Genotyping of Benzimidazole-Resistant and Dicarboximide-Resistant Mutations in Botrytis cinerea Using Real-Time Polymerase Chain Reaction Assays

Abstract

Botrytis cinerea, an economically important gray mold pathogen, frequently exhibits multiple fungicide resistance. A fluorescence resonance energy transfer-based real-time polymerase chain reaction assay has been developed to detect benzimidazole- and dicarboximide-resistant mutations. Three benzimidazole-resistant mutations-(198)Glu to Ala (E198A), F200Y, and E198K-in beta-tubulin BenA were detected using a single set of fluorescence-labeled sensor and anchor probes by melting curve analysis. Similarly, three dicarboximide-resistant mutations-I365S, V368F plus Q369H, and Q369P-in the histidine kinase BcOS1 were successfully distinguished. Unassigned melting profiles in BenA genotyping assay resulted in the identification of a new benzimidazole-resistant BenA E198V mutation. This mutation conferred resistance to carbendazim as do E198A and E198K mutations. The isolates with BenA E198V mutation showed a negative cross-resistance to diethofencarb, but to a lesser extent than the E198A mutants. A survey of 210 B. cinerea field isolates revealed that most of benzimidazole-resistant isolates possessed the E198V or E198A mutation in the BenA gene, and the I365S mutation in the BcOS1 gene was also frequently observed in Japanese isolates. However, benzimidazole-resistant isolates with BenA F200Y or E198K mutations, which confer the diethofencarb-insensitive phenotype, were rare. Our BenA and BcOS1 genotyping is a rapid and reliable method that is suitable for monitoring the fungicide-resistant field population.

Phenotypic Analyses of Fenhexamid Resistant Botrytis cinerea Mutants

“…In Japan, although fenhexamid was registered in 2000, it is not widely used and thus, the existence of fenhexamid resistance in B. cinerea has not been reported in Japan. Molecular-based techniques have been developed to rapidly detect the sensitivity of fungi to several fungicides by taking advantage of point mutations in target genes (Oshima et al 2002;Paplomatas et al 2004;Banno et al 2008, Furuya et al 2009Saito et al 2009). A rapid method for the detection of fungicide resistance is necessary for viticulturists to better understand the incidence of resistance in B. cinerea populations in their vineyards.…”

Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance

“…Two single nucleotide mutations of CYP51 resulting in amino acid substitutions Y136F and K147Q in Blumeria graminis were also found (Wyand & Brown, 2005). Different mutations were also found in Tapesia sp (Albertini et al, 2003), Penicillium italicum (Joseph-Horne & Hollomon, 1997), Ustilago maydis (Butters et al, 2000) and Blumeriella jaapii (Ma et al, 2006) Similarly, mutations have been described in the cytochrome b gene that lead to change of its corresponding protein G143A, conferring resistance to strobilurin (Avila-Adame & Koller, 2003aGisi et al, 2000, Zheng et al, 2000, Zhang, Z. et al, 2009 or the amino acid substitution in the -tubulin target protein involved in development of resistance to benzimidazoles in Botrytis cinerea (Banno, 2008), Venturia inaequalis and Penicillium italicum (Koenraadt et al, 1992), Monilinia fructicola , M. laxa (Ma & Michailides, 2005), P. expansum (Baraldi et al, 2003) and P. digitatum (Sánchez-Torres & Tuset, 2011).…”

Detection of Fungicide Resistance

“…For most, smaller laboratories, a more realistic approach concerns the use of polymerase chain reaction coupled with cleaved amplified polymorphic sequences (PCR-CAPS). Restriction-fragment length polymorphisms due to small nucleotide polymorphisms (SNP) that co-segregate or are caused by fungicide resistance create or abolish restriction sites in PCR products, and can be exploited for detection of fungicide resistance through the careful selection of locus-specific oligonucleotide primers (Banno et al 2008;Lesemann et al 2007, Quello et al 2009, Fontaine et al 2009others).…”