Specific Detection of Phytophthora parasitica by Recombinase Polymerase Amplification Assays Based on a Unique Multicopy Genomic Sequence

Phytophthoraparasitica is a highly destructive oomycete plant pathogen that is capable of infecting a wide range of hosts including many agricultural cash crops, fruit trees, and ornamental garden plants. One of the most important diseases caused by P. parasitica worldwide is black shank of tobacco. Rapid, sensitive, and specific pathogen detection is crucial for early rapid diagnosis, which can facilitate effective disease management. In this study, we used a genomics approach to identify repeated sequences in the genome of P. parasitica by genome sequence alignment and identified a 203-bp P. parasitica-specific sequence, PpM34, that is present in 31 to 60 copies in the genome. The P. parasitica genome specificity of PpM34 was supported by PCR amplification of 24 genetically diverse strains of P. parasitica, 32 strains representing 12 other Phytophthora species, one Pythium species, six fungal species, and three bacterial species, all of which are plant pathogens. Our PCR and real-time PCR assays showed that the PpM34 sequence was highly sensitive in specifically detecting P. parasitica. Finally, we developed a PpM34-based high-efficiency recombinase polymerase amplification assay, which allowed us to specifically detect as little as 1 pg of P. parasitica total DNA from both pure cultures and infected Nicotiana benthamiana at 39 degrees C using a fluorometric thermal cycler. The sensitivity, specificity, convenience, and rapidity of this assay represent a major improvement for early diagnosis of P. parasitica infection.