Development of a PCR assay to quantify take-all pathogens of wheat

Abstract

Take-all is a disease in wheat which can cause significant yield losses. If it was possible to predict these losses, by competitive PCR, agronomic decisions could be made to reduce them. As the causal agent, Gaeumannomyces graminis, is found in the soil organic matter, soil DNA extraction methods were tested for their efficacy. An inhibition assay was designed to test the level of inhibition present in DNA extracted from soil. Of the extractions tested the CTAB extraction was the most successful at removing inhibitors and the commercially available Soil DNA Isolation Kit ™ was the least successful. All extraction methods tested required some dilution to enable the amplification of the inhibition standard. Soil DNA extraction was found to be time consuming so stem bases and roots were used with the competitive PCR assay. To create a quantitative competitive PCR assay published primers were amplified with known G. graminis isolates. They produced unexpected results which meant that they were unsuitable for use in a competitive PCR assay. Consequently HAGG primers were designed and successfully amplified known G. graminis var. tritici and var. avenae isolates, and isolated G. graminis from take-all infected wheat plants. The HAGG competitive PCR assay was used to quantify the presence of the take-all pathogen in a large number of plant samples ranging in age. Regressions of DNA concentration and visual symptoms or yield did not result in strong correlations. Analysis suggested the variability between the fields explained a large proportion of variability. A large scale experiment was completed to compare the impact of various seed treatments on pathogen DNA concentration, visual disease assessment, grain quality and yield. Early assessment of the plant material demonstrated the heterogeneous nature of this disease. When taking the pathogen DNA concentration at stubble into account a Latitude® (silthiofam) seed treatment achieved the greatest yield; Baytan® (triadimenol and fuberidazole) resulted in the lowest yield. The experiment demonstrated that this competitive PCR assay has the potential in field experiments to take account of the initial inoculum load