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

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.

Development of a Nested Quantitative Real-Time PCR for DetectingPhytophthora cinnamomiinPersea americanaRootstocks

“…This assay was highly sensitive, with detection limits as low as 20 fg of P. cinnamomi DNA. This is an improvement in sensitivity vs other DNA based detection methods for P. cinnamomi where 100 fg could be detected (15 as a normalization parameter as has been shown in various studies (3,4).…”

“…cinnamomi (4). Although conventional PCR has been useful for numerous Phytophthora species, it has not been successful when low levels of target DNA as with latent infections, are encountered.…”

Induction of defence responses by cinnamomins against Phytophthora cinnamomi in Quercus suber and Quercus ilex subs. rotundifolia

“…This assay, developed by Eshraghi et al (2011), was applied first to establish a temporal colonization profile of cork oak roots which showed a highly significant colonization rate between 18 and 24 hpi. For reasons of availability, we used as internal control a plasmid containing the myelocytomatosis oncogene (Myc) of mouse, instead of the mouse ScFvB1sequence used by the authors.…”

“…Phytophthora cinnamomi primers (forward 5′-GCTAGCAAGCACGTATGAGG-3′ and reverse 5′-CGCCCCAACTATACGACAAC-3′) were employed for the amplification of a PDN gene (FJ493007) fragment with an amplicon size of 90 bp (Eshraghi et al 2011). PCR reactions were carried out using 20 ng of gDNA as template in a total volume of 50 μl containing 5 μl 10× Dream Taq Buffer, 20 mM MgCl2,5 μl dNTPs (dATP, dCTP, dGTP and dTTP, 2 mM each), 10 mM of primers and 1.25 U of DreamTaqTM DNA polymerase (Fermentas).…”

“…A quantitative real-time PCR (QPCR) assay following a method described by Eshraghi et al (2011) was performed to study the effect of α-CIN and β-CIN on the colonization of Q. suber roots by P. cinnamomi. We assume that the proportion of pathogen and host biomass present at any given time during an infection is equivalent to the proportion of pathogen and plant DNA.…”