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

  • Leila Eshraghi
  • Nader Aryamanesh
  • Jonathan P. Anderson
  • Bryan Shearer
  • Jen A. McComb
  • Giles E. St. J. Hardy
  • Philip A. O’Brien
Article

DOI: 10.1007/s10658-011-9819-x

Cite this article as:
Eshraghi, L., Aryamanesh, N., Anderson, J.P. et al. Eur J Plant Pathol (2011) 131: 419. doi:10.1007/s10658-011-9819-x

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. thalianaP. cinnamomi) and susceptible (host Lupinus angustifoliusP. cinnamomi) interactions to allow accurate measurement of pathogen biomass even in the presence of substantial host cell necrosis.

Keywords

Arabidopsis thaliana Internal control Lupinus angustifolius Non-host resistance 

Copyright information

© KNPV 2011

Authors and Affiliations

  • Leila Eshraghi
    • 1
  • Nader Aryamanesh
    • 2
  • Jonathan P. Anderson
    • 3
    • 4
  • Bryan Shearer
    • 1
    • 5
  • Jen A. McComb
    • 1
  • Giles E. St. J. Hardy
    • 1
  • Philip A. O’Brien
    • 1
  1. 1.Centre for Phytophthora Science and Management, School of Biological Sciences and BiotechnologyMurdoch UniversityMurdochAustralia
  2. 2.School of Plant Biology, Faculty of Natural and Agricultural SciencesThe University of Western AustraliaCrawleyAustralia
  3. 3.CSIRO Plant IndustryCentre for Environment and Life SciencesWembleyAustralia
  4. 4.The University of Western Australia Institute of AgriculturePerthAustralia
  5. 5.Department of Environment and conservationScience DivisionKensingtonAustralia

Personalised recommendations