Following the dynamics of strobilurin resistance in Blumeria graminis f.sp. tritici using quantitative allele‐specific real‐time PCR measurements with the fluorescent dye SYBR Green I

Abstract

Strobilurin-resistant isolates of Blumeria ( Erysiphe ) graminis f.sp. tritici , the cause of wheat powdery mildew, were more than 10-fold less sensitive to azoxystrobin than sensitive isolates. In all resistant isolates, a mutation resulting in the replacement of a glycine by an alanine residue at codon 143 (G143A) in the mitochondrial cytochrome b gene was found. Allele-specific primers were designed to detect this point mutation in infected wheat leaves. Using quantitative fluorescent allele-specific real-time polymerase chain reaction (PCR) measurements, strobilurin-resistant A143 alleles could be detected amongst strobilurin-sensitive G143 alleles at a frequency of at least 1 in 10 000, depending on the amount of target and nontarget DNA. Most isolates tested were dominant homoplasmic for either the A143 or G143 allele, although mixed populations of alleles could be detected in some isolates. In some of these isolates, strobilurin resistance was not always stable when they were maintained for many generations in the absence of selection. The allele-specific real-time PCR assay was also used to follow the dynamics of A143 alleles in field populations of B . graminis f.sp. tritici before and after application of fungicides. As expected, the A143 allele frequency only increased under selection pressure from a strobilurin fungicide. After three sprays of azoxystrobin, a pronounced selection for the strobilurin-resistant allele, with an increase in average frequency from 2·2 to 58%, was measured. The use of quantitative real-time PCR diagnostics for early detection of fungicide resistance genes at low frequency, coupled with risk evaluation, will be invaluable for further resistance risk assessment and validation of antiresistance strategies.

Fenhexamid Resistance in the Botrytis Species Complex, Responsible for Grey Mould Disease

“…In most cases, the degree of sensitivity of fungal populations to one or more fungicides is assessed by biological methods. Sierotzki et al, 2000) and on the opposite some of them on populations, where resistance allele is quantify in gDNA pools by using Real time PCR technologies (Benzimidazole resistance in Sclerotinia sclerotiorum Chen et al, 2009 ; Strobilurins resistance on Blumeria graminis Fraaije et al, 2002 and Pyrenophora teres Kianianmomeni et al, 2007). However, one limitation of this method concerns the nonspecific amplification of alleles, which may affect precision.…”

Identification and Characterization of BenzimidazoleResistance in Monilinia fructicola from Stone Fruit OrchardsinCalifornia

“…In most cases, resistance has been shown to be associated with point mutations in the ␤-tubulin gene which result in altered amino acid sequences at the benzimidazole binding site (13). Studies with laboratory mutants of Aspergillus nidulans, Neurospora crassa, and Saccharomyces cerevisiae showed that changes at codons 6,50,134,165,198,200, and 241 in the ␤-tubulin gene were responsible for benzimidazole resistance (13). In contrast to laboratory mutants, most field isolates of plant-pathogenic fungi exhibit codon changes that, strikingly, seem to be restricted to positions 50 (22), 198, 200 (1,13), and 240 (1).…”

Mechanism of action and selectivity of a novel fungicide, pyribencarb

“…1) binds with a hydrogen bond to the amide group of glutamic acid at position 272 in domain C; therefore, this region plays an important role in accepting fungicides. Since substitution of glycine for alanine at position 143 (G143A) in domain B significantly lowers the affinity of strobilurin fungicides to cytochrome b, 7,8,13,14,[19][20][21][22][23] interaction between domain B and strobilurin fungicides must be strong. On the other hand, substitution of phenylalanine for leucine at position 129 (F129L) in domain A has been shown not to significantly lower the affinity of strobilurin fungicides to cytochrome b.…”