[Objective] To explore the changes and differences of gut microbiota of worker bee larvae of Apis cerana cerana and Apis mellifera ligustica after infection with Chinese sacbrood virus (CSBV) and evaluate the antiviral activity of Bacillus subtilis against CSBV. [Methods] Two-day-old larvae of A. c. cerana and A. m. ligustica were collected from colonies and artificially reared in an incubator (34 ℃, RH 85%). The 3-day-old larvae were inoculated with CSBV and samples were collected when the larvae were 4 and 7 days old for 16S rRNA gene sequencing. In addition, the 3-day-old larvae of A. c. cerana were fed with different concentrations of B. subtilis suspensions during CSBV inoculation. When the larvae were 7 days old, the antioxidant capacity indicators malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined. Additionally, the relative expression levels of four antimicrobial peptide genes (Abaecin, Apidaecin, Hymenoptaecin, and Defensin) and the CSBV gene were determined by real-time fluorescence quantitative PCR. [Results] The number of gut microorganisms in bee larvae significantly decreased after CSBV infection. The abundance of Bacillus in the gut of 7-day-old A. c. cerana larvae significantly decreased. The gut of A. m. ligustica larvae showed significantly decreased abundance of Streptomyces and significantly increased abundance of Brevundimonas. At 4 days old, CSBV-infected A. c. cerana larvae had significantly higher abundance of Melissococcus in the gut than the infected A. m. ligustica larvae. At 7 days old, CSBV-infected A. c. cerana larvae had significantly lower Chao1 index than the control group and the infected A. m. ligustica larvae, which indicated that the gut microbiota diversity of A. c. cerana larvae was more susceptible to CSBV. The SOD activity of 7-day-old A. c. cerana larvae fed with 1×10⁴ CFU/kg B. subtilis significantly increased, while other doses of B. subtilis had no significant effect on the SOD activity in the larvae. All doses of B. subtilis significantly reduced the MDA content in bees. The relative expression levels of CSBV in the three treatment groups of 1×10⁴, 1×10⁶, and 1×10⁸ CFU/kg were significantly down-regulated. Moreover, the expression level of CSBV in the 1×10⁶ CFU/kg group was the lowest and significantly lower than that in the 1×10⁸ CFU/kg group. However, there were no significant differences in the expression levels of the four antimicrobial peptide genes among different treatment groups. [Conclusion] CSBV infection has a more significant impact on the gut microbiota diversity of A. c. cerana larvae. B. subtilis has a certain inhibitory effect on CSBV.

[Objective] To investigate the metabolite differences among different varieties and grades of flue-cured tobacco, as well as the impacts of nicotine-degrading bacteria on the quality of different grades of tobacco leaves. [Methods] This study employed untargeted metabolomics to identify and analyze metabolites in fermented tobacco leaves of ‘Yunyan 87’ and ‘Yunyan 97’, specifically B2F and C2F grades, while also examining the influences of nicotine-degrading bacteria on the quality of different grades of flue-cured tobacco leaves. [Results] There were significant metabolite differences between tobacco leaves of different varieties and grades. A total of 131 differential metabolites were identified between the samples of ‘Yunyan 87’ and ‘Yunyan 97’, while 138 differential metabolites were identified between B2F and C2F grades. These differential metabolites mainly included amino acids, flavonoids, alkaloids, and their derivatives. Analysis of KEGG metabolic pathways and enrichment levels for differential metabolites across different varieties and grades all indicated that flavonoid biosynthesis pathways were the most prominent. Furthermore, this study successfully isolated two bacterial strains, Pseudomonas sp. TR9 and Pseudomonas sp. TR14, from tobacco-cultivated soil, both capable of utilizing nicotine as the sole carbon and nitrogen source. Inoculation of the strain combination into different grades of tobacco leaves significantly reduced the content of nicotine, protein, and starch in lower-grade tobacco leaves. [Conclusion] This study reveals the mechanism by which varieties and grades affect the quality of tobacco leaves through the flavonoid biosynthesis pathways, and verifies the improvement effects of nicotine-degrading bacteria on low-grade tobacco leaves, providing theoretical support for the improvement of flue-cured tobacco quality and the optimization of fermentation processes.

[Objective] To provide a theoretical basis for developing microbiome-based ecological control strategies against citrus Huanglongbing (HLB), a devastating bacterial disease seriously threatening the global citrus industry. [Methods] Rhizosphere soil samples from both HLB-infected and healthy citrus trees in Yizhang County, Hunan Province, were investigated. Using 16S rRNA gene and ITS region amplicon sequencing, we systematically analyzed the impact mechanism of HLB on the rhizosphere micro-ecosystem. [Results] The results showed that HLB infection significantly reduced the organic matter (6.65 g/kg) and available phosphorus (7.25 mg/kg) content of the rhizosphere soil compared with that of the healthy plants, and triggered a significant decrease in the alpha diversity of bacterial communities and a significant increase in the alpha diversity of fungal communities (P<0.05). Beta diversity analysis showed that HLB significantly altered the structure of the microbial communities. Specifically, the relative abundance of pro-biotic bacteria such as Pseudomonadota and Gemmatimonadota decreased, while oligotrophic Acidobacteriota and Chloroflexota were significantly enriched. In fungal communities, the abundance of saprophytic fungi in the phyla Ascomycota and Basidiomycota increased by 5.32% and 7.38%, respectively, while the phylas Rozellomycota and Mortierellomycota decreased by 12.30% and 3.23%, respectively. HLB disrupted the rhizosphere microbial balance by inhibiting Rozellomycota, leading to excessive proliferation of saprophytic fungi and weakening the system’s disease resistance. Analysis at the order level further revealed that beneficial bacterial groups such as Burkholderiales and Hyphomicrobiales were significantly depleted, whereas stress-adaptive groups like Ktedonobacterales showed significant proliferation. PICRUSt2 analysis revealed that HLB disturbed the structure of the citrus rhizosphere bacterial community via metabolic pathways and genetic information processing. HLB also utilized saprophytic and ectomycorrhizal fungi to maintain soil health. [Conclusion] This study revealed that HLB affects soil microecological balance by remodeling the structure and function of citrus rhizosphere microorganisms, and the results may provide a theoretical basis for the development of ecological prevention and control strategies for HLB based on microbiome regulation.

[Objective] To investigate the biostimulatory effects of Bacillus velezensis XZT106 on the tuber crop sweet potato (Ipomoea batatas) and elucidate the potential mechanisms underlying its yield increase. [Methods] Sweet potato plants were treated by foliar spraying with B. velezensis fermentation broth, with the plants treated with inactivated B. velezensis fermentation broth as the control. We analyzed the chloroplast content, chloroplast ultrastructure, and antioxidant enzyme activity as well as the structure and metabolite composition of endophyte communities in different ecological niches of sweet potato plants to delve into the mechanisms by which B. velezensis fermentation broth increases the sweet potato yield. [Results] Foliar application of B. velezensis increased the sweet potato yield, enhanced the antioxidant enzyme activity in the roots, induced changes of chloroplast ultrastructure, and led to a more compact matrix structure with enlarged intracellular starch granules. In addition, foliar application of B. velezensis caused significant changes of endophyte community structures in various parts of sweet potato plants, significantly reducing the relative abundance of Fusarium and increasing the relative abundance of Pantoea. Moreover, the foliar application significantly altered the metabolome profiles of leaves and soil. Riboflavin metabolism, zeatin biosynthesis, and isoflavone biosynthesis, which regulate growth and enhance stress resistance, were significantly upregulated in leaves. The axon regeneration pathway promoting lateral root development and the glycerophospholipid metabolism pathway promoting cell proliferation were significantly upregulated in soil. [Conclusion] B. velezensis fermentation broth exerts a plant growth-promoting effect by enhancing antioxidant capacity, improving leaf cell ultrastructure, reshaping the endophyte community structure, and activating key growth-promoting and stress response metabolic pathways in sweet potato plants. These findings provide a new theoretical foundation for the application of B. velezensis-based microbial inoculants in enhancing the sweet potato yield.

[Objective] By examining intracellular and extracellular metabolite changes in ectomycorrhizal fungi (ECMF) under acidic aluminum stress, we identified key resistance-related metabolites and pathways, aiming to elucidate the aluminum tolerance mechanisms from the perspective of metabolic physiology and offer a theoretical basis for using ECMF in restoring aluminum-contaminated forests. [Methods] Pisolithus tinctorius was cultured in vitro in the acidic medium (pH 3.8) containing 0.0 mmol/L or 1.0 mmol/L Al³⁺. Untargeted metabolomics was employed to analyze changes in intracellular and extracellular metabolite levels. [Results] Compared with that under the 0.0 mmol/L Al³⁺ treatment, the colony diameter of P. tinctorius under 1.0 mmol/L Al³⁺ stress decreased significantly by 23.67%. In addition, the intracellular levels of nucleotides including uridylic acid, cytidine monophosphate, uridine, uridine diphosphate, cytidine, and guanosine were upregulated under 1.0 mmol/L Al³⁺ stress. Extracellular levels of organic acids such as shikimic acid, fumaric acid, heptanoic acid, and tartaric acid, along with carbohydrates including l-arabinose, trehalose, sucrose, and glucose, were also upregulated. Pyrimidine metabolism and citric acid cycle pathways were enriched intracellularly, while ABC transporters and phosphotransferase system pathways were enriched extracellularly. The potential biomarkers identified in the intracellular environment was citric acid, and those identified in the extracellular environment were trehalose and tartaric acid. [Conclusion] Acidic aluminum stress inhibits the growth of P. tinctorius. Intracellularly, P. tinctorius maintains cellular homeostasis and energy supply through enhanced nucleotide accumulation and activation of the citric acid cycle. Extracellularly, P. tinctorius promotes organic acid secretion and carbohydrate efflux to resist aluminum toxicity and associated oxidative damage.

[Objective] As soil acidification in southwestern China becomes increasingly severe, the labile phosphorus pool is transformed into a non-labile phosphorus pool, which reduces the availability of soil phosphorus, affecting crop yield and wasting phosphate fertilizer resources. In this study, we prepared a biochar-immobilized phosphorus-solubilizing bacterial agent with biochar as the carrier and a strain capable solubilizing both organic phosphorus and inorganic phosphorus as the immobilized strain and then optimized the preparation conditions. Furthermore, this bacterial agent was evaluated in terms of the stability and the solubilizing effects on insoluble phosphorus. [Methods] Selective media were used for the isolation of phosphorus-solubilizing bacteria from plant rhizosphere soil. The molybdenum-antimony colorimetric method was employed to quantify the ability of bacteria to solubilize phosphorus. The bacterial strain was identified through physiological and biochemical tests and molecular biological analysis. The immobilized bacterial agent was prepared by the adsorption method, and the preparation conditions were optimized by single factor experiments. The prepared agent was characterized by Fourier transform infrared spectrometry and scanning electron microscopy. Furthermore, the metabolic spectrum of organic acids and phosphatase activity were qualitatively and quantitatively tested by HPLC and the fluorescence method, respectively. [Results] The strain Klebsiella sp. was isolated for immobilization, and its abilities to solubilize lecithin and tricalcium phosphate were 236.5 mg/L and 200.3 mg/L, respectively. Genome analysis showed that the strain N107 carried 27 genes related to organic and inorganic phosphorus solubilization. The optimized preparation conditions were biochar addition of 30.0 mg/mL, N107 inoculation amount of 6.0%, immobilization temperature of 30.0 ℃, and immobilization time of 12.0 h. The bacterial agent prepared under the optimal conditions increased the phosphorus-solubilizing capacity for lecithin and tricalcium phosphate by 24.0% and 22.5%, respectively, compared with the free bacterial strain. The biochar-immobilized phosphorus-solubilizing bacterial agent contained more oxygen-containing functional groups, compared with the original biochar, its total specific surface area and external surface area increased by 61.9% and 165.1%, respectively. The mechanism of phosphorus solubilization by the immobilized bacterial agent was preliminarily analyzed. The results showed that the levels of tartaric acid, citric acid, and total acids changed significantly and the activities of acid and alkaline phosphatases in the culture medium were effectively improved, although the types of organic acids secreted by the agent had no obvious changes. The structural equation model showed that pH value was closely related to phosphatase activity and organic acid content, and the immobilized bacterial agent can promote the activation of insoluble phosphorus by increasing phosphatase activity and organic acid content. [Conclusion] The immobilized phosphorus-solubilizing bacterial agent prepared in this study provides a good bioremediation material for the activation of insoluble phosphorus. This study provides an innovative perspective for developing green remediation strategies based on microbiomes.

[Objective] To investigate the antifungal activity of Kobusin against Trichophyton interdigitale and its underlying mechanisms. [Methods] The minimal inhibitory concentration (MIC) of Kobusin was determined by the broth microdilution assay. The inhibitory effect of Kobusin on spore germination was observed microscopically, while that on hyphal radial growth was assessed on the agar plates containing Kobusin. Scanning electron microscopy (SEM) was employed to examine the morphological alterations in hyphae. Fluorescence microscopy and nucleic acid and protein leakage assays were employed to evaluated cell membrane integrity. Malvern Zetasizer was used to measure the changes in Zeta potential. A microplate reader was used to measure transmembrane potential, alkaline phosphatase (AKP) activity, malondialdehyde (MDA) content, reactive oxygen species (ROS) accumulation, superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities, mitochondrial membrane potential (MMP), ATP levels, as well as succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) activities. [Results] Kobusin exhibited a MIC of 39 μg/mL against T. interdigitale, significantly inhibiting spore germination and hyphal growth. SEM revealed severe ultrastructural damage to hyphae. Fluorescence microscopy confirmed compromised membrane integrity, evidenced by increased nucleic acid and protein leakage and disrupted Zeta/transmembrane potentials. Meanwhile, Kobusin significantly increased the MDA content and ROS accumulation, inhibited the activities of AKP, SOD, CAT, and POD, markedly reduced MMP, decreased ATP synthesis, and weakened the activities of SDH and MDH. [Conclusion] Kobusin exerts antifungal effects by inhibiting spore germination and hyphal growth, disrupting cell membrane and cell wall integrity, interfering with membrane potential stability, inducing oxidative stress damage, and impairing mitochondrial energy metabolism.

Probiotic products have attracted increasing attention for their potential to modulate the microbiota. However, most commercial products are designed for oral administration, and their probiotic properties relevant to topical use in the reproductive tract remain insufficiently evaluated. [Objective] To assess the probiotic properties of lactic acid bacteria (LAB) derived from probiotic products, with a particular focus on their potential for topical application, thus providing scientific evidence for their use in vaginal health. [Methods] Seven common oral probiotic products (P1-P7) containing at least two different LAB species were selected from major e-commerce platforms via keyword screening, along with one clinical probiotic product (P8). LAB strains were isolated and identified from these products. We evaluated the acid tolerance, as well as the growth characteristics under different pH conditions, of the isolates by culturing them in the media of varying pH values. The antimicrobial activities of the isolates were determined via co-culture assays with pathogenic microorganisms, while hemolysis assays and genomic comparison were conducted to assess safety. [Results] The isolation rates of LAB strains from P1 to P8 were 50.0% (2/4), 0 (0/4), 66.7% (2/3), 12.5% (1/8), 33.3% (2/6), 40.0% (2/5), 0 (0/7), and 100.0% (1/1), respectively. Most strains grew well at pH 6.0-7.0, and some maintained growth at pH 4.0. Strains P4 and P8 exhibited superior acid tolerance to the others. The inhibitory effects of different strains against common vaginal pathogens varied significantly. Strains P1-2, P5-1, P6-1 and P6-2 demonstrated moderate to strong broad-spectrum inhibitory activity against all tested pathogens. Other isolated strains except P8 exhibited inhibitory activity against Gardnerellavaginalis, while strain P8 showed weak inhibitory activities against the tested pathogens. Strains P4, P5-2, P6-1, and P6-2 achieved inhibition rates exceeding 99.73% against Candidaalbicans across all three tested inoculum concentrations, and strain P5-1 reached an inhibition rate of over 94.64%. None of the strains exhibited β-hemolytic activity, and no antibiotic resistance or virulence genes were detected. [Conclusion] Several LAB isolates from commercial probiotic products exhibited notable inhibitory activities against pathogenic microorganisms and demonstrated good safety profiles. Topical administration may therefore offer greater practical value in promoting female reproductive tract health.

[Objective] To explore the influence of Acinetobacter pittii LSQ 3 on the biofilm formation of Bacillus velezensis LSQ 19 and analyze the genomic characteristics of strain LSQ 3. [Methods] The effect of the cell-free supernatant (CFS) of LSQ 3 on the biofilm formation of LSQ 19 was analyzed by crystal violet staining, cell surface property analysis, the phenol-sulfuric acid method, XTT reduction assay, and scanning electron microscopy (SEM). Whole-genome sequencing was employed to determine the taxonomic status of strain LSQ 3, and the biosynthetic gene clusters for secondary metabolites were predicted based on the whole-genome data. [Results] The CFS of strain LSQ 3 significantly inhibited the biofilm formation of strain LSQ 19, and the volume ratio of 10 µL bacterial suspension to 190 µL CFS was determined as the minimum inhibitory concentration (MIC). Compared with the control, the CFS of strain LSQ 3 at MIC significantly reduced the surface hydrophobicity, adhesion, extracellular polymeric substances (EPS) production rate, and biofilm metabolic activity, while significantly improving the self-aggregation ability of LSQ 19 cells. In the presence of the CFS at MIC, LSQ 19 failed to form a biofilm on the glass surface. Strain LSQ 3 was identified as A. pittii based on whole-genome sequencing data. Its genome size was 3 939 365 bp, with the G+C content of 38.82% and 3 601 DNA coding sequences. The genome contained multiple genes involved in biofilm formation and virulence factors. The antiSMASH analysis showed that the genome of strain LSQ 3 contained seven biosynthetic gene clusters for secondary metabolites. [Conclusion] The CFS of A. pittii LSQ 3 can inhibit the biofilm formation of B. velezensis LSQ 19. This study provides a theoretical basis and reference for the construction of synthetic bacterial communities from the perspective of biofilms.

As a widely conserved interspecies quorum sensing signaling molecule, autoinducer-2 (AI-2) is involved in regulating various crucial physiological processes such as bioluminescence, chemotaxis, and biofilm formation. However, the effects of AI-2 on Halomonas elongata and its underlying mechanisms remain unreported. [Objective] To reveal the receptor that regulates the chemotaxis and biofilm formation of H. elongata in response to AI-2. [Methods] The quantitative capillary assay was employed to examine the chemotactic response of H. elongata to AI-2. We conducted protein domain identification, sequence alignment, and molecular docking of methyl-accepting chemotaxis proteins to identify the key amino acid sites in Tar1, the potential AI-2 receptor. The ligand-binding domain (LBD) of Tar1 and single-point mutants were expressed and purified, and the binding between Tar1-LBD and AI-2 was measured by the Vibrio harveyi MM32 bioluminescence assay. tar1 was deleted by homologous recombination, and the effects of AI-2 on the chemotaxis and biofilm formation of H. elongata were evaluated by quantitative capillary and biofilm formation assays. [Results] The quantitative capillary assay revealed that H. elongata exhibited chemotaxis to AI-2. Four methyl-accepting chemotaxis proteins were identified in H. elongata. Protein domain identification, sequence alignment, molecular docking, and V. harveyi MM32 bioluminescence assay demonstrated that Tar1-LBD bound to AI-2. The tar1-deleted mutant of H. elongata was successfully constructed by homologous recombination. The deletion of tar1 impaired the chemotaxis of H. elongata to AI-2, whereas the complementation of this gene restored the chemotaxis to level comparable to that in the wild-type. Furthermore, biofilm formation assay revealed that AI-2 enhanced the biofilm formation in H. elongatavia Tar1. [Conclusion] H. elongata exhibits chemotaxis to AI-2, and this signal molecule binds to the LBD of Tar1, thereby modulating chemotaxis and biofilm formation.