In planta genotyping of Erysiphe graminis f. sp. tritici isolates for strobilurin-resistance using a fluorometric allele-specific PCR assay

摘要

Three strobilurin-resistant isolates of Erysiphe graminis f. sp. tritici from Germany and the UK were 100-fold less sensitive to azoxystrobin than sensitive isolates. A 675 base pair (bp) fragment of the mitochondrial cytochrome b gene was sequencedand a guanine to cytosine (G-to-C) change for one nucleotide was identified. The mutation resulted in the substitution of a glycine for an alanine residue at codon 143 (G143A) in the cytochrome b of resistant isolates. Allele-specific primers were designed to detect this point mutation in infected leaves by polymerase chain reaction (PCR) linked to dsDNA-specific fluorescence. The resistant A143 allele can be detected amongst sensitive G143 alleles at a frequency below 1:1000. Eight isolates were tested and no false positives were found, although the sensitive G143 allele could be detected, albeit in low amounts, in strobilurin-resistant isolates. This suggests that a mixed population of strobilurin-sensitive and resistant mitochondria could be present in these isolates.