This study was conducted to purify and identify metabolites of antimicrobial activity against phytopathogens from Xenorhabdus nematophila YL001. Three dipeptide compounds were purified from its cell-free cultural broth and identified as (±)-nematophin, cyclo (L-Pro-Gly), and N, N′-dimethyl-cyclo (L-Phe-L-Leu). Nematophin demonstrated a wider antifungal spectrum than the other two compounds. It also exhibited strong inhibitory effects on mycelial growth of Rhizoctonia solani and Phytophthora infestans with EC₅₀ values of 40.00 and 51.25 μg/ml, respectively. Its (S)-configuration structure [(+)-nematophin] was also synthesized and exhibited higher antimicrobial activity than the enantiomeric mixture. The detached leaf assay revealed that nematophin possessed significant preventive and curative efficacy against R. solani on broad bean leaves showing corresponding control efficacies of 93.01 and 94.93% at 1,000 μg/ml, comparable to those of a chemical fungicide (carbendazim) at 500 μg/ml. Additionally, the pot experiments indicated that nematophin could effectively inhibit the disease extension on rice and broad bean plants caused by R. solani. Nematophin also exerted some adverse influences on the sclerotial development of R. solani by dramatically suppressing their formation and maturation at 40.00 μg/ml, as well as their germination at 15.00 μg/ml. Morphological and ultrastructural observations showed that the hyphae of R. solani became twisted, shriveled, and deformed at the growing points after exposure to nematophin at 40.00 μg/ml, and that the subcellular fractions also became abnormal concurrently, especially the mitochondrial structure. These results indicate that nematophin has great potential to be used as a bio-pesticide in agricultural production.

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 (Xcn1), a major antimicrobial compound produced by Xenorhabdus nematophila , has great potential for use in agricultural productions. In this study, we evaluated the effects of CpxR, a global response regulator associated with the mutualism and pathogenesis of X . nematophila , on the antimicrobial activity and Xcn1 production. The mutation of cpxR could promote the production of Xcn1 significantly with its level in ΔcpxR mutant being 3.07 times higher than that in the wild type. Additionally, the expression levels of xcnA‐L genes, which are responsible for the production of Xcn1, were increased in ΔcpxR mutant while the expression levels of xcn MN , which are required for the conversion of Xcn1 into Xcn2 was reduced. Noticeably, Xcn2 was also enhanced on account of the conversion of excessive Xcn1 in spite of low expression levels of xcnM and xcnN in ΔcpxR mutant. The transcriptional levels of ompR and lrp , encoding the global response regulators OmpR and Lrp which negatively and positively regulate the production of Xcn1 were concurrently decreased and increased, respectively. Correspondingly, ΔcpxR mutant also exhibited increased antimicrobial activities in vitro and in vivo. Together, these findings suggest that CpxR negatively regulates xcnA‐L genes expression while positively regulating xcn MN expression in X . nematophila YL 001, which led to a high yield of Xcn1 in ΔcpxR mutant.

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.

The bacterial genus Streptomyces is an important source of antibiotics, and genome mining is a valuable tool to explore the potential of microbial biosynthesis in members of this genus. This study reports an actinomycete strain 135, which was isolated from Qinghai-Tibet Plateau in China and displayed broad antimicrobial activity. The fermentation broth of strain 135 displayed strong antifungal activity (>70%) against Sclerotinia sclerotiorum, Botrytis cinerea, Valsa mali, Phytophthora capsici, Glomerella cingulata, Magnaporthe grisea, Bipolaris maydis, Exserohilum turcicum in vitro, meanwhile possessed significant preventive and curative efficacy against S. sclerotiorum, Gaeumannomyces graminis, and P. capsici on rape leaves (54.04 and 74.18%), wheat (90.66 and 67.99%), and pepper plants (79.33 and 66.67%). X-14952B showed the greatest antifungal activity against S. sclerotiorum and Fusarium graminearum which the 50% inhibition concentration (EC₅₀) were up to 0.049 and 0.04 μg/mL, respectively. Characterization of strain 135 using a polyphasic approach revealed that the strain displayed typical features of the genus Streptomyces. 16S rRNA gene sequencing and phylogenetic analysis demonstrated that the isolate was most closely related to and formed a clade with Streptomyces huasconensis HST28^T (98.96% 16S rRNA gene sequence similarity). Average nucleotide identity (ANI) and DNA-DNA hybridization (DDH) values in strain 135 and related type strains were both below the threshold of species determination (91.39 and 56.5%, respectively). OrthoANI values between strain 135 and related type strains are under the cutoff of determining species (<95%). The biosynthetic gene cluster (BGC) designated to X-14952B biosynthesis was identified through genome mining and the possible biosynthesis process was deduced.

To avoid the widespread resistance of commercial fungicides, new broad-spectrum botanical fungicides need to be developed. In previous bioactive screening assays, extracts of Artemisia ordosica Krasch. (A. ordosica) had highly antifungal activities, but the responsible phytochemicals were unidentified. In this study, active compounds of A. ordosica extracts were identified using a bioassay-guided method, and antifungal assays were performed in vitro and in vivo. The bioactive compounds were dissolved in petroleum ether, and the best antifungal fraction contained four compounds: trans-dehydromatricaria ester (TDDE), 7, 4-demetylnringenin, capillarin, and stearic acid. Among them, TDDE exhibited the highest antifungal activity against six pathogenic fungi and five bacteria. It exhibited significant fungicidal activity against Thanatephorus cucumeris and Botrytis cinerea with EC₅₀ values of 0.464 μg/mL and 1.4 μg/mL, respectively. The living tissue bioassay results showed that the relative protection effects (RPE) of TDDE on tomato leaves, tomato fruit, and strawberry leaves infected with B. cinerea reached 76.78%, 86.2%, and 80.89%, respectively. In pot experiments, the RPE on tomato and strawberry plants infected with B. cinerea reached 84.11% and 96.37%, respectively. Morphological and physiological examination showed that TDDE had significant inhibitory effects on mycelial growth, including increased top offshoot, contorted hyphal tips, and extravasated cytochylema. Meanwhile, bactericidal activities of TDDE were significantly higher than kanamycin and streptomycin in five bacteria, and the plant tissue experiments further demonstrated that it had an 88.31% RPE on walnut leaves infected with Xanthomonas campestris pv. jugiandis, 72.18% RPE on potato infected with Erwinia carotovora subsp. carotovora, and 82.50% RPE on kiwifruit branches infected with Pseudomonas syringae pv. actinidiae. The active compounds isolated from A. ordosica in this study show great potential value for developing broad-spectrum fungicides, and also provide an important way to identify and isolate new bioactive products from medicinal plants.