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.

The fatty acid compound 2E, 4E-decadienoic acid (DDA) was obtained from coculture of Bacillus subtilis Tpb55 and Trichoderma asperellum HG1, which has a specific inhibitory effect on Phytophthora. The objective of this study is to evaluate the effects of DDA on tobacco black shank (caused by P. nicotianae) and on the microbial community of tobacco rhizosphere soil, and to provide a basis for the application of such natural products in the prevention and control of plant Phytophthora disease.

To evaluate the control efficacy of DDA against tobacco black shank, a pot experiment was performed, and the DNA copies of P. nicotianae in tobacco rhizosphere soil were detected by real-time fluorescence quantitative PCR technique. The bacterial, fungal and oomycetal communities in tobacco rhizosphere soil were analyzed through Illumina Hiseq high-throughput sequencing, and the correlation between soil nutrient factors and microbial community structure was assayed based on redundancy analysis and Mantel test.

Compared with the control treatment with 0.5% DMSO, the treatment with 1.25 g·L^-1 DDA for root irrigation showed 77.06% and 84.35% decrease for black shank disease index and P. nicotianae DNA copy number, respectively. DDA treatment also significantly increased the soil pH from 6.72 to 6.82, improved the soil conductivity by 102.11%, and decreased the soil organic matter by 13.05%. DDA treatment had no significant effect on the diversity and richness of fungi and bacteria in tobacco rhizosphere soil. However, the OTU level, Shannon index, Chao1 index, and ACE index of oomycetes significantly increased, while the Simpson index significantly decreased. The PCoA results showed that DDA treatment significantly affected the structure of tobacco rhizosphere oomycete community (r=0.667, P=0.028), but had no significant effect on fungi (r=0.259, P=0.305) and bacteria (r=0.593, P=0.098). The relative abundance of gram-positive bacterial genera such as Phycicoccus and Terrabacter significantly increased in DDA treatment, while the abundance of gram-negative bacterial genera including Devosia, Bordetella and Pseudoxanthomonas significantly decreased. Some beneficial fungi such as Aspergillus, Albifimbria, Arcopilus were enriched by DDA. For oomycetes, the relative abundance of Phytophthora was significantly decreased, but that of Globisporangium increased. The Mantel test results indicated that the changes in soil physicochemical properties caused by the application of DDA had no significant impact on the rhizosphere microbial community.

2E, 4E-decadienoic acid (DDA), as a novel natural fatty acid compound, can effectively prevent and control tobacco black shank, reduce the pathogen DNA copy number in tobacco rhizosphere soil, and specifically regulate the structure of oomycete community. It has the application potential in the prevention and control of crop oomycete diseases.