When time really is money: in situ quantification of the strobilurin resistance mutation G143A in the wheat pathogen Blumeria graminis f. sp. tritici

Abstract

BackgroundThere has been an inexorable increase in the incidence of fungicide resistance in plant pathogens in recent years. Control of diseases and the management of resistance would be greatly aided by rapid diagnostic methods. Quantitative allele specific PCR (ASqPCR) is an ideal technique for the analysis of fungicide resistance in the field as it can both detect and quantify the frequency of mutations associated with fungicide resistance. We have applied this technique to the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), an obligate biotrophic fungus that causes wheat powdery mildew and is responsible for up to 25% yield loss annually. In Australia, strobilurin resistant Bgt was first discovered in samples from Tasmania and Victoria in 2016. Molecular analysis revealed a nucleotide transversion in the cytochrome bc1 enzyme (cytb) complex, resulting in a substitution of alanine for glycine at position 143 (G143A) in Cytb. ResultsWe have developed an in-field ASqPCR assay that can quantify both the resistant (A143) and sensitive (G143) cytb alleles down to 1.67% in host and Bgt DNA mixtures within 90 min of sample collection. The in situ analysis of field samples collected during a survey in Tasmania revealed A143 frequencies ranging between 9-100%. We validated the analysis with a newly developed laboratory based digital PCR assay and found no significant differences between the two methods. ConclusionWe have successfully developed an in-field quantification method, for a QoI resistant allele, by pairing an ASqPCR assay on a lightweight qPCR instrument with a quick DNA extraction method. The deployment of this type of methodologies in the field can contribute to the effective in-season management of fungicide resistance.