A quantitative PCR assay for accurate in planta quantification of the necrotrophic pathogen Phytophthora cinnamomi

L. Eshraghi; N. Aryamanesh; J.P. Anderson; B. Shearer; J.A. McComb; G.E.St.J. Hardy; P.A. O’Brien

doi:10.1007/s10658-011-9819-x

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A quantitative PCR assay for accurate in planta quantification of the necrotrophic pathogen Phytophthora cinnamomi

Journal article

Peer reviewed

A quantitative PCR assay for accurate in planta quantification of the necrotrophic pathogen Phytophthora cinnamomi

L. Eshraghi, N. Aryamanesh, J.P. Anderson, B. Shearer, J.A. McComb, G.E.St.J. Hardy and P.A. O’Brien

European Journal of Plant Pathology, Vol.131(3), pp.419-430

2011

DOI: https://doi.org/10.1007/s10658-011-9819-x

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Abstract

A reliable method for measuring disease progression is important when evaluating susceptibility in host-pathogen interactions. We describe a sensitive quantitative polymerase chain reaction (QPCR) assay that enables quantitative measurement of in planta DNA of the necrotrophic pathogen, Phytophthora cinnamomi, that avoids problems caused by variation in DNA extraction efficiency and degradation of host DNA during host tissue necrosis. Normalization of pathogen DNA to sample fresh weight or host DNA in samples with varying degrees of necrosis led to overestimation of pathogen biomass. Purified plasmid DNA, containing the pScFvB1 mouse gene, was added during DNA extraction and pathogen biomass was normalized based on plasmid DNA rather than host DNA or sample fresh weight. This method is robust and improves the accuracy of pathogen measurement in both resistant (non-host A. thaliana-P. cinnamomi) and susceptible (host Lupinus angustifolius-P. cinnamomi) interactions to allow accurate measurement of pathogen biomass even in the presence of substantial host cell necrosis.

Details

Title: A quantitative PCR assay for accurate in planta quantification of the necrotrophic pathogen Phytophthora cinnamomi
Authors/Creators: L. Eshraghi (Author/Creator)
N. Aryamanesh (Author/Creator)
J.P. Anderson (Author/Creator)
B. Shearer (Author/Creator)
J.A. McComb (Author/Creator)
G.E.St.J. Hardy (Author/Creator)
P.A. O’Brien (Author/Creator)
Publication Details: European Journal of Plant Pathology, Vol.131(3), pp.419-430
Publisher: Kluwer Academic Publishers
Identifiers: 991005541807607891
Murdoch Affiliation: Centre for Phytophthora Science and Management; School of Biological Sciences and Biotechnology
Language: English
Resource Type: Journal article

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Collaboration types: Domestic collaboration
Citation topics: 3 Agriculture, Environment & Ecology; 3.97 Plant Pathology; 3.97.1439 Phytophthora Management
Web Of Science research areas: Agronomy; Horticulture; Plant Sciences
ESI research areas: Plant & Animal Science