Extracellular Cystatin-like Protease Inhibitor ( EPIC1 ) Gene Based PCR Primers for Specific Detection of Phytophthora nicotianae Infecting Citrus

摘要

Background: In case of genus Phytophthora , species identification and detection is a difficult task and requires use of taxonomic keys and knowledge of host range of the pathogen. Internal Transcribed Spacer (ITS) or intergenic mitochondrial DNA spacers (mtDNA- IGS), Ypt1 gene, Lpv gene, SCAR/ RAPD markers are very much in use in PCR- based molecular detection of Phytophthora spp. Though these genes are effective in detecting Phytophthora spp., but they all have their own limitations. Hence new genes are to be explored to broaden the molecular diagnostic tool box for Phytophthora spp., detection in infected samples. Studies on host-pathogen interaction carried out in last decade showed that the Irish potato famine pathogen, Phytophthora infestans secretes effector proteins viz., cystatin-like protease inhibitors (EPICs) targeting host proteases during infection. But potential of EPIC gene in PCR-based diagnosis of plant pathogens in infected samples was not assessed by any of the research groups earlier. Therefore, potential of EPIC1 gene for detection of P. nicotianae from infected citrus samples was assessed in the study. Materials and Methods: The sequence of EPIC1 region of Phytophthora nicotianae was retrieved from whole genomic data available at Broad institute of MIT and Harvard, Cambridge, MA website and specific primers were designed for its PCR-based identification and diagnosis. Results: Out of 4 primer pairs, F EPIC1 F/F EPIC1 R was found best suitable pair for its use in PCR-based species specific detection system. These primers were tested successfully on P. nicotianae infected citrus leaf, stem and root tissues. No cross reactivity of primers were observed with six other Phytophthora spp., viz., P. palmivora , P. citrophthora , P. boehmeriae , P. lacustris , P. insolita , P. tropicalis and three other oomycete/ fungi viz., Pythium sp., Colletotrichum sp. and Alternaria sp. Detection by these primers was done effectively up to 100 pg μL^?1 of DNA isolated from pure culture of P. nicotianae. Conclusion: The EPIC1 PCR assay was found to be robust and reliable technique to detect P. nicotianae in infected citrus samples and thus would be useful in restricting P. nicotianae mediated destruction in citrus. Present investigation, also reports for the first time, isolation and annotation of EPIC1 gene from P. nicotianae pathogenic to citrus.