Diagnostic assay for molecular detection of Bipolaris maydis and Stenocarpella maydis for safe exchange and long-term conservation of maize germplasm

Abstract

Maize is a versatile multi-purpose crop used as a feed and food crop and is one of the most extensively grown and traded crops globally. The maize cultivation is greatly influenced by several seed-borne fungal pathogens including Bipolaris maydis and Stenocarpella maydis. Keeping the potential risks associated with movement of seeds, a conventional and qPCR based reliable and sensitive method for detection of B. maydis and S. maydis was developed. Four sets of specific primers derived from kilbournase gene were developed for detection and quantification of S. maydis, whereas, six sets of specific primers were developed from internal transcribed spacer (ITS) region for detection of B. maydis. The specificity and sensitivity of the primers developed for both the fungal pathogens were tested and the primers were found specific to the target pathogens and are able to detect up to 0.1 pg of genomic DNA. A duplex PCR assay for simultaneous detection of these fungal pathogens was successfully established using highly specific and sensitive primer sets, BmayITS1F&5R specific to B. maydis and DMKB4F&4R specific to S. maydis. The primers produced specific size of amplicons in duplex as well as individually for respective target pathogens. The developed protocol in the present study could be utilized effectively for detection and quantification of these pathogens to facilitate the quick quarantine processing, monitor seed health and seed certification thereby preventing infected maize from entering into food and feed chain and also to facilitate their long-term safe conservation.