Development of a PNA-LNA-LAMP assay to detect a SNP associated with QoI resistance in Erysiphe necator

Authors: SHARMA, NANCY - Michigan State University; Neill, Tara; Yang, Hui-Ching; OLIVER, CHARLOTTE - Washington State University; Mahaffee, Walter - Walt; Naegele, Rachel; MOYER, MICHELLE - Washington State University; MILES, TIMOTHY - Michigan State University

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/11/2023
Publication Date: 10/25/2023
Citation: Sharma, N., Neill, T.M., Yang, H., Oliver, C., Mahaffee, W.F., Naegele, R.P., Moyer, M., Miles, T. 2023. Development of a PNA-LNA-LAMP assay to detect a SNP associated with QoI resistance in Erysiphe necator. Plant Disease. https://doi.org/10.1094/PDIS-09-22-2027-RE.
DOI: https://doi.org/10.1094/PDIS-09-22-2027-RE

Interpretive Summary: The QoIs (quinone outside inhibitor fungicides, strobilurins, FRAC11) have been widely used to manage grape powdery mildew disease. However, the repetitive use of these fungicides has led to development of resistance. Scientists have found that a genetic mutation, G143A, is the predominant mutation observed in QoI-resistant powdery mildew populations. Some molecular detection methods have been developed to detect and monitor the G143A mutation. However, most of the techniques are expensive and less flexible. This study successfully developed a new assay, which applied the PNA-LNA-LAMP (peptide nucleic acid-locked nucleic acid mediated loop-mediated isothermal amplification) method to differentiate populations with and without the mutation. This diagnostic tool is relatively inexpensive, easy to apply, and efficient, and therefore can be used to detect QoI resistance in powdery mildew populations.

Technical Abstract: The repetitive use of quinone outside inhibitor fungicides (QoIs, strobilurins; FRAC 11) to manage grape powdery mildew has led to development of resistance in Erysiphe necator. While several point mutations in the mitochondrial cytochrome b gene are associated with resistance to QoI fungicides, the one causing the substitution of glycine to alanine at codon 143 (G143A) is the predominant mutation observed in QoI-resistant 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 rapid detection of the G143A mutation in E. necator. 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 E. necator samples was determined by which reaction amplified first (A-143 or G-143, respectively). Sixteen single-spore QoI-resistant and sensitive E. necator 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 QoI-resistant E. necator isolates. This diagnostic tool was sensitive to 1 conidia worth of extracted DNA with an R2 value of 0.824 and 0.865, for G-143 and A-143 reactions, respectively. This diagnostic approach was also evaluated against a TaqMan probe-based assay using 92 E. necator samples collected from vineyards. The PNA-LNA-LAMP assay detected QoI resistance in 30 minutes and showed 100% agreement with the TaqMan probe-based assay for the QoI-sensitive and QoI-resistant isolates. There was 73.33% 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 and this technology was transferred successfully. The results showed 94.44% accuracy in one laboratory and 100% accuracy in two other laboratories. The PNA-LNA-LAMP diagnostic tool takes less time to complete and requires less expensive equipment relative to previously made TaqMan probe-based assay, making it accessible to a broader range of diagnostic laboratories for detection of QoI resistance in E. necator. To our knowledge, this is the first study using PNA-LNA-LAMP techniques in plant pathology to discriminate a SNP, therefore this study will potentially widen the horizon of point-of-care detections for specific pathogen genotypes.