Development of a PNA-LNA-LAMP Assay to Detect an SNP Associated with QoI Resistance in .

The repetitive use of quinone outside inhibitor fungicides (QoIs, strobilurins; Fungicide Resistance Action Committee [FRAC] 11) to manage grape powdery mildew has led to development of resistance in . While several point mutations in the mitochondrial cytochrome gene are associated with resistance to QoI fungicides, the substitution of glycine to alanine at codon 143 (G143A) has been the only mutation observed in QoI-resistant field populations. Allele-specific detection methods such as digital droplet PCR and TaqMan probe-based assays can be used to detect the G143A mutation. In this study, a peptide nucleic acid-locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA-LAMP) assay consisting of an A-143 reaction and a G-143 reaction, was designed for rapidly detecting QoI resistance in . The A-143 reaction amplifies the mutant A-143 allele faster than the wild-type G-143 allele, while the G-143 reaction amplifies the G-143 allele faster than the A-143 allele. Identification of resistant or sensitive samples was determined by which reaction had the shorter time to amplification. Sixteen single-spore QoI-resistant and -sensitive isolates were tested using both assays. Assay specificity in distinguishing the single nucleotide polymorphism (SNP) approached 100% when tested using purified DNA of QoI-sensitive and -resistant isolates. This diagnostic tool was sensitive to one-conidium equivalent of extracted DNA with an value of 0.82 and 0.87 for the G-143 and A-143 reactions, respectively. This diagnostic approach was also evaluated against a TaqMan probe-based assay using 92 samples collected from vineyards. The PNA-LNA-LAMP assay detected QoI resistance in ≤30 min and showed 100% agreement with the TaqMan probe-based assay (≤1.5 h) for the QoI-sensitive and -resistant isolates. There was 73.3% agreement with the TaqMan probe-based assay when samples had mixed populations with both G-143 and A-143 alleles present. Validation of the PNA-LNA-LAMP assay was conducted in three different laboratories with different equipment. The results showed 94.4% accuracy in one laboratory and 100% accuracy in two other laboratories. The PNA-LNA-LAMP diagnostic tool was faster and required less expensive equipment relative to the previously developed TaqMan probe-based assay, making it accessible to a broader range of diagnostic laboratories for detection of QoI resistance in . This research demonstrates the utility of the PNA-LANA-LAMP for discriminating SNPs from field samples and its utility for point-of-care monitoring of plant pathogen genotypes.