Identification and Characterization of Fungicide Resistance in Botrytis Populations from Small Fruit Fields in the Mid-Atlantic United States

From 2014 to 2019, 249 isolates of Botrytis spp. were collected from blackberry, black raspberry, grape, red raspberry, and strawberry showing gray mold symptoms. All isolates were phylogenetically characterized as Botrytis cinerea. A mycelial growth assay determined the following overall frequencies of resistance to fungicides: 92% to pyraclostrobin, 86% to cyprodinil, 71% to thiophanate-methyl, 48% to fenhexamid, 47% to iprodione, 26% to boscalid, 11% to fludioxonil, 8% to penthiopyrad, 7% to benzovindiflupyr, 4% to pydiflumetofen, and 4% to isofetamid. Isolates collected from blackberry, red raspberry, and strawberry had a higher median chemical class resistance value compared with isolates from black raspberry and grape. Resistance-conferring mutations were found in a selection of isolates characterized as resistant to thiophanate-methyl, iprodione, pyraclostrobin, fenhexamid, and boscalid, including E198A in β-tubulin; I365N/S, Q369P, and N373S in bos1; G143A in cytb; P238S, N369D, and F412I/S in erg27; and P225F and H272R/Y in sdhB, respectively. Also, multiple drug resistance (MDR) phenotypes MDR1 and MDR1h were identified by analyzing fludioxonil sensitivity and mrr1 sequences. MDR1 and MDR1h isolates had multiple amino acid variations and two insertions in mrr1 that resembled the group S genotype. A detached grape assay confirmed that the aforementioned mutations in isolates from different small fruit crops resulted in field-relevant resistance. An additional in vitro assay found that values for the effective concentration that inhibits mycelial growth by 50% of B. cinerea isolates to pydiflumetofen and inpyrfluxam averaged 0.4 and 1.0, 0.8 and 0.7, 149.8 and 23.2, 0.9 and 0.9, and 38.8 and 48.8 µg/ml for the wild-type, H272R, H272Y, N230I, and P225F genotypes, respectively. These results revealed widespread fungicide resistance in B. cinerea from Mid-Atlantic small fruit fields, highlighting the need for resistance management alternatives.