Specific and Sensitive Detection of Phytophthora nicotianae by Nested PCR and Loop-mediated Isothermal Amplification Assays

Phytophthora nicotianae is an important soilborne plant pathogen. It causes black shank in tobacco and other commercially important crop diseases. Early and accurate detection of P.nicotianae is essential for controlling these diseases. In this study, primers based on the Ras-related protein gene (Ypt1) of P.nicotianae were tested for their specific detection of the pathogen using nested PCR and LAMP assays. For specificity testing, DNA extracts from 47 P.nicotianae isolates, 45isolates of 16 different oomycetes and 25 isolates of other fungal species were used; no cross-reaction with other pathogens was observed. The sensitivity assay showed that the nested PCR and LAMP assays had detection limits of 100fg and 10fg genomic DNA per 25-l reaction, respectively. Furthermore, the nested PCR and LAMP assays were used for the detection of DNA from naturally P.nicotianae-infected tobacco tissues and soil. Our results suggest that the LAMP assay has the greatest potential for the specific detection of P.nicotianae in regions that are at risk of contracting tobacco black shank disease and that the Ypt1 gene is a novel and effective target of P.nicotianae LAMP visual detection.