In this experiment, we determined the occurrence of fungicide resistance of Botrytis cinerea causing gray mold in host plants such as strawberry, perilla, cucumber, and tomato ginseng to investigate inhibitory efficacy of fungicides inhibiting succinate dehydrogenase (SDHI fungicides) on the mycelial growth of the gray mold pathogen. Also, it was illustrated the relationship between the amino acid substitution of the target enzyme as a succinate dehydrogenase and the fungicide resistance of B. cinerea, the ability to adapt to the fields according to the genetic type of B. cinerea isolates resistant to the fungicides, and the competitive fitness of B. cinerea under the selection pressure as the application of SDHI fungicides in the fields.
Over the three years of 2011, 2015 and 2016. 436 isolates of B. cinerea were collected from the diseased plants Among them, 197 isolates were selected, and were used to determine the resistance to boscalid, fluopyram, fluxapyroxad, isopyrazam, penthiopyrad by using an agar dilution method. Based on the results of EC50 values, it was determined resistant isolate as showing an EC50 value of 100 ㎍/㎖ or more against boscalid including into a SDHI fungicide group.
To analyze the sdhB gene sequences of the resistant isolates, it was confirmed that there were the substitution of a amino acid at the 225th codon from proline (CCC) to phenylalanine (TTC), at the 230th codon from AAC (asparagine) to ATC (isoleucine) and at the 272nd codon from CAC to CGC (arginine) or CTC (leucine). In particular, isolates in which the 225th and the 272th amino acid were replaced with proline and phenylalanine (P225F) and histidine to arginine (H272L), respectively, were resistant to boscalid as well as the other four fungicides.
Because of the different degrees of resistance of B. cinerea to SDHI fungicides depending on where the amino acid substitutions appear in the sdhB, a resistance monitoring method is needed to search for the substitution of the amino acid in sdhB. It was conducted a multiplex allele-specific PCR method to develop a speedy and accurate assay to detect the resistance of B. cinerea isolates with different amino acid substitutions on the same gene. With six pairs of primers constructed based on the four mutation positions in sdhB, it was performed multiplex allele-specific PCR to detect all mutations such as P225F, N230I, H272L and H272R in sdhB of B. cinerea.
Among the strains of B. cinerea isolated in 2015, it was the same the isolation ratio of each mutation type resistant to SDHI fungicide, but in 2016, the isolation rate of the mutation type as P225F was greatly increased. The reason for the increased mutation type of P225F was probably due to its higher competitiveness than other mutation type under the selective pressure of fungicide. This high competitiveness demonstrates the high possibility that the mutant type of P225F resistant to SDHI fungicide could become a dominant group in the fields using SDHI fungicides. However, in the absence of the fungicide selection pressure, the competitiveness of the wild-type isolates was higher than that of fungicide-resistant isolates including into other mutation type. Therefore, in the case of SDHI fungicide resistance, it was thaught that the susceptible isolates of B. cinerea would be a dominant group of the pathogen population in the fields. These informations will be a very important information in managing the resistance of SDHI disinfectants in fields.