Objective Southern blight, caused by Sclerotium rolfsii Sacc., is a major soil-borne disease that limits peanut production. At present, its management relies mainly on chemical fungicides. To improve the field control efficacy, reduce fungicide application rates, and lower pesticide residues in peanuts, this study screened antagonistic microorganisms against S. rolfsii and evaluated their synergistic effects with chemical fungicides in the control of peanut southern blight. The results are expected to provide technical support for the green and sustainable management of this disease. Methods This study first used a plate-based screening assay to obtain biocontrol strains showing strong antagonistic activity against S. rolfsii and high compatibility with commonly used fungicides. Next, a fungicide screening assay was conducted to identify control agents that effectively inhibited S. rolfsii without adversely affecting the growth of the biocontrol strains. Subsequently, an in vitro combined toxicity assay was performed. The pathogen was inoculated onto PDA plates containing different concentrations of thifluzamide (0.15 and 0.30 μg/mL), sterile 10% fermentation broth of H02, or their mixtures. Fungal growth was observed, and the inhibition rate and inhibition ratio (IR) were calculated to evaluate the interaction type of the combined treatment. Finally, a pot experiment was carried out to compare the control efficacy against peanut southern blight among the fermentation broth, the full-dose fungicide (recommended field rate), and the combination of fermentation broth with a half-dose fungicide. Results A Burkholderia gladioli strain H02 with high antagonistic activity and good fungicide compatibility was screened out, exhibiting an inhibition rate of 76.03%. Concurrently, thifluzamide was identified as a fungicide with strong inhibitory activity against S. rolfsii and no negative impact on the growth of strain H02, exhibiting an EC₅₀ of 0.151 3 μg/mL. Results from the in vitro combined toxicity assay indicated that the combination of the H02 fermentation broth and thifluzamide showed an IR greater than 1, exhibiting a synergistic effect. Moreover, the combination of half the concentration of thifluzamide with the fermentation broth significantly demonstrated higher inhibition rates against pathogen mycelia and sclerotia (74.91% and 95.58%, respectively) than the full concentration of thifluzamide alone (68.04% and 83.67%, respectively). Pot experiment results showed that the combination of H02 fermentation broth and half dosage of thifluzamide had the control efficacy comparable to that of the full dosage of thifluzamide (68.94% vs. 66.63%), whereas the single application of H02 fermentation broth provided the control efficacy of 61.17%. Conclusion The combined application of B. gladioli H02 and thifluzamide can achieve synergistic control of peanut southern blight and reduce chemical fungicide usage while maintaining effective disease control, thus showing promising potential for practical application.

Meloidogyne incognita is one of the most destructive plant-parasitic nematodes worldwide, causing severe economic losses in agricultural production. Biocontrol bacteria can effectively control M. incognita, with significant differences in control efficacy among different strains. However, the mechanisms underlying differences in control efficacy remain unclear. Objective To explore the mechanisms responsible for the different efficacy of various biocontrol bacteria against nematodes. Methods The differences in nematicidal activity between two biocontrol bacterial strains, B133 and B104, were analyzed. Comparative genomics and metabolomics techniques were employed to investigate the genetic composition and metabolic mechanisms influencing the nematicidal activity of the two strains. Result From 24 h to 120 h of fermentation, the nematicidal activity of strain B133 was significantly higher than that of strain B104, reaching peaks of 77% and 54%, respectively, at the time point of 60 h. Whole-genome comparative analysis revealed that strain B133 possessed a larger genome size and a greater number of coding genes than strain B104. The phylogenetic trees conducted based on 16S rRNA gene or the housekeeping gene gyrB indicated that strains B133 and B104 were two different subspecies of Priestia megaterium. Predictions based on the virulence factors database (VFDB) and Kyoto encyclopedia of genes and genomes (KEGG) database showed that strain B133 harbored 22 unique virulence genes and 75 unique metabolism genes compared with strain B104. Meanwhile, the metabolites in the fermentation filtrate (60 h) were determined. Principal component analysis demonstrated significant differences in metabolite profiles between the two strains. Compared with that of strain B104, the fermentation filtrate of strain B133 had 40 increased metabolites (P<0.05), such as galactinol, 4-aminobenzoic acid, lumichrome, anthranilic acid, trehalose, and 3-methylthiopropionic acid. Moreover, through integrated genomics-metabolomics analyses, cysteine and methionine metabolism was identified as a key pathway influencing nematicidal activity. This pathway involves an L-lactic dehydrogenase (LDH) gene unique to strain B133 and 3-methylthiopropanoic acid with an elevated level and a positive correlation with the nematicidal effect of the strain. Conclusion By coupling genomics and metabolomics, this study reveals the different functional gene clusters and potential related metabolites of different subspecies of P. megaterium, laying a theoretical foundation and a practical basis for the targeted screening, modification, and industrial development of efficient biocontrol agents for nematodes.

A reverse genetics platform for foot-and-mouth disease virus (FMDV) is an indispensable tool for studying the pathogenic mechanism, protein function, and vaccine development. However, the conventional method of constructing infectious clones of FMDV is usually laborious, time-consuming, and costly. Objective To establish a new reverse genetics platform for rapid rescue of FMDV based on infectious subgenomic amplicons (ISA), which can avoid in vitro ligation and bacterial cloning. Methods The whole gene of FMDV O/GDLeiZh/2020 strain was divided into five overlapping fragments and then individually amplified by high-fidelity PCR. The T7 promoter sequence was added to the 5′-end gene and the poly(A) tail was introduced at the 3′-end. At the same time, the poly(C) sequence and molecular markers were introduced by fusion PCR. Two large fragments covering the whole gene of FMDV were obtained by multiple rounds of fusion PCR amplification and co-transfected into BSR/T7 cells expressing T7 RNA polymerase. The cell supernatant was collected 72 h post-transfection. The rescued virus was identified and characterized by RT-PCR, indirect immunofluorescence, electron microscopy, plaque assay, and one-step growth curve assay. Results The typical cytopathic effect of FMDV was observed 60 h post-transfection. Sequencing, immunofluorescence, and electron microscopy collectively confirmed that infectious FMDV was successfully rescued. Furthermore, one-step growth curve and plaque assays demonstrated that the rescued virus retained replication kinetics and biological characteristics comparable to those of the wild-type virus. Conclusion This study successfully establishes a new method for rapid and efficient rescue of FMDV based on ISA, which will lay a solid foundation for further improving FMDV rescue technology and rapidly expanding its application in the future.

Objective To investigate the antagonistic activity of Bacillus amyloliquefaciens X60 against tobacco phyllosphere microorganisms and its effects on the phyllosphere microbial community of tobacco. Methods Bioactivity assays were conducted to evaluate the antagonistic effects of B. amyloliquefaciens X60 against 20 species of pathogenic fungi, 15 species of non-pathogenic fungi, 2 specialized forms of pathogenic bacteria, and 15 species of non-pathogenic bacteria. Amplicon sequencing was employed to assess the influence of this strain on the phyllosphere microbial community structure. Results B. amyloliquefaciens X60 exhibited strong antagonistic activity (inhibition rates of 60.00%-80.00%) against 13 species of pathogenic fungi (e.g., Rhizopus oryzae) and 12 species of non-pathogenic fungi (e.g., Trichoderma harzianum). Moderate antagonism (inhibition rates of 10.00%-59.00%) was observed against 7 species of pathogenic fungi (e.g., Alternaria tenuissima) and 3 species of non-pathogenic fungi (e.g., Thielavia microspora). Significant antibacterial activity (inhibition zone diameter >20 mm) was detected against 2 specialized forms of pathogenic bacteria (Pseudomonas syringae pv. tabaci and pv. angulata) and 7 non-pathogenic bacteria (e.g., Exiguobacterium). After application, X60 showed the control efficacy of 52.35% against tobacco leaf spot. Following treatment, the relative abundance of Pantoea—a genus of opportunistic bacteria dominating the infected tissue—increased, whereas bacterial diversity and richness initially declined and then recovered. Fungal richness decreased throughout the observation period, while fungal diversity exhibited a transient decrease followed by a rebound. The relative abundance of phytopathogenic fungi declined from 44.87% to 6.71%. Conclusion B. amyloliquefaciens X60 possesses a broad antimicrobial spectrum and exerts strong antagonistic activity against 25 fungal and 9 bacterial species colonizing the tobacco phyllosphere. Under field conditions, the strain provided 52.35% control of tobacco leaf spot and significantly reduced the abundance of foliar phytopathogens, demonstrating the potential as a biocontrol agent for the management of this disease.

Objective To address the problems of rhizospheric microenvironment deterioration and medicinal quality decline caused by continuous cropping obstacles of Fritillaria unibracteata Hsiao et K. C. Hsia, this study explored the regulatory effects of different concentrations of salicylic acid (SA) under continuous and non-continuous cropping patterns and clarified the optimal SA concentration and underlying mechanism for alleviating continuous cropping obstacles, aiming to provide a theoretical basis for optimizing cultivation techniques. Methods A pot experiment was conducted with two cultivation patterns (continuous cropping and non-continuous cropping) and six SA concentration gradients (0, 20, 50, 100, 200, and 500 μmol/L). The changes in root exudates, soil physicochemical properties, soil enzyme activities, alkaloid content, and microbial community structure were determined. Correlation analysis and redundancy analysis (RDA) were performed to elucidate the regulation mechanism. Results SA exerted significant concentration-specific regulatory effects on the rhizospheric microenvironment and alkaloid biosynthesis of F. unibracteata, showcasing a significant interaction effect with cultivation patterns. Total phenolic acids in root exudates increased under 20 μmol/L and 500 μmol/L SA treatments (P<0.05), and organic acids reached the peak under 200 μmol/L SA treatment. The soil organic carbon and soil organic matter in the non-continuous cropping group were significantly higher than those in the continuous cropping group. SA at 50 μmol/L optimized soil pH, increased the supply of available phosphorus and ammonium nitrogen, and enhanced the activities of urease and acid phosphatase. Pseudomonadota and Ascomycota were the dominant phyla in bacterial and fungal communities, respectively. SA at 500 μmol/L enriched beneficial microorganisms such as Streptomyces, inhibited pathogens, and specifically increased the content of peimisine and sipeimine. RDA results showed that SA remodeled the microbial community by regulating the composition of root exudates, thereby mediating alkaloid biosynthesis. Conclusion SA at 50 μmol/L SA is suitable for optimizing rhizosphere nutrient supply and enzyme activities, and that at 500 μmol/L is suitable for effectively alleviating continuous cropping obstacles and promoting the accumulation of medicinal alkaloids. SA achieves rhizospheric ecological restoration and medicinal quality improvement through a synergistic pathway of regulating root exudate composition, remodeling microbial community structure, and repairing rhizospheric interaction network. This study provides a new approach for the management of continuous cropping obstacles for F. unibracteata.

Excessive nitrogen input caused by eutrophication in nearshore waters is a major environmental stressor driving the global degradation of seagrass beds. Objective To screen and identify efficient aerobic denitrifying bacteria from seagrass bed ecosystems and elucidate their nitrogen removal performance and mechanisms, thus providing microbial resources for alleviating nitrogen loading and restoring eutrophic seagrass beds. Methods Aerobic denitrifying bacteria were isolated and screened from seagrass rhizosphere sediments in Zhifu Bay, Yantai by enrichment-domestication culture and bromothymol blue assay. The taxonomic status of the strains was determined by 16S rRNA gene sequencing. On the basis of nitrogen removal performance, an efficient aerobic denitrifying strain was selected. Single-factor and orthogonal experiments were conducted to optimize its denitrification conditions, and nitrogen balance experiments and whole-genome sequencing were employed to elucidate its nitrogen removal pathways and key functional genes. Results A total of 34 denitrifying strains were isolated from seagrass rhizosphere sediments in Zhifu Bay, Yantai. The dominant genera were Pseudomonas and Acinetobacter. A strain designated as Pseudomonas sp. S22 with high denitrification performance was selected. The denitrification conditions of this strain were optimized as follows: sodium succinate as the carbon source, C/N=15, pH 9.0, salinity (S)=30‰, and T=28 ℃. Under these conditions, the strain achieved a removal rate of 99.99% for 140 mg/L nitrate nitrogen within 36 h, demonstrating excellent nitrogen removal efficiency. Nitrogen balance analysis revealed that approximately 59.64% of the initial nitrate nitrogen was converted to gaseous nitrogen, confirming that denitrification was the dominant nitrogen removal pathway. Genomic sequencing revealed that strain S22 carried key functional genes for aerobic denitrification, including napA and nirS, providing a genetic basis for its denitrification phenotype at the molecular level. Conclusion This study systematically isolated and identified aerobic denitrifying bacteria from seagrass beds in northern China. Strain S22 exhibits outstanding nitrogen removal performance and environmental adaptability. Nitrogen balance and genomic analyses confirm that denitrification is its primary nitrogen removal pathway and the strain carries key functional genes for aerobic denitrification. Strain S22 can serve as a potential microbial resource for reducing nitrogen loading in seagrass beds. This study provides both a valuable strain and a theoretical basis for the future development of microbe-seagrass synergistic remediation technologies.

Sugarcane smut is a severe fungal disease caused by Sporisorium scitamineum, resulting in yield reduction and economic losses. Reversible protein phosphorylation plays a crucial role in the sexual mating and pathogenicity of S. scitamineum. Protein phosphatases, as key regulators of reversible protein phosphorylation, remain poorly characterized in S. scitamineum. Objective To elucidate the biological functions of the protein phosphatase SsPpe1 in S. scitamineum, providing a potential target for effective control of sugarcane smut. Methods We constructed overexpression mutants OE-Ssppe1 by Agrobacterium-mediated genetic transformation technology and analyzed the sporidium morphology, sexual mating ability, stress tolerance, and pathogenicity. Results The OE-Ssppe1 sporidia exhibited pseudohyphal morphology with multiple nuclei and abnormal chitin accumulation. The OE-Ssppe1 mutants showed reduced tolerance to NaCl and SDS, sexual mating, and pathogenicity. RT-qPCR and RNA-seq analyses revealed that Ssppe1 overexpression affected the expression of genes related to pheromone response, MAPK, and cAMP-PKA signaling pathways. In addition, Ssppe1 overexpression affected protein synthesis and folding process. Conclusion The protein phosphatase SsPpe1 is involved in regulating the sporidium morphology, stress responses, sexual mating, and pathogenicity of S. scitamineum. These findings provide a theoretical basis for thoroughly elucidating the pathogenic mechanisms of S. scitamineum and developing targeted disease control strategies.

Objective To achieve the targeted isolation of the cyclooctapeptides, surugamides, from the deep-sea-derived Streptomyces sp. NA13 and explore their biological activities. Methods An approach integrating genome mining and LC-MS/MS molecular networking was employed to discover cyclopeptides from Streptomyces sp. NA13. Through systematic natural product isolation and characterization, these compounds were identified as surugamides. Their growth-promoting effects on Oryza sativa and Zea mays were assessed. Results Four cyclooctapeptides (surugamides A, B, D, and E) were isolated and identified. They had significant effects of promoting root growth in Z. mays and O. sativa seedlings. Notably, surugamide A at a concentration of 0.1 µmol/L demonstrated particularly outstanding growth-promoting effects on Z. mays roots. Conclusion This study uncovers the novel plant growth-promoting activity of surugamides, offering lead compounds for the development of innovative marine microbial-derived plant growth regulators.

Objective Cyanocobalamin (CN-CbI) requires to be converted into adenosylcobalamin (Ado-CbI) to exert neuroprotective effects, yet its conversion efficiency is impaired under conditions such as chronic sleep deprivation (CSD). This study aimed to obtain a bacterial strain with high efficiency in converting CN-CbI to Ado-CbI that could enhance neuroprotective effects. Methods Lactobacillus paragasseri CCFM1526, a strain capable of converting CN-CbI to Ado-CbI, was isolated via UPLC. A mouse model of CSD was established, and the cognitive functions of mice were evaluated by the novel object recognition and Morris water maze tests. The neuronal structure in the hippocampal dentate gyrus (DG) region was observed via histological staining. Tissue vitamin B₁₂ levels were measured, and the ERK/mTOR signaling pathway along with related neural protein expression was analyzed to assess the neuroprotective mechanism of the fermentation broth. Results Compared with CN-CbI supplementation alone, the fermentation broth of L. paragasseri CCFM1526 significantly improved the cognitive function and alleviated the structural damage in the hippocampal DG region of CSD mice. Intervention with the fermentation broth increased the total vitamin B₁₂ content in the liver, serum, and brain by 10.2%, 16.3%, and 29.0%, respectively (P<0.05). Meanwhile, it activated the ERK/mTOR signaling pathway, leading to increases of 21.5%, 52.4%, 17.3%, and 19.7% in the content of myelin basic protein, postsynaptic density protein 95, brain-derived neurotrophic factor, and nerve growth factor, respectively (P<0.05). Conclusion L. paragasseri CCFM1526 converts CN-CbI into Ado-CbI through fermentation, subsequently activating the ERK/mTOR signaling pathway and upregulating the expression of neurotrophic and myelin repair-related proteins, thereby alleviating CSD-induced nerve injury.

Objective To overcome the limitations such as high costs and restricted substrate utilization of mono-culture fermentation, bacterium-alga co-culture based on resource complementarity offers a promising new avenue for ectoine production. This study investigated the co-culture conditions of Dunaliella pseudosalina ZBY-1 and Halomonas campaniensis XH26 and the variations in ectoine yield, aiming to elucidate the metabolic regulation mechanism of ectoine biosynthesis in the co-culture system. Methods Strains XH26 and ZBY-1 were co-cultured at different inoculation ratios (1/0, 1/5, 1/10, 1/15, and 1/20) to screen the ratio yielding the highest ectoine production. Targeted metabolomics analysis was performed on the co-culture group [H group (H)], the bacterial control group [XH26 group (X)], and the algal control group [ZBY-1 group (D)] to identify significant differential metabolites. Results The highest ectoine yield was achieved at a bacterium-to-alga ratio of 1:15, while the pigment content of the algal strain was lower than that of the control group. Metabolomics analysis identified 15 (H vs. D), 16 (H vs. X), and 16 (X vs. D) significant differential metabolites, including L-alanine, L-asparagine, L-aspartic acid, L-phenylalanine, malic acid, and pyruvic acid. Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis revealed that alanine, aspartate and glutamate metabolism, glyoxylate and dicarboxylate metabolism, and arginine biosynthesis were the significantly altered metabolic pathways. Conclusion The co-culture system exhibited an asymmetric pattern characterized by bacterial proliferation and algal inhibition. The co-culture system significantly activated the central carbon metabolic network of the bacteria. Notably, aspartic acid and glutamic acid were significantly accumulated in cells, serving as the direct carbon skeleton and amino donor, respectively, to directly promote the efficient synthesis of ectoine.