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

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.

KEYWORDS:

• GBS
• detection
• enrichment
• Asperello
• Hypocreaceae

Acknowledgements

The authors thank Fran Focquet, Xian Notredame, Zoë Madder, and Kristof Maenhout for technical assistance. We thank Dr. Maria Isabel Trillas Gay (University of Barcelona) for her encouragement of this research, for a critical review of the manuscript and for providing T. asperellum isolates 247, 655, 875, 2094, 2170, 2206, 2212, 2724, 3540, 3911, 4190 and 4288 via Dr. I. Druzhinina (Vienna University of Technology). We would also like to thank Riccardo Liguori of Gowan Company for providing Trichoderma asperellum strain ICC012. We thank Miriam Levenson for English-language editing.

Disclosure statement

Authors KVP, AH, KH declare they have no conflict of interest (financial or non-financial). Authors SCF and EC were sponsored by private companies, one of which co-sponsored the research (see author affiliations and declaration regarding sources of funding). However, both SCF and EC declare that publication of the manuscript does not entail a conflict of interest in the form of a financial interest given that a negligible commercial benefit is expected from the publication. The tool described in the manuscript is intended for research purposes, to help identify cropping systems and environmental conditions where colonization by the biocontrol agent is suboptimal. Authors SCF and EC declare they have no relevant non-financial interests.

Data availability statement

The data that support the findings of this study are available from the corresponding author (KH), upon reasonable request.

Additional information

Funding

KVP, AH, and KH were funded by the Flemish Government, Belgium. SCF was funded by Biobest Group, Belgium. EC was funded by Biocontrol Technologies, Spain. Materials, GBS sequencing and technical assistance was partially sponsored by Biobest and partially by the Flemish Government.