Rapid Detection of Phytophthora nicotianae in Infected Tobacco Tissues and Soil Samples Based on Its Ypt1 Gene

摘要

This paper describes the development of a polymerase chain reaction (PCR) assay for the detection of Phytophthora nicotianae, the causal agent of Phytophthora blight of tobacco and other plants. The PCR primers were designed based on a Ras-related protein (Ypt1) gene, and 115 isolates representing 26 species of Phytophthora and 29 fungal species of plant pathogens were used to test the specificity of the primers. PCR amplification with species-specific (Pn) primers resulted in a product of 389 bp only from isolates of P. nicotianae. The detection sensitivity with Pn primers was 1 ng of genomic DNA. Using Ypt1F/Ypt1R as first-round amplification primers, followed by a second round using the primer pair Pn1/Pn2, a nested PCR procedure was developed, which increased the detection sensitivity 100-fold to 10 pg. PCR with the Pn primers could also be used to detect P. nicotianae from naturally infected tobacco tissues and soil. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.