Optimized Sample Processing Pipeline for PCR-Based Fungicide Resistance Quantification of Stubble-Borne Fungal Pathogens.

Globally, yield losses associated with failed crop protection due to fungicide-resistant pathogens present an increasing problem. For stubble-borne pathogens, assessment of crop residues during the off-season could provide early fungicide resistance quantification for informed management decisions to mitigate yield losses. However, stubble assessment is hampered by assay inhibitors that are derived from decaying organic matter. To overcome assay inhibition from weathered stubble samples, we used a systems approach to quantify the frequency of resistance to demethylase inhibitor fungicides of the barley pathogen Pyrenophora teres f. teres. The system canvassed (i) 10 ball-milling conditions; (ii) four DNA extraction methodologies; and (iii) three column purification techniques for the provision of sufficient yield, quality, and purity of fungal DNA for a PCR-based fungicide resistance assay. Results show that DNA quantity and purity differed within each of the above three categories, with the optimized pipeline being (i) ball-milling samples in a 50-ml stainless steel canister for 5 min using a 20-mm ball at 30 revolutions s-1; (ii) a modified Brandfass method (extracted 64% more DNA than other methods assessed); and (iii) use of silica resin columns for the highest DNA concentration with optimal DNA purity. The chip-digital PCR assay, which quantified fungicide resistance from field samples, was unaffected by the DNA extraction method or purification technique, provided that thresholds of template quantity and purity were satisfied. In summary, this study has developed molecular pipeline options for pathogen fungicide resistance quantification from cereal stubbles, which can guide management for improved crop protection outcomes.

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

As the incidence of fungicide resistance in plant pathogens continues to increase, 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 in-field analysis of fungicide resistance as it can quantify the frequency of mutations in fungicide targets. We have applied this technique to the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), the causal agent of wheat powdery mildew. In Australia, strobilurin-resistant Bgt was first discovered in 2016. Molecular analysis revealed a nucleotide transversion in the cytochrome b (cytb) gene in the cytochrome bc1 enzyme complex, resulting in a substitution of alanine for glycine at position 143 (G143A). We 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 samples collected during a survey in Tasmania revealed A143 frequencies ranging between 9-100%. Validation of the analysis with a newly developed laboratory based digital PCR assay found no significant differences between the two methods. We have successfully developed an in-field quantification method, for a strobilurin-resistant allele, by pairing the ASqPCR assay on a lightweight qPCR instrument with a quick DNA extraction method. The deployment of these type of methodologies in the field can contribute to the effective in-season management of fungicide resistance.

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

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.

Genetic variability in the coat protein gene of Potato virus X and the current relationship between phylogenetic placement and resistance groupings.

The complete coat protein nucleotide sequences of 11 Potato virus X isolates from Australia and two from Britain were compared to those of 72 others. On phylogenetic analysis, clade I contained all 11 Australian sequences, and sub-clade II-1 contained the two new British sequences. Clade I isolates were from six different continents, but those in sub-clades II-1 and II-2 were only from Europe and the Americas, respectively. Clade I contained isolates in strain groups 1, 3 and 4, and sub-clades II-1 and II-2, isolates in strain groups 2 and 4. Thus, strain group 4 now occurs within both clades.

Genetic variability in the coat protein gene of Potato virus S isolates and distinguishing its biologically distinct strains.

The complete coat protein (CP) nucleotide sequences of 13 Potato virus S (PVS) isolates from Australia and three from Europe were compared to those of 37 others. On phylogenetic analysis, the Australian sequences were in PVS(O) sub-clades III and IV, and the European isolates were in sub-clades I and VII. The European isolates invaded Chenopodium spp. systemically, but eight Australian isolates did not. Amino acid sequence differences at the N-terminal ends of the CPs were unrelated to the ability to invade Chenopodium spp. systemically. The acronym PVS(O-CS) is suggested for isolates that invade Chenopodium spp. systemically but are not within clade PVS(A).