Microbial network and composition changes according to tobacco varieties and interferes differently in black shank disease defense.

AIMS: The soil-borne oomycete pathogen Phytophthora parasitica can cause black shank disease in tobacco plants. The use of resistant varieties can be used to control black shank disease. The potential relationships of the composition of the rhizosphere microbiome to resistance to black shank disease are poorly understood. This work aims to compare the rhizosphere microbial community and network of the tobacco resistant variety HB202 with the susceptible variety XY3. METHODS AND RESULTS: Rhizospheric soils were collected from tobacco plants of HB202 and XY3 in the fields with same soil types and agricultural operations. The compositions of the rhizosphere microbial communities were revealed by Illumina sequencing of bacterial 16S rRNA genes and fungal spacer (ITS) sequences and analysed with molecular ecological network pipeline. The alpha diversity of fungal communities of the two varieties was significantly different. The structure and composition of bacterial and fungal communities in the resistant variety in the rhizosphere was different from the susceptible variety. Relative abundances of beneficial genera in the HB202 microbiota were higher than in the XY3. Conversely, the XY3 microbiota exhibited a higher abundance of deleterious genera compared to the HB202 microbiota. The resistant variety influences the topological properties and microbial interactions in the rhizosphere against the disease. The network of the HB202 was more complex and had higher connectivity compared to the XY3 network. CONCLUSIONS: The rhizosphere microbial communities and networks of two tobacco varieties are very different. These changes in the microbial communities and their interactions may play an important role in tobacco resistance to black shank disease.

Application of nematicide avermectin enriched antibiotic-resistant bacteria and antibiotic resistance genes in farmland soil.

The extensive use of antibiotics in medicine and agriculture has resulted in the accumulation of antibiotic-resistant microorganisms and antibiotic resistance genes (ARGs) in environments, which threaten human health and contaminate environment. Nematicide avermectin is widely applied to control root-knot nematodes. The effect of five-years application of avermectin on rhizosphere microbiome and resistome of sick tobacco plants in farmland were investigated in present study. The environmental risks of avermectin was assessed adequately. Metagenomic method was used to analyze antibiotic-resistant bacteria and antibiotic resistance genes in the avermectin-treated soil. The abundance and distribution of antibiotic-resistant bacteria and their antibiotic resistance genes were affected by avermectin application. The antibiotic resistant Proteobacteria occupied the highest percentage (36%) in rhizosphere soil and carried 530 ARGs. Opportunistic human pathogens carrying antibiotic resistance genes were enriched in the avermectin-treated soil. Avermectin application increased the counts of many types of antibiotic resistance genes. The relative abundances of genes adeF, BahA, fusH, ileS, and tlrB in the avermectin-treated soil were significantly greater than in the untreated control soil. Different resistance mechanisms were revealed in the avermectin-treated soil. The efflux of antibiotic (670 ARGs), inactivation of antibiotic (475 ARGs), and alteration of antibiotic target (267 ARGs) were the main resistance mechanisms. Rigid control the avermectin dose and use frequency and other pesticides can decrease soil antibiotic resistance genes and protect agricultural products' safety and public health. Overall, application of nematicide avermectin enriched antibiotic-resistant bacteria and antibiotic resistance genes in farmland soil, which should be on the alert for environment protection.

The cellular redox state in Bacillus amyloliquefaciens WH1 affects biofilm formation indirectly in a surfactant direct manner.

Surfactin is a signal to trigger biofilm formation against harsh environments. Generally, harsh environments can result in change of the cellular redox state to induce biofilm formation, but we know little about whether the cellular redox state influences biofilm formation via surfactin. Here, the reductant glucose could reduce surfactin and enhance biofilm formation by a surfactin-indirect way. The oxidant H2 O2 led to a decrease of surfactin accompanying with weakened biofilm formation. Spx and PerR were both necessary for surfactin production and biofilm formation. H2 O2 improved surfactin production but inhibited biofilm formation by a surfactin-indirect manner in Δspx, while it reduced surfactin production without obvious influence on biofilm formation in ΔperR. The ability against H2 O2 stress was enhanced in Δspx, but weakened in ΔperR. Thereby, PerR was favorable for resisting oxidative stress, while Spx played a negative role in this action. Knockout and compensation of rex also supported that the cells could form biofilm by a surfactin-indirect way. Collectively, surfactin is not a unique signal to trigger biofilm formation, and the cellular redox state can influence biofilm formation by a surfactin-direct or -indirect way in Bacillus amyloliquefaciens WH1.

Plant growth-promoting and antibacterial activities of cultivable bacteria alive in tobacco field against Ralstonia solanacearum.

Bacterial wilt disease caused by Ralstonia solanacearum leads to decrease of crops yield. Investigation of cultivable bacteria diversity provides more microbial species for screening antagonistic bacteria. In the present study, a variety of cultivation methods were used to investigate the diversity of cultivable bacteria alive in tobacco field. A total of 441 bacterial strains were obtained that belonged to four phyla, 49 genera and 146 species. Actinobacteria and Proteobacteria were the dominant phyla. Agrobacterium, Arthrobacter, Bacillus, Klebsiella, Paenarthrobacter, Pseudomonas and Pseudarthrobacter were the dominant genera. Some rare genera were discovered including Bosea, Cedecea, Delftia and Dyella. Diversity, species and abundances of bacteria altered under different cultivation conditions. One hundred three bacterial strains showed plant growth-promoting attributes. Twenty Bacillus strains showed high antibacterial activity against R. solanacearum. In field experiments, individual strain and consortia of Bacillus subtilis, B. siamensis and B. vallismortis effectively inhibited bacterial wilt. The core genes that controlled synthesis of secondary metabolites were knocked out in B. vallismortis SSB-10. Difficidin, which was synthesized by dif operon and controlled by sfp gene, was the antibacterial substance produced by SSB-10. Difficidin destroyed cell wall and cell membrane of R. solanacearum and inhibited its motility, production of extracellular polysaccharides and cellulase activity.

Cations and surfactin serving as signal molecules trigger quorum sensing in Bacillus amyloliquefaciens.

Microorganisms including Bacillus can produce signal molecules such as surfactin, resulting in the variation of membrane potential to trigger quorum sensing such as biofilm formation and sporulation in response to the environment stresses. However, biosynthesis of surfactin requires multiple resources such as huge enzyme complex, amino acids, fatty acids, and energy. Insufficient resources in the natural soil environment restrain biosynthesis of surfactin. When surfactin is inadequate, cations in soil might serve as substitutes to regulate quorum sensing. Our results showed that both surfactin and cations could lead to the variation of membrane potential, thus providing signals to trigger the quorum sensing such as growth, biofilm formation, and sporulation in Bacillus amyloliquefaciens. Neither KinC nor Abh was essential for surfactin or cations to trigger quorum sensing. The cation signaling pathway is only partially dependent on Spo0A, but the surfactin signaling pathway is fully dependent on this global regulator. Compared to surfactin, cations are less effective in promoting biofilm formation, but more effective to trigger sporulation in B. amyloliquefaciens. This study reveals a pathway through which cations regulate the quorum sensing in B. amyloliquefaciens in the case of insufficient surfactin in environment.

Protecting tobacco plants from O3 injury by Bacillus velezensis with production of acetoin.

Plant growth-promoting rhizobacteria (PGPRs) confer benefits to crops by producing volatile organic compounds (VOCs) to trigger induced systemic tolerance (IST). Here we show that Bacillus velezensis GJ11, a kind of PGPRs, produce VOCs such as 2,3-butanediol and acetoin to trigger IST and cause stomatal closure against O3 injury in tobacco plants. Compared to 2,3-butanediol, acetoin was more effective on triggering IST against O3 injury. The bdh-knockout strain GJ11Δbdh with a blocked metabolic pathway from acetoin to 2,3-butanediol produced more acetoin triggering stronger IST against O3 injury than GJ11. Both acetoin and GJ11Δbdh effectively enhance the antioxidant enzymes activity (e.g. superoxide dismutase and catalases) that is favorable for scavenging the reactive oxygen species like H2 O2 in leaves after exposure to O3 . Consequently, less H2 O2 accumulation was observed, and reasonably less chlorophylls and proteins were damaged by H2 O2 in the tobacco leaves treated with acetoin or GJ11Δbdh. The field experiment also showed that both acetoin and GJ11Δbdh could protect tobacco plants from O3 injury after application by root-drench. This study provides new insights into the role of rhizobacterial B. velezensis and its volatile component of acetoin in triggering defense responses against stresses such as O3 in plants.

Microbial Network and Soil Properties Are Changed in Bacterial Wilt-Susceptible Soil.

Bacterial wilt disease is a devastating disease of crops, which leads to huge economic loss worldwide. It is hypothesized that the occurrence of bacterial wilt may be related to changes in soil chemical properties and microbial interactions. In this study, we compared the soil chemical properties and microbial network structures of a healthy soil (HS) and a bacterial wilt-susceptible soil (BWS). The contents of available nitrogen, potassium, and phosphorus and the soil pH in the BWS were significantly lower than those in the HS. BWS showed nutrient deficiency and acidification in comparison with the HS. The structure and composition of the BWS network were quite different from those of the HS network. The BWS network had fewer modules and edges and lower connectivity than the HS network. The HS network contained more interacting species, more key microorganisms, and better high-order organization and thus was more complex and stable than the BWS network. Most nodes and module memberships were unshared by the two networks, while the ones that were shared showed different topological roles. Some generalists in the HS network became specialists in the BWS network, indicating that the topological roles of microbes were changed and key microorganisms were shifted in the BWS. In summary, the composition and structure of the microbial network of the BWS were different from that of the HS. Many microbial network connections were missing in the BWS, which most likely provided conditions leading to higher rates of bacterial wilt disease.IMPORTANCE Bacterial wilt disease is caused by the pathogen Ralstonia solanacearum and is a widespread devastating soilborne disease leading to huge economic losses worldwide. The soil microbial community is crucial to the capacity of soils to suppress soilborne diseases through complex interactions. Network analysis can effectively explore these complex interactions. In this study, we used a random matrix theory (RMT)-based network approach to investigate the changes in microbial network and associated microbial interactions in a bacterial wilt-susceptible soil (BWS) in comparison to a healthy soil (HS). We found that the structure and composition of the microbial network in BWSs were quite different from those of the HS. The BWS network had fewer modules, edges, and key microorganisms and lower connectivity than the HS network. In the BWSs, apparently the topological role of microbes was changed and key microorganisms were shifted to specialists.

Embedding Bacillus velezensis NH-1 in Microcapsules for Biocontrol of Cucumber Fusarium Wilt.

Cucumber Fusarium wilt, caused by Fusarium oxysporum, is a devastating disease of cucumber and leads to enormous economic losses worldwide. The antagonistic bacterium Bacillus velezensis NH-1 suppresses F. oxysporum For a higher biological control effect, control-released microcapsules of NH-1 were prepared using cell immobilization technology. NH-1 cells were embedded in combinations of the biodegradable wall materials sodium alginate, chitosan, and cassava-modified starch to prepare control-released microbiological microcapsules. For the preparation of alginate single-layer microcapsules, the highest embedding rate of 72.60% was obtained by applying 3% sodium alginate and 2% calcium chloride. After the application of monolayer alginate microcapsules in soil, the number of bacterial cells corresponded to a sustained release curve, and the survival rate of NH-1 was higher than the control in which soil was directly irrigated with NH-1 broth. The use of 0.8% chitosan (pH 3.0) and 0.5% cassava-modified starch in the preparation of double-layer and triple-layer microcapsules changed the performance of the microcapsules and increased the embedding rate. After dry storage for 65 days, the number of NH-1 cells was at the highest level in the monolayer microcapsules. In the field experiment, the control efficiency of alginate-coated monolayer microcapsules on Fusarium wilt was 100%, which was significantly higher than for the NH-1 culture and double-layer and triple-layer microcapsules. Collectively, sodium alginate is an ideal wall material for preparing slow-release bacterial microcapsules to control cucumber Fusarium wilt. Monolayer alginate microcapsules retard the release of B. velezensis NH-1 in soils and significantly improve its biocontrol efficiency on cucumber Fusarium wilt.IMPORTANCEBacillus species are often used for the biocontrol of various plant pathogens, but the control efficiency of Bacillus is usually unstable in field experiments. To improve the control efficiency of Bacillus, in this study, microcapsules of Bacillus velezensis strain NH-1 were prepared using different wall materials (sodium alginate, chitosan, and cassava-modified starch). It was found that the control efficiency of alginate-coated monolayer microcapsules on Fusarium wilt was 100% in field experiments, which was higher than for NH-1 culture and double-layer and triple-layer microcapsules. This study provides a new approach for preparing a biocontrol agent against Fusarium wilt with high biocontrol efficiency.

Nematodes avoid and are killed by Bacillus mycoides-produced styrene.

Root-knot nematodes are obligate parasites that feed on plant roots and cause serious crop losses worldwide. Bacillus species (Bacilliaceae) can produce nematicidal metabolites and have shown good potential for biological control of nematodes. In this study, Bacillus mycoides strain R2 isolated from rhizosphere soil of tomato plants exhibited high nematicidal activity against the free-living nematode Caenorhabditis elegans and the root-knot nematode Meloidogyne incognita. In a pot experiment, control efficiency of B. mycoides R2 on M. incognita was as high as 90.94%. The nematicidal compound was isolated and identified as styrene. The median lethal concentration of styrene against M. incognita was 4.55 µg/ml (m/v). The volatile styrene caused avoidance and killed nematodes primarily by the olfactory neuron and G protein signal pathway. C. elegans detected styrene with the AWB neuron; the signal was then transmitted to the downstream G protein coupled receptors CHE-3, DOP-3, and STR-2. Then signal activated G protein GPA-3 and GPA-7. The signal was then transmitted to ion channels (CNGs channel and TRPV channel), causing calcium ion internal flow and a stress response towards the increased concentration of intracellular calcium. Styrene should be registered as a nematode repellent and biocontrol agent for protection of crops against root-knot nematode attack.

Production of optically pure 2,3-butanediol from Miscanthus floridulus hydrolysate using engineered Bacillus licheniformis strains.

2,3-Butanediol (2,3-BD) can be produced by fermentation of natural resources like Miscanthus. Bacillus licheniformis mutants, WX-02ΔbudC and WX-02ΔgldA, were elucidated for the potential to use Miscanthus as a cost-effective biomass to produce optically pure 2,3-BD. Both WX-02ΔbudC and WX-02ΔgldA could efficiently use xylose as well as mixed sugars of glucose and xylose to produce optically pure 2,3-BD. Batch fermentation of M. floridulus hydrolysate could produce 21.6 g/L D-2,3-BD and 23.9 g/L meso-2,3-BD in flask, and 13.8 g/L D-2,3-BD and 13.2 g/L meso-2,3-BD in bioreactor for WX-02ΔbudC and WX-02ΔgldA, respectively. Further fed-batch fermentation of hydrolysate in bioreactor showed both of two strains could produce optically pure 2,3-BD, with 32.2 g/L D-2,3-BD for WX-02ΔbudC and 48.5 g/L meso-2,3-BD for WX-02ΔgldA, respectively. Collectively, WX-02ΔbudC and WX-02ΔgldA can efficiently produce optically pure 2,3-BD with M. floridulus hydrolysate, and these two strains are candidates for industrial production of optical purity of 2,3-BD with M. floridulus hydrolysate.

Characterization of a novel disease-causing mutation in exon 1 of SH2D1A gene through amplicon sequencing: a case report on HLH.

BACKGROUND: Hemophagocytic lymphohistocytosis (HLH) is a rare but fatal hyperinflammatory syndrome caused by uncontrolled proliferation of activated macrophages and T lymphocytes secreting high amounts of inflammatory cytokines. Genetic defect is a common cause of HLH. HLH is complicated to be diagnosed as there are many common symptoms with other disorders. CASE PRESENTATION: Here we report on an HLH case caused by 1 bp deletion in gene SH2D1A. Patient was a 3-years-old boy and had fever for more than 8 days. Splenomegaly and hemophagocytosis in bone marrow were observed in examination. The results of the blood analysis suggested the diagnosis of HLH. Genetic test based on high throughput amplicon sequencing was then conducted by targeting all six known HLH-causing genes simultaneously. It took only one single day to accomplish the amplicon sequencing library preparation, sequencing and data analysis. Finally, a novel 1 bp deletion in gene SH2D1A was discovered. The result was also confirmed by Sanger sequencing. The result of the genetic test served as a good basis for further diagnosis of HLH. CONCLUSION: This is the first case that the disease-causing genetic defect of HLH was quickly determined by high throughput amplicon sequencing. This diagnosis was also confirmed by Sanger sequencing and cross-validated by blood analysis and other clinical criteria. This case suggests that genetic test based on amplicon sequencing is a powerful tool for diagnosis of HLH and other diseases caused by genetic defect.

Construction of random tumor transcriptome expression library for creating and selecting novel tumor antigens.

Novel tumor antigens are necessary for the development of efficient tumor vaccines for overcoming the immunotolerance and immunosuppression induced by tumors. Here, we developed a novel strategy to create tumor antigens by construction of random tumor transcriptome expression library (RTTEL). The complementary DNA (cDNA) from S180 sarcoma was used as template for arbitrarily amplifying gene fragments with random primers by PCR, then ligated to the C-terminal of HSP65 in a plasmid pET28a-HSP for constructing RTTEL in Escherichia coli. A novel antigen of A5 was selected from RTTEL with the strongest immunotherapeutic effects on S180 sarcoma. Adoptive immunotherapy with anti-A5 sera also inhibited tumor growth, further confirming the key antitumor roles of A5-specific antibodies in mice. A5 contains a sequence similar to protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1). The antisera of A5 were verified to cross-react with PCMT1 by Western blotting assay and vice versa. Both anti-A5 sera and anti-PCMT1 sera could induce antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity toward S180 cells by in vitro assay. Further assay with fluorescent staining showed that PCMT1 is detectable on the surface of S180 cells. Summary, the strategy to construct RTTEL is potential for creating and screening novel tumor antigens to develop efficient tumor vaccines. By RTTEL, we successfully created a protein antigen of A5 with significant immunotherapeutic effects on S180 sarcoma by induction of antibodies targeting for PCMT1.

Enhancement of L-valine production in Bacillus licheniformis by blocking three branched pathways.

OBJECTIVES: Bacillus licheniformis WX-02 is used for the production of many valuable chemicals. Here, we have sought to improve L-valine production by blocking the metabolic pathways related to branched-chain amino acids. RESULTS: The synthesis genes of L-leucine (leuA) and L-isoleucine (ilvA) were deleted to obtain mutant strains. L-Valine yields of WX-02ΔleuA and WX-02ΔilvA reached 33.2 and 21.1 mmol/l, respectively, which are 22 and 14 times higher than the wild-type WX-02 (1.53 mmol/l). After further deletion of L-lactate dehydrogenase gene (ldh) from WX-02ΔleuA, the productivity reached 0.47 mmol/l h, an increase of 19 %. CONCLUSION: We provide a possibility to over-produce L-valine using genetically-modified B. licheniformis using remodeling of the biosynthetic pathway to L-valine.

Efficient expression of nattokinase in Bacillus licheniformis: host strain construction and signal peptide optimization.

Nattokinase (NK) possesses the potential for prevention and treatment of thrombus-related diseases. In this study, high-level expression of nattokinase was achieved in Bacillus licheniformis WX-02 via host strain construction and signal peptides optimization. First, ten genes (mpr, vpr, aprX, epr, bpr, wprA, aprE, bprA, hag, amyl) encoding for eight extracellular proteases, a flagellin and an amylase were deleted to obtain B. licheniformis BL10, which showed no extracellular proteases activity in gelatin zymography. Second, the gene fragments of P43 promoter, Svpr, nattokinase and TamyL were combined into pHY300PLK to form the expression vector pP43SNT. In BL10 (pP43SNT), the fermentation activity and product activity per unit of biomass of nattokinase reached 14.33 FU/mL and 2,187.71 FU/g respectively, which increased by 39 and 156 % compared to WX-02 (pP43SNT). Last, Svpr was replaced with SsacC and SbprA, and the maximum fermentation activity (33.83 FU/mL) was achieved using SsacC, which was 229 % higher than that of WX-02 (pP43SNT). The maximum NK fermentation activity in this study reaches the commercial production level of solid state fermentation, and this study provides a promising engineered strain for industrial production of nattokinase, as well as a potential platform host for expression of other target proteins.

Rhizospheric microbiota of suppressive soil protect plants against Fusarium solani infection.

BACKGROUND: Fusarium infection has caused huge economic losses in many crops. The study aimed to compare the microbial community of suppressive and conducive soils and relate to the reduction of Fusarium wilt. RESULTS: High-throughput sequencing and microbial network analysis were used to investigate the differences in the rhizosphere microbiota of the suppressive and conducive soils and to identify the beneficial keystone taxa. Plant pathogens were enriched in the conducive soil. Potential plant-beneficial microorganisms and antagonistic microorganisms were enriched in the suppressive soil. More positive interactions and keystone taxa existed in the suppressive soil network. Thirty-nine and 16 keystone taxa were identified in the suppressive and conducive soil networks, respectively. Sixteen fungal strains and 168 bacterial strains were isolated from suppressive soil, some of which exhibited plant growth-promotion traits. Thirty-nine bacterial strains and 10 fungal strains showed antagonistic activity against F. solani. Keystone taxa Bacillus and Trichoderma exhibited high antifungal activity. Lipopeptides produced by Bacillus sp. RB150 and chitinase from Trichoderma spp. inhibited the growth of F. solani. Microbial consortium I (Bacillus sp. RB150, Pseudomonas sp. RB70 and Trichoderma asperellum RF10) and II (Bacillus sp. RB196, Bacillus sp. RB150 and T. asperellum RF10) effectively controlled root rot disease, the spore number of F. solani was reduced by 94.2% and 83.3%. CONCLUSION: Rhizospheric microbiota of suppressive soil protects plants against F. solani infection. Antagonistic microorganisms in suppressive soil inhibit pathogen growth and infection. Microbial consortia consisted of keystone taxa well control root rot disease. These findings help control Fusarium wilt. © 2024 Society of Chemical Industry.

Cooperative Action of Fulvic Acid and Bacillus paralicheniformis Ferment in Regulating Soil Microbiota and Improving Soil Fertility and Plant Resistance to Bacterial Wilt Disease.

Excessive continuous cropping and soil degradation, such as acidification, hardening, fertility decline, and the degradation of microbial community, lead to the epidemic of soilborne diseases and cause great loss in agriculture production. Application of fulvic acid can improve the growth and yield of various crops and effectively suppress soilborne plant diseases. Bacillus paralicheniformis strain 285-3 producing poly-gamma-glutamic acid is used to remove the organic acid that can cause soil acidification and increase the fertilizer effect of fulvic acid and the effect of improving soil quality and inhibiting soilborne disease. In field experiments, the application of fulvic acid and Bacillus paralicheniformis ferment effectively reduced the incidence of bacterial wilt disease and improved soil fertility. Both fulvic acid powder and B. paralicheniformis ferment improved soil microbial diversity and increased the complexity and stability of the microbial network. For B. paralicheniformis ferment, the molecular weight of poly-gamma-glutamic acid became smaller after heating, which could better improve the soil microbial community and network structure. In fulvic acid and B. paralicheniformis ferment-treated soils, the synergistic interaction between microorganisms increased and the number of keystone microorganisms increased, which included antagonistic bacteria and plant growth-promoting bacteria. Changes in the microbial community and network structure were the main reason for the reduced incidence of bacterial wilt disease. Application of fulvic acid and Bacillus paralicheniformis ferment improved soil physicochemical properties and effectively controlled bacterial wilt disease by changing microbial community and network structure and enriching antagonistic and beneficial bacteria. IMPORTANCE Continuous cropping tobacco has led to soil degradation and caused soilborne bacterial wilt disease. Fulvic acid as a biostimulator was applied to restore soil and control bacterial wilt disease. For improving its effect, fulvic acid was fermented with Bacillus paralicheniformis strain 285-3 producing poly-gamma-glutamic acid. Fulvic acid and B. paralicheniformis ferment inhibited bacterial wilt disease, improved soil quality, enriched beneficial bacteria, and increased microbial diversity and microbial network complexity. Some keystone microorganisms in fulvic acid and B. paralicheniformis ferment-treated soils had potential antimicrobial activity and plant growth-promoting attributes. Fulvic acid and B. paralicheniformis 285-3 ferment could be used to restore soil quality and microbiota and control bacterial wilt disease. This study found new biomaterial to control soilborne bacterial disease by combining fulvic acid and poly-gamma-glutamic acid application.

Transcription Factor Spo0A Regulates the Biosynthesis of Difficidin in Bacillus amyloliquefaciens.

Bacillus amyloliquefaciens WH1 produces multiple antibiotics with antimicrobial activity and can control bacterial wilt disease caused by Ralstonia solanacearum. Antibacterial substances produced by WH1 and the regulation mechanism are unknown. In this study, it was found that difficidin, and to a minor extent bacillibactin, exhibited antibacterial activity against R. solanacearum. Lipopeptides, macrolactin, bacillaene, and bacilysin had no antibacterial activity. Ferric iron uptake transcriptional regulator Fur bound the promoter region of the dhb gene cluster of bacillibactin biosynthesis. Mutant Δfur showed a higher bacillibactin production and its antibacterial activity increased by 27% than wild-type WH1. Difficidin inhibited R. solanacearum growth and disrupted the integrity of the cells. Lack of transcription factor Spo0A abolished difficidin biosynthesis. Spo0A bound the promoter region of the dfn gene cluster of difficidin biosynthesis. Changing phosphorylation levels of Spo0A via deletion of phosphatase gene spo0E and histidine kinases genes kinA and kinD affected the biosynthesis of difficidin. Deletion of spo0E increased the phosphorylation level of Spo0A and consequently improved the difficidin production. The antibacterial activity of mutant Δspo0E and ΔkinA increased by 12% and 19%. The antibacterial activity of mutant ΔkinD decreased by 28%. Collectively, WH1 produced difficidin to disrupt the cell of R. solanacearum and secreted siderophore bacillibactin to compete for ferric iron. Spo0A regulated difficidin biosynthesis. Spo0A regulates quorum-sensing responses and controls the biosynthesis of secondary metabolites in B. amyloliquefaciens. This study has important findings in the regulation mechanism of antibiotic synthesis and helps to improve antibiotic yield in Bacillus. IMPORTANCE Pathogen R. solanacearum causes bacterial wilt disease in many crops. There is no chemical bactericide that can control bacterial wilt disease. It is vital to find antagonistic microorganisms and antibacterial substances that can efficiently control bacterial wilt disease. B. amyloliquefaciens WH1 could inhibit the growth of R. solanacearum. Via genetic mutation, it was found that difficidin and to a minor extent bacillibactin produced by WH1 acted efficiently against R. solanacearum. The transcription factor Spo0A regulated the synthesis of difficidin. Phosphorylation of Spo0A affected the production of difficidin. Increasing the phosphorylation level of Spo0A improved the difficidin production and antibacterial activity. In-depth analysis of the regulation mechanism of antibiotic difficidin is meaningful for enhancing the control efficiency of WH1. B. amyloliquefaciens WH1 and the antibacterial substances have vast application potential in controlling bacterial wilt disease.

The Endophytic Root Microbiome Is Different in Healthy and Ralstonia solanacearum-Infected Plants and Is Regulated by a Consortium Containing Beneficial Endophytic Bacteria.

Plant bacterial wilt disease caused by Ralstonia solanacearum leads to huge economic losses worldwide. Endophytes play vital roles in promoting plant growth and health. It is hypothesized that the endophytic root microbiome and network structure are different in healthy and diseased plants. Here, the endophytic root microbiomes and network structures of healthy and diseased tobacco plants were investigated. Composition and network structures of endophytic root microbiomes were distinct between healthy and diseased plants. Healthy plants were enriched with more beneficial bacteria and bacteria with antagonistic activity against R. solanacearum. R. solanacearum was most abundant in diseased plants. Microbial networks in diseased plants had fewer modules and edges, lower connectivity, and fewer keystone microorganisms than those in healthy plants. Almost half of the nodes were unique in the two networks. Ralstonia was identified as a key microorganism of the diseased-plant network. In healthy plants, abundant bacteria and biomarkers (Pseudomonas and Streptomyces) and keystone microorganisms (Bacillus, Lysobacter, and Paenibacillus) were plant-beneficial bacteria and showed antibacterial and plant growth-promoting activities. The endophytic strain Bacillus velezensis E9 produced bacillaene to inhibit R. solanacearum. Consortia containing keystone microorganisms and beneficial endophytic bacteria significantly regulated the endophytic microbiome and attenuated bacterial wilt by inducing systemic resistance and producing antibiotic. Overall, the endophytic root microbiome and network structure in diseased plants were different from those in healthy plants. The endophytic root microbiome of diseased plants had low abundances of beneficial bacteria and an unstable network and lacked beneficial keystone microorganisms, which favored infection. Synthetic microbial consortia were effective measures for preventing R. solanacearum infection. IMPORTANCE Bacterial wilt disease causes heavy yield losses in many crops. Endophytic microbiomes play important roles in control of plant diseases. However, the role of the endophytic root microbiome in controlling bacterial wilt disease is poorly understood. Here, differences in endophytic root microbiomes and network structures between healthy and diseased tobacco plants are reported. A synthetic microbial consortium containing beneficial endophytic bacteria was used to regulate the endophytic microbiome and attenuate bacterial wilt disease. The results could be generally used to guide control of bacterial wilt disease.

Genome sequence of Bacillus licheniformis WX-02.

Bacillus licheniformis is an important bacterium that has been used extensively for large-scale industrial production of exoenzymes and peptide antibiotics. B. licheniformis WX-02 produces poly-gamma-glutamate increasingly when fermented under stress conditions. Here its genome sequence (4,270,104 bp, with G+C content of 46.06%), which comprises a circular chromosome, is announced.

Role of surface proteins SspA and SspB of Streptococcus gordonii in innate immunity.

Streptococcus gordonii, a normal inhabitant of the human oral cavity, is a potential live vaccine vehicle. Several pathogen-associated molecular patterns from S. gordonii that are recognized by antigen-presenting cells have recently been identified. In this study, we have identified that the cell-wall-anchored proteins SspA and SspB are immunostimulatory components of S. gordonii. SspA and SspB are members of the antigen I/II family of proteins widely expressed by viridans oral streptococci. The results showed that the mutant (OB219) lacking SspA and SspB had a reduced ability to induce cytokine/chemokine production in epithelial cells and bone-marrow-derived dendritic cells as compared with the parent strain (DL1). Purified SspA induced interleukin-6 and monocyte chemotatic protein-1 production from human lung epithelial A549 cells. The induction could be inhibited by a function-blocking anti-ß1 integrin mAb and the purified SspA could bind to ß1 integrin precoated on microtitre plates, suggesting that the induction was effected by SspA-ß1 integrin interactions. The role of SspA and SspB in innate immunity was further demonstrated in a mouse intranasal challenge experiment, which showed that the clearance of OB219, the recruitment of neutrophils (as indicated by myeloperoxidase activity), and chemokine and cytokine production in the lungs of OB219-inoculated mice were delayed or reduced as compared with the DL1-inoculated mice. In addition to the above, S. gordonii OB219 was more sensitive to polymyxin, nisin and histatin-5 than DL1, suggesting that SspA and SspB also play a role in susceptibility to cationic antimicrobial peptides. Collectively, the results indicate that SspA and SspB are immunostimulatory components of S. gordonii and play an important role in modulating the host's innate immunity.