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Trichoderma is a biological control agent used to improve the resistance to diseases, which may also estimulate plant growth. Commercial products with Trichoderma are available in different countries, but most of them are based on conidial suspension. This study aimed at evaluating the efficiency of native Trichoderma populations collected from different production systems and applied to the soil by using two methods: conidial suspension and inoculated oat grains. The efficiency of native Trichoderma populations collected from conventional crop and agropastoral and agrosilvopastoral systems in a long-term field experiment was evaluated. The populations efficiencies were evaluated by in vivo tests that assessed the control of soybean damping-off caused by Rhizoctonia solani, plant height and soil colonization with the antagonist. In vitro tests, such as dual culture and assessment of volatile and non-volatile compounds, were conducted to study the mode of action of the populations. Some native Trichoderma populations were as efficient as those from a commercial product in all tests. Compared to conidial suspension, Trichoderma spp. inoculated through oat grains promoted a greater damping-off control, higher plants and more colony-forming units per gram of soil after 3 months of application. Native populations performed equally well or even better than the commercial strain, and the use of a substrate that supports the Trichoderma growth was more efficient than the conidial suspension method.