Abstract Berkeleyomyces rouxiae is a highly destructive soil borne fungal pathogen causing a pandemic black root rot (BRR) disease of cotton seedlings in New South Wales, Australia. Accurate identification and differentiation of B. rouxiae from its morphological twin, B. basicola was solely based on DNA sequence analyses. In this study, we for the first time developed a duplex PCR assay for simultaneous detection and identification of B. rouxiae and B. basicola without the need for sequencing. Based on nucleotide variation in the MCM7 fragment, a new primer was designed to pair with the one developed by Nakane and Usami (2020) to specifically amplify a 159 bp amplicon of B. basicola . Subsequently, a duplex PCR assay was developed to simultaneously detect the two pathogens at a limit of an approximately 0.05 ng/µL. The duplex PCR was sensitive enough to detect B. rouxiae from crude extracts prepared from a 5-min-microwave-based DNA extraction protocol. Additionally, when used together with a nested PCR, the duplex PCR assay successfully detected the BRR pathogen in DNA extracts obtained directly from a small amount (15–30 mg) of diseased cotton tissue. This duplex PCR provides valuable diagnostic tool for detecting and monitoring the prevalence and distribution of BRR pathogens in cotton in Australia.
Nguyen et al. (Fri,) studied this question.