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

To provide a high-throughput, efficient, and accurate method to monitor multiple-fungicide resistance of 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, Die, Bos, and Ipr, 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 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 -E198, -198A, -H272, -272Y, -I365, -365S, -F412, and -412S, which were associated with resistance toward carbendazim or diethofencarb, boscalid, iprodione, and fenhexamid (Fen), respectively. Most of the benzimidazole-resistant isolates (81.55%) possessed the E198V mutation in the gene. Ninety-three isolates with dual resistance to carbendazim and diethofencarb showed the E198V/K mutation. All Bos isolates carried the H272R mutation in the gene. The I365S and Q369P+N373S (66.99%) mutations in the gene were more frequently observed. No mutation was detected in the gene in Hebei isolates. There were 13 resistance profile phenotypes. Phenotypes with triple resistance were the most common (83.50%), and CarDieBosIprFen was the major type. Car 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 isolates that possessed H272R mutation were lowly resistant (LR). Ipr isolates were all LR or MR. The distribution of half maximal effective concentrations of Car isolates with E198V/K mutations and Ipr 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 in the field.