Soil microbes play important roles in plant growth and health. Little is known about the differences of soil microbes between healthy and bacterial wilt infected soils with Ralstonia solanacearum. By Illumina-MiSeq sequencing of 16S rRNA and 18S rRNA gene amplicons, we found the soil microbial composition and diversity were distinct between healthy and bacterial wilt infected soils. Soil microbial community varied at different plant growth stages due to changes of root exudates composition and soil pH. Healthy soils exhibited higher microbial diversity than the bacterial wilt infected soils. More abundant beneficial microbes including Bacillus, Agromyces, Micromonospora, Pseudonocardia, Acremonium, Lysobacter, Mesorhizobium, Microvirga, Bradyrhizobium, Acremonium and Chaetomium were found in the healthy soils rather than the bacterial wilt infected soils. Compared to bacterial wilt infected soils, the activities of catalase, invertase and urease, as well as soil pH, available phosphorous and potassium content, were all significantly increased in the healthy soils. In a conclusion, the higher abundance of beneficial microbes are positively related the higher soil quality, including better plant growth, lower disease incidence, and higher nutrient contents, soil enzyme activities and soil pH.

Continuous cropping of the same crop leads to land degradation. This is also called the continuous-cropping obstacle. Here, we investigated how long-term continuous cropping of tobacco influences soil biochemical properties and bacterial networks in the mountain lands of China. Two different fields were sampled: one with 25 years of continuous cropping tobacco and one with noncontinuous cropping tobacco. Soil chemical and biological properties were measured including available phosphorus and potassium, soil organic matter, pH, alkali-hydrolysable nitrogen, micronutrients contents, and activity of urease, catalase, invertase, and phosphatase as well as tobacco agronomic characteristics. Bacterial communities of the two different soils were sequenced by metabarcoding of the 16S ribosomal RNA, and, with these data, network analysis was done. Soil chemical properties and tobacco agronomical properties were negatively affected by the continuous-cropping obstacle, and this treatment has a less complex network (less modules, nodes, and connectivity) than the soil with noncontinuous cropping treatment. For continuous cropping, there were less generalists, which were key species that connect network, than noncontinuous cropping.Moreover, the taxonomic composition of bacterial network was different in the two different treatments. In the continuous-cropping network, 40% nodes had negative interactions, suggesting that more competition or antagonism existed among bacterial species. It concluded that continuous cropping has a detrimental effect on soil chemical and that the bacterial network properties under continuous cropping are more sensitive to soil variables (so more unstable and inefficient) because there are less bacterial species that interact each other and this is due to limited nutrients or excessive toxic nutrient.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Plant-parasitic nematodes cause serious crop losses worldwidely. This study intended to discover the antagonistic mechanism of Bacillus cereus strain S2 against Meloidogyne incognita. Treatment with B. cereus strain S2 resulted in a mortality of 77.89% to Caenorhabditis elegans (a model organism) and 90.96% to M. incognita. In pot experiment, control efficiency of B. cereus S2 culture or supernatants were 81.36% and 67.42% towards M. incognita, respectively. In field experiment, control efficiency was 58.97% towards M. incognita. Nematicidal substances were isolated from culture supernatant of B. cereus S2 by polarity gradient extraction, silica gel column chromatography and HPLC. Two nematicidal compounds were identified as C16 sphingosine and phytosphingosine by LC-MS. The median lethal concentration of sphingosine was determined as 0.64 μg/ml. Sphingosine could obviously inhibit reproduction of C. elegans, with an inhibition rate of 42.72% for 24 h. After treatment with sphingosine, ROS was induced in intestinal tract, and genital area disappeared in nematode. Furthermore, B. cereus S2 could induce systemic resistance in tomato, and enhance activity of defense-related enzymes for biocontrol of M. incognita. This study demonstrates the nematicidal activity of B. cereus and its product sphingosine, as well provides a possibility for biocontrol of M. incognita.

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BackgroundD-2,3-butanediol has many industrial applications such as chiral reagents, solvents, anti-freeze agents, and low freezing point fuels. Traditional D-2,3-butanediol producing microorganisms, such as Klebsiella pneumonia and K. xoytoca, are pathogenic and not capable of producing D-2,3-butanediol at high optical purity. Bacillus licheniformis is a potential 2,3-butanediol producer but the wild type strain (WX-02) produces a mix of D- and meso-type isomers. BudC in B. licheniformis is annotated as 2,3-butanediol dehydrogenase or acetoin reductase, but no pervious experiment was performed to verify this hypothesis.ResultsWe developed a genetically modified strain of B. licheniformis (WX-02 ΔbudC) as a D-2,3-butanediol producer with high optimal purity. A marker-less gene deletion protocol based on a temperature sensitive knock-out plasmid T2-Ori was used to knock out the budC gene in B. licheniformis WX-02. The budC knock-out strain successfully abolished meso-2,3-butanediol production with enhanced D-2,3-butanediol production. No meso-BDH activity was detectable in cells of this strain. On the other hand, the complementary strain restored the characteristics of wild strain, and produced meso-2,3-butanediol and possessed meso-BDH activity. All of these data suggested that budC encoded the major meso-BDH catalyzing the reversible reaction from acetoin to meso-2,3-butanediol in B. licheniformis. The budC knock-out strain produced D-2,3-butanediol isomer only with a high yield of 30.76 g/L and a productivity of 1.28 g/L-h.ConclusionsWe confirmed the hypothesis that budC gene is responsible to reversibly transfer acetoin to meso-2,3-butanediol in B. licheniformis. A mutant strain of B. licheniformis with depleted budC gene was successfully developed and produced high level of the D-2,3-butanediol with high optimal purity.

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.

Soil degradation is a serious global problem, but little is known about how soil microbial communities respond to soil degradation as well as their feedback to ecosystem functioning. In this study, we found the microbial community composition, structure and functional potential significantly altered in the degraded soils with bacterial wilt (termed as degraded soils). Compared with healthy soils, OTU richness of beneficial microorganisms were significantly decreased, but OTU richness of pathogenic microorganisms were significantly increased in the degraded soils. Functional gene array (GeoChip 5.0) analysis showed the functional metabolic potential of genes involved in stress, virulence, sulfur cycle, metal resistance, degradation of plant cell wall was significantly increased in the degraded soils. Increased functional metabolic potential of these genes may be related to the acidification and severe plant disease of degraded soils. Biological activity of degraded soils was obviously decreased with weakened soil enzyme activities when compared to the healthy soils. Soil pH and enzyme activities were negatively correlated with the abundance of genes involved in sulfur cycle, virulence, and stress responses. This study provides new insights into our understanding of soil microbial community responses to soil degradation.

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.