IJSRP, Volume 7, Issue 2, February 2017 Edition [ISSN 2250-3153]
Rajapaksha R.W.P.M, Vivehananthan K, Warshamana I.K
The majority of countries today follow stringent quarantine regulations to prevent the entry of pests into their countries especially with the seed stocks. Pectobacteriumcaratovorum is one of the significant bacterial pathogens of various horticultural crops around the world and a quarantine pathogen of seed potato in Sri Lanka. This research focused on detection of quarantine pathogens associated with imported seed potato consignments. Seed potato samples were collected from the intercepted consignments at the entry ports. Microbial DNA extraction protocol was optimized. Extracted DNA was sequenced using Ion Torrent Next Generation Sequencing technology by amplifying 400 bp V1-V2 region of 16S rRNA gene for the detection of bacterial pathogens. The recovered sequences were aligned with 16s rDNA in NCBI, Greengenes and SILVA databases for the detection of bacteria associated with the samples. Further, sub species level of the quarantine pathogen was identified by PCR amplification. The optimized DNA extraction protocol enabled faster isolation of microbial DNA from seed potato tubers and DNA was sufficient for PCR amplification and Next Generation Sequencing (NGS) analysis. Out of twenty four samples analysed by NGS, the quarantine pathogen Pectobacteriumcaratovorum was detected only in one sample. In addition, subspecies level was confirmed as Pectobacteriumcaratovorum subsp. caratovorum. As this pathogen is the causal organism of soft rot and black leg diseases of potato, detection of the pathogen in asymptomatic potato tubers indicate the risk of latent infections associated with the imported seed lots.