Combination of Suspension Array and Mycelial Growth Assay for Detecting Multiple-Fungicide Resistance in Botrytis cinerea in Hebei Province in China

Abstract

To provide a high-throughput, efficient, and accurate method to monitor multiple-fungicide resistance of Botrytis cinerea in the field, we used the suspension array, sequencing, and mycelial growth assay in our research. Discriminating-dose bioassays for detecting carbendazim, diethofencarb, boscalid, and iprodione resistance (Car^R, Die^R, Bos^R, and Ipr^R, respectively) were used to analyze 257 isolates collected from Hebei Province in China during 2016 and 2017. High resistance frequencies to carbendazim (100%), diethofencarb (92.08%), and iprodione (86.59%) were detected. Bos^R isolates accounted for 11.67% of the total. In addition, 103 isolates were randomly selected for phenotype and genotype detection. The high-throughput suspension array was utilized to detect eight genotypes simultaneously, including BenA-E198, BenA-198A, SdhB-H272, SdhB-272Y, BcOS1-I365, BcOS1-365S, erg27-F412, and erg27-412S, which were associated with resistance toward carbendazim or diethofencarb, boscalid, iprodione, and fenhexamid (Fen^R), respectively. Most of the benzimidazole-resistant isolates (81.55%) possessed the E198V mutation in the BenA gene. Ninety-three isolates with dual resistance to carbendazim and diethofencarb showed the E198V/K mutation. All Bos^R isolates carried the H272R mutation in the SdhB gene. The I365S and Q369P+N373S (66.99%) mutations in the BcOS1 gene were more frequently observed. No mutation was detected in the erg27 gene in Hebei isolates. There were 13 resistance profile phenotypes. Phenotypes with triple resistance were the most common (83.50%), and Car^RDie^RBos^SIpr^RFen^S was the major type. Car^R isolates that carried E198V/K/A were all highly resistant (HR) and only one F200Y mutant was moderately resistant (MR) to carbendazim. Isolates that possessed E198V/K were MR or HR to diethofencarb. Bos^R isolates that possessed H272R mutation were lowly resistant (LR). Ipr^R isolates were all LR or MR. The distribution of half maximal effective concentrations of Car^R isolates with E198V/K mutations and Ipr^R isolates with Q369P+N373S mutations significantly increased from 2016 to 2017. Combined with our observations, a combination method of the high-throughput suspension array and the mycelial growth assay was suggested to accurately monitor multiple resistance of B. cinerea in the field.