Identifying the Routes of Infection of Eutypa Dieback among Vineyards, Orchards, and Riparian Areas in California

Our objective was to determine if Prunus orchards (stonefruits, almond) and riparian areas are sources of Eutypa inoculum (sexual spores, ascospores) for vineyards. To address this goal, we identified the direction of spore dispersal among vineyards,orchards, and riparian areas. We gathered 216 isolates from four locations in California (San Benito Co., Solano Co., Napa Co., Merced Co.), and five hosts (grapevine, apricot, cherry, almond, willow). In all locations, 30 to 50{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} of apricot trees and grapevines sampled were infected with Eutypa. This is in contrast with the low recovery of Eutypa from cherry orchards in Napa (3{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}) and Solano (0{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}), as well as from willow in all locations (4-24{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}). We obtained a genetic fingerprint for each isolate with a combination of nine microsatellite loci in order to examine the distribution of genetic diversity among host plants and locations. Bayesian analysis did not reveal any genetic grouping of isolates based on location or host of origins, thus suggesting that the collection of 216 isolates forms a large, genetically-homogenous population in California. Almost all isolates had a unique genetic fingerprint, supporting the important role of ascospores in infection of all hosts examined. There were no differences in genetic diversity among locations. In contrast, Eutypa populations from willow were significantly less diverse than those from grape (mean number of alleles per locus = 3.1 vs 2.6; P = 0.007). Both the low incidence of Eutypa in riparian areas and the lower genetic diversity in samples from willow suggest that Eutypa populations from riparian areas may not constitute important sources of inoculum for infections of cultivated crops. However, hierarchical analysis of molecular variance indicated that there were no significant genetic differences among samples collected either from different locations or from different host plants. Indeed, 99{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} of the total genetic variation found among all isolates was actually present within a single field. Furthermore, genetic differentiation among all samples was very low and non-significant (Fst = 0.008; P = 0.12), suggesting high levels of spore dispersal among the different host plants. Our findings suggest that Eutypa spores from Prunus orchards serve as inoculum reservoirs for vineyard infections (and vice versa), whereas infections in riparian areas likely originate from vineyards and orchards. In spite of the lack of genetic evidence of host specialization, we continued with the 2nd stage of the work, which is to test the hypothesis of host specialization in Eutypa with complimentary, cross-pathogenicity tests. Results of such tests will be acquired in the winter 2012-2013.

Identifying the Routes of Infection of Eutypa Dieback Among Vineyards, Orchards, and Riparian Areas in California

Our goal is to determine if Prunus orchards and riparian areas are sources of Eutypa lata inoculum for vineyards. We examined evidence of spore dispersal (gene flow) among vineyards (wine-grape cultivars), orchards (stone fruits apricot and cherry), and riparian areas (wild host willow). We gathered 151 strains from four locations in California (Merced, Napa, San Benito, and Solano Counties), representing larger collections from grapevine, apricot, cherry, and willow, and a small subset of strains from both almond and pear. Incidence of Eutypa dieback was consistently high in all vineyards and apricot orchards (40 to 50{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}). In contrast, incidence was low in cherry orchards and from willow. We obtained a genetic fingerprint for each strain (haplotype) with a set of nine microsatellite loci, and examined the distribution of genetic diversity among hosts and locations. Genetic diversity indices were similar among strains, although those of Napa were slightly more diversified than those of San Benito. Our finding of high haplotypic richness suggests that sexual reproduction is the mode of reproduction that shapes the genetic structure of the pathogen on all hosts examined, and supports the important role of wind-dispersed sexual spores (ascospores), and not asexual spores (conidia) as infectious propagules. Hierarchical analysis of molecular variance indicated that genetic differences in Eutypa populations among locations were greater than those among hosts. When examined at a local spatial scale (< 65 km), we identified an isolation-by-distance pattern in the genetic structure of the pathogen. This finding has important consequences for the epidemiology of Eutypa dieback in California. First, ascospore dispersal may be limited to distances less than 65 km. Second, ascospores may be exchanged among grape, cherry, and apricot. In contrast, the low incidence of Eutypa on willow in riparian areas and the presence of unshared alleles in strains from willow may indicate limited ascospore exchange between riparians areas and the cultivated hosts in vineyards and stone fruit orchards. Therefore, Eutypa populations from willow are not likely to constitute important sources of inoculum for primary infections of cultivated crops. Additional sampling and pathogenicity tests (Objective 2, proposed for funding cycle 2011-2012) are required to confirm these findings.

Grapevine Canker Diseases in California

Since Eutypa lata (Diatrypaceae) was first identified in California in 1975, cankers and consequently dieback of grapevines have been attributed mainly to “Eutypa dieback” in the State. However, in the past few years, our research has shown that dieback of grapevines in California is a much more complex situation than originally thought, and grapevine cankers can be caused by at least 20 different fungi in the Diatrypaceae, Botryosphaeriaceae, and Valsaceae families. Furthermore, our studies have indicated Botryosphaeriaceae fungi to constitute the main pathogens isolated from grapevine cankers statewide. In vitro pathogenicity studies in the laboratory as well as in vivo studies in commercial vineyards have shown all Botryosphaeriaceae spp. to be pathogenic. Moreover, 4 out of the 9 Botryosphaeriaceae spp. found in California appeared to be much more pathogenic than E. lata. In addition, species of Eutypella and P. viticola were commonly isolated from diseased vines from the table and raisin grape-growing regions of Southern San Joaquin Valley and Coachella Valley. Preliminary pathogenicity tests have suggested that these fungi constitute new pathogens in table grape areas capable of colonizing wood and producing cankers. Identification work suggested that these fungi may constitute new species and more work is being conducted to characterize these fungi.

Spore trapping studies conducted for the family Botryosphaeriaceae have allowed us to better understand the epidemiology of this new group of fungi in California. Spore trap studies have shown Botryosphaeriaceae spores to be mainly trapped following rainfall events and overhead and/or drip irrigation. Botryosphaeriaceae spores were trapped frequently after the first rainfall in September-October to March-April. These studies have allowed us to characterize low infection risk periods throughout the growing season and therefore, improving appropriate timing periods for pruning. Results from the spore trapping study conducted in Coachella Valley showed a high incidence of Eutypella spp. In this case it was documented that spore release occurred during sprinkler irrigation and also by drip irrigation. Surveys for the perithecia of Botryosphaeriaceae in California have shown various grapevine cultivars with perithecia of B. dothidea suggesting that the sexual stage could also play an important role in the epidemiology of the disease. More work is being done to understand the role of native and ornamental trees adjacent to vineyards in the epidemiology and disease cycle of canker diseases.

Our laboratory has developed chemical, cultural and organically acceptable control methods to reduce infections caused by these fungi. Double pruning was shown to be an effective cultural practice which completely eliminates canker formation by Diatrypaceae spp. We have now proved that this practice is also effective against the more rapidly moving Botryosphaeriaceae fungi. Finally, we showed that dormant application of Rally alone or in combination with Enable (another DMI fungicide) with a bark penetrant reduced infection by E. lata. Also, Rally treatments significantly also reduced Phaeoacremonium infection. More work is being done to evaluate single and combined applications of different active materials to control canker diseases. Finally, dormant application of fungicides with a penetrating surfactant was not a significant phytotoxicity hazard to grapevines.

Grapevine Canker Diseases in California

Until recently, grapevine dieback in California was attributed mainly to the fungus Eutypa lata (Diatrypaceae). This fungus is known as the causal agent for Eutypa dieback. However, we have shown that dieback of grapevines in California is also caused by other fungi in the family Diatrypaceae (Eutypa leptoplaca, Cryprovalsa, Diatrype, Eutypella and Diatrypella) as well as by several species of Botryosphaeria, which appeared to constitute the main pathogens isolated from grapevine cankers statewide (Urbez-Torres, et al., 2006; Urbez- Torres, et al., 2007). This year, more species of Diatrypaceae have been identified from diseased grapevine. These included 5 species in the genus Eutypella collected in the table grape areas of the Coachella Valley. The common occurrence of these species in cankers as well as preliminary pathogenicity tests has suggested that these fungi constitute new pathogens in table grape areas. Identification work suggested that these fungi may constitute new species and more work is being conducted to characterize these fungi.

Isolations from cankers collected in the San Joaquin Valley have shown Phomopsis viticola to be the most common pathogen in table and raisin cultivars (Urbez-Torres, et al., 2006). Results from this year pathogenicity test have shown that Phomopsis viticola looks to be a grapevine wood pathogen capable of colonizing wood and producing cankers.

Results of spore trapping studies have shown that Botryosphaeria spores were mainly trapped following rainfall events in Mendocino, Napa, Colusa, San Joaquin, Monterey, and San Luis Obispo counties, and following overhead and/or drip irrigation in the Coachella Valley. Botryosphaeria spores were trapped frequently after the first rainfall in September-October to March-April. Interestingly, Botryosphaeria spores were sometimes trapped without rainfall or irrigation in Colusa County, suggesting that other environmental factors may contribute to spore release and/or spores were brought from other areas by wind. Results from the spore trapping study in Coachella Valley showed a high incidence of other fungi such as Eutypella species which were trapped together with B. rhodina. In this case it was documented that spore release occurred during sprinkler irrigation and also by drip irrigation.

Development of Control Methods for Eutypa Dieback Disease

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{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} 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{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} 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{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} 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{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} boric acid in vitro the product is formulated at 5{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} 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.

Understanding the Sexual Life-Cycle of Eutypa lata

A start has been made on determining the basis for sexual reproduction and hence of variability in the fungal pathogen Eutypa lata. This will contribute to a better understanding of the basis of pathogenic variation and of the basis for selecting stable forms of resistance in breeding new varieties and of other approaches to managing this major, worldwide disease problem.

PDF: Understanding the Sexual Life-Cycle of Eutypa lata

Development of Control Methods for Eutypa Dieback Disease

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.

PDF: Development of Control Methods for Eutypa Dieback Disease

Understanding the Sexual Life-Cycle of Eutypa lata

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 NcHMG1 & 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 (580C) 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 NcHMG1&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 N. 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 NcHMG1) 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 (NcHMG1 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 NcHMG1 + 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.

PDF: Understanding the Sexual Life-Cycle of Eutypa lata

Development of Control Methods for Eutypa Dieback Disease

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

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.