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Molecular Characterization and Diagnosis of QoI Resistance in Cucumber and Eggplant Fungal Pathogens

First, seventh, and eighth authors: National Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki 305-8604, Japan; second author: Kochi Agricultural Research Center, Nankoku, Kochi 783-0023, Japan; third author: Agricultural Experiment Station, Okayama Prefectural General Agriculture Center, Sanyo, Okayama 709-4602, Japan; fourth author: Saga Prefectural Agricultural Research Center, Saga, Saga 840-2205, Japan; and fifth and sixth authors: BML Inc., Kawagoe, Saitama 350-1101, Japan; and ninth author: Oita Prefectural Agricultural Research Center, Usa, Oita 872-0103, Japan.

The molecular mechanism of QoI fungicide resistance was studied using isolates of cucumber Corynespora leaf spot fungus (Corynespora cassiicola) and the eggplant leaf mold (Mycovellosiella nattrassii). In both pathogens, a mutation at position 143 from glycine to alanine (G143A) was detected in the cytochrome b gene that encodes for the fungicide-targeted protein. Moreover, the nucleotide sequence at amino acid position 143 was converted from GGT or GGA in sensitive (wild-type) to GCT or GCA in resistant (mutant-type) isolates. The methods of polymerase chain reaction restriction fragment length polymorphism commonly used for QoI resistance monitoring were employed successfully, leading to the amplified gene fragment from resistant isolates being cut with the restriction enzyme ItaI. However, heteroplasmy (the coexistence of wild-type and mutated alleles) was found when the resistant isolates of C. cassiicola, M. nattrassii, and Colletotrichum gloeosporioides (strawberry anthracnose fungus) were subcultured in the presence or absence of QoI fungicides. QoI resistance of cucumber powdery and downy mildew isolates persisted for a few years following the removal of the selection pressure imposed by the fungicide under both laboratory and commercial greenhouse conditions. The proportion of mutated sequences in cytochrome b gene decreased over time in the pathogen population. The protective efficacy of the full dose of azoxystrobin decreased when the populations of powdery and downy mildews contained resistant isolates at 10%. Using FMBIO, a fluorescence bio-imaging analyzer, the mutant allele from the QoI-resistant isolates could be detected at the level of 1%, whereas the detection sensitivity of ethidium-bromide-stained gels was ≈10 times lower.

Additional keywords:mitochondrial DNA, point mutation.