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

Abstract

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 GCT) 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 azoxystrobinsensitive, 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.

Exploring Mechanisms of Resistance to Respiratory Inhibitors in Field Strains of Botrytis cinerea , the Causal Agent of Gray Mold

“…However, other mutations conferring lower levels of resistance may be selected in these fungi, such as F129L in P. teres (30). These findings may account for the presence of the G143A substitution only in strains lacking the intron in Botrytis group II in our sample and in Californian and Chinese isolates (40) and in Japanese strains isolated from various host plants for which no fitness cost associated with strobilurin resistance could be found (7). In our population data set, G143A was not found in any of the 27 group I strains.…”

“…Some shifts in susceptibility to multisite fungicides and fluazinam have also been recorded in ornamental plants and vegetables (44). Moreover, field strains of B. cinerea resistant to inhibitors of mitochondrial complexes II and/or III have recently been detected (7,38,40,54,65). Finally, strains displaying MDR have been isolated from French and German vineyards (42,44).…”

Deleterious Effect of the Q o Inhibitor Compound Resistance-Conferring Mutation G143A in the Intron-Containing Cytochrome b Gene and Mechanisms for Bypassing It

“…Botrytis cinerea is particularly interesting, since the same species presents two types of cytochrome b gene; some field isolates contain intron bi2, while others do not. The mutation G143A has been reported only in the latter populations (1,17). These observations suggested that the presence or absence of intron bi2 might affect the occurrence of the G143A mutation.…”

The QoI Fungicides, the Rise and Fall of a Successful Class of Agricultural Fungicides

“…The case of Botrytis cinerea is different; two types of CYTB gene were found in Japanese populations of the grey mould pathogen, one with three introns and the other with an additional intron (Bcbi-143/144 intron) inserted between the 143rd and 144th codons. All QoI-resistant isolates showed the G143A mutation whereas the isolates possessing the Bcbi-143/144 intron were QoI-sensitive (Banno et al, 2009). The possession of such an intron may guarantee a low risk of QoI resistance but it might not always be fixed in pathogen populations.…”