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

被引：18
|
作者：
Eshraghi, Leila [1 ]
Aryamanesh, Nader [2 ]
Anderson, Jonathan P. [3 ,4 ]
Shearer, Bryan [1 ,5 ]
McComb, Jen A. [1 ]
Hardy, Giles E. St J. [1 ]
O'Brien, Philip A. [1 ]
机构：
[1] Murdoch Univ, Ctr Phytophthora Sci & Management, Sch Biol Sci & Biotechnol, Murdoch, WA 6150, Australia
[2] Univ Western Australia, Sch Plant Biol, Fac Nat & Agr Sci, Crawley, WA 6009, Australia
[3] CSIRO Plant Ind, Ctr Environm & Life Sci, Wembley, WA 6913, Australia
[4] Univ Western Australia, Inst Agr, Perth, WA 6009, Australia
[5] Dept Environm & Conservat, Div Sci, Kensington, WA 6983, Australia
来源：
EUROPEAN JOURNAL OF PLANT PATHOLOGY | 2011年 / 131卷 / 03期
关键词：
Arabidopsis thaliana; Internal control; Lupinus angustifolius; Non-host resistance; REAL-TIME PCR; ARABIDOPSIS-THALIANA; ALTERNARIA-BRASSICICOLA; DEFENSE RESPONSES; FUNGAL; BIOSECURITY; DIAGNOSTICS; EXPRESSION; RESISTANCE; INFECTION;
D O I：
10.1007/s10658-011-9819-x
中图分类号：
S3 [农学（农艺学）];
学科分类号：
0901 ;
摘要：
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.
引用
收藏
页码：419 / 430
页数：12
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