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.

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DNA and RNA detected in soil using molecular techniques may originate from a living or dead organism. It is therefore of interest to know how long the DNA and RNA from a decaying organism can persist in soil, and how environmental conditions such as soil temperature, moisture, and microbial populations impact on the survival time. This study determined the difference between the persistence of Phytophthora cinnamomi mRNA and DNA in different soil types. DNA and RNA were extracted from P. cinnamomi and 10 ng/250 mg of soil was applied to five different soil types that were either air‐dried or maintained at 70% field capacity. The persistence of DNA at 20°C was tested after intervals of 0, 3, 7, 14, 90, 241, and 378 days, and for RNA at 0, 1, 3, and 7 days using qPCR and RT‐qPCR techniques, respectively. Persistence was longer in dry than moist soil, P. cinnamomi DNA could be readily detected in dry soil conditions for up to 90 days and was found at extremely low levels at 241 and 378 days. RNA was detected only on day 1, except for dry river sand, and moist sandy loam in which it persisted for 3 days; it was not detected after seven days. These results confirm that RNA degrades very quickly, making it a valuable tool for determining the presence of viable Phytophthora in soil. In contrast, DNA can be remarkably stable in some environments, and positive results could be obtained even after the death of the organism for a year or more prior to the test. For diagnostics, the use of an RNA‐based test avoids the possibility of such false positive results. In the context of the research project, this study is relevant to determining how long viable Phytophthora remains in soil after the eradication protocols have been instigated. In a broader context, the persistence of DNA is relevant to any study using environmental DNA for diagnostics or for metabarcoding when undertaking community ecology or microbiome studies. These results are relevant for studies using detection of P. cinnamomi nucleic acids in soils for purposes of diagnostics, ecological research, or projects on eradication.