Abstract:Proper pH is crucial for the survival and functions of microorganisms, whether in the environment or within cells. Under acidic or alkaline stress, microorganisms have evolved diverse strategies, such as proton transport, production of acidic or alkaline substances, and cell membrane protection, to maintain intracellular pH homeostasis. Moreover, microorganisms have evolved the ability to actively change the extracellular pH. This article reviews the mechanisms by which microorganisms maintain intracellular pH homeostasis under acid or alkaline stress and alter extracellular pH. It aims to enhance our understanding of the interaction between microorganisms and the environment and provide a reference for further research on the synergistic mechanisms between microorganisms and the environment.

Abstract:Fatty acids are not only the components of cell membrane but also the raw materials for the synthesis of bioactive molecules. Unsaturated fatty acids (UFAs) with low phase-transition temperatures are essential molecules for bacteria to regulate cell membrane fluidity. Therefore, the synthetic pathways of UFAs are key targets for the screening of antibacterial agents. Bacteria can adopt the anaerobic pathway to synthesize UFAs. For example, Escherichia coli, a model organism, synthesizes UFAs via the classic FabA-FabB pathway. However, the anaerobic pathways for the synthesis of UFAs vary in different bacteria, and the catalytic enzymes are also different. Bacteria can synthesize UFAs via aerobic pathways, in which fatty acid desaturase directly converts saturated fatty acids (SFAs) into UFAs. Different desaturases introduce double bond to form UFAs with different structures, which play roles in stress responses, pathogenicity and other aspects. Other enzymes involved in the synthesis of fatty acids can also participate in the synthesis of UFAs or regulation of different UFAs. Some bacteria can use monooxygenase to convert capryl-ACP (acyl carrier protein) in the fatty acid synthesis pathway into cis-3-decenyl ACP to synthesize UFAs. We comprehensively reviewed the research progress in the synthesis of UFAs in bacteria, aiming to provide theoretical support for deciphering the mechanism of bacterial synthesis of UFAs and developing the targeted antibacterial drugs.

Abstract:The α-herpesviruses are a large class of enveloped double-stranded DNA viruses characterized by neurotropic infection and latent infection, posing a serious threat to human and animal health. The α-herpesvirus genome encodes a variety of proteins. UL24, a major virulence gene of α-herpesviruses, encodes a highly conserved protein and play a key role in regulating viral infection. This paper introduced the basic characteristics of UL24 and the encoded protein and summarized the regulatory roles of UL24 in the virus assembly, replication, infection, pathogenicity, and inhibition of host innate immunity, aiming to provide a theoretical reference for understanding the functions of α-herpesvirus proteins and further preventing and controlling α-herpesvirus infection.

Abstract:Retinoid acid-inducible gene-I-like receptor (RLR) signaling pathways, the immune signaling pathways in response to infections, play a regulatory role in the production of pro-inflammatory cytokines, chemokines, and type I interferons. Ubiquitination as one of the post-translational modifications refers to the process of ubiquitin binding to different amino acid sites on the target proteins, which regulates the fates of proteins. For example, it initiates the proteasome pathway to degrade the target protein or activating the protein transport. The ubiquitination of RLR signaling pathways is a way of regulating multiple effectors and one of the classical pathways through which viruses induce major diseases in animals, autoimmune diseases, and chronic inflammation. This paper introduces the typical structural features and the ubiquitination types of key effectors in the RLR signaling pathways. Furthermore, it expounds the roles of ubiquitination in the regulation of key molecules in the RLR signaling pathways, aiming to provide a reference for the intervention or treatment of related diseases.

Abstract:With the evolution of bacteria and the overuse of certain antibiotics, drug-resistant bacterial infections have emerged as a major public health challenge in the 21st century. Klebsiella pneumoniae, in particular, has aroused wide concern due to its drug-resistant nature. Bacteriophages have demonstrated the potential and advantages in treating bacterial infections caused by drug-resistant strains, while there are no clinical guidelines for phage therapy. Notably, even though a few successful cases of bacteriophage therapy have been documented in the treatment of K. pneumoniae infections, bacteriophages are used in combination with antibiotics in most cases and the role bacteriophages play remains unclear. This article provides an overview of bacteriophage therapies for K. pneumoniae infections, including their characteristics and the factors influencing their efficacy. We compiled and analyzed data from available studies, with the intention of offering valuable insights for the application of bacteriophage therapy in combating K. pneumoniae and other drug-resistant bacteria.

Abstract:The term “microorganism” refers to tiny organisms such as archaea, bacteria, protists, fungi, and viruses, and the term “microbiome” refers to a collection of microorganisms. Although they share the body space of the host, their roles as determinants in host health and diseases are ignored. As a collection of information, the microbiome includes the genomic data, structural elements, metabolites, and environmental conditions of microorganisms. Studies have demonstrated that the microbiome plays an essential role in maintaining host homeostasis and regulating host phenotypes. With the advent of new technologies, including next-generation sequencing (NGS) and sequencing-based microbiome profiling, researchers have probed into the relationship between the microbiome and host phenotypes. By an overview of microbiome, this paper elaborated on the microbiome-host genetics interactions based on genome-wide association analysis and made an outlook on the future of this field.

Abstract:Inhibiting the synthesis of fungal cell wall is a safe and effective strategy for preventing and treating fungal infections. Chitin synthases are the key enzymes to catalyze the synthesis of chitin, an important structural component of fungal cell wall and septa. The roles of chitin synthases vary in regulating the synthesis of fungal chitin. This review outlines the roles of chitin synthases in regulating fungal cell proliferation, morphogenesis, interactions with hosts, and compensatory effect induced by cell wall damage in the three major pathogenic fungal species: Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans, aiming to understand the importance of chitin synthases in fungal pathogenicity. Furthermore, this paper proposes a new antifungal strategy and the future research directions about the chitin synthases of fungi.

Abstract:[Objective] Bacillus thuringiensis (Bt), characterized by the massive production of insecticidal crystal proteins (ICPs) during sporulation, serves as the main strain resource for the commonly used and safe microbial insecticides. To further explore the mechanisms of sporulation and parasporal crystal formation and lay a theoretical foundation for the construction of efficient strains, we compared the transcriptomes of Bt at three important stages. [Methods] The transcriptomes of the hypervirulent strain Bt4.0718 at the middle vegetative growth stage (T1-10 h), the early sporulation stage (T2-20 h), and the late sporulation stage (T3-32 h) were compared. The representative differentially expressed genes (DEGs) were verified by real-time fluorescence quantitative PCR (qRT-PCR), and the phenotypes of the mutant strains with the knockout of specific functional genes were examined. [Results] The number of DEGs was 2 147 (T2/T1), 1 861 (T3/T1), and 1 708 (T3/T2), respectively. At T1, the medium was rich in nutrients, which served the sporulation and parasporal crystal formation. The high transcription levels of kinA/D, spo0A/F, and sigE regulating sporulation played a role in the growth and development of the cells. The transcription of Cry1Ac, poly-hydroxybutyric acid (PHB), and hydroxybutanone (acetoin) were started at this time. The substantial formation of ICPs and spores occurred at T2 and T3, and the transcript levels of the regulatory genes were higher at T2 than those at T3. The genes associated with spore core/coat/cortex, germination protein, and spoII–spoVI began to be transcribed in large amounts at T2, with the highest levels among the three stages. The corresponding complex networks of carbohydrate, amino acid, and lipid metabolism, energy, nucleic acid, and peptide metabolism, secondary metabolite production, and environmental adaptation showed differences. In addition, as the physiological processes stimulated by nutrient signals, the two-component signal transduction system (TCS) and ABC transport system played an essential role in the process of sporulation and ICP transcription and expression, and their transcription levels were significantly different. [Conclusion] With the production of ICPs and sporulation, nutrients are gradually consumed, and the high expression of sigB, sigW, and sigM contributed to the stability of cell wall and the resistance to environmental changes. Meanwhile, the small heat shock proteins Hsp20 and Hsp20B, as molecular chaperones, were also important for maintaining intracellular homeostasis and may facilitate the sporulation and ICP production.

Abstract:[Objective] To investigate the regulatory role of the transcription factor BldM in the morphological development and antibiotic synthesis of Streptomyces pactum Act12, a biocontrol strain with multiple effects. [Methods] The bldM-deleted mutant strain ∆bldM and the bldM-overexpressing mutant strain OE-bldM were constructed by genetic engineering. The scanning electron microscopy, antibacterial experiment, high performance liquid chromatography, and real-time quantitative PCR were employed to compare the morphological development, growth rate, oligomycin yield, and resistance to pathogens, respectively, between ∆bldM, OE-bldM, and the wild-type strain Act12. [Results] The sequencing results proved that ∆bldM and OE-bldM were successfully constructed. ∆bldM showed significantly reduced production of oligomycin D and was incapable of forming aerial hyphae. OE-bldM presented dense aerial hyphae and active sporulation. Compared with the wild type, OE-bldM showed an increase of 23% in the yield of oligomycin D and the up-regulation of 2–3 times in the transcriptional levels of the genes encoding oligomycin core synthetase. Moreover, the antimicrobial activity of OE-bldM remarkably enhanced. [Conclusion] The global transcriptional regulator BldM can not only affect the formation of aerial hyphae and sporulation but also participate in the positive regulation of oligomycin synthesis in Act12. The results of this study supplement the knowledge about the regulatory function of BldM and provide a reference for further research on the growth, metabolism, and regulation mechanism of S. pactum Act12.

Abstract:[Objective] To explore the reasons for differences in the C-methylation programming of non-reducing polyketide synthases (NR-Pkss). [Methods] We used bioinformatics tools and AlphaFold 2 to compare the domain sequences and structures of the NR-Pkss involved in the synthesis of Monascus pigment and citrinin in Monascus ruber M7, i.e., Mr-PksPT and Mr-PksCT. Furthermore, we employed molecular docking to compare the binding of C-methyltransferase domains (CMeTs) with other domains and the intermediates of the two NR-Pkss. [Results] The large differences of the overall structure and the high similarity of domain sequence and structure between the two NR-Pkss suggested that the differences of C-methylation programming between NR-Pkss may be resulted from domain interactions. The CMeT of Mr-PksCT was more likely to bind to the acyl carrier protein (ACP) carrying the substrate than that of Mr-PksPT, making the intermediate more easily catalyzed by CMeT. Moreover, CMeT had lower binding free energy to methyl receptor substrate than the β-ketosynthase domain (KS). [Conclusion] The CMeTs of NR-Pkss can affect the C-methylation of the products by competing with KS. The findings provide a new idea for the study of C-methylation programming of Pkss.

Abstract:[Objective] To screen out a fungal strain that can efficiently assimilate ammonia nitrogen, reveal the metabolome differences of the strain in different media and the changes in the amino acid content of the feed fermented with the strain, and clarify the mechanism of its ammonia assimilation. [Methods] Seven strains of Trichoderma, 7 strains of Aspergillus niger, and 9 strains of Aspergillus oryzae were cultured in the media with (NH₄)₂SO₄ as the only nitrogen source. The strains with high ammonia nitrogen use efficiency and glutamine synthetase (GS) activity were selected for comparison of the metabolic differences in potato dextrose agar (PDA) plates and inorganic nitrogen plates by non-targeted metabonomics. Furthermore, the crude protein and organic nitrogen content in the feed fermented with different strains was determined, and the changes in amino acid content in the fermented feed extract were measured by amino acid-targeted metabolomics. [Results] The utilization rate of ammonia nitrogen and the glutamine synthetase activity of A. oryzae MQ28 were 54.46% and 0.61 μmol/(h·g), respectively, which were higher than those of other strains (P<0.05). The comparative metabonomics analysis suggested that MQ28 was associated with the metabolism of multiple amino acids during ammonia assimilation. MQ28 fermentation increased the crude protein and organic nitrogen in the feed by 22.25% and 35.83% (P<0.05), respectively. Furthermore, MQ28 fermentation increased the total amino acid content in feed extract from 31.86 mmol/100 g to 57.69 mmol/100 g (P<0.05). Specifically, it increased the content of 14 amino acids such as threonine, lysine, and arginine, glutamic acid (by 3.46 folds), and glutamine (by 99 folds) (P<0.05). [Conclusion] To sum up, A. oryzae MQ28 has high ammonia nitrogen utilization capacity. It may regulate the ammonia assimilation process through the synthesis of glutamine to regulate amino acid metabolism and can be used as an elite strain for the production of single-cell protein.

Abstract:[Objective] To reveal the mechanisms of Pseudomonas putida Y-9 in actively regulating the extracellular and intracellular pH homeostasis during ammonia oxidation. [Methods] Y-9 was cultured in the nitrification media with initial pH 7.19 and 9.40, respectively, for 48 h. Metabolomics was employed to compare the differential metabolites and predict dissociation constant (pKa) during the ammonia oxidation. Transcriptomics was employed to compare the genes regulating. [Results] In the medium with initial pH 7.19, Y-9 produced maltitol to raise extracellular pH, and up-regulated the expression of the genes related to deaminase, deiminase, and cation transport to maintain intracellular pH stability. In the medium with initial pH 9.40, Y-9 produced acidic substances such as 5-aminovaleric acid 3 and oxamic acid to lower extracellular pH and regulated the expression of the genes associated with NADH dehydrogenase, cytochromes, ATP synthase, and amino acid transport to maintain intracellular acidity. [Conclusion] This study revealed the novel phenomenon of Y-9's extracellular pH stabilizing capacity and investigated its intracellular pH homeostasis mechanism. The findings enrich our knowledge about microorganism-environment interactions, and provide a theoretical basis for further understanding the pH stabilization mechanism in microbial denitrification processes.

Abstract:[Objective] Cyanophages, the viruses specifically infecting cyanobacteria, are ubiquitous in water environments. They play a role in regulating the population dynamics and density of cyanobacteria and promote the biogeochemical cycling of the aquatic ecosystem. This study aims to isolate and identify a cyanophage. [Methods] A novel cyanophage Yong-L2-223 was isolated from fresh water samples with marine Synechococcus sp. PCC 7002 as the indicator host. The host range, genome sequence, open reading frames (ORFs), and phylogenetic relationship of Yong-L2-223 were studied. [Results] The host range tests against 31 strains of cyanobacteria showed that Yong-L2-223 could infect the indicator host PCC 7002 (Synechococcales) and two freshwater strains Microcystis viridis FACHB-1342 (Chroococcales) and Aphanizomenon flos-aquae FACHB-1209 (Nostocales) from the Dianchi Lake. The infection of the cyanobacterial strains from both the seawater and freshwater samples indicated that Yong-L2-223 was a euryhaline cyanophage. Yong-L2-223 was myovirus-like, consisting of an icosahedral head (about 60 nm in diameter) and a contractile tail (about 136 nm in length). The genome (double-stranded DNA) of Yong-L2-223 had a length of 65 725 bp, with the G+C content of 58.6% and 100 ORFs. It was predicted to carry the Cas4 gene, gene transfer factor (GTA) gene, auxiliary metabolic genes (AMGs), and a gene cluster for the synthesis of pre-Q0. These genes may contribute to the adaptation and infection of the cyanophage in cyanobacteria of three orders. The pairwise sequence comparison (PASC) illustrated that the highest similarity sharing by cyanophage Yong-L2-223 and all the viruses in the current genome databases was only 3.78%, far below the genus boundary cut-off of 70% defined by the International Committee on Taxonomy of Viruses. In the phylogenetic tree based on the whole proteomes, Yong-L2-223 formed an independent branch, with long evolutionary distances from other phages. [Conclusion] Yong-L2-223 is a new genus of the Caudoviricetes class. For the first time, we used a marine cyanobacterial strain as the indicator host to isolate and obtain a novel cyanophage from freshwater, which broadened the understanding of cyanophages, enriched cyanophage genome database, and laid a foundation for the development of cyanophage resources.

Abstract:Gray mold caused by Botrytis cinerea is one of the major diseases affecting tomato production. The currently used fungicides are gradually restricted due to residues, pathogen resistance, and food safety. Therefore, screening out antagonistic microorganisms has gradually become an effective approach for the biocontrol of gray mold. [Objective] We screened out endophytic strains capable of endowing plants with disease resistance and promoting plant growth from tomato plants and evaluated their biocontrol potential, aiming to provide a theoretical basis for developing a new approach for the biocontrol of tomato gray mold. [Methods] The endophytic bacteria and fungi were isolated from different parts of tomato plants by the tissue culture method, and the candidate strains were preliminarily identified by 16S rRNA and ITS sequence analysis. The endophytes with antagonistic activity against B. cinerea were screened by confrontation culture and fruit inoculation in vitro. Furthermore, we examined the abilities of the strain to secrete indole-3 acetic acid (IAA), protease, and siderophores and the promoting effects on the growth of Arabidopsis thaliana and tomato seedlings.[Results] A total of 72 endophytic bacterial strains and 31 endophytic fungal strains were isolated from different parts of tomato plants. An endophytic bacterium FQ-G3 with strong inhibitory activities against several pathogens was screened out and was identified as Bacillus velezensis. FQ-G3 showed the inhibition rate of 80.93% against B. cinerea in vitro and inhibited the mycelial expansion on tomato fruits in vivo. The strain could secrete IAA, protease, and siderophores, and promote the growth of A. thaliana and tomato seedlings. [Conclusion] The endophytic strain FQ-G3 isolated from tomato plants endows plants with disease resistance and promotes plant growth, and thus can serve as a candidate for the prevention and control of gray mold. The findings enrich the tomato endophyte resources and provide support for the control of gray mold and the growth promotion of tomato.

Abstract:[Objective] Bacillus velezensis SH-1471 (CCTCC No. M 2022923, Patent No. ZL 2022 1 1479280.X) is a strain that can control soil-borne diseases of crops and promote soil nutrient conversion and crop growth. This study aims to explore its potential biological activity and the optimum fermentation conditions, so as to promote the industrialization and commercial development of this strain.[Methods] A strain SH-1471 was identified based on the morphological, physiological, and biochemical characteristics and the 16S rRNA gene and gyrB-based phylogenetic trees. PCR was carried out to detect antibiotic synthesis genes in the strain. The inhibitory spectrum of the strain was measured by the plate confrontation assay and the fermentation liquid inhibition test. The abilities of the strain to produce enzymes, solubilize phosphorus and potassium, fix nitrogen, and secrete siderophores in vitro were measured. The fermentation conditions were optimized by single factor tests and response surface methodology with OD₆₀₀ value and inhibition rate as indicators. The growth-promoting effect of the fermentation liquid on tomato plants and the control effect of the fermentation liquid on tomato Fusarium wilt before and after optimization were determined by indoor pot experiments. [Results] Strain SH-1471 was identified as B. velezensis, carrying the antibiotic synthesis genes srfA, fenB, ituA, ituD, and bymA. It had strong antagonistic effects on 8 pathogenic microorganisms such as Fusarium oxysporum, Alternaria alternate, and Exserohilum turcicum. Moreover, the strain was capable of producing protease and cellulase, solubilizing phosphorus, fixing nitrogen, and secreting siderophores. The optimal medium formula for the fermentation of SH-1471 was composed of 17.92 g/L sucrose, 16.95 g/L soybean powder, 2.88 g/L magnesium sulfate, and 5.0 g/L yeast extract. The optimum fermentation conditions were pH 7.5, 33 ℃, 220 r/min, and 20–24 h. After optimization, the inhibition rate, OD₆₀₀, and potted control efficiency of the fermentation liquid reached 94.08% (increasing by 15.6%), 3.28 (increasing by 36.7%), and 93.8%, respectively. Moreover, strain SH-1471 significantly improved plant height, stem circumference, root length, root weight, and fresh weight and dry weight of the aboveground part of tomato seedlings. [Conclusion] B. velezensis SH-1471 carries rich genes for antibiotic synthesis, has the abilities of producing protease and cellulase, solubilizing phosphorus, fixing nitrogen, and secreting siderophores, and demonstrates strong inhibitory effects on a variety of pathogens. It can significantly reduce the incidence of tomato Fusarium wilt and improve the agronomic traits of tomato seedlings. Therefore, the strain has broad application prospects in the biocontrol of plant diseases and the promotion of plant growth.

Abstract:[Objective] This study aims to observe the changes of the fecal microbial communities in the dairy cows with subacute rumen acidosis (SARA) induced by a high-concentrate diet and modulated by the addition of vitamin E (VE). The potential effects on the metabolism of dairy cows were evaluated to provide data for exploring the physiological mechanisms of SARA. [Methods] Seven multiparous Holstein cows with rumen fistulas were selected for this trial which was carried out in three phases of 18 days each. The first phase was the control (CON) phase, with a concentrate-to-forage ratio of 50:50 in the diet (dry matter basis). The second phase was the induction (HG) phase, in which the forage was replaced with wheat flour at 15% of the diet (dry matter basis) to induce SARA. The third phase was the regulation (HGE) phase, in which VE was added at 12 000 IU/d/cow on the basis of the diet in the HG phase. The feces samples were collected on day 18 in each phase, and the microbial communities in the samples were examined. [Results] The fecal microbial community structure showed significant differences between three phases and the Shannon index in the HG phase was lower than that in the CON phase (P<0.05). The HG phase had higher relative abundance of Proteobacteria and Blautia and lower relative abundance of unidentified_bacteria, Euryarchaeota, Desulfobacterota, Rikenellaceae_RC9_gut_group, and Alistipes than the CON phase (P<0.05). The relative abundance of Blautia in the HGE phase was higher than that in the HG phase (P<0.05). The functional prediction results showed that SARA caused metabolic disorders in the dairy cows, while VE regulated the intestinal microbiota and health by improving the stability of microbial growth and promoting microbial reproduction. [Conclusion] The SARA induced by a high-concentrate diet led to reduced intestinal microbial diversity and metabolic disorders in dairy cows. VE can regulate intestinal health and maintain intestinal homeostasis by promoting the proliferation of beneficial intestinal microorganisms.

Abstract:[Objective] This study aims to improve the bactericidal ability of phages against carbapenem-resistant Klebsiella pneumoniae, a major pathogen in clinical practice, and reduce host resistance to phages by developing a novel method of phage training. [Methods] Phages were isolated from municipal wastewater by the double-layer agar plate method with clinical carbapenem-resistant K. pneumoniae strain Kp2092 as the host, and their host ranges were analyzed. The morphological and genetic characteristics of a phage strain with strong lysis ability and a broad host range were analyzed by transmission electron microscopy and whole-genome sequencing. Phage training was performed by phage-host co-culture for generations, and the phages before and after training were compared in terms of biological properties such as bactericidal activity, optimal multiplicity of infection, one-step growth curve, and stability under different stress conditions. [Results] A total of 9 phage strains were isolated, in which P55anc was a short-tailed phage with the strongest lysis ability and the broadest host range. The genome (40 301 bp) of P55anc included 51 coding sequences, of which 27 showed possible functions involving nucleic acid metabolism, virion morphogenesis, DNA packaging, and host lysis. Three evolved phages of P55anc were obtained after 9 days of phage training. These evolved phages showed significantly enhanced bactericidal activities, manifested by the increased burst size, broadened host range, and reduced host resistance. Moreover, the evolved phages maintained stable when being exposed to heat, ultraviolet, and serum treatments. [Conclusion] Phage training by phage-host co-culture can be employed to obtain evolved phages with enhanced bactericidal effects. Furthermore, the evolved phages reduced the host resistance and remained stable under different stress conditions.

Abstract:[Objective] Streptomyces is a genus of Gram-positive aerobic bacteria characterized by complex morphological differentiation and potent secondary metabolite-producing ability. SapB, a class III lanthipeptide, promotes the morphological differentiation of Streptomyces coelicolor, which suggests that SapB-like peptides might be developed as targets for engineering of morphological differentiation. In this study, we characterized the effects of SapB-like peptides on the morphological differentiation of multiple Streptomyces species, aiming to provide a theoretical basis for the engineering of these peptides. [Methods] Bioinformatics tools were used to analyze the gene clusters for the synthesis of SapB-like peptides in the genomes of Streptomyces spp.. The plasmids for heterologous expression were constructed and introduced into Streptomyces spp. through conjugation. The colony and mycelial morphology were compared to reveal the effects of these peptides on the morphological differentiation of Streptomyces. [Results] SapB-like peptides promoted the differentiation of Streptomyces from vegetative to aerial mycelia. Specifically, they increased the aerial mycelia and accelerated the differentiation, thus shortening the morphological differentiation cycle. [Conclusion] SapB-like peptides can help shorten the morphological differentiation cycle of Streptomyces, demonstrating the potential for the morphological differentiation engineering of Streptomyces.

Abstract:[Objective] To mitigate the threat of heavy metal pollution in wastewater to global food safety and human health, reduce the accumulation of lead (Pb) in soil, plants, and animals, and improve the removal rate of heavy metals by immobilizing microbial strains. [Methods] We carried out mixed strain test to select the white rot fungal strains with strong Pb²⁺ removal effects and excellent compatibility and explored the optimal strains and ratio for combination. Furthermore, we optimized the formula of the fungal strain composite and explored the optimal adsorption conditions of the composite in application. [Results] Phanerochaete chrysosporium, Coriolus versicolor, Lentinus sajor-caju, and Pleurotus ostreatus with good compatibility were selected for subsequent experiments. C. versicolor mixed with L. sajor-caju at a volume ratio of 1:1 outperformed the single strains in removing Pb²⁺. The fungal strain composite composed of 20.0 g/L sodium alginate, 15.0 g/L biochar, 2.0×10₆ CFU/mL white rot fungi, silica, and zeolite showed the Pb²⁺ removal rate of 90.63% within 96 h. Moreover, this composite had higher mechanical strength and strong resistance to mechanical shear. At the addition amount of 8.35 g/L and pH 5.64, the composite demonstrated the Pb²⁺ removal rate of 97.45% within 96 h. Moreover, this composite can be reused 7 times after adsorption-desorption-readsorption and maintained high Pb²⁺ removal capacity. [Conclusion] The immobilized white rot fungal strain composite can significantly improve the microbial utilization rate and wastewater treatment efficiency compared with single strains. It can greatly adsorb Pb²⁺ in wastewater under appropriate conditions within a short time and reduce the environmental threat caused by heavy metal pollutants. Therefore, the promotion of environmental protection greatly benefits from the use of immobilized mixed white rot fungal strains in the treatment of heavy metal-contaminated wastewater.

Abstract:[Objective] To investigate Bacillus velezensis XRD006 in terms of the inhibitory effects on diseases of postharvest green walnuts, the fresh-keeping effect on green walnuts, genetic characteristics, secondary metabolites, and antifungal mechanism. [Methods] The activities of XRD006 against pathogens of postharvest green walnuts were determined by the inhibition experiments. In vivo fungal inhibition and storage quality experiments were conducted to investigate the inhibitory effects of the strain on pathogens and the effect of the strain on the storage quality of postharvest green walnuts. The genomic characteristics and potential antifungal genes of XRD006 were investigated by whole genome sequencing. The secondary metabolites of XRD006 were predicted by antiSMASH, and the collinearity and differences of the secondary metabolite gene clusters between strains XRD006, FZB42, and SQR9 were analyzed by comparative genomics. The secondary metabolites of XRD006 were identified by high performance liquid chromatography (HPLC) and mass spectrometry. The antagonistic ability of the strain was tested by the oxford cup method. [Results] The inhibition rates of XRD006 against Colletotrichum aenigma, Colletotrichum siamense, Botryosphaeria dothidea, and Fusarium fujikuroi of postharvest green walnuts were 49.22%, 50.61%, 53.83%, and 58.71%, respectively. In vivo antifungal experiments showed that XRD006 effectively inhibited the infection and growth of pathogenic fungi on the fruits. The fermentation supernatant of XRD006 significantly retarded the weight loss, inhibited the microbial growth, and reduced changes in peroxidase (POD) and polyphenol oxidase (PPO) activities while maintaining the kernel quality. The whole genome sequencing showed that the genome of XRD006 was 4 371 975 bp in length, containing 46.07% GC and 4 362 protein-coding genes (including antifungal and plant growth-promoting genes such as extracellular hydrolase and biofilm genes). The antiSMASH predicted that XRD006 had nine known and five unknown gene clusters of secondary metabolites. XRD006 was closely related to FZB42 and SQR9, and they shared eight secondary metabolite gene clusters, which showed varied location and coding genes. XRD006 produced two families of lipopeptides: iturin and fengycin. Compared with C13-iturin, C14-iturin, C15-iturin, and C17-fengycin C, C16-fengycin B of the fengycin family had the strongest inhibitory effect on C. siamense HT12. [Conclusion] B. velezensis XRD006 has good biocontrol effects on diseases of postharvest green walnuts and the potential for application in production.