Characterization of QoI resistance in Botrytis cinerea and identification of two types of mitochondrial cytochrome b gene

• Published in: Plant pathology 2009-02, Vol.58 (1), p.120-129
• Main Authors: Banno, S, Yamashita, K, Fukumori, F, Okada, K, Uekusa, H, Takagaki, M, Kimura, M, Fujimura, M
• Format: Article
• Language: English
• Subjects: Botryotinia fuckeliana; Botrytis cinerea; cytochrome b; fluorescent probe; grey mould; QoI fungicides; real-time PCR; Sclerotinia fuckeliana
• ISSN: 0032-0862; 1365-3059
• DOI: 10.1111/j.1365-3059.2008.01909.x

Botrytis cinerea field isolates collected in Japan were screened for resistance to Qo inhibitor fungicides (QoIs). Of the 198 isolates screened, six grew well on a medium containing azoxystrobin, a QoI, when salicylhydroxamic acid, an alternative oxidase inhibitor, was present. The resistance mutation in the cytochrome b gene (cytb) was characterized. All QoI-resistant isolates had the same mutation (GGT to G CT) in cytb that led to the substitution of glycine by alanine at position 143 of cytochrome b, which is known to confer QoI resistance in plant pathogens. To detect this mutation, a hybridization probe assay based on real-time PCR amplification and melting curve analysis was developed. Using DNA samples prepared from aubergines coinfected with QoI-resistant and QoI-sensitive B. cinerea isolates, two similar peak profiles with their corresponding melting temperatures were obtained. This result suggests that QoI-resistant and QoI-sensitive isolates may compete equally in terms of pathogenicity, and the assay may be used to assess the population ratio of mutant and wild-type isolates. However, the hybridization probe did not anneal to PCR products derived from the DNA samples of some QoI-sensitive isolates. Structural analysis of cytb revealed that B. cinerea field isolates could be classified into two groups: one with three introns and the other with an additional intron (Bcbi-143/144 intron) inserted between the 143rd and 144th codons. All 88 isolates possessing the Bcbi-143/144 intron were azoxystrobin-sensitive, suggesting that the QoI-resistant mutation at codon 143 in cytb prevents self-splicing of the Bcbi-143/144 intron, as proposed in some other plant pathogens.