Molecular detection of Phytophthora pluvialis, the causal agent of red needle cast in P. radiata.

Abstract

BACKGROUND\nPhytophthora pluvialis was first described in 2013 and is the causal agent of red needle cast (RNC) in P. radiata as well as infection in Douglas fir (Pseudotsuga menziesii). A species-specific PCR is necessary for detection of this pathogen and diagnosis of RNC.\n\n\nOBJECTIVE\nTo design and validate a species-specific molecular assay for P. pluvialis using isolates from infected pine needles.\n\n\nMETHODS\nSpecies-specific PCR primers were generated from the ras-related GTP-binding protein 1 gene (Ypt1) gene sequence, concentrating on DNA regions unique to P. pluvialis, and real-time and quantitative polymerase chain reaction (qPCR) were used to detect P. pluvialis from both artificially inoculated and naturally infected samples.\n\n\nRESULTS\nThe species-specific PCR assay was generated following P. pluvialis DNA sequence analysis. In vitro tests of the specificity of the probe-based, quantitative, polymerase chain reaction (qPCR) assay showed that no amplification was observed with other Phytophthora species including other closely-related clade 3 species, or with fungal species associated with pine or with pine DNA. The limit of detection of the qPCR assay was 2\u202fpg/μl. When the qPCR assay was used to detect P. pluvialis in artificially-inoculated and naturally infected P. radiata needles, a PCR product was detected in all inoculated samples; the mean concentration ranges of P. pluvialis DNA in the inoculated and naturally infected samples tested were 5.9-124.5\u202fpg/μl and 8.1-340.2\u202fpg/μl, respectively. The assays described herein were used with serological diagnostic strips, providing the ability to identify to species level.\n\n\nCONCLUSIONS\nThe assay described herein detects P. pluvialis with high specificity and sensitivity from a range of DNA samples, including those extracted from infected plant material and serological diagnostic strips. The ability to detect and identify P. pluvialis, from infected tissues directly, provides value and practicality to diagnostics, biosecurity and research.