In this section:
Diagnosis and management of eutypa dieback
Supported by the CRCV and GWRDC, July 2003 - June 2006
A project involving SARDI, University of Adelaide, Monash University, United States Department of Agriculture, Australian Wine Research Institute and Taylors Wines.
Summary
Eutypa dieback is a degenerative disease of mature vines which threatens the sustainability of premium Australian vineyards. Vines become infected when ascospores of the fungus Eutypa lata infect wounds made in the wood. The fungus grows slowly in the wood and produces foliar symptoms several years after infection. At the start of this project, no fungicides or biological agents were registered for the control of eutypa dieback in Australia, however, Vinevax (Trichoderma spp.) has since been registered. Remedial surgery is often undertaken to restore vines to productivity. There is a need to improve strategies for the management of eutypa dieback, based on a sound understanding of epidemiology. Also, rapid diagnostic techniques will facilitate studies of epidemiology and control, and improve the prospects for management of this intractable disease.
Foliar symptoms in naturally infected, mature Shiraz vines varied from year to year, and there was a constant relationship between the severity of foliar symptoms and yield. Variation in rainfall and temperature was related to the incidence of foliar symptoms and further work is required to confirm if these factors influence disease development. Foliar symptoms were induced on young grapevine cuttings within 8 months of inoculation with mycelium in a shadehouse. This was considerably faster than that in mature field vines, which did not express foliar symptoms 4 years after inoculation. The absence of foliar symptoms was not a reliable indicator that a vine was healthy, as shown by the presence of discoloured wood in vines examined during re-working trials. Discoloured wood, which contained the pathogen, was observed in below-ground parts of the trunk, but not in the roots. Such vines would not be candidates for reworking. E. lata grew at up to 50 mm/yr in mature Cabernet Sauvignon and Shiraz vines in the field, while in the shadehouse a maximum rate of 115 mm/yr was recorded on Grenache rootlings. The staining of wood typically associated with eutypa dieback was not correlated with the spread of the fungus, and E. lata was isolated up to 80 mm in advance of staining in young vines. Growth rate of E. lata and foliar symptoms of the disease varied significantly among cultivars.
A survey of the Hunter Valley and Mudgee regions of New South Wales showed that eutypa dieback was not responsible for the dead arms prevalent on mature vines, as isolation yielded Botryosphaeria spp. which cause the disease known as Bot canker or Black dead arm. These trunk diseases may be associated with the warm to hot, sub–tropical climate of the Hunter Valley whereas the Mediterranean climate of southern Australia is more suited to eutypa dieback. This distribution reflects research in California.
The benzimidazole fungicides Benlate (benomyl) and Bavistin (carbendazim) were the most effective of the materials evaluated for protection of pruning wounds from infection following artificial inoculation. Application of Benlate to pruning wounds did not result in residues in the fruit. Because Benlate and related products have been withdrawn from the market, steps have been taken to extend the label registration of Bavistin for use on grapevine wounds to control eutypa dieback. Other fungicides, such as Cabrio (pyraclostrobin), BASF 516 (boscalid + pyraclostrobin), Nustar (flusilazole), Shirlan (fluazinam), Scala (pyrimethanil) and Fungaflor (imazalil) were effective but require further evaluation to obtain registration for this purpose. Application of fungicides using commercial spray equipment would reduce the time involved in application and reduce the cost of protecting pruning wounds. Preliminary trials showed that spray application can be as effective as application with a paint brush, but further research is required to develop an efficient method suitable for adoption by growers.
None of the biological products tested was effective. However, other reports from Australia and abroad claim that Trichoderma spp. (as Trichoseal or Vinevax) applied to wounds reduces infection by E. lata and Vinevax is now registered for this use in Australia. Further testing is necessary to determine the reasons for the variable control. Physical barriers such as paints and pastes were as effective as fungicides. Acrylic paint prevented infection by E. lata and paste products such as Garrison and Nectec, which contain fungicides, were also effective. Steps are being taken to register Garrison for this purpose.
Remedial surgery to restore eutypa dieback-affected vines to productivity is a labour intensive and costly process. In the short–term, remedial surgery is an effective strategy to manage affected vines. The success of this method is affected by cultivar and the extent of infection. Growers are advised to monitor vines in early spring for foliar symptoms and to tag symptomatic cordons for removal by remedial surgery in winter and that, when removing stained tissue, growers should cut back a further 10 cm or more to ensure removal of E. lata mycelium in advance of the staining. Finally, wounds need to be protected by application of acrylic paint, paste or fungicide. Long–term evaluation is required to confirm the benefits of remedial surgery.
The injection of fungicides or Trichoderma spp. into the trunks of infected vines had no influence on grape yield or symptoms of eutypa dieback. Foliar application of Brotomax increased yield of infected vines, only after 3 years of treatment, but with no reduction in disease symptoms. This approach is not recommended for the control of eutypa dieback in grapevine.
DNA markers for use in PCR (polymerase chain reaction) and Southern hybridisation assays were developed to identify E. lata in culture and detect the fungus in infected grapevine wood. Once validated, the PCR assay was used routinely throughout the project to confirm the identity of isolates suspected to be E. lata. Only the Bio-101 soil DNA extraction kit yielded DNA of a quality suitable for PCR amplification without inhibition by the phenolic compounds which occur in discoloured, diseased wood. However, this kit costs approximately A$1,000 for 50 DNA extractions, and is not cost–effective for use in routine detection of E. lata DNA. Southern hybridisation, a technique much less susceptible to inhibition by phenolic compounds, allowed the reliable detection of E. lata DNA in artificially inoculated and naturally infected wood. Detection of E. lata DNA in mature wood and annual canes using Southern hybridisation was more sensitive than re-isolation into culture and provided an accurate and efficient means of assessing the efficacy of potential control agents.
Analysis of secondary metabolites produced by E. lata showed that the production of potentially phytotoxic acetylenic secondary metabolites was most consistent in culture media containing grapevine wood and sucrose. Eutypine, previously implicated as the toxin responsible for the foliar symptoms of eutypa dieback, was not produced by all isolates. The related compound, eutypinol, was produced by all isolates except one and was selected for use as a biochemical marker to detect the pathogen in infected vines. Eutypinol was detected in pure cultures of the pathogen using HPLC (high performance liquid chromatography), TLC (thin layer chromatography) and UV spectroscopy. HPLC analysis also identified eutypinol in micropropagated grapevines inoculated with mycelium of E. lata. However, neither eutypinol nor any other metabolites of E. lata could be detected in grapevines naturally infected with E. lata using HPLC analysis, TLC or Raman spectroscopy. Phytotoxicity tests showed that extracts from cultures of E. lata were toxic to grapevine leaf discs and indicated that compounds other than acetylenic phenols may contribute to the foliar symptoms characteristic of eutypa dieback. In vitro studies showed that secondary metabolite production by E. lata is strongly influenced by temperature and water availability, which may have implications for managing vines infected with the pathogen.
For more information please contact Mark Sosnowski
