the most common species found in the survey, representing more than 50% of the isolates.

Summary. Esca and Petri disease, two of the most important fungal trunk diseases of grapevine, are responsible for significant losses by causing premature decline and dieback in vineyards worldwide. The Portuguese Dão wine region is no exception. Local winegrowers’ knowledge on Grapevine Trunk Diseases (GTD) in general and of esca and Petri disease in particular is incomplete. The real scope of those problems has been based largely on individual perceptions rather than on a methodical evaluation of the situation. In order to get a full picture of the diseases impact, a leaflet with color pictures was produced and issued to winegrowers, accompanied by a simple questionnaire. The results of this survey represent a first indication of the extent of grapevine trunk diseases in the Dão wine region, specifically its economic impact and relevance to the local wine industry. In conjunction with the survey, several samples of wood collected from esca and Petri disease symptomatic vines, identified during the survey throughout the entire region, were processed and a collection of isolates of Phaeomoniella chlamydospora obtained. To determine the intra-specific variability among these isolates, morphological, cultural and molecular characteristics were evaluated. A protocol to study the pathogenicity with P. chlamydospora was conducted, consisting on the inoculation of cv. Touriga Nacional’s spurs with a previously studied P. chlamydospora isolate.

Black-foot disease of grapevine has been associated with two closely related species of the genus Cylindrocarpon, C. destructans and C. obtusisporum. However, only C. destructans isolates could be obtained from young vines and diseased grapevine rootstock nurseries in Portugal. In the present study, an alternative to traditional methods of detection of Cylindrocarpon spp. fungi from infected grapevine is described. In 1996, Hamelin et al. designed species-specific primers (Dest1 and Dest4) for detection of C. destructans ITS variants from conifer seedlings. With these primers, a DNA fragment of 400 bp was produced by direct PCR using DNA extracted from cultures of C. destructans (60 isolates) obtained from grapevine plants. Whereas no fragments were detected when cultures of common wood grapevine fungi were analysed, an amplicon of the same size was obtained for isolates of C. obtusisporum revealing the failure of these primers to distinguish Cylindrocarpon species. The 400 bp fragment mentioned could also be produced by direct PCR after adding C. destructans to healthy grapevine tissue (cv. Periquita), followed by DNA extraction using frozen plant tissue in liquid nitrogen and polyvinylpyrrolidone (PVP) treatment. However, amplification failed to detect C. destructans in artificially inoculated grapevine plants (cv. Periquita). Consequently, a nested PCR was carried out by modifying the procedure described by Hamelin et al. The universal primer ITS4 and the fungus-specific primer ITS1F were used in a first-stage fungus-specific amplification, followed by a second-stage amplification with the primers Dest1 and Dest4 using the PCR products from stage one. This approach was found to be a simple and reliable method for collective detection of Cylindrocarpon spp. directly from infected grapevine tissues.

The isolates of Cylindrocarpon destructans used in the present study were selected from a collection of isolates from rootstock nurseries and grapevines showing symptoms of decline in Portugal during the last few years. These isolates were compared on the basis of gross cultural morphology, micromorphology and pathogenicity. The most variable parameter was colony colour. Analysis of micro- and macroconidial size of cultures on “Spezieller Nährstoffarmer Agar” plus 0.1% yeast extract medium (SNAY) revealed that all isolates except one, could be clustered in a single group. On carnation leaf agar (CLA) medium, however, all isolates clustered in a single group. All C. destructans isolates were pathogenic on rooted grapevine cuttings of cv. Periquita under greenhouse conditions. Internal black-foot symptoms were seen in inoculated plants and the pathogen was reisolated from 60 to 100% of these plants. All C. destructans isolates caused a significant (P=0.05) decrease in the height of inoculated plants, and the majority also caused a significant (P=0.05) reduction in the number of roots.