Quantification of necrotrophic pathogen biomass using an internal control in a real-time PCR assay
Eshraghi, L., Aryamanesh, N., Anderson, A.J., McComb, J., Hardy, G.E.S.J., Shearer, B. and O'Brien, P.A. (2011) Quantification of necrotrophic pathogen biomass using an internal control in a real-time PCR assay. In: Asian Association of Societies for Plant Pathology (AASPP) and the Australasian Plant Pathology Society Conference, 26 - 29 April, Darwin, Australia.
A common approach for measuring the susceptibility of a plant to infection by a pathogen is to measure the pathogen biomass in planta using quantitative PCR. However, the processes such as the production of phenolics, or degradation of cellular DNA and cell collapse associated with necrosis process in plant tissue can lead to variation in DNA extraction efficiency, inhibition of PCR, or overestimation of pathogen biomass thereby affecting accurate measurement of pathogen biomass. Our approach to this is to add a control DNA (plasmid) to the plant tissue before the DNA extraction. By measuring the amount of plasmid DNA recovered in the DNA extract, and by normalising the amount of pathogen DNA to plasmid DNA we can allow for differences in DNA extraction efficiency and avoid overestimation of pathogen biomass. In both lupin–Phytophthora cinnamomi and Arabidopsis–Phytophthora cinnamomi pathosystems, cell collapse and tissue necrosis were observed and led to overestimation of pathogen biomass when normalised to host plant DNA. In lupin we observed up to 17 fold overestimation of pathogen biomass compared to three fold overestimation found in infected A. thaliana 72 h after inoculation with P. cinnamomi. Our results suggested that by increasing the degree of necrosis due to necrotrophic infection, the level of overestimation of pathogen biomass increases if normalised based on host DNA. We demonstrated the capability and robustness of this developed technique in two different plant‐pathogen interactions with various levels of resistance. This method can also be adapted in quantification of pathogen biomass in other pathosystems.
|Publication Type:||Conference Item|
|Murdoch Affiliation:||Centre for Phytophthora Science and Management|
School of Biological Sciences and Biotechnology
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