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.

In agriculture, soil-borne fungal pathogens, especially Fusarium oxysporum strains, are posing a serious threat to efforts to achieve global food security. In the search for safer agrochemicals, silica nanoparticles (SiO₂NPs) have recently been proposed as a new tool to alleviate pathogen damage including Fusarium wilt. Hollow mesoporous silica nanoparticles (HMSNs), a unique class of SiO₂NPs, have been widely accepted as desirable carriers for pesticides. However, their roles in enhancing disease resistance in plants and the specific mechanism remain unknown. In this study, three sizes of HMSNs (19, 96, and 406 nm as HMSNs-19, HMSNs-96, and HMSNs-406, respectively) were synthesized and characterized to determine their effects on seed germination, seedling growth, and Fusarium oxysporum f. sp. phaseoli (FOP) suppression. The three HMSNs exhibited no side effects on cowpea seed germination and seedling growth at concentrations ranging from 100 to 1500 mg/L. The inhibitory effects of the three HMSNs on FOP mycelial growth were very weak, showing inhibition ratios of less than 20% even at 2000 mg/L. Foliar application of HMSNs, however, was demonstrated to reduce the FOP severity in cowpea roots in a size- and concentration-dependent manner. The three HMSNs at a low concentration of 100 mg/L, as well as HMSNs-19 at a high concentration of 1000 mg/L, were observed to have little effect on alleviating the disease incidence. HMSNs-406 were most effective at a concentration of 1000 mg/L, showing an up to 40.00% decline in the disease severity with significant growth-promoting effects on cowpea plants. Moreover, foliar application of HMSNs-406 (1000 mg/L) increased the salicylic acid (SA) content in cowpea roots by 4.3-fold, as well as the expression levels of SA marker genes of PR-1 (by 1.97-fold) and PR-5 (by 9.38-fold), and its receptor gene of NPR-1 (by 1.62-fold), as compared with the FOP infected control plants. Meanwhile, another resistance-related gene of PAL was also upregulated by 8.54-fold. Three defense-responsive enzymes of POD, PAL, and PPO were also involved in the HMSNs-enhanced disease resistance in cowpea roots, with varying degrees of reduction in activity. These results provide substantial evidence that HMSNs exert their Fusarium wilt suppression in cowpea plants by activating SA-dependent SAR (systemic acquired resistance) responses rather than directly suppressing FOP growth. Overall, for the first time, our results indicate a new role of HMSNs as a potent resistance inducer to serve as a low-cost, highly efficient, safe and sustainable alternative for plant disease protection.

Extensive efforts have been made to discover new biofungicides of high efficiency for control of Fusarium oxysporum f. sp. cubense race 4, a catastrophic soilborne phytopathogen causing banana Fusarium wilt worldwide. We confirmed for the first time that aureoverticillactam (YY3) has potent antifungal activity against F. oxysporum f. sp. cubense race 4, with effective dose for 50% inhibition (EC₅₀) of 20.80 μg/ml against hyphal growth and 12.62 μg/ml against spore germination. To investigate its mechanism of action, we observed the cellular ultrastructures of F. oxysporum f. sp. cubense race 4 with YY3 treatment and found that YY3 led to cell wall thinning, mitochondrial deformities, apoptotic degradation of the subcellular fractions, and entocyte leakage. Consistent with these variations, increased permeability of cell membrane and mitochondrial membrane also occurred after YY3 treatment. On the enzymatic level, the activity of mitochondrial complex III, as well as the ATP synthase, was significantly suppressed by YY3 at a concentration >12.50 μg/ml. Moreover, YY3 elevated the cytosolic Ca²⁺ level to promote mitochondrial reactive oxygen species (ROS) production. Cell apoptosis also occurred as expected. On the transcriptome level, key genes involved in the phosphatidylinositol signaling pathway were significantly affected, with the expression level of Plc1 increased approximately fourfold. The expression levels of two apoptotic genes, casA1 and casA2, were also significantly increased by YY3. Of note, phospholipase C activation was observed with YY3 treatment in F. oxysporum f. sp. cubense race 4. These findings indicate that YY3 exerts its antifungal activity by activating the phospholipase C calcium-dependent ROS signaling pathway, which makes it a promising biofungicide.

Self-emulsifying solid nanodispersion technology is emerging as an attractive strategy to prepare new eco-friendly and efficient nano-formulations due to its simple, energy efficient and easy scale-up process. However, it is still unknown whether this technology can be employed to cope with the drawbacks of botanical insecticides including poor water solubility, rapid photodegradation and limited targeting efficiency. In this study, rotenone (Rot) was selected as a model of botanical insecticides, and its solid nanodispersion (Rot–SND) was prepared by a self-emulsifying method combined with parameter optimization. Our target nano-formulation, consisting of 5% Rot, 20% surfactant complexes of 8% Ethylan 992 and 12% EL–80, and 75% lactose, exhibited excellent storage stability and significantly improved the pseudo-solubility of Rot by at least 250 times. The average particle size and polydispersity index (PDI) of Rot–SND were determined to be 101.19 nm and 0.21, respectively. Rot–SND displayed smaller contact angles and greater retention on both cucumber and cabbage leaves than those of a commercial emulsifiable concentrates (ECs). Rot–SND was also more resistant to photodegradation, with a degradation rate reduced by 27.01% as compared with the ECs. In addition, the toxicity of Rot–SND towards Aphis gossypii was 3.01 times that of the ECs, with a median lethal concentration (LC₅₀) of 1.45 µg a.i./mL. Under the field conditions, Rot–SND showed a prolonged duration for A. gossypii control, with a significantly higher control efficacy (88.10%) on the 10th day than that of the ECs (77.02%). Moreover, a 2.34-fold decline in the toxicity towards nontarget mosquito larvae was observed for Rot–SND as compared with the EC. Overall, for the first time, our results indicate the role of Rot–SND as an eco-friendly and efficient way to improve the solubility, foliar affinity, photostability, bioactivity and eco-safety of Rot. This research also provided a feasible strategy to prepare more eco-friendly botanical pesticide formulations of high efficiency.

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.

Self-emulsifying solid nanodispersion technology is emerging as an attractive strategy to prepare new eco-friendly and efficient nano-formulations due to its simple, energy efficient and easy scale-up process. However, it is still unknown whether this technology can be employed to cope with the drawbacks of botanical insecticides including poor water solubility, rapid photodegradation and limited targeting efficiency. In this study, rotenone (Rot) was selected as a model of botanical insecticides and its solid nanodispersion (Rot–SND) was prepared by a self-emulsifying method combined with parameter optimization. Our target nano-formulation, consisting of 5% Rot, 20% complex surfactants of 8% Ethylan 992 and 12% EL–80, and 75% lactose, exhibited excellent storage stability and significantly improved the pseudo-solubility of Rot by at least 250 times. The average particle size and polydispersity index (PDI) of Rot–SND were 101.19 nm and 0.21, respectively. Rot–SND displayed smaller contact angles and greater retention on both cucumber and cabbage leaves than those of the commercial emulsifiable concentrates (EC). Rot–SND was also more resistant to photodegradation with a degradation rate reduced by 27.01% as compared with the EC. In addition, the toxicity of Rot–SND towards Aphis gossypii was 3.25 times that of the EC, with a median lethal concentration (LC50) of 1.26 µg a.i./mL. Under the field conditions, Rot–SND showed a prolonged duration for A. gossypii control, with a significantly higher control efficacy (88.10%) on the 10th day than that of the EC (77.02%). Moreover, a 2.88-fold decline in the toxicity towards nontarget mosquito larvae was observed for Rot–SND as compared with the EC. Overall, for the first time, our results indicated the role of Rot–SND as an eco-friendly and efficient way to improve the solubility, foliar affinity, photostability, bioactivity and eco-safety of Rot. This research provides a feasible strategy to prepare more eco-friendly botanical pesticide formulations of high efficiency.