Development of Control Methods for Eutypa Dieback Disease

AVF Proj. ID: 194 / /Cat.: ,
Primary Investigator(s): Nancy Irelan, David Block, Lynn Epstein, Sanliang Gu, Douglas Gubler, Russell Molyneux, Jean VanderGheynstby

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.GublerA 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. The role of taxonomically closely related fungi and epidemiology of Eutypa dieback is being investigated. Phylogenetic analyses based on morphological studies and molecular tools are being done at the species and genus level of all the isolates collected.VanderGheynstA nucleic acid-based procedure has been developed to detect E. lata in grapevine tissue. This procedure involves DNA extraction directly from wood tissue and PCR amplification of E. lata DNA using Eutypa specific PCR primers. Since compounds co-purified with the DNA may inhibit the assay, experiments are underway to determine the sensitivity of PCR to these compounds.

DEVELOP AND IMPLEMENT IMPROVED CONTROL METHODS BASED ON CULTURAL PRACTICES

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. 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 or machine-pruned vines either with or without hand follow-up displayed lower level of Eutypa dieback development. Hand follow-up increased Eutypa incidence on mechanically pruned vines. There were no symptoms of Eutypa dieback in the minimally-pruned treatment. Soil fertility and training/pruning interacted to affect the severity of the Eutypa dieback.EpsteinAlthough analysis of our data is not yet complete, it is clear that at least some of the infections at the UC Davis plot were initiated by ascospores and not be either vegetative propagation or by conidia. Our 2001 milestones were all completed. We demonstrated that ascospores that are siblings can be differentiated by AFLP and that AFLP is sufficiently reproducible to identify clones as such.In addition, AFLP and vegetative compatibility tests were performed on a collection of isolates that allows a rigorous testing of our hypothesis that the pathogen is transmitted with vegetative propagation. We are on schedule to complete this analysis of the genetics of the isolates in the Davis plot in 2002. After that is finished, we propose to elucidate the distribution of Eutypa in symptomatic and asymptomatic wood. We are particularly interested in the possible movement of Eutypa through phloem. For our study, we will use both quantitative PCR and traditional plating. However, as indicated above, there are advantages of quantitative PCR over traditional plating, particularly for detection of Eutypa in phloem; any Eutypa in phloem would probably be killed by disinfection. The technical issues for quantitative PCR, particularly for minimization of inhibitor activity, are largely the same as indicated in VanderGheynst?s project in Objective I (Optimization of current PCR-based diagnostic test procedures). Both of these projects will be done in close collaboration.GublerField trials were set up to find out how fast the disease could progress in plant tissue after infection had occurred throughout the dormant season using machine pruning prior to hand pruning. A trial was set up on Merlot, another on Chardonnay. The protocol of the experiment includes six different periods of pruning: each month from October to March. An average of 320 shoots (i.e. 320 replications) per trial were pruned and inoculated each month from October to March. Pruning was done above the wire and shoots were inoculated with 10 ul of a suspension of E.lata ascospores at 200 spores/ul. Ascospores were obtained from E.lata stromata collected in the vineyard of California. Stromata were soaked in water for one to two hours, and placed in inverted petri plates. Masses of ascospores were then collected and diluted in sterile water.

DEVELOP AND IMPLEMENT IMPROVED CHEMICAL CONTROL METHODS

Gubler – Benlate has been withdrawn from the market with no alternative treatment to replace it and prevent the spread of the Eutypa dieback in California. In the past few years, our lab extensively tested the fungitoxic activity of several potential treatments towards E. lata by developing an in vitro assay. Boron formulated as boric acid (17.5%a.i) showed excellent disease control. Benlate was reported in the literature to have little activity overtime by not sticking onto the pruning wounds after being washed off by rain. Our lab was concerned about similar effects using boric acid as a chemical treatment in the field. We also were concerned about the long-term use of boric acid as a liquid resulting in poisoning of the soil. Therefore, products were formulated in order to be more stable and stick longer onto the pruning wounds in order to prevent infection of Eutypa during the entire period of susceptibility of pruning wounds. Thus, boric acid was formulated in two products. The first product named ?biopaste? is a mixture of 5%boric acid and a chemical paste. The physical property of the paste has been shown to stick better in rainy weather. The second product named ?bioshield? is a mixture of 5%boric acid with a spore suspension of Cladosporium herbarum. C. herbarum is an epiphyte of grapevine wounds and can prevent E. lata infection by niche displacement. However, Eutypa ascospores can still infect when the biocontrol population is low. Mixing C. herbarum with boric acid prevents early infection. Even though C. herbarum was shown to tolerate up to 10%boric acid in vitro the product is formulated at 5%to avoid phytotoxicity. The following results are the first results obtained in natural conditions showing disease control using biopaste as an alternative treatment for the control of Eutypa dieback. However, the long term efficacy of both biopaste and bioshield need to be further tested under field conditions as well as boron toxicity before being recommended to growers for chemical treatment of their vineyard. The objective of this research is to offer alternative treatments to growers for the control Eutypa dieback.

DEVELOP AND IMPLEMENT IMPROVED BIOLOGICAL CONTROL METHODS

VanderGheynst and Block – To date, we have progressed in our studies geared toward determining a mechanism of action of the Eutypa control by F. lateritium. To examine the possibility of production of antifungal agents by F. lateritium, we have developed a new in vitro assay for assessing activity of cell-free fermentation broth. This assay involves mixing of filtered F. lateritium broth with actively growing liquid culture of E. lata for a fixed time. Subsequently, the mixture is plated on Potato Dextrose Agar and incubated for one week to allow growth of E. lata. Controls with water and uninoculated medium (in place of the filtered broth) are also performed. Simple image analysis is used to quantify the extent of coverage of the plate after incubation. Using this assay, we have seen a maximum effect of cell-free broth in samples taken from a six to ten day Fusarium culture with a slight reduction in activity for older cultures. Preliminary fractionation by the USDA group with subsequent bioassay has indicated that more of the activity is in an organic phase than in aqueous phase, but that the aqueous phase does contain some activity compared to the control. Further fractionation leading to isolation of active compounds is currently being planned.Respiration studies have also been completed to examine the existence of competition as a mechanism of control of Eutypa by F. lateritium. Respiration bioassays on grapevine canes have shown that F. lateritium begins cane colonization before E. lata. However, cumulative respiration results show that E. lata consumes more of the cane than F. lateritium. F. lateritium completely controlled E. lata when E. lata was inoculated 5 days after F. lateritium, but not when they were inoculated simultaneously. The results suggest that the wound needs to be colonized by F. lateritium in order to prevent growth of E. lata.

DEVELOP AND IMPLEMENT IMPROVED PLANT-RELATED CONTROL METHODS

Gubler –The process of canker formation in wood tissue by Eutypa dieback is being understood. Degradation of the cell wall of grape wood tissue is being determined in vitro and in vivo. Pectin, lignin, cellulose and cross-linking glycans composition is being quantified in sterile wood blocks of Cabernet Sauvignon prior and after degradation by E. lata. Similar analyses will also occur in artificially inoculated grape wood cuttings and in naturally infected grapevines of Cabernet Sauvignon. Enzymes produced by the fungus will also be determined in vitro. Because of the difficulty of identifying phenolic compounds involved in the tolerance of cvs. To Eutypa dieback, research will focus essentially on the process of canker formation in wood tissue of grapevines. Understanding the biochemical degradation caused by Eutypa and the pathogenicity factors involved in canker formation and grapevine?s death could lead to breeding programs for genetic resistance towards pathogen attack.MolyneuxDetermine the various factors that influence pathogenicity. Previously developed analytical methods (GC/MS, TLC, HPLC) will be applied to the study of metabolite biosynthesis by E. lata. The techniques will be used to screen a larger population of Eutypa isolates to determine the variability in toxin production within a cross-section of the species. Additional pathogenic and non-pathogenic strains of E. lata, including samples from Australia, will be examined for the presence of specific metabolites. Those compounds which correlate with phytotoxicity will be isolated and completely identified. Productive strains will be examined for the metabolite profile generated on a grapewood-based medium. Once optimum toxin production is established, large quantities of specific toxins will be isolated by preparative HPLC and provided for biological evaluation. As an alternative, if sufficient quantities of metabolites for biological experiments are not produced by the fungus, individual toxins will be synthesized. These compounds will be evaluated for toxicity in a grapeleaf-based phytotoxicity assay which is capable of providing a quantitative measure of toxicity.