A simple method of DNA isolation from jute (Corchorus olitorius) seed suitable for PCR-based detection of the pathogen Macrophomina phaseolina (Tassi) Goid

Abstract

A simple method was developed for isolating DNA from jute seed, which contains high amounts of mucilage and secondary metabolites, and a PCR protocol was standardized for detecting the seedborne pathogen Macrophomina phaseolina. The cetyl trimethyl ammonium bromide method was modified with increased salt concentration and a simple sodium acetate treatment to extract genomic as well as fungal DNA directly from infected jute seed. The Miniprep was evaluated along with five other methods of DNA isolation in terms of yield and quality of DNA and number of PCR positive samples. The Miniprep consistently recovered high amounts of DNA with good spectral qualities at A260/A280. The DNA isolated from jute seed was found suitable for PCR amplification. Macrophomina phaseolina could be detected by PCR from artificially inoculated as well as naturally infected jute seeds. The limit of PCRbased detection of M. phaseolina in jute seed was determined to be 0.62 9 10-7 CFU g -1 seed.