Specific SCAR markers and multiplex real-time PCR for quantification of two Trichoderma biocontrol strains in environmental samples
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Several strains from the genus Trichoderma (Ascomycetes, Hypocreales) are commercially used as biocontrol agents, e.g. in formulations containing the two Trichoderma strains IMI206039 (Hypocrea parapilulifera B.S. Lu, Druzhinina & Samuels) and IMI206040 (T. atroviride P. Karst). To quantify the presence of the two isolates after application, we developed primers for SCAR markers (Sequence-Characterised Amplified Region). In order to quantify both fungal strains simultaneously, we also designed fluorophore-labelled probes distinguishing the two strains, to be used in combination with the SCAR primers. In incubations of two different soils, artificially inoculated and maintained under controlled conditions, the quantification through amplification with the SCAR markers in qPCR and through colony-forming units from plate counting correlated well. Further tests of the markers on samples taken from a golf green treated with a product containing both strains indicated that the two biocontrol strains did not establish, either on the golf green or in the surrounding area.
KeywordsAscomycetes Golf Hypocrea parapilulifera Hypocreales Trichoderma atroviride Trichoderma polysporum
The authors would like to thank Per Hansson, greenkeeper at the St. Arild golf course, for letting us taking golf green samples and Leticia Pizzul for supplying the Ultuna soil and giving practical suggestions for soil treatment in the laboratory. This work was largely funded by MISTRA (Foundation for Strategic Environmental Research), with additional support from BINAB Bio-Innovation AB.
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