Rapid in situ quantification of the strobilurin resistance mutation G143A in the wheat pathogen Blumeria graminis f. sp. tritici
Kejal N. Dodhia, Belinda A. Cox, Richard P. Oliver & Francisco J. Lopez‐Ruiz|Centre for Crop and Disease Management, School of Molecular and Life Sciences, Curtin University, Bentley, Perth, WA 6102, Australia; Faculty of Science and Engineering, Curtin University, Perth, WA 6102, Australia. Curtin University, Perth, WA 6102, Australia|Scientific Report|2021|11:4526 https://doi.org/10.1038/s41598-021-83981-9
Fungicide resistance in plant pathogens is a common incidence that occurs in the agricultural industry. It lowers the efficacy of chemical control methods; lowers yields and quality due to increased disease pressure. It is important to determine what chemistries that the crop is resistant to and then use more efficacious chemicals instead. Fungicide resistance can be functionally linked to specific changes in target genes. Blumeria graminis [DC.] E.O. Speer f. sp. tritici Em. Marchal (synonym Erysiphe graminis DC) (Bgt) is an obligate biotrophic pathogen of wheat that causes wheat powdery mildrew (WPM) which is very common in many crops. Bgt can be controlled with strobilurins which target the cytochrome bc1 enzyme complex and the selection of genotypes resistant to this group of chemicals is facilitated by Bgt’s large population size and the fungicide use to control it. An ideal solution is to develop an assay which can be performed rapidly, in situ, to quantify the population of the fungal pathogens that are resistant to a given fungicide with high confidence results. In this study, an allele-specific qPCR (ASqPCR) combined with a 2-step crude DNA extraction to quantify Bgt with genetic changes correlated with fungicide resistance was developed to be performed on a portable thermal cycler (Mic qPCR Cycler) and completed within 90 minutes of sample collection. To differentiate between the mutant and wild-type, which can be detected within the same reaction, sensitive and resistant Bgt sequences were aligned to find where the mutations lie. It was determined that a single nucleotide polymorphism (SNP) of guanidine to cytosine at position 428 was found between sensitive and resistant isolates. A master mix that contained a ‘fast’ and robust polymerase was added to the reaction to tolerate the use of crude extracts as well as a portable, battery powered qPCR instrument (Mic qPCR Cycler) to allow for in-field analysis within 90 minutes of sample collection and prevent the use of strobilurins that can increase funigicide resistance. Validation of the newly developed on-site test was compared to laboratory based digital PCR found no significant differences between the two methods.