In Situ Detection of Benzimidazole Resistance in Field Isolates of Venturia inaequalis in Indiana

Abstract

Venturia inaequalis, the causal agent of apple scab, infects both commercial apples and ornamental crabapples. We found four classes of benzimidazole fungicide sensitivity in the Indiana population: sensitive (S) isolates unable to grow on 0.5 μg active ingredient (a.i.)/ml; low resistant (LR) isolates that grew at 0.5 μg a.i./ml, but not at 5 μg a.i./ml; moderately resistant (MR) isolates that grew at 5 μg a.i./ml, but not at 50 μg a.i./ml; and very highly resistant (VHR) isolates that grew rapidly at 50 μg a.i./ml. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of the β-tubulin gene with two restriction enzymes, BstUI and Cac8I, enabled us to rapidly identify benzimidazole resistance among all tested isolates. Sixty-nine percent of the resistant isolates tested possessed the BstUI RFLP at codon 198 that corresponds to VHR, and the remaining LR and MR isolates possessed the Cac8I RFLP corresponding to a newly identified resistance allele at codon L240F. Combined, PCR-RFLP correctly identified the resistance status of all isolates tested to date. The preponderance of benzimidazole-resistant isolates from commercial apple orchards and their absence in the landscape on ornamental crabapple suggests that two distinct populations of V. inaequalis coexist in Indiana.

Venturia inaequalis resistance in local Spanish cider apple germplasm under controlled and field conditions

“…In particular, the benzimidazoles and inhibitors of the biosynthesis of ergosterol (IBE) have been intensively used in the past. The first cases of resistance to benzimidazoles were detected in those countries where these fungicides were most intensively used, and various resistant strains to IBE were found several years ago in France and other European countries (Schnabel and Parisi 1997), and later in commercial apple orchards in Indiana, USA (Quello et al 2010). Also, V. inaequalis strains resistant to sterol demethylation inhibitors were isolated already nearly a decade ago (Schnabel and Jones 2001;Koller et al 2004;Ma and Michailides 2005).…”

Detection of Fungicide Resistance

“…Finally, the calculated the cost of 96 reactions, excluding labor and plate reader, was found to be under $4 per isolate for AB assays and over $15 per isolate for mycelial growth assays (Cox et al 2009) making this affordable for multiple labs and diagnostic clinics to screen for fungicide resistance as a service. Despite the lack of intellectual appeal, there are other significant advantages of fungicide resistance phenotype screening, chief amongst which is the low cost, accuracy and reliability of the screening for the fungicide resistant phenotype over the genotype, because, unless all known genes and alleles are screened are known and screened, the risk of under-reporting actual resistances exists (Quello et al 2010), and is rarely discussed. This is not to say that molecular detection of fungicide resistance is without merit.…”

“…Genome-sequencing efforts have resulted in technological advances in DNA sequencing and led to the improvement of longer sequencing length, done faster and with less expense. It has also permitted the rapid sequencing of isolates (in part or into total) that did not conform to expected genotype: phenotype relationships (Lesniak et al 2011;Quello et al 2010;Leroux et al 1999, and many others). Metzker (2009) provides an excellent review of 'next generation sequencing' technology, although the rapid pace of technology marches on.…”

“…Resistance is correlated with point mutations in the -tubulin gene, with different mutations resulting in altered amino acid changes at the benzimidazole-binding site. These various mutations at different codon sites also result in different levels of resistance: In V. inaequalis, mutations at codon 198 resulted in medium resistance, at codon 200 it resulted in very high resistance (Koenraadt et al 1992a), and at 240 it resulted in low resistance (Quello et al 2010). In Monilinia, only low and high resistance to benomyl and thiophanate-methyl has been observed for field isolates of M. fructicola (Ma and Michelides, 2005).…”

Disease Decision Support Systems: Their Impact on Disease Management and Durability of Fungicide Effectiveness

“…Resistance to multisite inhibitors, DMIs, anilinopyrimidines and SDHIs are still monitored with biological tests. For benzimidazoles and QoIs, resistance was found to be determined by single mutation within the target genes (β-tubulin and cytochrome b, respectively) (Quello et al, 2010;Fontaine et al, 2008). These mutations were detected after partial PCR amplification of these genes and digestion of the PCR product by a restriction enzyme that recognizes the restriction site generated (or lost) by the mutation determining resistance (PCR-RFLP or CAPS test).…”