Effects of ROS-Ca2+ signaling crosstalk on the biosynthesis of dendrobine‑type total alkaloids in Trichoderma longibrachiatum UN32 under cobalt stress

Trichoderma longibrachiatum UN32 produces dendrobine-type total alkaloids (DTTAs), and the addition of Co ²⁺ significantly increased the production of DTTAs by elevating reactive oxygen species (ROS). Unexpectedly, the Co ²⁺ supplementation also increased intracellular Calcium ions (Ca ²⁺ ) and Nitric oxide (NO) levels. Therefore, this study aimed to investigate the potential interactions among Co ²⁺ -induced changes in intracellular ROS, Ca ²⁺ , and NO levels in regulating DTTAs biosynthesis. Using Ca ²⁺ signaling inhibitors and NOS inhibitors under cobalt stress, it was confirmed that Co ²⁺ -induced Ca ²⁺ accumulation promoted DTTAs production. The increase in Ca ²⁺ is driven by a burst of ROS, whereas NO likely acts as a related stress-response signal without directly regulating DTTAs production. The addition of neomycin (Neo) and LaCl ₃ led to a decrease in Ca ²⁺ accumulation, which consequently reduced DTTAs production. Treatment with 6 mM Neo significantly reduced Ca ²⁺ accumulation by 59.86% (P < 0.01) and DTTAs production by 33.70% (P < 0.01). In addition, Neo supplementation under cobalt stress decreased the H ₂ O ₂ level by 25.50%. Subsequently time-course analysis of intracellular DTTAs, Ca ²⁺ and H ₂ O ₂ levels revealed that under normal fermentation conditions, ROS level fluctuated whereas Ca ²⁺ level remained relatively stable. Under cobalt stress, intracellular Ca ²⁺ and ROS exhibited reciprocal positive regulation. Results obtained with exogenous CaCl ₂ and H ₂ O ₂ suggest that excessive ROS and Ca ²⁺ trigger a protective negative-feedback loop that affects hyphal growth and DTTAs production. Overall, this study demonstrates that signal crosstalk between ROS and Ca ²⁺ under cobalt stress regulates the DTTAs biosynthesis, providing a theoretical basis for enhancing DTTAs production in Trichoderma longibrachiatum UN32 through signal regulation and fermentation optimization.