Journal article
A qPCR assay for the detection of phytophthora cinnamomi including an mRNA protocol designed to establish propagule viability in environmental samples
Plant Disease, Vol.103(9), pp.2443-2450
2019
Abstract
Phytophthora cinnamomi causes root and collar rot in many plant species in natural ecosystems and horticulture. A species-specific primer and probe PCIN5 were designed based on a mitochondrial locus encoding subunit 2 of cytochrome c oxidase (cox2). Eight PCR primers, including three forward and five reverse, were designed and tested in all possible combinations. Annealing temperatures were optimized for each primer pair set to maximize both specificity and sensitivity. Each set was tested against P. cinnamomi and two closely related clade 7 species, P. parvispora and P. niederhauseri. From these tests, five primer pairs were selected based on specificity and, with a species-specific P. cinnamomi probe, used to develop quantitative real-time PCR (qPCR) assays. The specificity of the two most sensitive qPCR assays was confirmed using the genomic DNA of 29 Phytophthora isolates, including 17 isolates of 11 species from clade 7, and representative species from nine other clades (all except clade 3). The assay was able to detect as little as 150 ag of P. cinnamomi DNA and showed no cross-reaction with other Phytophthora species, except for P. parvispora, a very closely related species to P. cinnamomi, which showed late amplification at high DNA concentrations. The efficiency of the qPCR protocol was evaluated with environmental samples including roots and associated soil from plants artificially infected with P. cinnamomi. Different RNA isolation kits were tested and evaluated for their performance in the isolation of RNA from environmental samples, followed by cDNA synthesis, and qPCR assay. Finally, a protocol was recommended for determining the presence of P. cinnamomi in recalcitrant environmental samples.
Details
- Title
- A qPCR assay for the detection of phytophthora cinnamomi including an mRNA protocol designed to establish propagule viability in environmental samples
- Authors/Creators
- M.B. Kunadiya (Author/Creator) - Murdoch UniversityW.D. Dunstan (Author/Creator) - Murdoch UniversityD. White (Author/Creator) - Murdoch UniversityG.E.St.J. Hardy (Author/Creator) - Murdoch UniversityA.H. Grigg (Author/Creator) - Alcoa (Australia)T.I. Burgess (Author/Creator) - Murdoch University
- Publication Details
- Plant Disease, Vol.103(9), pp.2443-2450
- Publisher
- The American Phytopathological Society
- Identifiers
- 991005540569407891
- Copyright
- © 2019 The American Phytopathological Society.
- Murdoch Affiliation
- Centre for Phytophthora Science and Management; School of Veterinary and Life Sciences
- Language
- English
- Resource Type
- Journal article
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95 Record Views
InCites Highlights
These are selected metrics from InCites Benchmarking & Analytics tool, related to this output
- Collaboration types
- Industry collaboration
- Domestic collaboration
- Citation topics
- 3 Agriculture, Environment & Ecology
- 3.97 Plant Pathology
- 3.97.1439 Phytophthora Management
- Web Of Science research areas
- Plant Sciences
- ESI research areas
- Plant & Animal Science