Development of Control Methods for Eutypa Dieback Disease

AVF Proj. ID: 13 / /Cat.: ,
Primary Investigator(s): Nancy Irelan, David Block, Sanliang Gu, Doug Gubler, Russell Molyneux, Jim Prince, Jean Vandergheynst, Lynn Epsteinby

OBJECTIVE 1: IMPROVED DISEASE DIAGNOSTICS AND FORECASTING.

Work has progressed on establishing sensitivity thresholds for the PCR-based diagnostic tools. Detection thresholds are being established for Eutypa DNA, mycelia and wood samples. Research has indicated that both mycelia and wood contain PCR-inhibitory substances. Current focus is on optimizing extraction procedures and sample size for effective and accurate test results. Molecular tools are currently being applied to genetically compare Eutypa isolates from different countries. Research is also underway to determine the key factors that influence spore germination. Thus far, there is good correlation of Eutypa sporulation with rain and temperature criteria.

OBJECTIVE 2: EVALUATE VITICULTURAL PRACTICES FOR IMPROVED CONTROL OF EUTYPA DISEASE.

Preliminary vineyard trial analyses indicate that higher soil fertility may lead to higher incidence of Eutypa disease. Hand cane pruning and mechanical pruning treatments are displaying lower disease incidence; as opposed to the hand spur pruning treatment which showing a higher incidence of Eutypa. The minimal pruning treatment is currently showing no signs of the disease.

OBJECTIVE 3: EVALUATION AND IMPROVEMENT OF CHEMICAL AND BIOLOGICAL CONTROL AGENTS.

Progress has been made in determining how Fusarium lateritium controls Eutypa growth. Research has confirmed that F. lateritium produces “Enniatins” which are a class of proven anti-fungal agents. Work is underway to assess the impact of these compounds on Eutypa growth and development.

OBJECTIVE 4: MECHANISMS OF HOST SUSCEPTIBILITY AND RESISTANCE.

Techniques for analysis of lignin, pectin, cellulose, and hemicellulose content were established for grape wood. Wood composition of 2 susceptible (Sauvignon Blanc and Cabernet Sauvignon) and 2 tolerant (Semillon and Merlot) were determined. Two phenolic compounds (cinnamic acid and benzoic acid) were shown to have an inhibitory effect on ascospore germination and mycelial growth of Eutypa in vitro.

OBJECTIVE 5: MECHANISMS OF FUNGAL PATHOGENICITY AND VIRULENCE.

Research has been focused on finding perithecia and on isolating axenic colonies from individual ascospores. Finding Eutypa perithecia was more difficult than anticipated. None were found in the San Joaquin Valley. Perithecia were found in a few grape vineyards in Sonoma County, and a relatively extensive collection of perithecia was made from one of these vineyards. Eutypa perithecia were not found in great quantity on native hosts (e.g. Ceanothus and Toyon) in the coastal mountains. Current efforts are on axenic isolation of ascospores, and on selection of clean, clonal progeny from the ascospores. Progress was also made in identifying new bioactive compounds from Eutypa. These potential toxins are currently being examined for possible roles in pathogenicity and virulence.

Understanding the Sexual Life-Cycle of Eutypa lata

AVF Proj. ID: 42 / /Cat.: ,
Primary Investigator(s): Dr. Peter G. Long, Dr. Rosie E. Bradshawby

Neurospora crassa is a model pyrenomycete fungus for which there are MAT-2 (= mat a) HMG box primers available, hence it is an ideal positive control. Isolates of both mating types {mt a and mt A) were obtained from the Fungal Genetics Stock Center (FGSC), genomic DNA was extracted and used in PCR with the primers NcHMGl & NcHMG2. PCR conditions were optimised to yield a product of the expected size (300 bp) with the mt a strain. DNA sequence analysis of the 300 bp product confirmed its identity as part of the MAT {mt a) gene. In Southern blots (58°C) the 300 bp fragment hybridised to N. crassa mt a genomic DNA but not to N. crassa mt A or E. lata DNA. This confirmed our expectation that the overall nucleotide sequence identity between the N. crassa and E. lata MAT-2 HMG boxes would not be sufficiently high to allow identification of the E. lata MAT-2 by hybridisation. However, the PCR conditions developed for N. crassa provided a starting point for PCR using E. lata as a template. Four NZ isolates of E. lata were initially available in our laboratory. It was not known whether these were mating type 1 or 2 (or heterothallic). However in order to increase the probability that at least one of them carried a MAT-2 gene it was important to dismiss the possibility that they were genetically identical to one another (clonal), as found with other fungal plant pathogens (e.g. Dothistroma pini) in New Zealand (Hirst et al 1999; NZ J Forest Sci. 29:459). Molecular profiles were generated with microsatellite-based primers (anchored PCR; Ganley 2000, Massey University Masterate thesis). The profiles showed extensive polymorphisms between each of the isolates. The NcHMGl &2 primers were tested with the four isolates of E. lata. It was not known what size intron (if any) would be present in the E. lata MAT-2 HMG box, hence the PCR products were expected to be > 215 bp. Despite extensive testing with different PCR conditions, no abundant distinctive PCR products of the expected size were seen. Two limitations of this work were that (a) the E. lata sequences might differ from those of TV. crassa and (b) the isolates tested so far could all be MAT-1 rather than MAT-2. Further strategies were therefore developed to address these limitations. MAT-2 HMG box amino acid sequences of pyrenomycete fungi were obtained from the GenBank database and aligned using Gene Jockey 2. The positions of the Neurospora crassa primers designed by Arie et al 1997 (Fungal Genetics and Biology 21:118) are indicated. The upstream (5′) region (primer NcHMGl) is highly conserved between all the pyrenomycetes for which sequence information is available. However, the downstream (3′) region (primer NcHMG2) is variable in amino acid sequence between the different species. Although Eutypa species are most closely related to Sordaria and Neurospora species, new primers were designed for use with E. lata based on the sequences of two species of Gibberella (primer GfHMG2) and Podospora anserina (primer PaHMG2) to maximise the probability of successful PCR amplification. Use of the new primer combinations (NcHMGl with GfHMG2 or PaHMG2) with N. crassa genomic template gave an amplified product of the expected size as well as additional products. Several of the E. lata isolates gave abundant products in the expected size range with the NcHMGl + GfHMG2 combination. Amplification with single primers (e.g. GfHMG2 only) was also carried out to identify which products were only produced in the presence of both primers (and hence were more likely to be genuine MAT gene products). Direct sequencing of one of these products yielded a mixed sequence, hence this and other PCR products ranging in size from 215 – 300 bp are currently being cloned in E. coli prior to further sequence analysis.

Control of Eutypa Dieback of Grape

AVF Proj. ID: 9912 / /Cat.: ,
Primary Investigator(s): Doug Gublerby

In 1997, an in vitro assay was set up to screen and quickly evaluate a fungicide potential prior to field trials. This method allows the testing of fungicide efficacy to Eutypa lata infection by looking at the colonization of grape wood blocks treated with specific fungicides. This test does not require the use of ascospores, no differences were observed in controlling the disease when the inoculation was done with E. lata mycelium or an ascospore suspension. A good correlation was generally observed between in vitro and in vivo trials for fungicide testing. Nectec paste and 1%Benlate in vitro prevented Eutypa colonization of grape wood blocks and also resulted in a high rate of protection of pruning wounds in field trials. However, Nectec paste formulation has changed in 1999 because propiconazole became unavailable for Janssen. Techniques have been set up to extract lignin, cellulose and hemicellulose from wood to measure their composition in different grapevine cultivars before and during infection and subsequent deterioration by E.lata and secondary fungi. Thus, the understanding of Eutypa pathogenicity is starting to be better understood. The fungus was shown to use glucose to grow in woody tissue. The epidemiology of E. lata in California is better known. Optimum temperature of ascospore germination and mycelial growth were established to be 20°C. Ascospore release was recorded at 4 different locations after rainfall. Fungi presenting characteristic ascospore morphology and mycelial growth of E. lata were found in these sites. The molecular probe that has been developed to identify E. lata from other grape pathogens, also identified these fungi to be different from E. lata. Work is underway to screen these fungi to see if they are pathogenic or are saprobes because their presence confounds the picture in terms of number of spores produced and conditions of their release.

Control of Eutypa Dieback of Grape

AVF Proj. ID: 9815 / /Cat.: ,
Primary Investigator(s): Doug Gublerby

In 1997, an in vitro assay was set up to screen and quickly evaluate a fungicide potential prior to field trials. This method allows the testing of fungicide efficacy to Eutypa lata infection by looking at the colonization of grape wood blocks treated with specific fungicides. This test does not require the use of ascospores, no differences were observed in controlling the disease when the inoculation was done with E. lata mycelium or an ascospore suspension. In 1997, a good correlation was observed between in vitro and in vivo trials for fungicide testing. Nectec paste and 1%Benlate in vitro prevented Eutypa colonization of grape wood blocks and also resulted in a high rate of protection of pruning wounds in field trials. Techniques have been set up to extract lignin, cellulose and hemicellulose from wood to measure then-composition in different grapevine cultivars before and after infection and subsequent deterioration by Elata. Moreover, techniques of DNA extraction from E. lata isolates and other grape pathogens was established as well as amplification of the ITS region of the DNA and restriction of this region with Alul enzyme.

Influence of Vine Trellis Training Systems on Eutypa Lata in a Cabernet

AVF Proj. ID: 9842 / /Cat.: ,
Primary Investigator(s): Dr. R. Keith Strieglerby

During the 1998 season the emphasis of this project shifted from intensive viticultural data collection to an investigation of the impact of training system on incidence and severity of Eutypa infection and management of vines infected with Eutypa. Baseline viticultural data (to maintain treatments and monitor the impact of Eutypa incidence) consisting of yield, fruit composition and vegetative growth assessment were collected. This season produced significantly greater yields for the minimally-pruned treatment than for any other treatment. The machine-pruned and hand-pruned treatments had yields that were comparable. A significantly greater number of clusters were found on minimally-pruned vines. Increased yield for minimally-pruned vines appeared to be directly related to higher cluster number. Mechanized treatments (minimal and machine pruning) significantly reduced cluster weight when compared to hand-pruned treatments. Planting vines on a low capacity soil type produced lower yield and numbers of clusters per vine. Fruit composition was significantly affected by treatment in 1998. Soluble solids data were grouped in a narrow range of values that although significantly different, would all be acceptable for the production of a medium-bodied red wine. The minimally-pruned treatment displayed a delay in maturity that has been a characteristic of this treatment during the course of the study. Vines planted in a low capacity soil type showed that they were able to produce more soluble solids and higher anthocyanin content than vines planted in the high capacity soil type. A significantly greater number of shoots per vine were observed for the minimally-pruned treatment. Measurement of mature nodes is an estimation of the capacity of a vine to grow and produce crop in a sustainable manner. There were no significant differences in mature node data so it can be said that treatments had comparable levels of growth over the length of the season. Symptoms of Eutypa infection were observed in the plot for the third consecutive season. Suspected vines were marked and later sampled for analysis with DNA-based diagnostic tools at the Molecular Marker Laboratory in the Viticulture and Enology Research Center at California State University, Fresno. The result of this analysis will be presented in a supplementary report. Minimally-pruned vines did not display symptoms of Eutypa infection. Visual ratings of Eutypa incidence were confirmed with assistance from Dr. Doug Gubler, Extension Plant Pathologist, UC. Cooperative Extension.

Control of Eutypa Dieback of Grapes

AVF Proj. ID: 9614 / /Cat.: ,
Primary Investigator(s): Douglas Gublerby

Several objectives were investigated in 1996-97, the primary short term objective of this research is to find an effective control of Eutypa dieback, caused by the fungus Eutypa lata. In the field, fungicides (Benomyl, Cinnamyl Aldehyde, Boric acid, Soap, Lime Sulfur, Nectec Paste, HCA CARN, HCA EEE, Proguard A,B,C and Gel, Fusarium lateritium, Cladosporium herbarum and Agriquest microbe) were applied in 4 locations (Davis, Lodi, Sonoma Co. and Yolo Co.) on different grape cvs. (Cabernet Sauvignon, Grenache, Petite Sirah, Semillon). The percent of infected spurs will be correlated to the fungicide and the control. Susceptibility of grapevine cvs. will also be compared by measuring lesion expansion after inoculation of the spur or the cordon of different grapes cvs. (Cabernet Sauvignon, Merlot, Malbec, Viognier, Sirah). Phenotypic differences will be noted and associated to time of budbreak and shoot lignification in each cvs. In vitro experiments showed that the fungicides (Benomyl, Soap, Break…) and biocontrols {Fusarium lateritium) have an effect on the mycelial growth of the fungus. Combination of biocontrol fungi and fungicides have been tested in the lab in order to obtain better protectant activity. A new in vitro technique has been developed and is being used to compare the efficacy of fungicides in preventing colonization of the wood by Eutypa lata mycelium. Moreover, a lab and a greenhouse model is being established in order to test fungicide trials on living plants as a pre vineyard screen and to determine cultivar differences. A new part of this research concerns the physiological and biochemical aspect of the host-pathogen interaction. We have shown that some phenolic compounds have an effect on the mycelial growth of the fungus (on PDA), this effect being associated with the pH of the media. Finally a technique of DNA extraction on Eutypa lata has been established, and the determination of a wide range of the Eutypa population will be done by PCR to evaluate the heterogeneity of the California Eutypa population in order to try to pinpoint sources of inoculum.

Control of Eutypa Dieback of Grapes

AVF Proj. ID: 9508 / /Cat.: ,
Primary Investigator(s): Doug Gublerby

The objective of this research is to find an effective control for Eutypa dieback, caused by the fungus Eutypa lata. Specific objectives this year were:

  1. Continue testing efficacy of fungicides.
  2. Study colonization of wounds by applied fungi.
  3. Determine how the biological control agents inhibit infection.
  4. Test the combination of fungicide and biological control treatment.
  5. Monitor the natural flora of pruning wounds of grapevines.
  6. Determine the relationship between vine training system and disease susceptibility.
  7. Determine the susceptibility differences between cultivars.

Fusarium, Cladosporium, Aureobasidium and Trichoderma were tested for their Eutypa control effectiveness relative to benomyl and for their ability to colonize the grape wound surface. The biologicals were often as effective as benomyl with Fusarium lateritium being the most successful wound colonizer. A dual treatment of F. lateritium and benomyl is being investigated as a control measure. The natural rate at which F. lateritium is benomyl resistant was determined to be 1 ppm. Screening for F. lateritium mutants which grow at higher benomyl concentrations is taking place. The total wound area of vines of different training systems was assessed and the nonspur wounds, those made directly on the cordon, accounted for over half of the total wound area. If nonspur growth was removed in the summer, the total wound surface area could be diminished in half and this would make the vine less susceptible to Eutypa infection.

Control of Eutypa Dieback of Grapes

AVF Proj. ID: 9408 / /Cat.: ,
Primary Investigator(s): James J Marois, Douglas Gublerby

The objective of our research is to find an effective control for Eutypa dieback, caused by the fungus Eutypa lata. Specific objectives were to test efficacy of the fungicides Benlate 50 WP, Rally 40 WP, and Nustar 20 DF, to determine whether multiple applications provide greater control than one application, and to test efficacy of the fungi Cladosporium herbarum, Fusarium lateritium, Aureobasidium pullulans, Trichoderma viride as biological control agents. Other objectives were to study colonization of wounds by applied fungi, to determine how the fungi inhibit infection by Eutypa, to test the combination of Benlate and Fusarium applied together, and to monitor the natural flora of pruning wounds of grapevines. We are testing the fungicides mentioned above and biological control agents in vineyards for their ability to prevent infection by Eutypa. This disease is difficult to study, because several years can elapse between the time of infection and the appearance of symptoms. By reisolating Eutypa from treated spurs after only nine months, we can assess efficacy long before symptoms appear. Our studies have shown that all four fungi can successfully colonize the wound surface when spore suspensions are applied immediately after pruning. Colonization is less successful when application of the spore suspensions is delayed for a period of one or two weeks. All four fungi are effective in reducing infection by Eutypa, but those studies must be conducted over many years to observe results over a variety of weather conditions. Studies on the natural wound colonization showed that numbers of filamentous fungi, yeasts, and bacteria are initially zero, and increase rapidly for 7-14 days after pruning, then remain constant.

Control of Eutypa Dieback of Grapes

AVF Proj. ID: 9307 / /Cat.: ,
Primary Investigator(s): James J Marois, Douglas Gublerby

The objective of our research is to find an effective control for Eutypa dieback, caused by the fungus Eutypa lata. This disease is difficult to study, because several years can elapse between the time of infection and the appearance of symptoms. Specific objectives this year were to continue testing efficacy of the fungicides Benlate 50 WP (multiple applications). Rally 40 WP, and Nustar 20 DF, to study colonization of wounds by applied fungi, to determine how the biological control agents inhibit infection by Eutypa, to test the combination of Benlate and Fusarium applied together, and to monitor the natural flora of pruning wounds of grapevines. The fungicide experiments are still in progress, but data obtained thus far indicate that applications of benlate at 1 and 14 days after pruning is more effective than only one application at pruning. The second application appears to protect the wound during the later half of the wound healing process which ultimately protects the plant from infection. Thus far, none of the inoculated vines that recieved two applications of benomyl have become infected with Eutypa. The biological control agents are spore suspensions of the fungi Fusarium, Clado.sporium, Aureobasidium, Trichoderma, and PeniciIlium. Our studies have shown that these fungi can successfully colonize the wound surface when we apply them as spore suspensions. These fungi also are effective in reducing infection by Eutypa, but those studies must be conducted over many years to observe results over a variety of weather conditions Studies on the natural wound colonization showed that numbers of filamentous fungi, yeasts, and bacteria are initially zero, and increase rapidly for 7-14 days after pruning, then remain constant.

Control of Eutypa Dieback of Grapes

AVF Proj. ID: 9207 / /Cat.: ,
Primary Investigator(s): James J. Marois, W. Douglas Gublerby

The ultimate objective of this research is to develop practical measures for the control of Eutypa dieback of grapevines by modifying the pruning wound so that infection cannot take place. The original five objectives were 1) investigate the potential of experimental and registered fungicides to control disease, 2) determine the mechanism by which wounds become less susceptible over time 3) determine the potential for chemicals which increase the rate of wound healing to decrease the period of susceptibility of fresh wounds, 4) screen fungi which commonly occur on wounds for their potential to inhibit germination of Eutypa and therefore control disease, 5) test for the ability of nonpathogenic fungi to increase the wound healing response of the vine. Due to progress made to date, we have terminated objectives 2, 3, and 4 and added three new objectives. Objective 6 is to determine the potential role of Central Valley cherry orchards to serve as inoculum sources for Eutypa. The second new objective (Obj. 7) is to quantify the relationship between vineyard age and incidence of Eutypa dieback. We found that wound healing was dependent on a degree day relationship, which explains why late spring pruning results in a reduced period of susceptibility to infection. Using historical yield data and current disease assessment of different aged vineyards, it was found in vineyards with susceptible varieties of grapes that peak Eutypa dieback was (90%of vines infected with 40%of the spurs diseased) after 20 years correlated with a decrease in yield commonly observed beginning at about the same age vineyard. This relationship was not found in a resistant variety. Cross inoculations resulted in typical dieback symptoms of both grapes and cherries when inoculated with Eutypa fungus originating from either host. Thus, cherry orchards in the Central Valley are a likely source of inoculum for vineyards. For ongoing research, we established two plots to determine the efficacy of Rally and multiple applications of benlate (0 and 14 days after inoculation) to control disease. Degree of control achieved will be determined in the winter of 1994. We also established two field plots to test our most promising biological control agents (2 saprophytic fungi originally isolated from wounds) at two rates. We also are conducting extensive ecological studies of the microbial community on the wound service to further increase the efficacy of the biological control agents.