Improved Detection and Monitoring of Fungicide Resistance in Blumeria graminis f. sp. hordei With High-Throughput Genotype Quantification by Digital PCR

Abstract

The increased occurrence of triazole fungicide resistant strains of Blumeria graminis f. sp. hordei (Bgh) is an economic concern for the barley industry in Australia and elsewhere. High levels of resistance to triazoles in the field are caused by two separate point mutations in the Cyp51 gene, Y136F and S509T. Early detection of these mutations arising in pathogen field populations is important as this allows time for changes in fungicide practices to be adopted, thus mitigating potential yield losses due to fungicide failure and preventing the resistance from becoming dominant. A digital PCR (dPCR) assay has been developed for the detection and quantification of the Y136F and S509T mutations in the Bgh Cyp51 gene. Mutation levels were quantifiable as low as 0.2% in genomic DNA extractions and field samples. This assay was applied to the high throughput screening of Bgh field and bait trial samples from barley growing regions across Australia in the 2015 and 2016 growing seasons and identified the S509T mutation for the first time in the Eastern states of Australia. This is the first report on the use of digital PCR technology for fungicide resistance detection and monitoring in agriculture. Here we describe the potential application of dPCR for the screening of fungicide resistance mutations in a network of specifically designed bait trials. The combination of these two tools constitute an early warning system for the development of fungicide resistance that allows for the timely adjustment of management practices.

Sterol 14α-Demethylase Ligand-Binding Pocket-Mediated Acquired and Intrinsic Azole Resistance in Fungal Pathogens

“…Blumeria graminis, the most common ascomycetous fungal pathogen infecting barley crops, causes a powdery mildew that reduces barley yields by 20–40% [ 51 , 119 ]. In Blumeria graminis , SDM Y136F has been found together with the S509T mutation [ 51 ]. Isolates with this genotype are resistant to TBC [ 51 ].…”

“…In Blumeria graminis , SDM Y136F has been found together with the S509T mutation [ 51 ]. Isolates with this genotype are resistant to TBC [ 51 ].…”

Rapid in situ quantification of the strobilurin resistance mutation G143A in the wheat pathogen Blumeria graminis f. sp. tritici

“…For novel field failures, where the target gene is known, capillary sequencing methods are used to determine if any known mutations are the cause 3 , 40 . Once a mutation has been confirmed, PCR and LAMP are commonly used for its detection, whereas for quantification qPCR and more recently digital PCR are used 18 , 26 , 41 – 43 . Except for LAMP, none of the other methods have been used in the field for the detection of fungicide resistance, therefore currently the quantification of mutations associated with fungicide resistance is exclusively laboratory based 20 .…”

The Inhibitory Potential of Selected Essential Oils on Fusarium spp. Growth and Mycotoxins Biosynthesis in Maize Seeds

“…Fusarium fungi are widespread in many ecological niches, including cereal cultivation environments [7][8][9]. The control of mycotoxigenic fungi and their metabolites are high priorities for the cereal production industry, therefore different strategies are taken into account, such as suitable agrotechnical practices, the use of resistant varieties, or monitoring of fungal populations [45]. However, usually fungicides are used to reduce or prevent serious losses caused by phytopathogenic fungi.…”