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

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.