Influence of Bacillus subtilis strain Z-14 on microbial ecology of cucumber rhizospheric vermiculite infested with fusarium oxysporum f. sp. cucumerinum.

Fusarium oxysporum (FO) is a typical soil-borne pathogenic fungus, and the cucumber wilt disease caused by F. oxysporum f. sp. cucumerinum (FOC) seriously affects crop yield and quality. Vermiculite is increasingly being used as a culture substrate; nevertheless, studies exploring the effectiveness and mechanisms of biocontrol bacteria in this substrate are limited. In this study, vermiculite was used as a culture substrate to investigate the control effect of Bacillus subtilis strain Z-14 on cucumber wilt and the rhizospheric microecology, focusing on colonization ability, soil microbial diversity, and rhizosphere metabolome. Pot experiments showed that Z-14 effectively colonized the cucumber roots, achieving a controlled efficacy of 61.32% for wilt disease. It significantly increased the abundance of Bacillus and the expression of NRPS and PKS genes, while reducing the abundance of FO in the rhizosphere. Microbial diversity sequencing showed that Z-14 reduced the richness and diversity of the rhizosphere bacterial community, increased the richness and diversity of the fungal community, and alleviated the effect of FO on the community structure of the cucumber rhizosphere. The metabolomics analysis revealed that Z-14 affected ABC transporters, amino acid synthesis, and the biosynthesis of plant secondary metabolites. Additionally, Z-14 increased the contents of phenylacetic acid, capsidol, and quinolinic acid, all of which were related to the antagonistic activity in the rhizosphere. Z-14 exhibited a significant control effect on cucumber wilt and influenced the microflora and metabolites in rhizospheric vermiculite, providing a theoretical basis for further understanding the control effect and mechanism of cucumber wilt in different culture substrates.

Transcriptomic analysis of humic acid in relieving the inhibitory effect of high nitrogen on soybean nodulation.

Introduction: Nitrogen fertilizer intake promotes soybean growth before the formation of nodules, but excess nitrogen has an inhibitory effect on soybean nodulation. It is important to balance nitrogen levels to meet both growth and nodulation needs. Methods: the nitrogen level suitable for soybean growth and nodulation was studied, the role of humic acid (HA) in alleviating the inhibition of high nitrogen on soybean nodulation was analyzed, and transcriptomic analysis was performed to understand its mechanism. Results: The results showed that a lower level of nitrogen with 36.4 mg urea per pot could increase the number of nodules of soybean, and a higher level of nitrogen with 145.9 mg urea per pot (U4 group) had the best growth indicators but inhibited nodulation significantly. HA relieved the inhibitory effect at high nitrogen level, and the number of nodules increased by 122.1% when 1.29 g HA was added (H2 group) compared with the U4 group. The transcriptome analysis was subsequently performed on the H2 and U4 groups, showing that there were 2995 differentially expressed genes (DEGs) on the 25th day, accounting for 6.678% of the total annotated genes (44,848) under the test conditions. These DEGs were enriched in mitogen-activated protein kinase signaling pathway-plant, flavonoid biosynthesis, and plant hormone signal transduction based on the -log10 (P adjusted) value in the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG). Discussion: HA balanced the nitrogen level through the above pathways in soybean planting to control the number of nodules.

Metabolomic and Transcriptome Analysis of the Inhibitory Effects of Bacillus subtilis Strain Z-14 against Fusarium oxysporum Causing Vascular Wilt Diseases in Cucumber.

Controlling cucumber Fusarium wilt caused by Fusarium oxysporum f. sp. cucumerinum (FOC) with Bacillus strains is a hot research topic. However, the molecular mechanism of Bacillus underlying the biocontrol of cucumber wilt is rarely reported. In this study, B. subtilis strain Z-14 showed significant antagonistic activity against FOC, and the control effect reached 88.46% via pot experiment. Microscopic observations showed that strain Z-14 induced the expansion and breakage of FOC hyphae. The cell wall thickness was uneven, and the organelle structure was degraded. The combined analysis of metabolome and transcriptome showed that strain Z-14 inhibited the FOC infection by inhibiting the synthesis of cell wall and cell membrane, energy metabolism, and amino acid synthesis of FOC mycelium, inhibiting the clearance of reactive oxygen species (ROS) and the secretion of cell wall-degrading enzymes (CWDEs), thereby affecting mitogen-activated protein kinase (MAPK) signal transduction and inhibiting the transport function.

Transcriptomic and Metabolomic Analysis of Soybean Nodule Number Improvements with the Use of Water-Soluble Humic Materials.

Water-soluble humic materials (WSHMs) can enhance the nodule numbers of soybean plants. In this study, targeted metabolomics and transcriptomics were used to understand this mechanism. Results showed that 500 mg/L WSHM increased the adsorption and colonization of rhizobia in soybean roots. High-performance liquid chromatography and targeted metabolomics showed that WSHMs could regulate the content and distribution of endogenous hormones of soybean plants at the initial stage of soybean nodulation. Transcriptomic analysis showed a total of 2406 differentially expressed genes (DEGs) by the 25th day, accounting for 4.89% of total annotation genes (49159). These DEGs were found to contribute primarily to the MAPK signaling pathway, glycolysis/gluconeogenesis, and plant hormone signal transduction according to the -log 10 (Padjust) value in the KEGG pathway. Subsequently, DEGs related to these hormones were selected for verification using quantity-PCR. The WSHM increased the number of nodules by regulating the expression of endogenous hormones in soybean plants.

Anaerobic solid-state fermentation with Bacillus subtilis for digesting free gossypol and improving nutritional quality in cottonseed meal.

Microbial fermentation is an effective method to degrade free-gossypol, which is a toxic substance restricting the utilization of cottonseed meal in animal husbandry. However, there are few researches on the nutritional effect and the change of bacterial community on cottonseed meal fermented with anaerobic solid-state fermentation. This study evaluated the effects of fermentation with Bacillus sp. on gossypol degradation and nutritional quality improvement in cottonseed meal (CM), as well as the changes of bacterial community structure during fermentation. The strains with high activity for digesting free gossypol were screened from high protease-producing strains preserved in the laboratory. Then the strains which had both the gossypol degradation activity and protease producing activity were selected to degrade macromolecular protein and free gossypol in CM. The unsterilized SSF medium was inoculated with 109 CFU/kg Bacillus culture and fermented at room temperature for 14 days. Each group had three parallels. And the effects of anaerobic solid-state fermentation on unsterilized CM was evaluated. Results showed that for the seven strains with high activity for digesting free gossypol and producing protease that were screened, free gossypol content in fermented cottonseed meal (FCM) decreased and acid-soluble protein (ASP) contents increased. Among them, strain M-15 had the best fermentation effect, with the free gossypol degradation rate of 93.46% and acid soluble protein content of 13.26%. M-15 was identified as Bacillus subtilis. During fermentation with M-15, the bacterial diversity in CM was reduced, but not significant and the community structure was simpler significantly. The strain M-15 selected in this experiment reduced the free gossypol content and improved the nutritional quality of CM through anaerobic solid-state fermentation, which can be used for industrial large-scale production.

Influence of Bacillus subtilis strain Z-14 on microbial communities of wheat rhizospheric soil infested with Gaeumannomyces graminis var. tritici.

Wheat take-all disease caused by Gaeumannomyces graminis var. tritici (Ggt) spreads rapidly and is highly destructive, causing severe reductions in wheat yield. Bacillus subtilis strain Z-14 that significantly controlled wheat take-all disease effectively colonized the roots of wheat seedlings. Z-14 increased the metabolic activity and carbon source utilization of rhizospheric microorganisms, thus elevating average well-color development (AWCD) values and functional diversity indexes of soil microbial communities. Z-14 increased the abundance of Bacillus in the rhizosphere, which was positively correlated with AWCD and functional diversity indexes. The Z-14-treated samples acquired more linkages and relative connections between bacterial communities according to co-occurrence network analyses. After the application of Ggt, the number of linkages between fungal communities increased but later decreased, whereas Z-14 increased such interactions. Whole-genome sequencing uncovered 113 functional genes related to Z-14's colonization ability and 10 secondary metabolite gene clusters in the strain, of which nine substances have antimicrobial activity. This study clarifies how bacterial agents like Z-14 act against phytopathogenic fungi and lays a foundation for the effective application of biocontrol agents.

Anaerobic Solid-State Fermentation of Soybean Meal With Bacillus sp. to Improve Nutritional Quality.

The study evaluated the impact of fermentation with Bacillus sp. on the nutritional quality of soybean meal (SBM) and the changes of bacterial community structure during fermentation. High protease-producing strains were screened to degrade SBM macromolecular protein and anti-nutritional factors (ANFs). Unsterilized SBM then underwent an anaerobic solid-state fermentation method to evaluate the effects of fermentation. Results showed that for the nine high-producing protease strains that were screened, acid-soluble protein (ASP) contents in fermented SBM increased, with the highest value found to be 13.48%, which was fermented using strain N-11. N-11 was identified as Bacillus subtilis. N-11 fermentation reduced ANFs such as glycinin and ß-conglycinin by 82.38 and 88.32%, respectively. During N-11 fermentation, the bacterial richness and diversity in SBM increased but not significantly. The high-yield protease strain B. subtilis N-11 selected in this experiment improved the nutritional quality of SBM through fermentation, and it can be used for industrial large-scale production.

Water-Soluble Humic Materials Modulating Metabolism and Triggering Stress Defense in Sinorhizobium fredii.

Bacteria have evolved a series of mechanisms to maintain their survival and reproduction in changeable and stressful environments. In-depth understanding of these mechanisms can allow for better developing and utilizing of bacteria with various biological functions. In this study, we found that water-soluble humic materials (WSHM), a well-known environment-friendly plant growth biostimulant, significantly promoted the free-living growth and survival of Sinorhizobium fredii CCBAU45436 in a bell-shaped, dose-dependent manner, along with more-efficient carbon source consumption and relief of medium acidification. By using RNA-Seq analysis, a total of 1,136 genes significantly up-/downregulated by external addition of WSHM were identified under test conditions. These differentially expressed genes (DEGs) were enriched in functional categories related to carbon/nitrogen metabolism, cellular stress response, and genetic information processing. Further protein-protein interaction (PPI) network analysis and reverse genetic engineering indicated that WSHM might reprogram the transcriptome through inhibiting the expression of key hub gene rsh, which encodes a bifunctional enzyme catalyzing synthesis and hydrolysis of the "magic spot" (p)ppGpp. In addition, the root colonization and viability in soil of S. fredii CCBAU45436 were increased by WSHM. These findings provide us with new insights into how WSHM benefit bacterial adaptations and demonstrate great application value to be a unique inoculant additive. IMPORTANCE Sinorhizobium fredii CCBAU45436 is a highly effective, fast-growing rhizobium that can establish symbiosis with multiple soybean cultivars. However, it is difficult to maintain the high-density effective viable cells in the rhizobial inoculant for the stressful conditions during production, storage, transport, and application. Here, we showed that WSHM greatly increased the viable cells of S. fredii CCBAU45436 in culture, modulating metabolism and triggering stress defense. The root colonization and viability in soil of S. fredii CCBAU45436 were also increased by WSHM. Our results shed new insights into the effects of WSHM on bacteria and the importance of metabolism and stress defense during the bacteria's whole life. In addition, the functional mechanism of WSHM may provide candidate genes for improving environmental adaptability and application potential of bacteria through genetic engineering.

Inhibitory Abilities of Bacillus Isolates and Their Culture Filtrates against the Gray Mold Caused by Botrytis cinerea on Postharvest Fruit.

Botrytis cinerea, a major phytopathogenic fungus, has been reported to infect more than 200 crop species worldwide, and it causes massive losses in yield. The aim of this study was to evaluate the inhibitory abilities and effects of Bacillus amyloliquefaciens RS-25, Bacillus licheniformis MG-4, Bacillus subtilis Z-14, and Bacillus subtilis Pnf-4 and their culture filtrates and extracts against the gray mold caused by B. cinerea on postharvest tomato, strawberry, and grapefruit. The results revealed that the cells of Z-14, culture filtrate of RS-25, and cells of Z-14 showed the strongest biocontrol activity against the gray mold on the strawberry, grape, and tomato fruit, respectively. All the strains produced volatile organic compounds (VOCs), and the VOCs of Pnf-4 displayed the highest inhibition values. Based on headspace solid-phase microextraction in combination with gas chromatography-mass spectrometry, esters accounted for the largest percentage of the VOCs produced by RS-25, MG-4, Z-14, and Pnf-4 (36.80%, 29.58%, 30.78%, and 36.26%, respectively). All the strains showed potent cellulase and protease activities, but no chitinase activity. RS-25, Z-14, and MG-4, but not Pnf-4, grew on chrome azurol S agar, and an orange halo was formed around the colonies. All the strains showed biofilm formation, fruit colonization, and lipopeptide production, which may be the main modes of action of the antagonists against B. cinerea on the fruit. This study provides the basis for developing natural biocontrol agents against the gray mold caused by B. cinerea on postharvest fruit.

Degradation of the phenolic ß-ether lignin model dimer and dyes by dye-decolorizing peroxidase from Bacillus amyloliquefaciens.

OBJECTIVE: The dye-decolorizing peroxidase from Bacillus amyloliquefaciens, BaDyP, was identified to be an efficient catalyst for the degradation of phenolic ß-ether lignin model dimer guaiacylglycerol-ß-guaiacyl ether (GGE) and dyes. RESULTS: Efeb gene encoding BaDyP from B. amyloliquefaciens MN-13 consisted of 1257 bp and the open reading frame encoded 418 amino acids. The efeb gene was expressed in Escherichia coli BL21 and a recombinant BaDyP of 50 kDa was achieved. The BaDyP exhibited activity in oxidizing GGE and decolorizing azo and triphenylmethane dyes. At pH 4.5 and 30 °C the BaDyP not only completely degraded GGE by the cleavage of ß-O-4 ether bond and Cα-Cß bond, and Cα oxidation without any oxidative mediator, but also decolorized four synthetic dyes, including congo red, bromine cresol green, eriochrome black T and crystal violet. This was achieved with decolorization rates of 65.7%, 70.62%, 80.06% and 62.09%, respectively, after 72 h of incubation. CONCLUSIONS: BaDyP was identified as a bacteria peroxidase with great potential for the degradation of lignin and bioremediation of dye-contamination.

Biodegradation of Leonardite by an alkali-producing bacterial community and characterization of the degraded products.

In this study, three bacterial communities were obtained from 12 Leonardite samples with the aim of identifying a clean, effective, and economic technique for the dissolution of Leonardite, a type of low-grade coal, in the production of humic acid (HA). The biodegradation ability and characteristics of the degraded products of the most effective bacterial community (MCSL-2), which degraded 50% of the Leonardite within 21 days, were further investigated. Analyses of elemental composition, (13)C NMR, and Fourier transform infrared revealed that the contents of C, O, and aliphatic carbon were similar in biodegraded humic acid (bHA) and chemically (alkali) extracted humic acid (cHA). However, the N and carboxyl carbon contents of bHA was higher than that of cHA. Furthermore, a positive correlation was identified between the degradation efficiency and the increasing pH of the culture medium, while increases of manganese peroxidase and esterase activities were also observed. These data demonstrated that both alkali production and enzyme reactions were involved in Leonardite solubilization by MCSL-2, although the former mechanism predominated. No fungus was observed by microscopy. Only four bacterial phylotypes were recognized, and Bacillus licheniformis-related bacteria were identified as the main group in MCSL-2 by analysis of amplified 16S rRNA genes, thus demonstrating that Leonardite degradation ability has a limited distribution in bacteria. Hormone-like bioactivities of bHA were also detected. In this study, a bacterial community capable of Leonardite degradation was identified and the products characterized. These data implicate the use of such bacteria for the exploitation of Leonardite as a biofertilizer.