Development and validation of a species-specific marker for Trichoderma atroviride

Abstract

Biocontrol agents can use a range of strategies to control pathogens, such as plant growth promotion, induced resistance, mycoparasitism, antibiosis and competition. The mechanisms of biocontrol used by any particular agent vary depending on the conditions they experience. In recent years, it has become more apparent how complex these mechanisms are in respect to a combination of abiotic and biotic factors such as soil type, pH, temperature, host plant and pathogen. In order to study biocontrol activity by Trichoderma atroviride in a natural environment, it is important to distinguish it from other Trichoderma species present and to track and quantify the strains in the environment. Here we present the development and validation of a species-specific molecular marker for T. atroviride. The Trichoderma endochitinase-42 encoding gene chit42 is a single-copy gene and was selected as it has only low sequence homology to chitinases in other fungi. We looked for sequence variations in chit42 that distinguished T. atroviride from other Trichoderma species. Genomic sequences of chit42 from 66 Trichoderma isolates, representing 9 species, were compared to identify T. atroviride-specific polymorphisms. Based on these sequence polymorphisms, primers were designed that only amplify T. atroviride. A species-specific PCR amplification of chit42 using these primers was optimised to find the most stringent PCR conditions. Using these primers in real-time PCR, standard curves were generated to quantify the amount of T. atroviride chit42 DNA in natural non-sterilised soil inoculated with spores or mycelial tissue. This species-specific marker will allow us to study T. atroviride’s mode of action in more detail in pot and field trials and natural ecosystems.