Spray-Induced Silencing of Grape Powdery Mildew Genes to Reduce Powdery Mildew Growth

AVF Proj. ID: 2366 / /Cat.: ,
Primary Investigator(s): Mary Wildermuthby

Powdery mildews are widespread pathogens of grapevine that are difficult to control. Resistance has emerged against all current fungicides and health consequences associated with extensive use of sulfur are beginning to surface. Therefore, the grape industry is in great need of new methods for limiting powdery mildew disease. The potential of spray induced gene silencing (SIGS) in agricultural pest control has been recently realized. The method is also useful in characterizing gene function. The efficiency of SIGS has been demonstrated to control the growth of viral, fungal, insect and nematode pathogens in several plant host species. With the first year of AVF funding support, we showed that SIGS can also be effective in silencing powdery mildew genes, resulting in reduced powdery mildew growth and reproduction. We optimized dsRNA design, application method, dosage, timing of application and powdery mildew growth assessment for testing of SIGS against gene targets in both Golovinomyces orontii-Arabidopsis and Erysiphe necator-grapevine systems. With the continued support of AVF in Year 2, we screened powdery mildew genes prioritized to impact metabolic and regulatory pathways critical to powdery mildew colonization, growth, and reproduction. All selected G. orontii target genes had homologs in E. necator and are conserved among powdery mildews. dsRNA against individual target genes were designed, applied exogenously and the growth of powdery mildew was quantified. We had a 60% success rate in identifying efficacious novel targets, with reductions in powdery mildew proliferation ranging from 2- to 5-fold compared to the controls. The initial screening of target genes was done using the Arabidopsis-powdery mildew system as it was faster than grapevine. We then selected 6 genes that had shown significant reduction in G. orontii growth on Arabidopsis via SIGS and tested them in grapevine-powdery mildew system. SIGS against each of these six E. necator genes showed significant and reproducible reduction in powdery mildew growth and reproduction on grapevine. This year (Year 3),we refined our target prediction pipeline using multidimensional bioinformatic and modeling. Fifteen out of 16 novel gene targets tested, identified using this pipeline, showed significant reduction in powdery mildew growth in G. orontii-Arabidopsis system that increased our success rate of target prediction to 94% from 60% in the previous year. Five new grapevine powdery mildew genes were tested via SIGS and all of them successfully reduced powdery mildew infection. We are currently testing the effects of multiplexing SIGS 2 targets for further increase in SIGS effectiveness in controlling disease. Through the NSF-funded i-Corps customer discovery program, and interviews with 100 grapevine industry personnel, we learned of the needs and specific requirements for new powdery mildew control products. It appears our RNAi-based biopesticide has the potential to meet these needs. dsRNAs are biodegradable, flexible, and specific, with reduced negative environmental and health impacts compared with existing fungicides giving them excellent potential as future powdery mildew disease mitigation agents. We are optimizing this technology for effective powdery mildew control in the field, while testing new targets and multiplexing already tested targets. We completed one vineyard field trial in the 2020 growing season with Dr. Akif Eskalen and are conducting field tests at two locations this coming 2021 growing season.

Botrytis Bunch Rot: Who, Where, When, And What to Use

AVF Proj. ID: 2491 / /Cat.:
Primary Investigator(s): Walter Mahafeeby

In the 2020 field season, nine field sites from eight different vineyards (seven commercial vineyards and one research vineyard) in the Willamette Valley were sampled for Botrytis from June to September. Grape clusters, vineyard floor debris, and nearby wild blackberries were collected, incubated, and then visually accessed for Botrytis. Botrytis incidence on clusters over the field season ranged from about 3% to over 30%. Botrytis isolates generated were screened for fungicide resistance to Benomyl (FRAC 1), Iprodione (FRAC 2), Difenoconazole (FRAC 3), Boscalid (FRAC 7), Cyprodinil (FRAC 9), Trifloxystrobin (FRAC 11), and Fenhexamid (FRAC 17). Some level of tolerance was seen in all fungicide classes among isolates with tolerance to more than one fungicide class observed in 22% of the isolates. Monitoring for sources of inoculum was done by sampling dead grape rachis and cane tissue from vineyard floor and wild blackberries adjacent to the vineyard. Vineyard floor debris decreased as the season progressed but for field sites with wild blackberries (3 vineyard sites), Botrytis on blackberry flower parts and berries was found throughout the season and at one site increased dramatically at the end of the season near harvest after a significant rainfall. These results suggest that vineyard Botrytis resistance levels are of concern and should be continued to be monitored along with the changes of Botrytis inoculum to better time applications of fungicides and other integrative pest management tools.

The Role of Low-Molecular Weight Fungal Metabolites in Eutypa Dieback Grapevine Trunk Disease (GTD)

AVF Proj. ID: 2275 / /Cat.: ,
Primary Investigator(s): Barry Goodellby

This project focuses on the fundamental chemistries that GTD fungi causing Eutypa canker/dieback employ in infecting and causing damage to grapevines. We also are developing antioxidant/chelator (A/C) treatments for these GTD fungi which block their chemistries. Additionally, we are disseminating our findings on GTD causal systems and potential treatments to vineyard owners/managers and other parties. Our three Objectives for this two-year project are:

A) Understand the role of iron-binding compounds produced by the mixed-consortia fungi involved in Eutypa dieback, with particular focus on how these compounds generate hydroxyl radicals, the most damaging form of ‘free radical’ found in biological systems.

B) Use our enhanced understanding of Eutypa dieback to develop treatments and management strategies for the control of Eutypa GTD.

C) To disseminate and interpret our research results to vineyards personnel to allow a better practical understanding of how Eutypa dieback chemistries function in causing the disease, and disseminate results particularly related to treatments developed.

Findings to Date:
A New Causal Mechanism for Eutypa Dieback, which also has Potential for Involvement in Esca Disease: The three fungi in this study, Eutypa lata (Elata), Phaeomoniella chlamydospora (Pch), Phaeoacremonium minimum (Pmin) were all found to produce low molecular weight (LMW) polyphenolic compounds that reduce iron. Pch produced the greatest amount of polyphenolics and, in consortia growth with Pmin, also produced the greatest amount of iron reduction. The extracted LMW metabolites from these fungi were also found to nonenzymatically produce hydrogen peroxide. This previously unreported finding is important because hydrogen peroxide will react with reduced iron to generate highly destructive hydroxyl radicals. In other fungi, this type of chelator-mediated Fenton (CMF) reaction has been found to be responsible for aggressive damage and decay of plant tissue (wood cell walls). Our data provides the first evidence that Eutypa dieback, and also potentially esca disease, is not caused by enzymatic or toxin production by fungi. Rather, a CMF mechanism is active which causes both a canker and decay of grapevine wood. We postulate that as the canker/decay develops, fungal LMW metabolites may also diffuse to the foliage to produce end-stage scion/foliar symptoms. CMF mechanisms are known to be active in fungal attack of wood, causing common brown rot decay, but the mechanism has not previously been demonstrated to be causal in GTDs. Clear evidence for production of hydroxyl radicals suggests that CMF chemistry promoted by the pathogenic fungi is responsible for decay of the wood related to canker production in Eutypa dieback.

Development of Potential Treatments for Eutypa/Esca Based on Knowledge of New CMF Disease Mechanism: Laboratory testing of both A/Cs and biocontrol agents continues. However, one of the A/Cs (butylated hydroxyanisole or BHA) is a low-cost, food-safe antioxidant that shows suitability for potential treatments. BHA is showing positive results in completely preventing growth of all three fungal pathogens at relatively low concentrations (≥ 0.5 mM). We will continue testing and, although not part of AVF funded research, plan to conduct limited testing of BHA in pathogen-inoculated grapes in summer.

Developing a GMO-Free RNA Interference Approach to Mitigate Red Blotch Negative Impacts on Grape Berry Ripening

AVF Proj. ID: 2503 / /Cat.: ,
Primary Investigator(s): Laurent Delucby

RNA interference (RNAi) has been extensively used in crop protection platforms. Currently most approaches are based on the conventional use of transgenic plants expressing double[1]stranded RNAs (dsRNAs) against selected targets (endogenous plant genes, virus, fungi, bacteria). However, the use of Genetically Modified Organisms (GMOs) has raised scientific and public concerns. Approaches alternative to the production of Genetically Engineered material that could enable the direct exogenous application of RNA molecules to trigger RNAi has the potential to address public and industry concerns with regards to RNAi field-based technologyInterim_Report_Laurent_Deluc_February 2021 2 (Dalakouras et al., 2020). There are several methods to deliver RNA in plants. Our project proposes to develop dsRNAs application tools to mitigate the negative effects of Red Blotch Virus on grape berry composition. As a first step of a long-term project, we propose to identify through a genome wide study the genomic regions of the Red Blotch Virus that are targeted by grapevine during the early stages of viral infection. Since the acceptance of the project in June 2020, the PI has recruited a graduate student from Italy who will be working on the project. Through cooperation, the PL Deluc has been issued by the USDA-APHIS services a permit to import two bitmer infectious clones containing two variant strains (NY175 and NY358) of the Red Blotch virus (USDA -APHIS Permit #: 20-176-103m) These infectious clones will be essential to study the early phases of grapevine GRBV infection through two main methodologies that are describe below.

Layperson summary* RNA interference (RNAi) is a conserved biological response across living organisms (animal or plant cells) initiated by the presence of double-stranded RNA molecules from various pathogens, including viruses. The RNA interference mechanism initiated in the plants will lead to a cascade of molecular events that are meant to repress the activity of the virus and its propagation within the plant. Once infected, the plants will recognize and produce specific nucleic regions of the viral genome to activate the RNA silencing machinery. These regions are named “hot spots” regions. The main goal of this project is to identify these “hot spots” for the Red Blotch Virus. In the long-term, this knowledge could help developing innovative technology tools like ectopic RNA molecules application in vineyards to mimic the presence of the virus and to make the plants immune or “primed” to further infections like a vaccine will do. This might limit the propagation of the Red Blotch Virus from plants to plants and could potentially mitigate its negative effect on grape berry ripening of already infected plants. The current project will use Next Generation Sequencing technologies to identify these “hot spots” during the early stages of Red Blotch Virus infection in tissue culture material. Once the nucleic regions identified, the continuum of the project will involve trials for systemic application, through spray, of these RNA molecules to either infected plants to mitigate the negative effects of Red Blotch on ripening, or to non-infected plants, to trigger their immune responses to viral infection. If confirmed in a greenhouse setting, the next step will be to assess the ds-RNA formulation in field-trails.

Control of Vine Mealybug (Planococcus ficus Signoret) in Organic Viticulture

AVF Proj. ID: 2507 / /Cat.: ,
Primary Investigator(s): Luca Brillanteby

Vineyard mealybugs (Hemiptera:Pseudococcidae) are an increasing matter of concern for grape growers, as the economic losses resulting from their infestations continue to increase. Several species of mealybugs are present in California, but the vine mealybug (Planococcus ficus, Signoret) is the one creating most problems. It is an invasive species introduced ~20 years ago, and not yet under control. The control is complex in conventional viticulture but is even harder in organic production where systemic insecticides (e.g. spirotetramate) are not available and only contact products are allowed. These products cannot easily reach the insect population often located under the bark. This project aimed to compare on a large vineyard trial the efficacy of most common and best-suited products commercially available in organic production: pyrethrins, neem oil, diatomaceous earth, Chromobacterium subtsugae. The project also controlled for side effects on grapevine physiology and grape composition related to spraying dusts and oils on leaf and berry surfaces. The experiment was set up as a randomized complete block design with 4 treatments plus control and 4 replicates. Each replicate was 1 acre large for a total size of the trial of 20 acres. The vineyard was planted with Pinot Noir and moderately infested with mealybugs.

None of the products were effective in controlling mealybugs on the trunk and performed similarly. Pyrethrins were the least effective in controlling mealybugs on leaves, while diatomaceous earth and neem oil were most effective, but not significantly different from the control. An inverse relationship appeared between efficacy on the leaves and presence in the cluster. The most effective products had larger amounts of mealybugs inside the clusters, and the least effective products on leaves had fewer mealybugs in the clusters. Plants treated with neem oil had significantly lower stem water potentials. Treatments did not have any significant effect on gas exchange measurements, except for one date, when azaguard had cooler leaves and higher photosynthesis and stomatal conductance. Diatomaceous earth never significantly reduced photosynthesis or stomatal conductance, even though it well covered the leaves with dust. Although not statistically significant, we observed a strong tendency in sugar content. Brix level was lower in the control than in all other treatments and reached up to 1.9 °Bx of difference in the second measurement date. This delay in ripening was significant at the pH level when the control had a significantly lower pH than most of the other treatments.

This project provides new and unbiased information that will help organic growers to choose the right products to apply for controlling mealybugs in vineyards.

Spray-Induced Silencing of Grape Powdery Mildew Genes to Reduce Powdery Mildew Growth

AVF Proj. ID: 2366 / /Cat.: ,
Primary Investigator(s): Mary Wildermuthby

Powdery mildews are widespread pathogens of grapevine that are difficult to control. Resistance has emerged against all current fungicides and health consequences associated with extensive use of sulfur are beginning to surface. Therefore, the grape industry is in great need of new methods of limiting powdery mildew disease. The potential of spray induced gene silencing (SIGS) in agricultural pest control has been recently realized. The method is also useful in characterizing gene function. The efficiency of SIGS has been demonstrated to control the growth of Fusarium in barley and Botrytis in several plant host species. With the first year of AVF funding support, we showed that SIGS can also be effective in silencing powdery mildew genes, resulting in reduced powdery mildew growth and reproduction. We optimized dsRNA design, application method, dosage, timing of application and powdery mildew growth assessment for testing of SIGS against powdery mildew target genes in both powdery mildew G. orontii -Arabidopsis and powdery mildew E. necator-grapevine systems. With the continued support of AVF in Year 2, we screened powdery mildew genes prioritized to impact metabolic and regulatory pathways critical to powdery mildew colonization, growth, and reproduction. All selected G. orontii target genes had homologs in E. necator and are conserved among powdery mildews. dsRNA against individual target genes were designed, applied exogenously and the growth of powdery mildew was quantified. We had a 60% success rate in identifying efficacious novel targets, with reductions in powdery mildew proliferation ranging from 2- to 5-fold compared to the controls. The initial screening of target genes was done using the Arabidopsis-powdery mildew system as it was faster than grapevine. We then selected 6 genes that showed significant reduction in G. orontii growth on Arabidopsis via SIGS and tested them using the grapevine system. SIGS against each of these six E. necator genes showed significant reduction in powdery mildew growth and reproduction on grapevine. This growing season, we are testing SIGS against two genes that showed maximum reduction in powdery mildew growth on grapevine in research field trials, performed with the assistance of cooperator UCCE viticulture advisor George Zhuang. dsRNAs are biodegradable, flexible, and specific, with reduced negative environmental and health impacts compared with existing fungicides giving them excellent potential as future powdery mildew disease mitigation agents. Introduction of this research to vineyard managers, growers, owners and other stakeholders at the UC Davis Continuing and Professional Education course ‘Current Wine and Winegrape Research’, held in February 2019, and the ‘8th Annual Vineyards & Wineries Continuing Education Class Series’ organized by Napa County Farm Bureau Foundation and the Ag Commissioner’s Office in November 2019 was well received, and productive discussions ensued. We have successfully developed the methodology to identify novel powdery mildew targets that when silenced reduce powdery mildew disease of grapevine and are translating and testing this approach in the vineyard in a highly timely manner.

Persistence of Fungicide Resistance in Grape Powdery Mildew

AVF Proj. ID: 2199 / /Cat.: ,
Primary Investigator(s): Walt Mahafeeby

The fitness of Erysiphe necator resistant to QoI/FRAC 11 fungicides was examined in terms of growth under various environmental stresses and overwintering survival and persistence. Eight Oregon E. necator isolates from the 2017 growing season were used. Four isolates were G143A mutants/QoI resistant and four were wild type/QoI sensitive, with two isolates each of MAT1-1 and MAT1-2 mating types. To analyze fitness under stress, a series of experiments were run where isolates were exposed to 10-32oC and germination, generation time, and mating were measured. The G143A mutant isolates had significantly greater germination at 14oC while all other temperatures had similar germination and growth. These results suggest that there may be a higher probability of G143A mutants infecting grapes in the early season and that QoIs should not be used during the early spring. The interaction of temperature and UVA and UVB radiative stress was examined, with results currently inconclusive as to whether there is a growth difference between wild type and G143A isolates. The same isolates were used in mating studies. Resulting chasmothecia have not fully matured and are still being observed. Previously designed real-time polymerase chain reaction (RT-PCR) primers for mating types were modified and optimized and determined to be poor at identifying mating type ratios in mixed samples. A digital droplet PCR was developed to quantify mating type ratios in mature chasmothecia, and will be used for future mating type analysis. Leaf swab samples were collected over 2018 and 2019 from 11 commercial vineyards untreated with QoIs. Samples showed increased G143A frequency from May to September. Chasmothecia sampling showed decreased G143A frequency in spring, suggesting fitness costs associated with overwintering.

The Role of Low-Molecular Weight Fungal Metabolites in Eutypa Dieback Grapevine Trunk Disease

AVF Proj. ID: 2275 / /Cat.: ,
Primary Investigator(s): Barry Goodellby

This project focuses on understanding the fundamental chemistries that grapevine trunk disease fungi causing Eutypa canker and dieback employ in infecting and causing damage to grapevines. We will also develop treatments for GTDs by blocking the chemistries used by the pathogenic fungi. As part of this two-year project we will also disseminate our findings on both GTD causal systems and the treatments we develop to vineyard owners/managers and other interested parties. Our three Objectives for the two-year project are:
A) To better understand the role of iron-binding compounds produced by the mixed-consortia fungi involved in Eutypa dieback, with particular focus on how these compounds generate hydroxyl radicals – the most damaging form of ‘free radical’ found in biological systems – which we propose leads to stem/wood necrosis.
B) To use our enhanced understanding of Eutypa dieback in Year 2 of this research to develop treatments and management strategies for the control of Eutypa GTD.
C) To disseminate and interpret our research results to vineyards personnel to allow a better practical understanding of how Eutypa dieback chemistries function in causing the disease, and also to disseminate results of the treatment systems we will be designing to counteract the chemistries of the iron-binding compounds produced by Eutypa-consortia fungi.

We currently have been able to conduct research on the project for only 6 months, because of a funding start in June of 2019, and the time needed to ramp-up equipment and recruit a graduate student with appropriate background for the project. To date we have been able to grow three Eutypa consortia fungi under conditions of low nutrients and low iron, and demonstrate that these fungi will produce low molecular weight compounds iron-binding compounds. To produce these compounds, which have been shown to be capable of generating highly damaging hydroxyl radicals, it was necessary to simulate the low-iron and low nutrient condition inside the grapevine trunk. This has not been demonstrated previously because prior research on ironbinding compounds from these fungi was conducted using artificial media containing conventional laboratory media containing high sugar and high iron. Continuing in Year 1, we will chemically characterize the compounds and conduct assays for redox-cycling and hydroxyl radical generation. Our preliminary evidence so far indicates that several of the isolated compounds have these important properties, and thus they are ideal candidates for GTD pathogenic factors. We are also working currently with grapevine wood block assays to investigate soft rot activity as part of the pathogenicity process. These investigations are important because they provide us with targets for developing the chelating/antioxidant treatments that are the focus of our Year 2 research on controlling GTD fungi. Once these treatments have been developed and successfully tested in the lab, we are already preparing to move them into test in experimental vineyard tests we are currently developing this spring as an extension of this project. Dissemination of our results and outreach activities will continue throughout the project period.

Controlling Grapevine Trunk Diseases in California

AVF Proj. ID: 2332 / /Cat.: ,
Primary Investigator(s): Akif Eskalenby

A total of 20 vineyards belonging to 10 counties in California were sampled during summer 2019. Cordon, trunk and root tissue were taken from vines using non-destructive methods in order to isolate, analyze and study endophytic bacterial communities between healthy and diseased vines exhibiting typical trunk disease symptoms. A collection of over 1,750 isolates was obtained, from which 27.43% has been screened for their potential antifungal effect against the main GTD-causing pathogens in vitro. A set of 80 bacterial isolates was selected after a first screening against Neofusicoccum parvum, exhibiting >40% of inhibition of the pathogen mycelium. Phylogenetic analyses showed that 57 isolates belong to the genus Bacillus, 8 isolates to Variovorax, 6 isolates to Pseudomonas, 3 isolates to Stenotrophomonas, 2 isolates to Pantoea, 2 isolates to Lysobacter, 1 isolate to Lysinibacillus and 1 to Klebsiella. These isolates are currently being tested using the same methodology against other important GTD pathogens (Diplodia seriata, Eutypa lata, Diaporthe ampelina, Phaeomoniella chlamydospora, Phaeoacremonium minimum and Ilyonectria liriodendri) and a second selection will be performed with bacterial isolates exerting the higher percentages of inhibition against the majority of pathogens in order to be tested in greenhouse and field experiments. Furthermore, a fungicide trial was set over the summer of 2019 in three different nurseries (Winters, Wasco and Bakersfield), including chemical and biological products, using a vacuum chamber to infiltrate the fungicides through the vessels of dormant cuttings prior the grafting process. Fourteen treatments were included, and vines were grafted. Callus formation was evaluated 18 days after the treatments and results indicate frequencies of callusing ratings were similar among treatments, being in average 18.1% of rootstocks showing 100% of callusing, 48.0% showing 80-99%, 22,4% showing 60-79%, 7.7% showing 40-59% and 4.9% showing below 40%. Plants were further planted in pots according to each nursery protocols and kept in the facilities for approximately 3 months. In October 2019, actively growing vines were transported to the UC Davis Department of Plant Pathology field station located in Davis, CA, to be planted in the field under a completely randomized block design. In one of the nurseries, treated vines were planted in the ground after the callusing. The rooting of these plants will be evaluated in March during their transplantation to the field. Trunk disease incidence and severity will be evaluated yearly during the summer season.

Deep Sequencing for Trunk Disease Diagnostics

AVF Proj. ID: 1798 / /Cat.: ,
Primary Investigator(s): Dario Cantuby

The aim of this multi-year project was to develop rapid and cost-effective diagnostic methods for detection, identification, and quantification of trunk pathogens in asymptomatic and symptomatic grape wood. Healthy vines are essential for the successful establishment and sustainability of all grape production systems. Since wood pathogens may remain asymptomatic in young, non-stressed vines, propagation material may contain latent fungal infections and may become symptomatic after planting and serve as a source of inoculum for further infections of potentially clean plants. Methods of virus detection and eradication have been crucial in ensuring that the material in germplasm repositories and clean plant programs is free of known viruses. There remains much to be developed in terms of fungal pathogen detection. Our laboratories have developed comprehensive genomic information on several ascomycetes associated with the most common and aggressive trunk diseases, which provides the unprecedented opportunity for the implementation of new sequencing based diagnostic tools that take advantage of Next Generation Sequencing (NGS) technologies. By allowing the testing of mother plants in foundation blocks and propagation material in nurseries, we expect that the applications of deep sequencing diagnostics will help establish a certification program for trunk pathogen-free germplasm and reduce the amount of trunk pathogens introduced into vineyards at planting as well as the incidence of young vine decline. Deep-sequencing diagnostics will also help identify disease-causing organisms associated with diseased vines in older vineyards.

In the 1st year of the project (2015 – 2016) we collected diseased wood material from commercial vineyards and characterized the associated fungal pathogen species using traditional methods, such as morphological and sequence-based identification of purified fungal colonies. We used these samples to determine how effective ITS-sequencing, meta-genome sequencing and metatranscriptome sequencing approaches are in identifying and quantifying pathogenic species directly in planta. Data simulations allowed us to determine what mapping algorithm was the most specific and sensitive in detecting trunk pathogens both qualitatively and quantitatively. All NGS methods we tested were in agreement with traditional diagnostic methods, but also allowed us to detect simultaneously multiple pathogen species with no need of hands-on sample culturing and colony purification. Additionally, unlike traditional diagnostics, which are strictly qualitative, NGS approaches allowed us to determine the relative abundances of the different infecting species. This work was published in Molecular Plant Pathology (Morales-cruz et al., 2017). Among all methods
tested, ITS-seq is still the most cost-effective until library preparation costs for RNA and DNA-seq do not decline significantly. For this reason, ITS-seq was chosen for further protocol optimization to improve sensitivity and specificity for diagnostics purposes. In the second year of the project (2016-2017), we (a) confirmed that NGS allows the detection with high specificity of actively infecting pathogens when vines are experimentally infected with individual pathogen strains; (b) established that NGS detection is quantitative and allows to differentiate between diseased and healthy vines; (c) developed a protocol for testing dormant cuttings and started testing cuttings provided by a commercial nursery. In the 2016-2017 funding cycle, we also developed a new DNA extraction protocol that reduced the time required for processing and the amounts of sample, reagents and waste. In the second phase on the project, our effort focused on the development and optimization of a new set of optimized primers for ITS-seq designed specifically to target the ITS of grapevine trunk pathogens. The primers as well as the method are publicly available and described in a peer-reviewed article published in December 2018 (Morales-cruz et al., 2018).

In summary, in these five years we have:
1. Applied NGS to trunk pathogen diagnostics and demonstrated that NGS provides qualitatively and quantitatively accurate simultaneous identification of multiple trunk pathogens directly from grapevine wood samples (Morales-Cruz et al., 2017 Mol Plant Pathol).
2. Developed a new protocol with optimized diagnostic markers for NGS ITS-seq diagnostics of trunk diseases, which is publicly available and described in detail in Morales-Cruz et al. (2018; BMC Microbiology).

We publish all protocols, which can be used freely by diagnostic and research labs. We are now seeking funds to survey propagation material and determine the association between pathogen contamination in propagation material and trunk disease incidence in young vineyards.