Development of conventional and real time pcr assay for the rapid detection and quantification of a biocontrol agent, Chaetomium globosum

Abstract

Chaetomium globosum is a potential biocontrol agent against various seed and soil-borne pathogens. To ensure proper use of C. globosum in agriculture, accurate data is essential for population monitoring. A PCR-based marker has been developed for detection of this biocontrol agent, which will help to detect the fungus at the place of its application. Out of twelve URP primers tested against 15 isolates of C. globosum and other Chaetomium species, URP 2R amplified a monomorphic band of 1,900 bp only in C. globosum isolates. This amplicon was cloned and sequenced, and based on the sequence obtained, four primer sets were designed, one of which in PCR assays amplified a region (SCAR; SCCgRA1900) of the expected size (1.9kb) in C. globosum isolates. The specific marker also detected the presence of C. globosum in soil, roots and leaves. The detection limit of marker in conventional PCR assay was 75 pg. The sensitivity and usefulness of SCAR marker was further enhanced by developing qPCR using the primer set SCCgQF/SCCgQR designed from SCCgRA1900, which detected as much as 1 pg of DNA (4.83×105 copy number of target DNA). The initial population of C. globosum in terms of target DNA in C. globosum-amended soil was equivalent to 2.5×108 copy number/g soil (0.51ng target DNA/g soil) which increased approximately 10 times after 15 days of application i.e., 2×109copy number/g soil (3.1ng/g soil). However, with, Bipolaris sorokiniana the quantity of C. globosum target DNA increased slowly reaching 4.32×108 copy number/g soil after 15 days. Conventional PCR-based detection using SCAR marker and subsequent qPCR provided a rapid and reliable tool for efficient detection and monitoring of C. globosum at the site of its application.