The objective of this study is to clarify the inhibitory effect of 2-octyl-3-isothiazolinone, a metabolite from the co-culture of Trichoderma asperellum HG1 and Bacillus subtilis Tpb55, on Alternaria alternata (the pathogen causing tobacco brown spot) and its impact on the physiological metabolism of the pathogen, and to provide a theoretical basis for the application of this natural product in the management of plant fungal diseases.

The antifungal activities of eight secondary metabolites from the co-culture against A. alternata were assessed using the mycelial growth rate method. The toxicity of 2-octyl-3-isothiazolinone was evaluated, and its EC₅₀ value was calculated. Field trials were conducted to determine its control efficacy against tobacco brown spot. Transcriptome sequencing was employed to analyze the gene expression profiles of A. alternata following treatment with 2-octyl-3-isothiazolinone. The impact on energy metabolism was assessed by measuring ATP content and ATPase activity. Propidium iodide (PI) staining and reactive oxygen species (ROS) detection were used to evaluate cell membrane integrity and oxidative stress levels, respectively.

At a concentration of 10 μg·mL^-1, 2-octyl-3-isothiazolinone exhibited 100% inhibition against A. alternata, which was significantly superior to that of the positive control dimethachlon (27.60%). In the field experiment, the control efficacies at concentrations of 100 and 200 μg·mL^-1 were 80.80% and 84.33%, respectively, which were equivalent to that of the positive control (40% dimethachlon diluted 400-fold, 80.15%), and there was no toxic effect on tobacco itself. Further studies showed that 2-octyl-3-isothiazolinone exerted its antifungal effect through a dual mechanism: on the one hand, it significantly inhibited the key enzymes of glycolysis and the tricarboxylic acid cycle, blocking the energy metabolism of the pathogen; on the other hand, the expression levels of genes related to ROS scavenging in the pathogen (AALT_g9032, AALT_g9033) were significantly down-regulated, eventually leading to the massive accumulation of ROS, causing oxidative damage and destroying the integrity and biological function of the cell membrane. Further verification demonstrated that 2-octyl-3-isothiazolinone significantly reduced ATP content and ATPase activity, confirming disruption of energy metabolism. PI staining revealed concentration-dependent damage to cell membrane integrity, while DCFH-DA fluorescence staining indicated substantial intracellular ROS accumulation, triggering oxidative stress and exacerbating cellular damage. These results were consistent with the transcriptomic findings.

As a green pesticide with broad application prospects, 2-octyl-3-isothiazolinone interferes with the normal life activities of A. alternata through the synergistic effect of energy blockage-oxidative damage. This study provides a theoretical basis and application support for its use as a new antifungal agent to control tobacco brown spot.