Numb-associated kinases (NAKs) are a family of evolutionarily conserved serine/threonine kinases, encompassing adaptor-associated kinase 1 (AAK1), cyclin G-associated kinase (GAK), bone morphogenetic protein 2-inducible kinase (BMP2K), and serine/threonine kinase 16 (STK16). NAKs are widely involved in various physiological processes, such as endocytosis, intracellular transport, cell differentiation, autophagy, and signal transduction. In recent years, studies have shown that NAKs play a key role in different life cycle stages including virus entry, assembly, release, and immune escape of various viruses. Furthermore, small-molecule inhibitors targeting NAKs have been applied in clinical research and treatment of related physiological or viral infectious diseases. This article systematically reviews the primary physiological functions of NAKs and their roles in viral infection, aiming to provide a theoretical foundation for elucidating the pathogenic mechanisms of viruses and developing novel therapeutic drugs targeting NAKs.

[Objective] To investigate the biocontrol potential of Penicillium sinense GS218, a new endophytic fungus, and to analyze the antifungal mechanism, so as to provide elite strain resources and lay a theoretical foundation for the biocontrol of Colletotrichum gloeosporioides in pepper. [Methods] Plate assays were employed to determine the hydrolase activity and siderophore production capacity of GS218. The inhibitory effects of GS218 on different phytopathogenic fungi were evaluated by the plate confrontation method. Whole genome sequencing was performed to obtain insights into the genetic information and physiological functions of the strain. The metabolome of strain GS218 co-cultured with C. gloeosporioides was analyzed to explore the potential active substances for the inhibitory effects. The medicated plate method was employed to validate the inhibitory activities of differential metabolites. [Results] Strain GS218 had hydrolase activity, produced siderophores, and exhibited strong inhibitory effects on five pathogenic fungi (with the inhibition rate of 72.76% on C. gloeosporioides in pepper). The sterile fermentation filtrate of strain GS218 demonstrated a good control effect on pepper anthracnose. The genome size of strain GS218 was 27.77 Mb, which has abundant metabolic pathways, and its genome contained 30 synthetic gene clusters for secondary metabolites. The metabolomics analysis showed that strain GS218 contained rich antimicrobial substances in organic acids and derivatives, phenylpropanoids and polyketides, and lipids and lipid-like molecules (including terpenoids). Compounds such as 7-ethoxycoumarin and pyruvic acid showed inhibitory effects against C. gloeosporioides in pepper. [Conclusion] The new strain, P. sinense GS218, has significant inhibitory effects on C. gloeosporioides and promising application prospects in the green development of agriculture. Whole genome sequencing and metabolomics analysis provide a theoretical basis for deciphering the biocontrol mechanism of strain GS218.

[Objective] This study examined the characteristics of the microbial communities and their associations with chemical components in aged tobacco leaves from different production areas in Yunnan Province, aiming to provide a theoretical foundation for the targeted exploitation of tobacco microbial resources and quality improvement of tobacco. [Methods] A systematic approach integrating metagenomic sequencing, GC/LC-MS, and Spearman’s correlation analysis was employed to investigate the microbial communities, chemical components, and their correlations of aged tobacco leaf samples collected from Dali, Wenshan, Honghe, Luoping, Pu’er, Zhaotong, and Lincang. [Results] The dominant bacterial phylum was Pseudomonadota in aged tobacco leaf samples. The dominant bacterial genera varied among different production areas. Specifically, Salinivibrio was identified as the core genus in the samples from Wenshan, while Methylobacterium and Sphingomonas exhibited significant enrichment in the samples from Luoping. For fungal communities, Ascomycota and Mucoromycota were the predominant phyla, and dominant fungal genera showed negligible variations. Alpha diversity analysis revealed the highest microbial richness and diversity in the samples from Lincang and the lowest in the samples from Wenshan. KEGG analysis revealed that metabolic pathways exhibited high relative abundance across samples from all regions, whereas environmental information processing pathways were more abundant in the samples from Wenshan. CAZy analysis showed that genes annotated as glycoside hydrolases (GHs) and glycosyl transferases (GTs) were the most prevalent, with both generally exhibiting lower abundance in the samples from Wenshan. Chemical profiling revealed that reducing sugar, total sugar and other conventional components were more concentrated in the samples from Dali, whereas total sugar-to-nicotine ratio and potassium-to-chloride ratio were elevated in the samples from Pu’er. Additionally, neutral aroma compounds, including benzyl alcohol, had significantly higher levels in the samples from Wenshan, while polyphenols such as anthocyanins exhibited markedly higher concentrations in the samples from Luoping. Correlation analysis further disclosed that Microbacterium had a significantly positive correlation with total nitrogen, while Aureobasidium showed a significantly negative correlation with total nitrogen. Leucosporidium exhibited significantly positive correlations with potassium ions and potassium-to-chloride ratio. Salinivibrio had significantly positive correlations with multiple neutral aroma components. In contrast, Methylobacterium and Friedmanniomyces showed significantly negative correlations with the majority of neutral aroma components. Additionally, Methylobacterium, Sphingomonas, Friedmanniomyces, and Metschnikowia had significantly positive correlations with multiple polyphenols. [Conclusion] Microbial communities and chemical components of aged tobacco leaves exhibited marked differences across different production areas in Yunnan. Notably, dominant genera are strongly correlated with aroma compounds, which lays a theoretical basis for targeted screening of functional microbes and the development of biotechnologies to improve tobacco quality.

[Objective] To explore the structural differences and associated environmental factors of bacterial communities in the water and sediment of river ecosystems during winter. [Methods] Fourteen sampling sites were established for Diannong River in winter. We employed high-throughput 16S rRNA gene sequencing to systematically analyze the bacterial community composition and diversity, constructed co-occurrence networks, and evaluated the roles of random processes in community assembly. Furthermore, we performed correlation analysis with environmental factors. [Results] The bacterial communities in the sediment had higher alpha diversity indexes than those in the water (P<0.001) and more stable community structures. The beta diversity decomposition showed that the community differences between water and sediment were mainly related to species turnover. Random processes dominated community assembly in both habitats. The co-occurrence network of bacteria in the sediment was more complex with stronger cooperation. Key species were primarily involved in carbon and sulfur cycles, and rare taxa played an important role in network stability. The bacterial communities in water were mainly influenced by environmental factors such as dissolved oxygen, chlorophyll a, and water temperature, while those in the sediment were influenced by pH, organic matter, and nitrogen factors. [Conclusion] This study systematically reveals the differences in the structure, co-occurrence network, and related environmental factors of bacterial communities in the water and sediment of Diannong River during winter, providing scientific evidence for a deeper understanding of the ecological adaptability of river bacterial communities in the freezing period and the different patterns of bacterial communities between water and sediment.

[Objective] To explore the control effects of Streptomyces TOR3209 and its volatile organic compounds (VOCs) on tomato Fusarium wilt and mine the differentially expressed genes related to disease resistance, thus providing effective strategies for the development of environmentally friendly biofungicides. [Methods] Strain TOR3209 suspensions of different concentrations (1.0×10¹, 1.0×10³, 1.0×10⁵, and 1.0×10⁷ CFU/mL) were co-cultured with tomato seedlings, and Fusarium equiseti was inoculated on the seedlings. The disease severity was graded. The co-culture experiment of VOCs from strain TOR3209 with tomato seedlings was conducted in a micro-greenhouse to evaluate the effect of VOCs on tomato seedlings infected by F. equiseti. Transcriptomic analysis was conducted on tomato seedlings with significant disease resistance to mine the differentially expressed genes induced by VOCs, which were then verified by RT-qPCR. [Results] The suspensions of strain TOR3209 at different concentrations all had control effects on tomato Fusarium wilt. Among them, the 1.0×10⁷ CFU/mL suspension had the best control effect (P<0.01). The biocontrol effects of different quantities of small dishes cultured with strain TOR3209 on tomato Fusarium wilt were significantly different from that of the control group. The group of 30 small dishes showed the best control effect (P<0.01). The transcriptomic analysis showed that the expression levels of disease-resistance genes encoding CXE17, LRR-RLK, F-box, TIP1-1 Aquaporin, and Peroxidase were upregulated. Fluorescence quantitative analysis indicated that co-culture of VOCs from the strain with tomato seedlings upregulated the expression levels of disease-resistance genes, indicating that the transcriptomic sequencing results were reliable. [Conclusion] The VOCs of strain TOR3209 effectively prevent and control tomato Fusarium wilt caused by F. equiseti infection by inducing the upregulated expression of disease-resistance genes in tomato seedlings. The findings lay a theoretical foundation for the research and development of biofungicides for the prevention and control of Fusarium wilt.

[Objective] The soil in the vegetable plantation suffered from fertility degradation, pH decrease, and heavy metal leaching, necessitating the exploration of the mechanism by which composite bacterial agents regulate the bacterial community structure, nitrogen composition, and heavy metal availability in the vegetable plantation soil. [Methods] The heavy metal-resistant bacterial strains Ralstonia Bcul-1 (R-B) and Bacillus cellulasensis Zn-B (BC-Z) were prepared with biochar as an immobilized bacterial agent and then applied to the acidic soil (pH 5.6) of a vegetable plantation under long-term tomato rotation. High-throughput sequencing of soil bacteria and the determination of soil composition were conducted to analyze the bacterial diversity, soil pH, nitrogen-carbon content, and heavy metal chemical speciation, on the basis of which the effects of the biochar composite bacterial agent on the bacterial community structure, nitrogen-carbon supply, and heavy metal activity in the soil were analyzed. [Results] Biochar immobilization facilitated the growth of exogenous bacteria R-B and BC-Z in the vegetable plantation soil contaminated with heavy metals and maintained long-term coexistence of R-B and BC-Z with the original highly resistant Bacillus (10.18%-11.88%) in the soil. Accordingly, it effectively improved the bacterial community structure, adjusted the distribution of differential bacteria (biomarkers), and restoratively increased the relative abundance of abundant bacteria (such as Streptomyces, Geopathophilus, and Nocardioids) in the soil. In addition, soil bacterial genera, partial abundant bacteria, and the exogenous bacterial strain R-B were closely related to heavy metal chemical speciation and nitrogen-carbon components. The application of biochar bacterial agents (BI+R-B, BI+BC-Z, and BI+R-B+BC-Z) increased the pH, EC, total nitrogen, nitrate nitrogen, organic matter, and total organic carbon of the soil by up to 0.41, 20.74%, 18.96%, 24.77%, 10.26%, and 21.56%, respectively, while decreasing the ammonium nitrogen residue by 13.91%, maintaining the nitrogen-carbon supply capacity of the soil. BI+R-B and BI+R-B+BC-Z reduced the content of exchangeable, reducible, and oxidizable heavy metals (Cd, Cr, Pb, Cu, and Zn) by 0.18%-12.33%, but increased the residual content of these heavy metals by 0.16%-14.59%, effectively passivating heavy metals in the soil. [Conclusion] The biochar composite bacterial agent (BI+R-B+BC-Z) improved the bacterial community structure, promoted R-B growth, increased the abundance of abundant bacteria, and maintained the long-term coexistence of exogenous bacteria R-B and BC-Z with the original highly resistant Bacillus in the vegetable plantation soil with heavy metal compound pollution. Moreover, it increased soil pH, EC, total nitrogen, nitrate nitrogen, total organic carbon, and organic matter, while reducing ammonium nitrogen residue and passivating soil heavy metals (Cd, Pb, and Cu). Therefore, it effectively regulated the bacterial community activity, exogenous bifunctional bacterial growth, nitrogen-carbon supply, pH, and heavy metal chemical speciation, with the potential to maintain the fertilizer supply capacity and control heavy metal compound pollution of vegetable plantation soil.

[Objective] To analyze the evolutionary conservation and structural characteristics of the heat shock protein GrpE from Mycoplasma bovis, elucidate its subcellular localization, and investigate its biological properties in mediating the adhesion process. [Methods] Primers were designed based on the GrpE gene sequence (GenBank accession number: CP002188.1) of Mycoplasma bovis PG45, and the prokaryotic expression vector pET-GrpE was constructed. Following gene sequencing, bioinformatics methods were employed to analyze the homology, phylogenetic relationships, physicochemical properties, and structural characteristics of GrpE. Following transformation of the recombinant plasmid and induced expression, the yielded recombinant GrpE protein was purified via nickel affinity chromatography, and then SDS-PAGE was conducted. The purified recombinant protein was used to immunize New Zealand White rabbits to generate polyclonal antibodies, with the antibody titer determined by ELISA and immunogenicity assessed via Western blotting. The subcellular localization of GrpE was examined via indirect indirect fluorescent antibody assay (IFA), ELISA, and Western blotting. The adhesion function of GrpE was validated through integrated IFA and ELISA. [Results] The prokaryotic expression vector pET-GrpE was successfully constructed in this study. Bioinformatics analysis revealed that the GrpE sequence was highly conserved in Mycoplasma bovis (with identity exceeding 95%). The encoded protein consisted of 341 amino acid residues, with no signal peptide and transmembrane domain but potential N-glycosylation and phosphorylation sites. SDS-PAGE results confirmed the successful expression of GrpE in a soluble form. Polyclonal antibodies generated via the purified recombinant protein exhibited a titer of 1:16 000. Western blotting analysis further verified the strong immunogenicity of the GrpE protein. Localization studies using IFA, ELISA, and Western blotting indicated that GrpE is distributed in both the cell membrane and the cytoplasm, with predominant distribution observed on the membrane surface. Importantly, the anti-GrpE polyclonal antiserum significantly inhibited the adhesion of Mycoplasma bovis to embryonic bovine lung (EBL) cells. Furthermore, binding assays demonstrated that the interaction between GrpE and host cell membrane proteins is dose-dependent, and this binding was inhibited by the polyclonal antibody (P<0.001). [Conclusion] GrpE is identified as a highly conserved novel adhesion of Mycoplasma bovis that directly participates in the adhesion to host cells, providing a key molecular target for elucidating the pathogenic mechanism of Mycoplasma bovis.

Monascus, as a genus of edible fungi used in fermentation, are widely used in various industries such as wine making, food colorants, and pharmaceuticals due to their abundant secondary metabolites. McrA, a global regulator discovered in Aspergillus nidulans, has the function of regulating the growth and secondary metabolism of filamentous fungi. We had identified and cloned mcrA in Monascus purpureus in the previous study. [Objective] On the basis of transcriptome analysis, we mined the differentially expressed genes (DEGs) of ΔmcrA and trpC:mcrA strains to explore the function of mcrA. [Methods] The knockout strain ΔmcrA and overexpression strain trpC:mcrA of M. purpureus were constructed by homologous recombination. The colonies and microscopic morphology on different media were observed. The yields of Monascus pigments and citrinin were determined. The metabolic pathways involving DEGs were analyzed by transcriptome sequencing. [Results] The yields of Monascus pigments and citrinin of ΔmcrA decreased. Transcriptome sequencing results showed that the ΔmcrA strain up-regulated 111 genes and down-regulated 47 genes. The metabolic pathways involving the DEGs of ΔmcrA were mainly glycolysis, pyruvate metabolism, fatty acid synthesis, tyrosine metabolism and so on. The trpC:mcrA strain up-regulated 1 199 genes and down-regulated 867 genes. The main metabolic pathways involving the DEGs of trpC:mcrA were tryptophan metabolism, sucrose and starch metabolism, arginine and proline metabolism, fatty acid degradation, etc. [Conclusion] McrA is a global transcriptional regulator, and the knockout and overexpression of its gene will affect carbohydrate, lipid, and amino acid-related metabolic pathways, thus affecting the production of secondary metabolites.

[Objective] To study the phylogenetic relationship and genomic diversity of intestinal obligate commensal bacteria in different populations from various regions of Xinjiang and provide a theoretical basis for developing personalized functional probiotics for different populations. [Methods] A total of 136 strains of Bifidobacterium longum subsp. longum were isolated from mother-infant populations of Uygur and Kazak ethnic groups in Kashgar and Yili regions of Xinjiang. Comparative genomic analysis was conducted with data of the strains from other regions in China that were available in public databases. [Results] The average genome size, G+C content, and the number of coding sequences of B. longum subsp. longum were 2.38 Mb, 59.91%, and 2 160, respectively. The phylogenetic tree constructed based on core genes showed that all strains from Xinjiang belonged to four clades in the phylogenetic tree. Strains from the same ethnic group but from different geographical regions were in different clades, and there was a certain degree of overlap between geographically closer and different population-derived strains. The analysis of a larger geographical range (China) showed that B. longum subsp. longum strains and their functional genes presented obvious geographical and ethnic distribution characteristics. The analysis of COG functional genes and carbohydrate hydrolyase-related genes showed that the functional gene spectra varied greatly among strains from the same ethnic group but in different regions. The carbohydrate hydrolyase-related gene families GH13 (α-amylases) and GH43 (β-amylases) were more abundant in the strains from Kashgar region. Conversely, even strains from different ethnic groups but from geographically close regions had similar spectra of COG functional genes and carbohydrate hydrolyase-related gene families. [Conclusion] The B. longum subsp. longum strains and their functional genes from different geographical regions and ethnic groups in Xinjiang showed obvious geographical and ethnic distribution characteristics. As the geographical scale becomes large, the geographical distribution characteristics of the strains become more obvious. The relationship between the geographical distribution scale of populations and the co-evolution and specificity of strains should be verified based on larger-scale genomic data of strains.

[Objective] Kiwifruit bacterial canker (KBC), caused by Pseudomonassyringae pv. actinidiae (Psa), has become the primary bottleneck restricting the sustainable development of the kiwifruit industry in China, highlighting an urgent need for eco-friendly and residue-free biocontrol strategies. [Methods] The kiwifruit variety ‘Hongyang’ was used to systematically evaluate the biocontrol efficacy of Bacillus pumilus H-46 through leaf disc and shoot inoculation assays. The active components were fractionated into three groups (small-molecule metabolites, proteins, and polysaccharides) via sequential extraction, with the major bioactive fraction identified through antimicrobial activity and disease control assessments. An integrated approach combining histochemical staining, antioxidant enzyme activity assays, and RT-qPCR of defense-related genes was employed to elucidate the mechanism of induced systemic resistance (ISR). Furthermore, pathogen migration and colonization assays were conducted to evaluate the inhibitory effects of the active components against Psa. [Results] B. pumilus H-46 showed excellent control effect against KBC, with the disease control efficacy of 86.54% in leaf disc assays. Its main active components (small-molecule metabolites, fraction A) achieved the control efficacy of 88.16% against KBC through non-antimicrobial mechanisms. The mechanisms included triggering early H₂O₂ accumulation and callose deposition, significantly increasing superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities, and activating the expression of key genes (AcMYC2, AcAOC, AcERF2, and AcEIN3) in jasmonicacid (JA) and ethylene (ET) signaling pathways to activate ISR. Consequently, the disease resistance of kiwifruit was enhanced, resulting in a reduction of 37.8% in Psa migration distance in leaf veins and a decrease of 96.6% in colonization ability. [Conclusion] Our findings demonstrate that the small-molecule metabolites (fraction A) of B. pumilus H-46 activates JA/ET-mediated ISR via non-antimicrobial mechanisms, offering a sustainable solution for the control against KBC and establishing a prototype for next-generation plant immunity activators in crop protection.