Advancing Novel Tactics and Triaging Fungicide Resistance for Botrytis cinerea and other Profit-limiting Pathogens

Goals / Objectives
1) Evaluate isolates of B. cinerea collected from floriculture greenhouses for sensitivity to common site-specific fungicides. 2) Characterize genetic mutations that are known to confer fungicide resistance. 3) Evaluate isolates for practical resistance to fludioxonil. 4) Evaluate genetic differentiation of greenhouse B. cinerea populations based on fungicide sensitivity, crop, and region. 5) Determine the efficacy of fungicide dips and lighting wavelength to protect cut flowers postharvest. 6) Characterize genetic mutations that may influence susceptibility to light treatment in B. cinerea isolates. 7) Test new plant-protection products, including reduced-risk fungicides, biopesticides, and biocontrols against root rot (e.g. Thielaviopsis basicola, Rhizoctonia solani, and Pythium spp.) and foliar spots and blights (e.g. downy and powdery mildew, Botrytis blight). 8) Provide results and recommendations directly to producers and allied industries via publication in trade magazines, website updates, and presentations.

Project Methods
Research will utilize Botrytis cinerea isolates collected from poinsettia, geranium, and petunia in Michigan production regions to generate whole-genome sequences from the collection. Objectives 1-4 will focus on fungicide resistance in B. cinerea greenhouse populations. Isolates will be screened for sensitivity to thiophanate-methyl, iprodione, boscalid, fluopyram, cyprodinil, pyraclostrobin, fludioxonil, and fenhexamid using a germination-based fungicide sensitivity assay. Target genes will be analyzed to reveal mutations conferring resistance to fungicides except fluopyram (newer fungicide), cyprodinil, and fludioxonil (common target site unknown). To evaluate practical resistance to fludioxonil, a subset of isolates (11) demonstrating in vitro resistance to fludioxonil will be incorporated into a greenhouse trial. At least 150 isolates will be evaluated for genetic differentiation based on fungicide sensitivity, crop, region, and year through comparative genomics tools. Objectives 5 and 6 will focus on post-harvest B. cinerea control in cut flowers. Trials with systemic fungicides will include cut roses and other cut flowers. Flowers will be dipped or sprayed with iprodione, benzovindiflupyr + azoxystrobin, boscalid, fluopyram + azoxystrobin, azoxystrobin, cyprodinil + fludioxonil, fludioxonil, and fenhexamid and inoculated with B. cinerea; controls will be included. Evaluation of disease, opening, vase life, and time to peak fresh weight will be made using established protocols. Flowers will be evaluated in cold storage in the dark or under white light, red light, green, or blue light and in boxes refrigerated for 3- or 10-days (no light). B. cinerea produces conidia under near UV and far-red light; blue, green, and red light are limiting. Some strains do not sense light, limiting light treatment efficacy. Regulatory genes (bcvel1, bclft1, bclft2, bcwcl1/bcwcl2, and bccry2) are implicated in the always conidia blind phenotype, but mutants have varying levels of virulence and a complete understanding of the near UV response system is unknown. Screening for single nucleotide polymorphisms in these loci will determine the effectiveness of lighting on natural populations. Objective 7 will focus on other significant diseases. Efficacy trials for downy and powdery mildew and Botrytis blight products will include single active ingredients and programs with tank-mixes that combine and alternate modes of action. Pythium, Rhizoctonia, and Thielaviopsis will be used to inoculate susceptible plants in individual trials. Replicated studies will include varying application rates and intervals of reduced-risk fungicides and biopesticides. Data will include plant quality, health, and size. Objective 8 ensures the results and recommendations are disseminated to extension educators, growers, florists, registrants, and allied industries via presentations and publications.