Development and evaluation of specific PCR and LAMP assays for the rapid detection of Phytophthora melonis
- 865 Downloads
Phytophthora melonis is a widespread and devastating pathogen for the Cucurbitaceae family. Early and accurate detection of P. melonis is essential to control the disease in the field. To establish a simple, visual, and rapid detection system for P. melonis, we developed nested polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) systems based on the Ras-related protein (Ypt1) gene. All 36 isolates of P. melonis, from geographically distinct counties in China, yielded positive detection results on LAMP or nested PCR assays. No cross reaction was observed with other oomycetes or fungal pathogens. A sensitivity assay showed that both methods had a detection limit of 10 fg genomic DNA. We also detected P. melonis in diseased cucumber tissues and soils, and evaluated positive detection rates using LAMP, nested PCR, and conventional isolation methods. The results suggest that the LAMP assay has the greatest potential for active detection of P. melonis in regions that are at risk of contracting the disease, and for use in resource-poor settings.
KeywordsLoop-mediated isothermal amplification (LAMP) Molecular detection Phytophthora melonis Ras-related protein (Ypt1) gene
This work was supported by grants from the Natural Science Foundation for Distinguished Young Scholars of Fujian Province (2011J06010), Doctoral Foundation of FAAS (2012DBS-2), and Special Fund for Agro-scientific Research in the Public Interest (201303018; 200903034).
- Ho, H., Gallegly, M., & Hong, C. (2007). Redescription of Phytophthora melonis. Mycotaxon, 102, 339–345.Google Scholar
- Katsura, K. (1976). Two new species of Phytophthora causing damping-off of cucumber and trunk rot of chestnut. Transactions of the Mycological Society of Japan, 17(3/4), 238–242.Google Scholar
- Mao, Z., Qiu, Y., Zheng, L., Chen, J., & Yang, J. (2012). Development of a visual loop-mediated isothermal amplification method for rapid detection of the bacterial pathogen Pseudomonas putida of the large yellow croaker (Pseudosciaena croacea). Journal of Microbiological Methods, 89, 179–184.PubMedCrossRefGoogle Scholar
- Schena, L., Duncan, J., & Cooke, D. (2008). Development and application of a PCR-based ‘molecular tool box’ for the identification of Phytophthora species damaging forests and natural ecosystems. Plant Pathology, 57, 64–75.Google Scholar
- Skottman, T., Piiparinen, H., Hyytiäinen, H., Myllys, V., Skurnik, M., & Nikkari, S. (2007). Simultaneous real-time PCR detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis. European Journal of Clinical Microbiology and Infectious Diseases, 26, 207–211.PubMedCrossRefGoogle Scholar
- Yamazaki, W., Kumeda, Y., Misawa, N., Nakaguchi, Y., & Nishibuchi, M. (2010). Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of the tdh and trh genes of Vibrio parahaemolyticus and related Vibrio species. Applied and Environmental Microbiology, 76, 820–828.PubMedCrossRefGoogle Scholar