DEVELOP AND IMPLEMENT IMPROVED DISEASE MONITORING METHODS
Molyneux Analytical methods have been developed which can be used routinely to screen different Eutypa strains for presence and absence of known metabolites. The time course for maximum production has been established as ca. 30 days and a number of metabolites have been isolated in moderate quantities for further biological evaluation.Gubler A PCR-based RFLP method has been developed to positively identify Eutypa dieback from other wood trunk pathogens and from taxonomically related fungi found in California on diseased grapevines or other hosts surrounding the vineyards. It is used now for fast diagnosis of E. lata when growers bring infected vines to the lab. Surveys for E. lata perithecia or sources of inoculum enabled to identify several new hosts for E. lata in California with plant species such as Malus, Pyrus, Acer and Salix. Willow species emerged as a significant natural reservoir for ascospores production. Inoculum was found in counties of Mendocino, Napa, Sonoma, San Joaquin, San Benito, El Dorado, Merced, Contra Costa and Stanislaus. Studies on the population of Diatrypaceae encountered in vineyards gave evidence of pathogenicity of such fungi when inoculated with ascospores into grapevine. Also, it proved that a second species of Eutypa: E. Leptoplaca occurs in vineyards of Sonoma and it has shown pathogenicity. VanderGheynst A Eutypa diagnostic procedure has been developed that involves direct extraction of DNA from wood and amplification of Eutypa DNA using Eutypa-specific primers in real-time PCR. With this method Eutypa can be detected in less than one day. Since the method has become routine in our labs, experiments on confirming method sensitivity and diagnosing Eutypa in multiple cultivars should be fairly straightforward.
DEVELOP AND IMPLEMENT IMPROVED CONTROL METHODS BASED ON CULTURAL PRACTICES
Epstein If there is transmission via vegetative propagation, pathogen dissemination could be readily controlled by using pathogen-free planting material. Although there may be some technical difficulties with adapting quantitative PCR methods for detection of the Eutypa in grapevines, it is ideally suited for monitoring the pathogen, particularly in phloem. Determination of the distribution of the fungus in asymptomatic tissue around the canker should be fairly straightforward, and consequently, the development of better recommendations for surgical removal of cankers should be too.Gu In general, Eutypa dieback symptoms continue to increase in the vineyard. Vines grown on the higher capacity soil had greater incidence and severity of Eutypa dieback as symptomatic shoots but lower as dead spurs. Vines trained to bilateral cordon and Sylvoz with hand spur pruning displayed greatest incidence and severity of Eutypa dieback. Head trained vines with hand cane pruning displayed lower level of Eutypa dieback development than those trained to bilateral cordon or Sylvoz with hand pruning. Hand follow-up increased Eutypa incidence on mechanically pruned vines. The incidence and severity of Eutypa dieback was the lowest in machine-pruned and minimally-pruned vines. Soil fertility and training/pruning interacted to affect the severity of the Eutypa dieback as symptomatic shoots.Light penetration into the fruiting zone was affected by soil type. Vines grown on San Joaquin loam (a low capacity soil) had higher level of light in fruiting zone from all directions than those grown on Columbia silt loam (a high capacity soil). Training systems did not significantly affect the light penetration into the fruiting zone. Soil capacity and training/pruning did not interact to affect light penetration into the fruiting zone from any direction except the North. Tarining/pruning systems had a significant effect on the number of clusters per vine and cluster weight. However, yield was not affected by either soil type or training/pruning systems. Fruit Brix, pH, and TA were affected by soil type and training/pruning systems at various times during maturation. At harvest, only TA was affected by training/pruning systems. Soil type affected petiole content of mineral nutrients N, P, K, Mn, Cu, B, and S at full bloom significantly. High capacity soil supported higher N, Mn, and S, but lower P and K content. Training systems had significant effect on petiole Ca and B content at full bloom. Must composition and wine chemistry will be reported upon the completion of analysis. Gubler Spore trapping studies on inoculum discharge revealed that ascospores of E. lata are released at all times during periods of rainfall of fall and winter season, although reductions of inoculum quantity are noticeable in late February and March. Temperature did not appear to be a strict limiting factor for ascospores release. However, no sporulation was recorded for a short period of time when temperature went down 40° F. The double pruning trial showed that late pruning is capable of reducing risk of cane contamination with E. lata. Also when contaminations of canes occurred in October, November or December after a long (5-6 buds) clean-up pruning, the lower 2 buds for the next vegetative year were clean of the pathogen when the second pruning was done in March.
DEVELOP AND IMPLEMENT IMPROVED CHEMICAL CONTROL METHODS
Gubler Investigating the influence of chemical showed that boron was effective in preventing infection and was formulated in two products, biopaste and bioshield. Biopaste is a mixture of 5%boric acid and a chemical paste and bioshield is a mixture of 5%boric acid and a spore suspension of Cladosporium herbarum. Both products were formulated to prevent infection of Eutypa overtime after treatment. Preliminary results
DEVELOP AND IMPLEMENT IMPROVED BIOLOGICAL CONTROL METHODS
VanderGheynst and Block We have made substantial progress towards identifying the mechanism of action of Fusarium lateritium against the Eutypa lata, both in terms of colonization kinetics and confirmation that diffusible factors (i.e. antifungal agents) are being producted by F. lateritium. Therefore, the next steps of fractionating the fermentation broth and identifying active fractions should be straightforward, with active ingredient indentification following. Once active compounds have been identified and analytical methods developed for them, it will be possible to optimize the fermentation production of these compounds using existing and novel methods already being developed in our labs. We have already begun investigating production of F. lateritium chlamydospores and their formulation. We have identified growth media and fermentation conditions that appear ideal for chlamydospore production. We have also identified several promising oil and surfactant pairs that may be used to formulate chlamydospores. Once we have established optimal growth and formulation procedures, it should be relatively easy to test products in grapevine bioassays.
DEVELOP AND IMPLEMENT IMPROVED PLANT-RELATED CONTROL METHODS
Gubler The analysis of the process of canker formation in wood tissue by Eutypa dieback showed In vitro that glucose in a non-cellulosic form was the first component of the cell wall targeted by the fungus for its metabolic activity. Understanding the biochemical nature of the canker induced by the pathogen could lead to a breeding program of improved resistance of grapevines towards Eutypa dieback.Molyneux Metabolite profiles have been established for 16 Eutypa strains from California, Italy and Australia and it has been shown that the metabolites conform to the same general structural class but that the specific profiles differ with strain and culture media. This establishes that the disease observed in different geographical locations has in common not only the presence of E. lata but also the metabolites produced. Information gained in one country should therefore be applicable to others. The fungus grows readily on grape-wood based media and analysis of metabolites produced is presently being conducted. Synthetic routes for the preparation of metabolites in larger quantity have been developed and individual compounds are now being bioassayed and used as reference standards. Preliminary experiments have led to a promising grapeleaf-based quantitative assay for phytotoxicity.