Objective To develop a PCR assay for rapid and accurate detection, epidemiology information, and integrated disease management on Phytophthora colocasiae, the pathogen of taro phytophthora blight.
Method A pair of species-specific primers, PCOF/PCOR, for P. colocasiae was designed based on the differences in Ras-related protein (Ypt1) gene sequence between P. colocasiae and other species in the same genus. The specificity, sensitivity and applicability of the primers were evaluated.
Result With the optimized reaction conditions and amplification, PCOF/PCOR amplified only a single band of 172 bp with genomic DNA extracted from all P.colocasiae strains, while the other tested pathogens had no corresponding band. The sensitivity of conventional PCR method using PCOF/PCOR as primers was 100 pg of genomic DNA in a 25 μL reaction solution. Whereas, the newly developed nested-PCR performed using Ypt1 gene universal primers ph1F/Yph2R for the first-round and PCOF/PCOR for the second-round increased 10 000-fold on the sensitivity to 10 fg. The nested-PCR methodology could positively detected P. colocasiae 100% in diseased leaves or 57.5% in symptom-free infected tissues.
Conclusion The newly established nested-PCR assay could be used for rapid, specific and sensitive detection of P. colocasiae.
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