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[EN] Since the early 1990s, grapevine trunk diseases (GTDs) have posed threats for viticulture. Esca complex, Eutypa- and Botryosphaeria-diebacks, mostly detected in adult vineyards, are currently responsible for considerable economic losses in the main vine-growing areas of the world. Other GTDs, such as Petri- (Esca complex) and Black-foot diseases, are emerging problems in grapevine nurseries (resulting in grafting failures and/or loss of saleable plants) and in young vineyards. The impacts of GTDs in modern viticulture depend on several factors, some related to their complexity, and others linked to host plant characteristics, changes in vineyard management and to the scarcity of simple tools for their control. For these reasons control of GTDs remains difficult, also depending on knowledge transfer from research to field and vice versa. This paper outlines the main preventive and curative techniques currently applied, scientifically tested or not that have resulted from the outcomes of "Winetwork", a European Union funded project with special emphasis on the promising and innovative approaches.

Black foot is an important disease of grapevines, affecting vines in nurseries as well as in young plantations. In recent years the disease has increased in incidence and severity throughout the world. Black foot is associated with at least two Campylocarpon and 12 Ilyonectria species, most of which have only recently been described. The recognition of previously unknown species, together with published reports of variability in virulence between and within species identified as I. macrodidyma and I. liriodendri, underlined the need to compare the virulence of isolates from these complexes. A further objective of this work was to determine the cross-infection potential of isolates of these species from other hosts to grapevine. Results from this study revealed recently described species such as I. lusitanica, I. estremocensis and I. europaea to be more virulent to grapevine than the species previously accepted as the main causal agents of black foot, such as I. liriodendri and I. macrodidyma. Furthermore, these results also provided support for isolates obtained from non-grapevine hosts to be as virulent to grapevines as isolates obtained from grapevine, underlying the cross-infection potential of these pathogens.

In an attempt to determine the aetiology of young vine decline in Portugal a study was carried out in rootstock nurseries and in young vineyards during the last few years. Rootstock nurseries located in the most important production areas of Portugal (Ribatejo-Oeste and Beira Litoral) were surveyed. The fungi most frequently isolated from discoloured wood at the base of the stem were Cylindrocarpon destructans and Phaeoacremonium spp., although Acremonium sp., Phomopsis sp., Fusarium spp., Fusicoccum sp., Gliocladium sp., Phoma sp. and Sphaeropsis sp. were also present. Young vines (2- to 8-year-old) showing decline symptoms were collected from various vinegrowing regions of Portugal. Disease incidence was variable but decline symptoms were present in several wine and table grapevine cultivars grafted onto different rootstocks (99R, 110R, 1103P, 101-14, 140Ru, 5BB and 161-49). Isolations from symptomatic internal tissues revealed that also C. destructans and P. chlamydosporum were the predominant fungi isolated from the basal end of the rootstocks and grafting tissues. In the pathogenicity tests carried out with C. destructans isolates the pathogen was reisolated from inoculated plants showing black-foot symptoms but never from the controls. Our results point out that both C. destructans and P. chlamydosporum might be involved with young grapevine decline in Portugal.

Previous surveys conducted in commercial nurseries found that different wood fungi, namely Cylindrocarpon spp., Botryosphaeriaceae, Phomopsis viticola and Phaeomoniella chlamydospora infect grapevine cuttings. Two fi eld trials were carried out to evaluate the effectiveness of cyprodinil + fl udioxonil, pyraclostrobin + metiram, fl udioxonil and cyprodinil to prevent or reduce natural infections caused by such fungi. Rootstock and scion cuttings were soaked in fungicidal suspensions for 50 min prior to grafting. After callusing, the grafted cuttings were planted in two commercial fi eld nurseries with and without a previous history of grapevine cultivation. After nine months in the nursery, the plants were uprooted and analysed for the incidence and severity of the wood fungi. Plants uprooted from the fi eld without a previous history of grapevine cultivation were generally less strongly infected by wood fungi. Under this condition, only the mixture cyprodinil + fl udioxonil simultaneously reduced the incidence of Cylindrocarpon and Botryosphaeriaceae fungi, as well as the severity of Cylindrocarpon infections. Treatments did not produce signifi cant differences in the incidence and severity of P. viticola, and Pa. chlamydospora. For plants grown in the fi eld with a grapevine history, all fungicides except cyprodinil signifi cantly reduced the incidence and severity of Cylindrocarpon fungi. Also, the incidence and severity of Botryosphaeriaceae pathogens were signifi cantly decreased both by cyprodinil + fl udioxonil and by cyprodinil. No signifi cant differences were noticed for P. viticola incidence and severity, and Pa. chlamydospora was not detected again. These results suggest that the practice of soaking grapevine cuttings in selected fungicides prior to grafting signifi cantly reduces Cylindrocarpon spp. and Botryosphaeriaceae infections, thus improving the quality of planting material.

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.

Black foot disease of grapevine is caused by Cylindrocarpon spp., with C. destructans being the main pathogen isolated from vine cuttings and young vineyards in Portugal. Few recommendations for black foot disease control are presently available, and they are not easy to implement within commercial nurseries. In this study, 14 fungicides were evaluated for their effect on the mycelial growth and conidium germination of four field isolates of C. destructans. Mycelial growth of the pathogen was inhibited by DMI fungicides, prochloraz (EC50 values

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.

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 of grapevine is an important disease caused primarily by Ilyonectria spp. and “Cylindrocarpon” pauciseptatum. These pathogens affect grapevine nurseries and young vineyards, causing the decline and death of plants. In the nursery, the primary infections of the grafted cuttings are mainly attributed to soil-borne inoculum, which could infect the roots and the basal end of rootstocks during the rooting stage. The aim of this research was to detect other possible sources of inoculum throughout the different nursery stages by classical and molecular techniques (nested-PCR and multiplex nested-PCR). Results revealed the presence of the I. liriodendri and/or I. macrodidyma complex in grapevine rootstock and scion cuttings, cutting tools, water from hydration tanks, well water, callusing medium, one indoor air sample and soils collected from mother fields and nurseries. “Cylindrocarpon” pauciseptatum was only detected in the callusing medium, nursery soils, rooted-graftlings and on the root pruning machine. Forty four isolates obtained from soils (mother fields and nurseries) and rooted graftlings (six grapevine cultivar/rootstock combinations) were sequenced for part of histone H3 gene to resolve the species. While I. liriodendri, I. macrodidyma and I. torresensis were identified from soil samples, from rooted graftlings it was also possible to detect I. liriodendri, I. macrodidyma, I. novozelandica, I. torresensis, Ilyonectria sp. 2, “C.“ pauciseptatum and four Ilyonectria isolates which are close to I. cyclaminicola. The results demonstrated that, in addition to nursery soils, mother field soils, rootstock and scion cuttings, water from wells and hydration tanks, callusing media, cutting tools and indoor air should be considered as potential sources of inoculum for black foot pathogens.