Trichoderma
species are known for their mycoparasitic activity against phytopathogenic fungi that cause significant economic losses in agriculture. During mycoparasitism,
Trichoderma
spp. recognize molecules produced by the host fungus and release secondary metabolites and hydrolytic enzymes to kill and degrade the host’s cell wall. Here, we explored the participation of the
Trichoderma atroviride
RNAi machinery in the interaction with six phytopathogenic fungi of economic importance. We determined that both Argonaute-3 and Dicer-2 play an essential role during mycoparasitism. Using an RNA-Seq approach, we identified that perception, detox, and cell wall degradation depend on the
T. atroviride
-RNAi when interacting with
Alternaria alternata, Rhizoctonia solani
AG2
,
and
R. solani
AG5. Furthermore, we constructed a gene co-expression network that provides evidence of two gene modules regulated by RNAi, which play crucial roles in essential processes during mycoparasitism. In addition, based on small RNA-seq, we conclude that siRNAs regulate amino acid and carbon metabolism and communication during the
Trichoderma
-host interaction. Interestingly, our data suggest that siRNAs might regulate allorecognition (
het
) and transport genes in a cross-species manner. Thus, these results reveal a fine-tuned regulation in
T. atroviride
dependent on siRNAs that is essential during the biocontrol of phytopathogenic fungi, showing a greater complexity of this process than previously established.
IMPORTANCE
There is an increasing need for plant disease control without chemical pesticides to avoid environmental pollution and resistance, and the health risks associated with the application of pesticides are increasing. Employing
Trichoderma
species in agriculture to control fungal diseases is an alternative plant protection strategy that overcomes these issues without utilizing chemical fungicides. Therefore, understanding the biocontrol mechanisms used by
Trichoderma
species to antagonize other fungi is critical. Although there has been extensive research about the mechanisms involved in the mycoparasitic capability of
Trichoderma
species, there are still unsolved questions related to how
Trichoderma
regulates recognition, attack, and defense mechanisms during interaction with a fungal host. In this work, we report that the Argonaute and Dicer components of the RNAi machinery and the small RNAs they process are essential for gene regulation during mycoparasitism by
Trichoderma atroviride
.