Summary of Final Project Report:

Powdery mildew is the dominant disease of CA grapevine.  It infects all grape varietals with vines treated ~9-11 times over the growing season.  Coupled with increased restrictions on synthetic chemical fungicide use and the demand for safer, more sustainable treatment options is the reduced efficacy of synthetic chemical fungicides due to the development of resistance by the powdery mildew fungi.  With AVF and complementary funding from the National Science Foundation, we developed a novel biological powdery mildew control approach and took it from initial proof-of-principle in the laboratory to multiple years of vineyard tests to a startup company, Varada Agriculture, and CDPR-funded IPM field trials.  Extensive communication and interaction with stakeholders facilitated our success.

The technology is called Spray Induced Gene Silencing (SIGS) and uses double-stranded (ds)RNA designed to specifically target powdery mildew genes required for growth and reproduction.  Using endogenous RNA interference machinery, the applied dsRNA is processed into multiple small interfering RNAs (siRNAs) that result in cleavage of the powdery mildew target gene transcript and its degradation, thereby reducing powdery mildew growth and reproduction.  As RNA is a naturally occurring molecule that is readily broken down in the environment and the designed dsRNA is specific to the targeted powdery mildew gene, SIGS poses minimal risks to humans or the environment.

After ascertaining powdery mildew can take up RNA, we developed SIGS using CYP51 (DMI target) as the powdery mildew target gene.  Using our multi-pronged method for selecting conserved powdery mildew genes likely to be critical to growth and reproduction, we screened these genes for their ability to control powdery mildew via SIGS.  We identified 31 powdery mildew targets that significantly reduce powdery mildew growth and reproduction via SIGS and patented 179 powdery mildew gene targets grouped by functional pathway/process.  We employed two pathosystems: Golovinomyces orontii-Arabidopsis for screening and Erysiphe necator-grapevine for commercial relevance.

Effective SIGS targets identified in Go-Arabidopsis were also effective against En on grapevine in growth chamber studies.  Furthermore, SIGS against all seven E.  necator targets tested limited powdery mildew in randomized block vineyard trials.  Vineyard trials (2020-2023) at multiple sites per season used commercial backpack sprayers for application, testing a variety of treatment regimes.  No phytotoxicity nor impact on canopy or berry development or berry chemistry was observed.

Treatment dosage, frequency, and formulation/delivery method were further explored for two dsRNA molecules (each targeting a distinct powdery mildew gene).  These RNA treatments decreased berry powdery mildew disease severity (DS, % berries per bunch with powdery mildew) for all six vineyard field trials included in our comparative analyses, despite very high prevalence of powdery mildew disease.  Furthermore, DS were highly correlated (r2>0.7) for the gold standard chemical (GS) and two dsRNA molecule treatments as a function of the untreated control (UTC) DS.  This indicates RNA, like GS, performed equally well across varietals (Chenin Blanc, Sauvignon Blanc, Carignan, Cabernet Sauvignon) and locations (Fresno, Yolo, and Stanislaus counties).  When DS <5% (desired by growers) was achieved for GS, it was also attained by RNA, despite UTC to 26%.

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.

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.