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.
/wp-content/uploads/2017/09/AFV-Header-Logo.png 0 0 AVF /wp-content/uploads/2017/09/AFV-Header-Logo.png AVF2021-03-17 11:32:092021-03-17 11:32:09Spray-Induced Silencing of Grape Powdery Mildew Genes to Reduce Powdery Mildew Growth