Spray-Induced Silencing of Grape Powdery Mildew Genes to Reduce Powdery Mildew Growth
Powdery mildew is the dominant disease of grapevine. It infects all grape varietals and vines are typically treated 9-11 times over the growing season. Despite well-planned treatments, infection with powdery mildew can still occur and tolerance levels on grapes is very low. Many commonly used fungicides for powdery mildew control are becoming less effective as powdery mildews develop resistance and there is a demand for safer treatments. In this multi-year project we are developing a novel biological powdery mildew control. The process or technology is called Spray Induced Gene Silencing (SIGS), where the RNAi molecules are designed to target powdery mildew genes that are essential for infection development and growth. Using endogenous RNAi machinery, the designed long double-stranded (ds) RNA is processed into multiple small interfering RNAs (siRNAs) that result in cleavage of the powdery mildew target gene transcript and its degradation resulting in reduced powdery mildew growth and reproduction. In previous funding years, we developed and optimized the method for SIGS assay using CYP51 as target gene, which is a target for FRAC group-3 fungicides. Using our optimized multi-pronged method for selecting conserved powdery mildew genes likely to be critical to powdery mildew growth and reproduction, we screened numerous target genes for their ability to control powdery mildew when silenced using SIGS, and have filed a provisional patent on >100 of these targets. We developed two pathosystems – the Arabidopsis-G. orontii powdery mildew system for ease of use and the grapevine-E. necator powdery mildew system for commercial relevance. Effective SIGS targets identified in the Arabidopsis powdery mildew system were also effective in the grapevine powdery mildew system. Furthermore, effective SIGS targets identified in growth chamber/greenhouse studies with Arabidopsis and grapevine powdery mildew systems were also effective in limiting powdery mildew in vineyard trials (performed 1 this year at 2 sites). Our results suggest SIGS treatment could be used as a replacement for systemic fungicides in mid-growing season. Furthermore, the SIGS treatment had no impact on canopy or berry development, or berry chemistry. In order to increase the efficacy of SIGS for powdery mildew control, we explored multiplexing different gene targets, predicted to act in the same or different functional processes. For the combinations and dosages tested, we have not seen further reduction in the effectiveness of treatment in controlling mildew infections. We are now focusing on testing the formulations/delivery of the dsRNA to enhance stability, uptake, and systemic action. Preliminary experiments confirm the predicted systemic action of topical dsRNA, with reduced powdery mildew proliferation in unsprayed distal tissue. Once we have a good formulation/delivery method in hand, we may reexamine multiplexing SIGS targets. Our 2022-23 proposal focuses on assessing formulation/delivery methods for efficacy and systemic action in the greenhouse and field.