Strain-specific and sensitive monitoring of the biocontrol agent Trichoderma asperellum T34 in growing medium via real-time PCR

Abstract

ABSTRACT Trichoderma asperellum Samuels, Lieckf. & Nirenberg is one of several Trichoderma species comprising effective biocontrol strains. One of these, Trichoderma asperellum T34, shows activity against soilborne pathogens such as Rhizoctonia, Fusarium, and Pythium. The activity of biocontrol strains such as T34 depends on their establishment in the rhizosphere. Monitoring of the population of T34 and other strains in the rhizosphere and the growing medium is therefore important for evaluating their potential in different crops and different growing systems. Until now, strain-specific monitoring has been very difficult as the main quantification methods, i.e. traditional dilution plating or standard molecular detection methods, are specific only at genus or species level, respectively. Genotyping-by-sequencing was used to identify genomic loci specific to T34. These were used to develop strain-specific Taqman real-time (qPCR) assays. After testing for specificity and sensitivity, two assays were combined in duplex format. The duplicate assay provided an internal control to avoid a false positive signal in case one of the target loci would be present in an as yet unidentified non-target organism. An optional enrichment step in semi-selective medium was included for increased sensitivity in cases of small T34 population size, theoretically allowing detection down to 0.1 spore equivalent per mL growing medium. The techniques were applied to 200 mL samples of growing medium analyzed 0, 19, and 112 days after spiking with different numbers and combinations of spores from target and non-target Trichoderma isolates. This technique shows potential for developing other strain-specific detection assays using an affordable genotyping tool.