A Novel LAMP Assay for the Detection of Phytophthora cinnamomi Utilizing a New Target Gene Identified from Genome Sequences

Abstract

Phytophthora cinnamomi is an ecologically and agriculturally significant plant pathogen. Early and accurate detection of P. cinnamomi is paramount to disease prevention and management. In this study, a loop-mediated isothermal amplification (LAMP) assay utilizing a new target gene Pcinn100006 identified from genomic sequence data was developed and evaluated for the detection of P. cinnamomi. This Pcinn100006 LAMP assay was found highly specific to P. cinnamomi. All 10 tested isolates of P. cinnamomi yielded positive results, whereas 50 isolates belonging to 16 other Phytophthora species, Globisporangium ultimum, and 14 fungal species lacked detection. This assay was 10 times more sensitive (100 pg in a 25-ml reaction mixture) than a conventional PCR assay (2 ng in a 50-ml reaction mixture) for detecting the genomic DNA of P. cinnamomi. In addition, it detected P. cinnamomi from artificially inoculated leaves of Cedrus deodara. Moreover, detection rates of P. cinnamomi using environmental DNAs extracted from 13 naturally infested rhizosphere samples were 100% in the Pcinn100006 LAMP assay versus 46% in the conventional PCR assay. Considering its higher accuracy and shorter time span, this Pcinn100006 LAMP assay is a promising diagnostic tool to replace conventional PCR-based and culture-dependent assays for screening of P. cinnamomi in regions at risk of infection or contamination.