耐高盐枯草芽孢杆菌XP合成球形纳米硒及其抑制草莓病原真菌生物活性

生物方法合成纳米材料具有低能耗、高安全性以及环境友好等优良特点,因而备受人们关注.利用细菌将硒酸盐或亚硒酸盐还原为单质硒,不仅可以降低硒毒性,而且还能获得价值更高的生物纳米材料.文中选用可耐受高盐环境胁迫的枯草芽孢杆菌亚种Bacillus subtilis subspecies stercoris strain XP构...     

Purification and kinetics of a protease-resistant,neutral, and thermostable phytase from Bacillus subtilis subsp. subtilis JJBS250 ameliorating food nutrition

Abstract

A novel protease-resistant and thermostable phytase from Bacillus subtilis subsp. subtilis JJBS250 was purified 36-fold to homogeneity with a combination of ammonium sulfate precipitation followed by Q-Sepharose and Sephadex G-50 chromatographic techniques. The estimated molecular mass of the purified phytase was 46?kDa by electrophoresis with optimal activity at pH 7.0 and 70?°C. About 19% of original activity was maintained at 80?°C for 10?min. Phytase activity was stimulated in presence of surfactants like Tween-20, Tween-80, and Triton X-100 and metal ions like Ca⁺², K⁺, and Co⁺² and it was inhibited by SDS and Mg⁺², Al⁺², and Fe⁺². Purified enzyme showed specificity to different salts of phytic acid and values of K_m and V_max were 0.293?mM and 11.49 nmoles s^?1, respectively for sodium phytate. The purified enzyme was resistant to proteases (trypsin and pepsin) that resulted in amelioration of food nutrition with simultaneous release of inorganic phosphate, reducing sugars, and soluble protein.     

Screening the Fusarium graminearum inhibitory mutant strain from Bacillus subtilis by atmospheric-pressure plasma jet

Aims:  The aim of this study was to improve the antagonistic activity of Bacillus subtilis JA towards Fusarium graminearum by screening high-yielding mutant using the atmospheric-pressure plasma jet (APPJ).
Methods and Results:  Atmospheric-pressure plasma jet was applied as mutagenic source for breeding high-yielding mutant strain. Helium was used as APPJ operating gas. The mutation effects of different treatment times of APPJ were studied. The mutant strain designated as B. subtilis B06 was successfully screened out, which showed higher antagonistic activity against F. graminearum in vitro . Its inhibition zone against the indicator fungus increased by 23% compared to the original one. HPLC and ESI (electrospray ionization) mass spectrometry analysis indicated that antifungal compounds produced by the mutant and original strain belonged to the lipopeptide, surfactin and iturin families. The mutant strain showed favourable properties of faster growth in the fermentation process and higher production of antibiotics. The lipopeptide production of the mutant was 2·3-fold as that of the original strain.
Conclusions:  A mutant strain with strong antagonistic activity and high yielding of antibiotics was obtained by APPJ in this study. The mutant could be used as a promising biocontrol agent in agriculture.
Significance and Impact of Study:  This study provides a novel mutagenic source for breeding high-yielding microbial mutant, which would be very useful in the application of some valuable metabolites from micro-organism.     

生防枯草芽孢杆菌B29菌株抗菌物质的初步研究

通过检测生防枯草芽孢杆菌除菌上清液对黄瓜枯萎病菌Fusarium oxysporium f．cumerinum菌丝生长和分生孢子萌发的抑制作用,初步研究了生防枯草芽孢杆菌B29菌株抗菌物质的活性。结果表明,枯草芽孢杆菌B29菌株分泌的抗菌物质不仅抑制病原菌的生长;并可抑制尖孢镰刀菌孢子的萌发,使分生孢子萌发畸形。研究确定了该菌株抗菌物质产生的最佳条件：培养温度30℃;培养基初始pH值7.5;装液量为250ml三角瓶装液75ml培养基;培养时间120h。经30%~70%硫酸铵沉淀获得的抗菌粗提物对60℃处理具有稳定性（活性达97.8%）;对蛋白酶K具有部分耐受性,对胰蛋白酶和胃蛋白酶较敏感。     

Formulation and Delivery of the Bacterial Antagonist Bacillus subtilis for Management of Lentil Vascular Wilt Caused by Fusarium oxysporum f. sp. lentis

Different formulations of Bacillus subtilis were prepared using standard laboratory protocols. Bacillus subtilis survived in glucose and talc powders at 8.6 and 7.8 log₁₀ CFU/g, respectively, for 1 year of storage at room temperature compared with 3.5 log₁₀ CFU/g on a peat formulation. Glasshouse experiments using soil and seed treatments were conducted to test the efficacy of B. subtilis for protecting lentil against the wilt disease caused by Fusariumoxysporum f. sp. lentis. Seed treatments with formulations of B. subtilis on glucose, talc and peat significantly enhanced its biocontrol activity against Fusarium compared with a treatment in which spores were applied directly to seed. The formulations decreased disease severity by reducing colonization of plants by the pathogen, promoting their growth and increased the dry weight of lentil plants. Of these treatments the glucose and talc‐based powder formulations were more effective than the peat formulation and the spore application without a carrier. It was shown that the B. subtilis spores applied with glucose were viable for longer than those applied with other carriers. Seed treatment with these formulated spores is an effective delivery system that can provide a conducive environment for B. subtilis to suppress vascular wilt disease on lentil and has the potential for utilization in commercial field application.     

Development of new formulations of Bacillus subtilis for management of tomato damping-off caused by Pythium aphanidermatum

Formulations of a strain of Bacillus subtilis AUBS-1 inhibitory to the growth of the damping-off pathogen, Pythium aphanidermatum, were developed for seed treatment. The formulations included a talc-based powder, lignite-based powder, lignite+fly ash-based powder, wettable powder, bentonite-paste, polyethylene glycol (PEG) paste and a water-dispersible tablet. Formulations were stored at room temperature for 2 years and frequently sampled to test their shelf life. Populations of bacteria in the formulations were stable for up to 2 years storage at room temperature (28°C). Viability of propagules in lignite, lignite+fly ash, bentonite paste, wettable powder and water dispersible tablet formulations was 100% for up to 1 year. However, the viability of propagules was significantly reduced in talc, wettable powder, PEG paste and tablet formulations beyond 1 year of storage. Seed treatment of tomato with these formulations resulted in effective control of damping-off caused by P. aphanidermatum, and also enhanced plant biomass under glasshouse and field conditions. Active rhizosphere colonization by the bacterium was observed on tomato plants grown from seeds treated with the above formulations.     

枯草芽孢杆菌TF26抗菌蛋白抑菌活性的初步研究

目的:研究枯草芽孢杆菌TF26抗菌蛋白的抑菌活性和生物稳定性,为菌株及抗菌蛋白的应用提供理论依据.方法:采用硫酸铵盐析方法提取抗菌蛋白,采用菌丝生长速率法检测其对13种植物病原真菌的抑菌活性,采用抑菌圈方法对其生物稳定性进行分析.结果:抗菌蛋白粗提物能够抑制13种植物病原真菌的生长,平皿抑制率为74.3％ ～91.3％,对葵花菌核病菌、番茄和黄瓜枯萎病菌、黄瓜菌核病菌和立枯病菌、水稻恶苗病菌和大豆根腐病菌抑制作用较强.抗菌蛋白在100℃以下,pH＜ 10范围内抑菌活性稳定,对紫外线照射不敏感,室温(20℃)和4℃储存150d抑菌活性稳定.结论:抗菌蛋白具有较强的热、酸碱、紫外和储存稳定性以及广谱的抑菌活性.     

Biocontrol of Rhizoctonia Damping-off of Tomato by Bacillus subtilis Combined with Burkholderia cepacia

Bacillus subtilis strain RB14‐C and Burkholderia cepacia strain BY were used in combination to control damping‐off of tomato plants caused by Rhizoctonia solani. Microcosm tests showed complete inhibition of R. solani growth on filter disks buried in soil added with the mixture of both bacteria. Single BY inhibited the fungus, but not completely, and RB14‐C had only slight inhibitory effect on pathogen growth. The efficacy of this combining treatment was checked in pot experiments, where bacteria were applied to the soil in several combinations: RB14‐C and BY together 4 days before seed planting, RB14‐C 4 days and BY 2 days before seed planting, RB14‐C 4 days and BY immediately before seeds. The effect of these treatments on population of R. solani in soil and infection of plants was compared with the activity of single application of each agent. All bacterial treatments significantly decreased damping‐off of tomato plants. The best control was obtained when BY was added 2 days after RB14‐C. In this treatment plant protection was significantly higher than that obtained in other combined applications and obtained by single strains, except BY added to the soil 4 days before seed planting. The lowest suppression indicated BY introduced to the soil before seed planting. RB14‐C only slightly decreased number of R. solani in the soil. In contrast, BY drastically reduced population of the pathogen. However, there was not a clear relation between decrease of pathogen density in soil and the rate of plant infection. The results show that combination of B. subtilis RB14‐C with B. cepacia BY can lead to greater damping‐off suppression than biocontrol exhibited by these strains used separately, but the effect of combining bacterial agents was clearly related to the order in which both agents were introduced.     

Identification of antifungal substances secreted by Bacillus subtilis Z-14 that suppress Gaeumannomyces graminis var. tritici

Bacillus subtilis strain Z-14 has biological control activity against the take-all fungus Gaeumannomyces graminis var. tritici (Ggt). In Petri dishes, the crude extract from B. subtilis Z-14 culture filtrate reduced take-all severity in roots of wheat seedlings by 91.3% and potted plants by 69.8% compared to the Ggt-inoculated control. Treatment with the crude extract also significantly (P?<?.05) increased growth of roots’ average length, and fresh weight in comparison with those of the Ggt-inoculated control. B. subtilis Z-14 culture filtrate was relatively thermally stable with 88.2% of the antifungal activity being retained after being heated at 100°C for 30?min. Meanwhile, the antifungal activity remained almost unchanged (>95%) when the culture filtrate was exposed to a pH ranging from 3 to 8, but significantly reduced in basic conditions. This activity was not transferred to the organic solvent phase after treatment with organic extraction agents. B. subtilis Z-14 culture filtrate exhibited a broad spectrum of antifungal activities against various phytopathogenic fungi. Three homologs of iturin A (C14–16) were characterised by liquid chromatography-mass spectroscopy (LC-MS) and electrospray ionisation mass spectrometry/mass spectrometry collision-induced dissociation (ESI-MS/MS CID).     

枯草芽孢杆菌磁场诱变株对采后番茄灰霉病的控制效果研究

对枯草芽孢杆菌(Bacillus subtilis)进行了磁场诱变,获得了39个诱变株,通过与番茄灰霉病菌(Botrytis cinerea Pers.)拮抗作用的研究,获得3株高效拮抗菌。实验结果表明,枯草芽孢杆菌BS0.25-1,BS1.25和BS0.10-1为高效拮抗菌株,其中BS1.25对采后番茄灰霉病防治效果较好,20℃下贮藏5天后,先接种BS1.25的处理防效达100%,显著好于对照,为较理想的拮抗菌。     

Integrated and biological control of gladiolus corm rot and wilt caused by Fusarium oxysporum f.sp. gladioli

An isolate of Trichoderma virens Miller, Giddens & Foster, carboxin and a combination of both were evaluated for the control of gladiolus corm rot and wilt caused by Fusarium oxysporum f.sp. gladioli in glasshouse and field experiments. All treatments significantly reduced disease incidence in both glasshouse and field conditions. T. virens gave control at least as good as carboxin in all experiments. Control was significantly improved in two field experiments by combining the biological and chemical treatments.     

An antifungal compound produced by Bacillus subtilis YM 10-20 inhibits germination of Penicillium roqueforti conidiospores

AIMS: To identify and characterize an antifungal compound produced by Bacillus subtilis YM 10-20 which prevents spore germination of Penicillium roqueforti. METHODS AND RESULTS: The antifungal compound was isolated by acid precipitation with HCl. This compound inhibited fungal germination and growth. Identification by HPLC and mass spectrometry analysis showed high similarity to iturin A. Permeabilization and morphological changes in P. roqueforti conidia in the presence of the inhibitor were revealed by fluorescence staining and SEM, respectively. CONCLUSOINS: The iturin-like compound produced by B. subtilis YM 10-20 permeabilizes fungal spores and blocks germination. SIGNIFICANCE AND IMPACT OF THE STUDY: Fluorescence staining in combination with flow cytometry and scanning electron microscopy are efficient tools for assessing the action of antifungal compounds against spores. Iturin-like compounds may permeabilize fungal spores and inhibit their germination.     

枯草芽孢杆菌HS-A38产抗菌脂肽Bacillomycin D的组分分析及鉴定

对一株枯草芽孢杆菌HS-A38产生的脂肽类物质进行分离鉴定及抑菌活性研究。通过酸沉淀分离和有机溶剂抽提的方法,从枯草芽孢杆菌HS-A38发酵液中得到脂肽粗提物LP,产率为1.956 g/L。利用薄层色谱和茚三酮染色法确定该脂肽粗提物中存在四个组分,分别为LP1、LP2、LP3和LP4;抑菌活性检测显示,组分LP3对两株海洋致病菌副溶血性弧菌和铜绿假单胞杆菌的活性较高。组分LP3经硅胶柱层析纯化分离后,应用反相高效液相色谱(RP-HPLC)和基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)对该组分做进一步纯化和鉴定。分析表明,LP3样品在保留时间20 min~28 min产生单峰团LP3-1,其纯度为85.24%;经MALDI-TOF-MS分析和数据比对,组分LP3-1中的主要成分为杆菌霉素Bacillomycin D。     

A formulation of neem cake seeded with Bacillus sp. provides control over tomato Fusarium crown and root rot

Fusarium crown and root rot (FCRR) caused by the fungus Fusarium oxysporum f. sp. radicis-lycopersici is a damaging soil-borne disease of tomato. A plant growth rhizobacterium, Bacillus sp. strain HN09 isolated from neem tree rhizosphere soil, was shown to inhibit the growth, germination and development of normal morphology of the FCRR pathogen. A substantial level of disease control was achieved in greenhouse trials by soil supplementation with a preparation of neem cake seeded with HN09. Dry sterilisation of neem cake before fermentation gained comparable disease control effect as those in the unsterilised treatment, whereas moist sterilisation treatment decreased the effect significantly. This bioformulation also led to significantly raised activities in tomato genes encoding pathogenesis-related proteins, hence providing an effective alternative for the control of FCRR, reducing the need for chemical fungicide and fertilisers that impact the environment.     

基于气相色谱-飞行时间质谱联用技术分析枯草芽孢杆菌对变异链球菌的抑制作用

【背景】龋病危害着人类健康,变异链球菌(Streptococcusmutans)是公认的主要致龋菌。我们前期的研究从口腔中分离出一株枯草芽孢杆菌(Bacillussubtilis),其能抑制S.mutans的生长,但尚未明确其抑菌作用的组分。【目的】利用非靶向代谢组学技术检测分析B. subtilis zh78抑制S. mutans生长可能的小分子代谢物,以评估其在龋病防治研究及应用中的前景。【方法】取B. subtilis zh78经生长0 h、7 h、12 h、5 d时的菌液,利用牛津杯法将冷甲醇萃取后的代谢物对S. mutans进行抑菌实验;采用气相色谱-飞行时间质谱联用技术(GC-TOF-MS)对代谢物进行检测;利用主成分分析(Principalcomponentanalysis,PCA)、正交偏最小二乘判别分析(Orthogonalpartialleastsquaresdiscriminantanalysis,OPLS-DA)和皮尔森相关分析(Pearson correlationanalysis)等方法进行数据分析。【结果】B.subtiliszh78生长5d时的代谢产物对S.mutans具有明显抑菌作用,其代谢物中木糖醇、氨基酸类及有机酸类等36种物质与其对S.mutans的抑制作用显著相关。【结论】B. subtilis zh78可能产生木糖醇、氨基酸类及有机酸类等可能促进B. subtilis zh78抑制S. mutans生长的小分子代谢产物,具有一定的防龋应用研究前景。     

Effect of a Bacillus subtilis strain on flax protection against Fusarium oxysporum and its impact on the root and stem cell walls

In Normandy, flax is a plant of important economic interest because of its fibres. Fusarium oxysporum, a telluric fungus, is responsible for the major losses in crop yield and fibre quality. Several methods are currently used to limit the use of phytochemicals on crops. One of them is the use of plant growth promoting rhizobacteria (PGPR) occurring naturally in the rhizosphere. PGPR are known to act as local antagonists to soil‐borne pathogens and to enhance plant resistance by eliciting the induced systemic resistance (ISR). In this study, we first investigated the cell wall modifications occurring in roots and stems after inoculation with the fungus in two flax varieties. First, we showed that both varieties displayed different cell wall organization and that rapid modifications occurred in roots and stems after inoculation. Then, we demonstrated the efficiency of a Bacillus subtilis strain to limit Fusarium wilt on both varieties with a better efficiency for one of them. Finally, thermo‐gravimetry was used to highlight that B. subtilis induced modifications of the stem properties, supporting a reinforcement of the cell walls. Our findings suggest that the efficiency and the mode of action of the PGPR B. subtilis is likely to be flax variety dependent.     

Identification of a quorum sensing pheromone posttranslationally farnesylated at the internal tryptophan residue from Bacillus subtilis subsp. natto

Bacillus subtilis subsp. natto produces poly-γ-glutamic acid under the control of quorum sensing. We identified ComX_natto pheromone as the quorum-sensing pheromone with an amino acid sequence of Lys-Trp-Pro-Pro-Ile-Glu and the tryptophan residue posttranslationally modified by a farnesyl group. ComX_natto pheromone is unique in the sense that the 5th tryptophan residue from the C-terminal is farnesylated.