Characterisation of QoI-resistant field isolates of Botrytis cinerea from citrus and strawberry

BACKGROUND: In 2004, field isolates of Botrytis cinerea Pers. ex Fr., resistant to strobilurin fungicides (QoIs), were first found in commercial citrus orchards in Wakayama Prefecture, Japan. Subsequently, QoI-resistant isolates of this fungus were also detected in plastic strawberry greenhouses in Saga, Ibaraki and Chiba prefectures, Japan. Biological and molecular characterisation of resistant isolates was conducted in this study.RESULTS: QoI-resistant isolates of B. cinerea grew well on PDA plates containing kresoxim-methyl or azoxystrobin at 1 mg L⁻¹, supplemented with 1 mM of n-propyl gallate, an inhibitor of alternative oxidase, whereas the growth of sensitive isolates was strongly suppressed. Results from this in vitro test were in good agreement with those of fungus inoculation tests in vivo. In resistant isolates, the mutation at amino acid position 143 of the cytochrome b gene, known to be the cause of high QoI resistance in various fungal pathogens, was found, but only occasionally. The heteroplasmy of cytochrome b gene was confirmed, and the wild-type sequence often present in the majority of resistant isolates, indicating that the proportion of mutated cytochrome b gene was very low.CONCLUSION: The conventional RFLP and sequence analyses of PCR-amplified cytochrome b gene are insufficient for molecular identification of QoI resistance in B. cinerea.