Yunfei Han

Xenorhabdus nematophila has excellent potential for application in both medicine and agriculture due to its various active secondary metabolites. The transcriptional regulator OmpR negatively regulates Xenocoumacin 1 (Xcn1), which has wide antimicrobial activity. Here, we expressed and purified OmpR and verified its binding activities to promoters via an electrophoretic mobility shift assay. RNA sequencing was used to analyze the relevance and difference of differentially expressed genes between X. nematophila and its mutant ΔompR. Compared with the WT, 1127 differentially expressed genes were found in ΔompR, while 4150 co-expressed genes were detected. RT-qPCR data validated the RNA-seq results with 20 randomly selected genes. OmpR positively regulates the process of porphyrin metabolism, quorum sensing, β-Lactam resistance and glyoxylate and dicarboxylate metabolism, while negatively regulating the phosphotransferase system, two-component system and bacterial chemotaxis. OmpR indirectly regulates the biosynthesis of Xcn1 by positively regulating the process of glyoxylate metabolism, which consumes energy and precursors, and negatively regulates biomacromolecules biosynthesis, which provides energy and precursors. Overall, this work revealed the indirect effects of OmpR on the biosynthesis of Xcn1, serving as a foundation for future research into the intricate regulatory network of X. nematophila.

Cucumber wilt, caused by Fusarium oxysporum f. sp. cucumerinum (FOC), is a soilborne disease that poses a significant threat to cucumber production, resulting in substantial yield losses. This study aimed to evaluate the biocontrol and growth-promoting effects of Bacillus velezensis, a highly active bacterial strain. In vitro assays revealed that B. velezensis F9 exhibited broad-spectrum antifungal activity against eight plant pathogenic fungi, with inhibition ratio ranging from 62.66% to 88.18%. Additionally, the strain displayed the ability to produce IAA (5.97 ± 1.75 µg/mL), fix nitrogen, produce siderophores, and form biofilms. In vitro growth promotion assays demonstrated that different concentrations of B. velezensis F9 significantly promoted cucumber seedling growth. Furthermore, two pot experiments revealed that the strain exhibited biocontrol efficacy against cucumber wilt, with disease control rates ranging from 42.86% to 67.78%. Notably, the strain significantly increased the plant height, fresh weight, and dry weight, with increases ranging from 20.67% to 60.04%, 40.27% to 75.51%, and 22.07% to 52.54%, respectively. Two field trials confirmed the efficacy of B. velezensis F9 in controlling cucumber wilt, with disease control rates of 44.95% and 33.99%, respectively. The strain effectively alleviated the dwarfing and wilting symptoms caused by the pathogen. Compared with the FOC treatment, the F9 + FOC treatment significantly increased the plant height, fresh weight, and dry weight, with increases of 43.85% and 56.28%, 49.49% and 23.70%, and 36.25% and 73.63%, respectively. Enzyme activity assays indicated that inoculation significantly increased SOD activity in cucumber leaves and neutral phosphatase, sucrase, and urease activity in rhizosphere soil. Correlation analysis revealed a negative correlation between the disease index and plant height, fresh weight, dry weight, and peroxidase activity, with correlation coefficients of −0.53, −0.60, −0.38, and −0.45, respectively. These findings suggest that plant height, fresh weight, and dry weight are significantly negatively correlated with the cucumber disease index, highlighting their importance as indicators for evaluating the biocontrol efficacy of B. velezensis F9. In conclusion, B. velezensis F9 is a highly effective plant growth-promoting rhizobacterium with excellent biocontrol potential, showcasing promising applications in agricultural production.

Xenocoumacin 1 (Xcn 1), antibiotic discovered from secondary metabolites of Xenorhabdus nematophila, had the potential to develop into a new pesticide due to its excellent activity against bacteria, oomycetes and fungi. However, the current low yield of Xcn1 limits its development and utilization. To improve the yield of Xcn1, response surface methodology was used to determine the optimal composition of fermentation medium and one factor at a time approach was utilized to optimize the fermentation process. The optimal medium composed of in g/L: proteose peptone 20.8; maltose 12.74; K₂HPO₄ 3.77. The optimal fermentation conditions were that 25 °C, initial pH 7.0, inoculum size 10%, culture medium 75 mL in a 250 mL shake flask with an agitation rate of 150 rpm for 48 h. Xenorhabdus nematophila YL001 was produced the highest Xcn1 yield (173.99 mg/L) when arginine was added to the broth with 3 mmol/L at the 12th h. Compared with Tryptic Soy Broth medium, the optimized fermentation process resulted in a 243.38% increase in Xcn1 production. The obtained results confirmed that optimizing fermentation technology led to an increase in Xcn1 yield. This work would be helpful for efficient Xcn1 production and lay a foundation for its industrial production.

Sclerotinia sclerotiorum (Lib.) de Bary, a polyphagous necrotrophic fungal pathogen, has brought about significant losses in agriculture and floriculture. Until now, the most common method for controlling S. sclerotiorum has been the application of fungicides. Xenocoumacin 1 (Xcn1) is a potential biopesticide having versatile antimicrobial activities, generated by Xenorhabdus nematophila. This study was intended to isolate Xcn1 from X. nematophila YL001 and clarify its efficacies for S. sclerotiorum control. Xcn1 demonstrated a wider antifungal spectrum against 10 plant-pathogenic fungi. It also exhibited a strong inhibitory effect on the mycelial growth of S. sclerotiorum with an EC₅₀ value of 3.00 μg/mL. Pot experiments indicated that Xcn1 effectively inhibited disease extension on oilseed rape and broad bean plants caused by S. sclerotiorum. Morphological and ultrastructural observations revealed that the hyphae of S. sclerotiorum became twisted, shriveled, and deformed at the growing points after treatment with Xcn1 at 3.00 μg/mL and that the subcellular fractions also became abnormal concurrently, especially the mitochondrial structure. Moreover, Xcn1 also increased cell membrane permeability and decreased the content of exopolysaccharide as well as suppressing the activities of polygalacturonase and cellulase of S. sclerotiorum, but exerted no effects on oxalic acid production. This study demonstrated that Xcn1 has great potential to be developed as a new biopesticide for the control of S. sclerotiorum.