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

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

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.

Assessing Fungicide Resistance of grape powdery mildew in Wine, Table and Raisin Grapes

The 2018 assessment campaign processed >3000 samples from the Western US grape production
regions. Analysis of these ToughSpot samples for resistance to quinone outside inhibitor (QoI)
fungicides (FRAC Group 11) using the G143A qPCR assay indicated there is still widespread
resistance throughout Western grape production regions (71% samples with the G143A) but that
resistance is decreasing. Examination of samples from the Willamette Valley of Oregon indicates
that when growers rotate away from using QoI and DMI, the G143A allele occurrence significantly
decreases in the population (31% with G143A). These results were confirmed when isolate and
field sample DNA underwent genotyping by sequencing analysis of the cytb gene. These results
are similar to the QoI resistance observed throughout Oregon in 2015 and 2016. Analysis of these
samples using various molecular techniques and fungicide resistance bioassays to determine
resistance to demethylation inhibitor (DMI) fungicides (FRAC group 3) and succinate
dehydrogenase inhibitor (SDHI) fungicides (FRAC group 7) is ongoing. We have identified 7 E.
necator isolates that are resistant to at least one SDHI fungicide with only one isolate resistant to
more than one SDHI. Sequencing results indicate only one isolate has a known genetic mutation,
thus further sequencing is required. The Y136F qPCR assay for DMI resistance readily detects
the Y136F mutation associated with DMI resistance and can be used to determine DMI resistance
in single spore isolates. Its use on field samples is difficult to interpret because of the multiple
genotypes associated with DMI resistance. A leaf sample collection method was developed that
decreases sample collection and processing time by 40% and is cheaper to process. In addition, a
bark sampling method was developed that agrees with early spring G143A frequency, thus
allowing growers to assess their risk to QoI resistance during the dormant season. Both assays
will be made available to the industry in 2019. Using this project and Oregon Wine board funding
as a foundation for demonstrating grower support, we obtained funding from USDA-SCRI
program to significantly expand research efforts to develop regional networks for managing and
mitigating fungicide resistance.

Assessing Fungicide Resistance of Grape Powdery Mildew in Wine, Table and Raisin Grapes

Throughout 2017, a field scouting campaign that covered Central and Northern California, Western Oregon, and Southern Washington yielded over 850 field samples and 64 isolates of Erysiphe necator. Analysis of these samples for resistance to quinone outside inhibitor (QoI) fungicides (FRAC Group 11) using the G143A qPCR assay indicated widespread resistance throughout all grape growing regions scouted (90% resistance among ToughSpot kit samples). These results were confirmed when isolate and field sample DNA underwent genotyping by sequencing analysis of the cytb gene. These results are similar to the QoI resistance observed throughout Oregon in 2015 and 2016. Analysis of these samples using various molecular techniques and fungicide resistance bioassays to determine resistance to demethylation inhibitor (DMI) fungicides (FRAC group 3) and succinate dehydrogenase inhibitor (SDHI) fungicides (FRAC group 7) is ongoing. A qPCR assay was developed to target a point mutation (Y136F) of the CYP51 gene that is a contributing factor to DMI resistance in E. necator; the mutation is present in 79.1% of tested 2017 samples (n=43). Analysis by sequencing of the SDHI gene complex has yielded 5 point mutations within two of the subunits in the SDH complex. Two of those mutations induce amino acid changes in their respective proteins and are being analyzed for potential contribution to SDHI resistance.

Durable Powdery Mildew Disease Resistance in Vitis Vinifera: Proof-of-Function and Discovery of Genetic Variation

For the past sixty years, mlo resistance to barley powdery mildew has remained durable and is now incorporated into over 50%of the European barley acreage. Related mlo genes in Arabidopsis, peach, pea, and tomato have recently been discovered and also confer resistance to powdery mildew via the same penetration resistance mechanism. Previously, we identified four strong candidates for mlo resistance in grapevine. To determine whether this gene could confer durable resistance in grapevine, we are pursuing a transgenic gene silencing approach. In 2009, we designed, cloned and confirmed 17 new artificial microRNA (amiRNA) silencing constructs. In 2010, we initiated a collaboration with Dr. Bruce Reisch to develop stably transformed Chardonnay grapevines. Plans are to silence four VvMlo candidate resistance genes individually, in pairs, and all at once in a thorough effort to confirm resistance gene function. In addition, we implemented a similar strategy to silence two candidates for a second resistance gene from Arabidopsis, Pmr6, individually and in pairs. Typical results for biolistic transformation experiments yielded hundreds of transiently-transformed Chardonnay cells per Petri dish. To-date we have regenerated a total of 58 transgenic plants that are ready for functional characterization and have over 600 total embryos after selection that may germinate for transfer to plant growth medium. While the transgenic assays described above could efficiently result in the development of transgenic V. vinifera cultivars with powdery mildew resistance, the information obtained could also be harnessed to identify V. vinifera breeding germplasm with previously undetected resistance alleles.

Revising the Gubler-Thomas Model for Powdery Mildew – Adding a Revised High Temperature Threshold, Varietal Susceptibility, and Expanding Public Access

The Gubler Thomas (GT) risk assessment model is a powerful tool that has been used to successfully control grape powdery mildew throughout California, as well as many other grape growing regions of the world. Proper use of the model has significantly reduced the number of fungicide applications significantly in most years while maintaining thorough disease control. Powdery mildew, caused by the fungus Erysiphe necator, can quickly spread throughout a vineyard. Controlling and reducing powdery mildew in grape crops can lead to higher yields and quality in subsequent grapes and wine. In California, powdery mildew germination, growth and reproduction is largely dependent on temperature. If the temperature remains above the pathogens optimal range for too long, the pathogens growth is inhibited. In previous years, our lab has shown that the pathogen can survive higher temperatures than previously thought (Backup, 2009). This led us to begin field trials testing whether a new high temperature threshold could be successfully incorporated into the model, with the goal of reducing fungicide sprays and improving disease control. We report here our finding on our second year of field trials and our continuing efforts in the laboratory to better understand the effects of heat, including a continuing study of the effects of multiple consecutive exposures to lethal and sub-lethal temperatures. In the last two years, we created two field trials that integrated our controlled environment laboratory studies with a vineyard and fungicide application regiment. These field sites were developed to test alternate high temperature thresholds for the GT model. In response to last years data, we have increased the duration that the pathogen must be exposed to 34?C, the lowest high temperature threshold. In addition, we have also included this year a new high temperature threshold modified by a cooperator in Oregon. All of the experimental high temperature models showed significant control in one of our vineyards, although none of the new models were significantly different from the original GT model. Our other vineyard had significantly greater disease pressure. All of the experimental models performed just as well in reducing disease incidence and severity compared to the GT original model. In the 2010 growing season, the unusual mild weather at our field sites made it difficult to test alterations to the high temperature threshold. The alternate models did not differ from one another in their subsequent fungicide spray schedules until the end of the season. Without higher temperatures, it is challenging to distinguish between these alterations. As part of the Western Weather Work Group (http://uspest.org/wea/index.html), we are collaborating with public and private scientists to help control disease in the western US by providing access to real time, virtual and forecasted weather data. We have completed 2 years of field data, and we are adjusting how the GT index accounts for observed delay in fungal growth. These modifications will ultimately be used to assist both public and private end users of the GT model.

A Microbiome-Based Approach to the Management of Grape Powdery Mildew

This report describes the progress we made during the first year of a two-year project that aims to describe the microbial communities (?microbiomes?) that are naturally associated with grape leaves and berries in vineyards. We are interested in these communities because we think they contain strains of bacteria, fungi and/or yeast that can be exploited to delay or predict the establishment in the vineyard of the fungal pathogen Erysiphe necator, which causes grape powdery mildew. Such strains are relevant to grape growers, because they can be incorporated into existing management practices or forecast models for powdery mildew in an effort to reduce the use of fungicides. To identify said strains, we are using a novel DNA-based method called pyrosequencing that is superior over older methods in revealing the identity and relative abundance of members that make up these microbial communities. So far, we have collected over 350 leaf and berry samples from 7 different vineyards in 2 states (California and Oregon), from 5 different clones of Pinot Noir and Chardonnay, and from 6 different stages of grape growth, ranging from flowering to harvest. This large number of samples is necessary to increase our chances of being able to pick out, from the huge number of microorganisms that are naturally present on grape leaves and berries, those that show a negative or positive correlation with the occurrence of E. necator and thus have true potential as challengers or foretellers of the establishment of this fungus in the vineyard. From the surfaces of all leaves and berries we collected, we have isolated bacteria, fungi, and yeasts and have quantified their abundances. We also extracted their DNA for pyrosequencing analysis which is currently underway. A preliminary analysis of a subset of DNA samples provided us with an early view into the bacterial diversity that is associated with Chardonnay leaves and berries. Clear from this analysis is the fact that leaves and berries differ in the types of bacteria that they carry on their surfaces. Does this difference correlate with levels of E. necator on the same leaves and berries? If so, what bacterial types underlie this difference, and can they be used for our purpose? These are some questions that will be addressed in the second year of the project. Our preliminary data set from the first year is the start of a database that will grow during the course of the second year into a depository of DNA-based information which we can use to identify strains with protective or predictive value against grape powdery mildew. The long-term goal of this research is to present grape growers with ?microbiome?-based options for management decisions relating to disease in their vineyards and quality of their product.

Development of Grower Performed LAMP PCR for Detection-Based Management Programs for Grapevine Powdery Mildew in Vineyards

Inoculum detection for timing fungicide applications against grape powdery mildew has been shown to work using qualitative and quantitative PCR approaches. However, these approaches require expensive equipment, specialized skills, and labor costs that impede implementation by viticulturist. Loop mediated isothermic PCR (LAMP) is a robust method for the detection of DNA that can be performed with minimal equipment and skill. A set of LAMP primers were designed against the ITS2 segment of the ribosomal DNA region of Erysiphe necator that are specific and can detect less than one spore or less than 5 copies of target DNA in a purified plasmid. Spores were trapped from vineyard air using by continuously running an impaction trap with 40 ? 1.5mm stainless steel rods coated with vacuum grease and replacing sample rods every 3 to4 days. DNA extraction was accomplished by placing rods in 100 ?l of TE buffer, centrifuging for 1 min, boiling for 5 min, vortexing for 10 sec and then placing 5 ?l DNA extract in PCR tube with mastermix. The PCR tube was then placed at 65?C for 45 min followed by 80?C for 5 min. Positive detection was determined by the formation of white precipitate. Grower implementation was tested by placing 3 traps at each vineyard with one processed by the grower using LAMP and the others processed in the lab for LAMP and quantitative PCR (qPCR). The results of the grower implementation was that participating growers were more than 50%accurate in detecting 1 or 10 spores and 100%accurate in detecting 100 spores in spiked samples. They had 74%agreement in detecting E. necator compared to our LAMP-PCR results, and our LAMP-PCR results were 96%in agreement with our qPCR results.

Durable powdery mildew disease resistance in Vitis vinifera: proof-of-function and discovery of genetic variation

For the past sixty years, mlo resistance to barley powdery mildew has remained durable and is now incorporated into over 50%of the European barley acreage. Related mlo genes in Arabidopsis and tomato have recently been discovered and also confer resistance to powdery mildew via the same penetration resistance mechanism. Previously, we identified four strong candidates for mlo resistance in grapevine. To determine whether this gene could confer durable resistance in grapevine, we are pursuing a transgenic gene silencing approach and are screening Vitis vinifera germplasm for natural and induced mutations. In 2009, we designed, cloned and confirmed 17 new artificial microRNA (amiRNA) silencing constructs. After showing that Agroinfiltration and protoplast transfection were inefficient transient assays for detecting gene silencing, we initiated a collaboration with Dr. Bruce Reisch to develop stably transformed Chardonnay grapevines. Plans are to silence four VvMlo candidate resistance genes individually, in pairs, and all at once in a thorough effort to confirm resistance gene function. In addition, we implemented a similar strategy to silence two candidates for a second resistance gene from Arabidopsis, Pmr6, individually and in pairs. Thus far, typical results for biolistic transformation experiments have yielded 1000 to 3000 transiently-transformed Chardonnay cells per Petri dish, with regeneration of in vitro plants scheduled for 2010. While the transgenic assays described above could efficiently result in the development of transgenic V. vinifera cultivars with powdery mildew resistance, the information obtained could also be harnessed to identify V. vinifera breeding germplasm with previously undetected resistance alleles. Having previously discovered a surprising absence of functional genetic variation in preliminary investigations of Mlo DNA sequences across diverse Vitis spp., we successfully adapted and tested capillary electrophoresis ecoTILLING for grapevine in 2009. However, we documented the inefficiency of the technology when searching for known mutations and finding that peaks for known mutations were often hidden by background noise and that most peaks were false positives – artifacts of the technology (86%). Therefore, we developed and implemented a strategy to sequence 9 Mb (million base pairs) of our Mlo and Pmr6 candidate genes in a Chardonnay M2 seedling population segregating for random, induced point mutations. This will allow us to identify V. vinifera seedlings with functional mutations for use in grape breeding programs along with molecular markers perfectly linked with the resistance gene.

Revising the Gubler-Thomas Model for Powdery Mildew – Adding Real Time PCR and a Revised High Temperature Threshold

Use of the Gubler-Thomas Model for powdery mildew risk assessment by California grape growers has already achieved goals of better disease control by targeting fungicide applications to high risk conditions over many of the temperature ranges seen in California grape growing areas. Use of the model in some years has reduced fungicide applications significantly. We report here our results to extend the predictive power of this model to higher temperature regimes. Better control of powdery mildew of grape has the potential to improve crop yield and quality as well as sustainability. Extending the high temperature range of the Gubler-Thomas Model would potentially allow for even fewer fungicide applications per season. With global climate change, the need for better understanding of the role of high temperatures on disease may increase in importance. We have conducted 2.years of controlled environment studies which have enabled us to understand the influence of high temperatures on grape powdery mildew (Backup 2009). Under controlled laboratory conditions, temperature and duration were carefully tested and their effects characterized on germination, growth and sporulation of the fungus. Our work shows that E. necator continues to germinate, infect, grow, and sporulate in the lab at higher temperatures than previously thought. In 2009, after controlled work with single heat exposures, we tested how multiple heat spikes affect fungal growth and reproduction. We found that temperature is increasingly lethal to the pathogen, and slows or reduces colony survival, delays spore production, and reduces the number of spores produced. The higher temperatures, such as 36 and 38° C for 4 and 2 hours, respectively, had a more pronounced effect than 34º C or the room temperature control, as did the higher number of consecutive heat treatments (1, 2 or 3), although to a lesser degree than temperature increases alone. However, repeated exposures to 4 hours of 36 and 38 C up to 3 times, which would total 12 hours, did not result in the same colony death and lack of spore production as one longer exposure of 12 hours straight had resulted. It appears that the fungus can and does recover with these shorter high temperature intervals. We have completed one year of testing the results of our integrating the lab work with our field studies. We are focusing on 36 °C and 38° C as important high temperatures and at 2 to 4 hours duration for the new high temperature threshold for the GT model. We are also adjusting how the index accounts for observed delays in fungal growth and reproduction due to the sublethal conditions experienced in the vineyard including not adding points to the index for several days after a high temperature spike to mimic the delays in growth observed in the lab. We are using the results from this work to assist both public and private end users of weather data and the model. With funding from the CDFA Specialty Crops Block grant only, we are developing vineyard spore trapping and molecular diagnostic techniques for the pathogen.