Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening

Alpha-lactalbumin(α-LA)is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants.In this study,Bacillus subtilis RIK1285 harboring AprE signal peptide(SP)was selected as the original strain for the production ofα-LA.It was found thatα-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation supernatant.The original strain most likely only producedα-LA intracellular,but not extracellular.To improve the expression and secretion ofα-LA in RIK1285,a library of 173 homologous SPs from the B.subtilis 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in RIK1285.SP YjcN was determined to be the best signal peptide.Bands in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal peptide.In addition,different promoters(P_(aprE),P_(43),and P_(glv))were compared and applied.The results indicated that the strain RIK1285-pBE-P_(glv)-YjcN-LALBA had the highestα-LA yield,reaching 122.04μg/mL.This study demonstrates successful expression and secretion of humanα-LA in B.subtilis and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.

Bacillus subtilis Z-5产果胶酶发酵条件的优化及其应用

为进一步提高果胶酶的产量,采用响应面法优化了Bacillus subtilis Z-5产果胶酶的发酵培养基和发酵条件,并将优化后得到的果胶酶应用于梨汁澄清实验。通过单因素实验探究9个因子对B.subtilis Z-5产果胶酶活力的影响,然后采用Plackett-Burman试验设计筛选出3个显著影响产酶的因素:酵母粉含量、pH、培养时间;再结合响应面设计法确定了最佳的发酵条件。结果表明最佳的发酵培养基成分是果胶10 g/L,酵母粉7.99 g/L,MgSO_(4)·7H2O 0.5 g/L;最佳的发酵条件为初始pH5.66,发酵温度37℃,培养时间72 h,接种量为6%(v/v),装液量50/250 mL,底物浓度为10 g/L;在此基础上,B.subtilis Z-5产果胶酶酶活力由879.00 U/mL提高到1549.62 U/mL,是优化前的1.76倍。在梨汁澄清实验中表明果胶酶的最适添加量为4 mL,最适酶解时间为2.5 h,最适酶解温度为65℃,最适酶解pH为6.0,此时透光率最大为79.77%,与商品酶相比,自制果胶酶是一种具有多种酶系复合酶,可作用于梨汁中含有的蛋白、淀粉等其他成分,使得梨汁更加澄清。本研究结果为果胶酶的工业化生产和应用提供了理论支撑。

Biotransformation of Shrimp Wastes by Bacillus subtilis OKF04 and Evaluation of Growth Promoting Effect in Crop Planting

In this study,we proposed a reliable and sustainable technique for the clean utilization of shrimp wastes,which can yield a solid inoculant of Bacillus subtilis OKF04 containing micronutrients at low cost without the risk of contamination.Study of the culture conditions revealed that the head of shrimp Litopenaus vannamei and the wheat bran acted as suitable substrates for the growth of B.subtilis OKF04.With 60%initial moisture content,30℃culture temperature,and 5%inoculation amount,followed by 48 hours of fermentation and 0.5%soluble starch added during the drying process(50℃for 6h),a solid B.subtilis OKF04 inoculant with a spore amount of 2.4×10^(10)CFU g^(-1)and a high amino acid content was obtained.The solid B.subtilis OKF04 inoculant was applied to cultivate pakchoi under pot experiment.As the result,of adding to,the size of stems and leaves,nutritional composition,and physiological activity of pakchoi were significantly(P<0.05)enhanced by solid B.subtilis OKF04 inoculant.B.subtilis OKF04 also significantly(P<0.05)increased the soil’s nutrient content and improved its microbial composition.Furthermore,pakchoi cultivated with a low dose of solid B.subtilis OKF04 inoculant(0.05 g kg^(-1)soil)resulted in the best results.This study provides a new method for the preparation of microbial inoculants with solid waste shrimp heads.

高产淀粉酶菌株Bacillus subtilis XC2的产酶条件及酶学性质

试验研究从高产淀粉酶菌株Bacillus subtilis XC2的产酶条件及酶学性质。通过单因素试验和正交试验,初步探究淀粉酶酶学性质并优化菌株发酵产酶条件。结果表明,菌株XC2产淀粉酶的反应最适温度为50℃,最适反应pH值为6.0,在30~50℃和pH值4.0~7.0时酶活稳定性较好,Fe^(2+)、Cu^(2+)和Zn^(2+)对酶活有促进作用,Na^(+)对酶活有抑制作用。菌株XC2产淀粉酶最佳条件为蔗糖添加量1.5%、牛肉膏添加量2.0%、K^(+)添加量0.6%、培养温度37℃、转速200 r/min、初始pH值7.0、发酵时间21 h。研究表明,对产酶条件优化后,Bacillus subtilis XC2产淀粉酶活力达984.5 U/mL。

耐盐性谷氨酰胺酶在Bacillus subtilis 168中的整合表达

微生物质粒过表达外源基因,会由于质粒分离稳定性低、种子培养基中需添加抗生素等原因,限制其在工业生产中的应用。为了得到一株可用于工业生产谷氨酰胺酶的菌株,选择食品安全级Bacillus subtilis 168作为宿主,将来源于Micrococcus luteus K-3的编码耐高浓度盐的谷氨酰胺酶基因(Mglu)插入到其染色体上进行整合表达。选择了2个内源性蛋白酶基因作为整合表达位点,通过lox序列的重组消除抗性基因,得到无抗性基因的重组谷氨酰胺酶表达菌株。遗传稳定性研究表明,在不添加抗生素的条件下,通过连续传代重组菌株并测定发酵酶活,发现重组菌株连续传42代时,酶活基本不变。随后,采用5 L发酵罐对重组菌株进行分批补料发酵,最高酶活达到了41.5 U/mL。本研究对提高谷氨酰胺酶重组菌株遗传稳定性提供了借鉴。

Efficacy of Bacillus subtilis MBI 600 Against Sheath Blight Caused by Rhizoctonia solani and on Growth and Yield of Rice

Rice sheath blight disease （ShB）, caused by Rhizoctonia solani, gives rise to significant grain yield losses. The present study evaluated the efficacy of Integral, the commercial liquid formulation of Bacillus subtilis strain MBI 600, against rice ShB and for plant growth promotion. In greenhouse studies, four log concentrations of Integral （from 2.2×10^6 to 2.2×10^9 cfu/mL） were used as seed treatment （ST）. After 25 d, seedlings were dipped （SD） into Integral prior to transplanting. At 30 d after transplanting （DAT）, leaf sheaths were inoculated with immature sclerotia of the pathogen. At 45 DAT, a foliar spray （FS） with Integral was applied to some treatments. The fungicide control was 50% carbendazim at 1.0 g/L, and a nontreated control was also included. Overall, there were 10 treatments, each with five replications. ShB severity was rated at 52 DAT, and seedling height and number of tillers per plant were rated at 60 DAT. In 2009, two field trials evaluated Integral at 2.2×10^8 and 2.2×10^9 cfu/mL. Integral was applied as ST, and seedlings were produced in a nursery bed. After 32 d, seedlings were treated with Integral as SD and transplanted into 10 m^2 blocks. Foliar sprays were given at 45 and 60 DAT. There were seven treatments, each with eight replications arranged as a factorial randomized complete block design. At 20 DAT, the plots were broadcast inoculated with R. solani produced on rice grains. Seedling height before transplanting, ShB severity at 90 DAT, and grain yield at harvest were recorded. Integral at 2.2×10^9 cfu/mL provided significant increase of seedling heights over other treatments under greenhouse conditions. The Integral treatments of ST ＋ SD ＋ FS at 2.2×10^9 cfu/mL significantly suppressed ShB over other treatments. In field studies, Integral provided significant increase of seedling height in nursery, and number of tillers per plant, compared with the control. ShB severity was significantly suppressed with higher concentrations of Integral compared to lower concentrations. Grain yield were the highest at an Integral concentration of 2.2×10^9 cfu/mL. Overall, Integral significantly reduced ShB severity, enhanced seedling growth, number of tillers per plant and grain yield as ST ＋ SD ＋ FS at the concentration of 2.2×10^9 cfu/mL under the conditions evaluated.

The PhoR/PhoP two-component system regulates fengycin production in Bacillus subtilis NCD-2 under low-phosphate conditions

Bacillus subtilis strain NCD-2 is an excellent biocontrol agent for plant soil-borne diseases, and the lipopeptide fengycin is one of the active antifungal compounds in strain NCD-2. The regulator PhoP and its sensor kinase PhoR compose a two-component system in B. subtilis. In this study, the phoR- and phoP-knockout mutants were constructed by in-frame deletion and the role of PhoR/PhoP on the production of fengycin was determined. Inactivation of phoR or phoP in B. subtilis decreased its inhibition ability against Botrytis cinerea growth in vitro compared to the strain NCD-2 wild type. The lipopeptides were extracted from strain NCD-2 wild type and its mutant strains by hydrochloric acid precipitate, and the lipopeptides from phoR-null mutant orphoP-null mutant almost lost the inhibition ability against B. cinerea growth compared to the lipopeptides from strain NCD-2 wild type. Fast protein liquid chromatography （FPLC） analysis of the lipopeptides showed that inactivation of phoR or phoP genes reduced the production of fengycin by strain NCD-2. The fengycin production abilities were compared for bacteria under low-phosphate medium （LPM） and high-phosphate medium （HPM）, respectively. Results indicated that the regulation of fengycin production by the PhoR/PhoP two-component system occurred in LPM but not in HPM. Reverse transcriptionaI-PCR confirmed that the fengycin synthetase gene fenC was positively regulated by phoP when cultured in LPM. All of these characteristics could be partially restored by complementation of intact phoR or phoP gene in the mutant. These data indicated that the PhoR/PhoP two-component system greatly regulated fengycin production and antifungal ability in B. subtilis NCD-2 mainly under low-phosphate conditions.

A Mutant of Bacillus Subtilis with High-Producing Surfactin by Ion Beam Implantation

In order to generate a mutant of Bacillus subtilis with enhanced surface activity through low energy nitrogen ion beam implantation, the effects of energy and dose of ions implanted were studied. The morphological changes in the bacteria were observed by scanning electron microscope （SEM）. The optimum condition of ions implantation, 20 keV of energy and 2.6 × 10^15N^＋/cm^2 in dose, was determined. A mutant, B.s-E-8 was obtained, whose surface activity of 50-fold and 100-fold diluted cell-free Landy medium was as 5.6-fold and 17.4-fold as the wild strain. The microbial growth and biosurfactant production of both the mutant and the wild strain were compared. After purified by ultrafiltration and SOURCE 15PHE, the biosurfactant was determined to be a complex of surfactin family through analysis of electrospray ionization mass spectrum （ESI/MS） and there was an interesting finding that after the ion beam implantation the intensities of the components were different from the wild type strain.

Optimization and Characterization of Nicosulfuron-Degrading Enzyme from Bacillus subtilis Strain YB1

A strain of Bacillus subtilis strain YB 1, isolated and preserved in our lab., showed a high nicosulfuron-degrading activity. Optimization of culture conditions on production of nicosulfuron-degrading enzyme from Bacillus subtilis strain YB 1 was carried out through mono-factor experiments. The characterization of degrading enzyme（s） was studied in this paper. The results showed that B. subtilis YB1 can use nicosulfuron as sole carbon source under aerobic condition. The key enzyme（s） involved in the initial biodegradation of nicosulfuron was localized to extracellular proteins and showed to be induced expressed. Enzyme-specific activity was up to 89.34 U mg-1 at pH 8.0 and 30℃, incubation for 96 h, inoculum 4.5x108 CFU mL-1 in Luria-Bertani liquid medium with nicosulfuron of 40 mg L-1. The maximum degradation rate of extracellular crude enzymes on nicosulfuron was 66% at pH 9.0, 35℃ in the enzymatic reaction system with nicosulfuron of 5 mg L-1. This degrading enzyme（s） was sensitive to high temperature, but kept high activity under alkaline conditions.

Effect of Bacillus subtilis and Pseudomonas fluorescens on Growth of Greenhouse Tomato and Rhizosphere Microbial Community

Bacillus subtilis （B. subtilis） and Pseudomonas fluorescens （P. fluorescens） are two of the most important plant growth promoting rhizobacteria （PGPR） in agriculture. An in situ trial was conducted on greenhouse tomato （Lycopersicum esculentum Mill.） to examine the effect of two bacterial strains, Bacillus subtilis （CGMCC 1.3343） and Pseudomonas fluorescens （CGMCC 1.1802）, on tomato growth, gray mold disease control, catabolic and genetic microbial features of indigenous rhizosphere bacteria under lownitrogen conditions. A commercial inoculant （ETS） was also tested as a comparison. Both B. subtilis and P. fluorescens promoted growth and biomass of seedlings, while only B. subtilis was efficient in reducing gray mold incidence in greenhouse tomato. The two bacterial strains could colonization in tomato rhizosphere soil at the end of experiment （10 days after the last inoculation）. Different AWCD trends and DGGE patterns were got in different bacterial treatments; however, analyses of microbial diversities showed that indigenous soil microbes did not seem to have significant differences at either the catabolic or genetic level among treatments. ETS, as a commercial microbial agent, promoted plant growth and gave a higher microbial diversity in rhizosphere soil.

Effect of Bacillus subtilis,Enterococcus faecium,and Enterococcus faecalis supernatants on serotonin transporter expression in cells and tissues

BACKGROUND Bacillus subtilis(B.subtilis),Enterococcus faecium(E.faecium),and Enterococcus faecalis(E.faecalis)are probiotics that are widely used in the clinical treatment of irritable bowel syndrome(IBS).Whether the supernatants of these three probiotics can improve gastrointestinal sensation and movement by regulating the serotonin transporter(SERT)expression needs to be clarified.AIM To investigate whether B.subtilis,E.faecium,and E.faecalis supernatants can upregulate SERT expression in vitro and in vivo.METHODS Caco-2 and HT-29 cells were stimulated with probiotic culture supernatants for 12 and 24 h,respectively.A male Sprague-Dawley rat model of post-infectious irritable bowel syndrome(PI-IBS)was established and the rats were treated with phosphate-buffered saline(group A)and three probiotics culture supernatants(groups B,C,and D)for 4 wk.The levels of SERT were detected by quantitative PCR and western blotting.RESULTS The levels of SERT at post-treatment 12 and 24 h were significantly elevated in Caco-2 cells treated with B.subtilis supernatant compared with those in the control group(aP<0.05).Those levels were markedly upregulated in Caco-2 cells stimulated with E.faecium and E.faecalis supernatants at 24 h(aP<0.05).In addition,SERT expression in groups B,C,and D was significantly higher than that in group A in the 2nd wk(aP<0.05).Increased SERT expression was only found in group D in the 3rd wk(aP<0.05).However,there was no significant difference in SERT expression between the groups in the last week(P>0.05).CONCLUSION The supernatants of B.subtilis,E.faecium,and E.faecalis can upregulate SERT expression in intestinal epithelial cells and the intestinal tissues in the rat model of PI-IBS.

Degradation of glucosinolates in rapeseed meal by Lactobacillus delbrueckii and Bacillus subtilis

Lactobacillus delbrueckii and Bacillus subtilis were employed as a new combination of strains to treat rapeseed meal by solid-state fermentation,aiming to efficiently degrade the glucosinolates,which are the main toxin in the meal.Single-factor tests and Response surface methodology(RSM)were used to optimize the fermentation parameters.Under the optimum fermentation parameters of 15%total injection volume of the mixture of Lactobacillus delbrueckii and Bacillus subtilis with a ratio of 2:1,bran content of16%,feed to water ratio of 1:1.5,fermentation temperature of 36°C and fermentation time of 72 h,the content of glucosinolates in rapeseed meal was decreased from 64.558μmol/g to 3.473μmol/g,reaching a high degradation rate(94.62%).The high detoxification rate by a consortium of Lactobacillus delbrueckii and Bacillus subtilis provides a bright application prospect in feed utilization of rapeseed meal.

Efficacy evaluation and mechanism of Bacillus subtilis EBS03 against cotton Verticillium wilt

Background:In our previous study,a strain EBS03 with good biocontrol potential was screened out of 48 strains of cotton endophyte Bacillus subtilis by evaluating the controlling effect against cotton Verticillium wilt.However,its mechanism for controlling Verticillium wilt remains unclear.The objective of this study was to further clarify its con-trolling effect and mechanism against cotton Verticillium wilt.Results:The results of confrontation culture test and double buckle culture test showed that the inhibitory effects of EBS03 volatile and nonvolatile metabolite on mycelium growth of Verticillium dahliae were 70.03%and 59.00%,respectively;the inhibitory effects of sporulation and microsclerotia germination were 47.16%and 70.06%,respec-tively.In the greenhouse test,the EBS03 fermentation broth root irrigation had the highest controlling effect at 87.11%on cotton Verticillium wilt,and significantly promoted the growth of cotton seedlings.In the field experi-ment,the controlling effect of EBS03 fermentation broth to cotton Verticillium wilt was 42.54%at 60 days after cotton sowing,and the boll number per plant and boll weight in EBS03 fermentation broth seed soaking,root irrigation,and spraying treatments significantly increased by 19.48%and 7.42%,30.90%and 2.62%,15.99%and 9.20%,respec-tively.Furthermore,EBS03 improved the resistance of cotton leaves against the infection of V.dahliae,and induced the outbreak of reactive oxygen species and accumulation of callose.In addition,the results of real time fluorescent quantitative polymerase chain reaction(RT-qPCR)detection showed that EBS03 significantly induced upregulation expression level of defense-related genes PAL,POD,PPO,and PR10 in cotton leaves,enhanced cotton plant resistance to V.dahliae,and inhibited colonization level of this fungal pathogen in cotton.Conclusion:Bacillus subtilis EBS03 has a good biological defense capability,which can inhibit the growth and coloni-zation level of V.dahliae,and activate the resistance of cotton to Verticillium wilt,thus increase cotton yield.

Effects of Bacillus subtilis CSL2 on the composition and functional diversity of the faecal microbiota of broiler chickens challenged with Salmonella Gallinarum

Background： The chicken gastrointestinal tract contains a diverse microbiota whose composition and structure play important roles in gut functionality. In this study, microbial shifts resulting from feed supplementation with Bacillus subtilis CSL2 were evaluated in broilers challenged and unchallenged with Salmonella Gallinarum. To analyse bacterial community composition and functionality, 454 GS-FLX pyrosequencing of 16 S r RNA gene amplicons was performed.Results： The Quantitative Insights into Microbial Ecology（QIIME） pipeline was used to analyse changes in the faecal microbiota over a 24-h period. A total of 718,204 sequences from broiler chickens were recorded and analysed. At the phylum level, Firmicutes, Bacteroidetes, and Proteobacteria were the predominant bacterial taxa. In Salmonellainfected chickens（SC）, Bacteroidetes were more highly abundant compared to control（NC） and Bacillus-treated（BT）chickens. At the genus level, in the NC and BT groups, Lactobacil us was present at high abundance, and the abundance of Turicibacter, unclassified Enterobacteriaceae, and Bacteroides increased in SC broilers. Furthermore, taxon-independent analysis showed that the SC and BT groups were compositional y distinct at the end of the 24-h period. Further analysis of functional properties showed that B. subtilis CSL2 administration increased gut-associated energy supply mechanisms（i.e. carbohydrate transport and metabolism） to maintain a stable microbiota and protect gut integrity.Conclusions： This study demonstrated that S. Gallinarum infection and B. subtilis CSL2 supplementation in the diet of broiler chickens influenced the diversity, composition, and functional diversity of the faecal microbiota. Moreover, the findings offer significant insights to understand potential mechanisms of Salmonel a infection and the mode of action of probiotics in broiler chickens.

Study on enzymatic hydrolysis of soybean β-conglycinin using alkaline protease from Bacillus subtilis ACCC 01746 and antigenicity of its hydrolysates

Due to its beneficial health effects,the use of soybean protein has shown a continuous increase,but concerns regarding the allergenicity of soybean antigenic protein have also increased.This study aimed to evaluate the hydrolytic effects of a non-commercial alkaline protease isolated from the Bacillus subtilis ACCC 01746 on soybeanβ-conglycinin and the allergenicity of its hydrolysates.Alkaline protease of the strain was separated by precipitation method of organic solvents,and theβ-conglycinin was separated by alkali-solution and acid-isolation and purified by use of gel column.Using the degree of hydrolysis(DH)and inhibition rate as evaluation indexes,the enzymatic hydrolysis parameters ofβ-conglycinin was optimized by single factor and L_(9)(3^(4))orthogonal tests,so as to explore the effect of the protease on the hydrolysis degree and the antigenicity ofβ-conglycinin hydrolysates.The results showed that the native enzyme existed as an 18.3 kDa monomer with a 430 U/g maximum activity.The purity ofβ-conglycinin was 84.8%.The single-factor test results showed that DH showed the oppostie trendency with the inhibition rate,and the increase of protein concentration causedmonotone increasing and monotone decreasing of the inhibition rate and the DH,and the optimal protein concentration was 30 mg/mL.The optimization results showed that pH had the largest impacts on both DH and the inhibition rate,followed by enzyme dosage,hydrolysis temperature and hydrolysis time.Under the optimum hydrolysis conditions of protein concentration 30mg/mL,enzymedosage0.7%,hydrolysis time40min,temperature 55°C and pH8.5,the DH reached the highest of 76.28%,and the inhibition rate was the lowest of 27.03%,which was reduced greatly compared with that before optimization.These results suggested that alkaline protease appeared to show a relatively high effeciency in lowering soybean allergenicity,making it possible to produce low-allergenicity soybean protein.

Biocontrol of Rhizoctonia solani K1 by Iturin A Producer Bacillus subtilis RB14 Seed Treatment in Tomato Plants

Bacillus subtilis RB14 was used as an antagonist against fungal pathogen Rhizoctonia solani K1 to control damping-off diseases in tomato plants. Tomato seeds were treated with B. subtilis RB14 culture. The concentration of bacterial cells for the treatment was about 108 cfu/ml. Treated tomato seeds showed 99% germination index similar to the untreated seeds. Scanning Electron Microscopic observations showed a clear evidence of the presence of B. subtilis RB14 on tomato seed surface. Clear inhibition zone was observed using treated seed in dual plate assay against R. solani K1. B. subtilis RB14 treated seed showed 80% reduction in disease incidence during in vivo plant experiments. B. subtilis RB14 produces lipopeptide antifungal antibiotic iturin A which could suppress R. solani K1. The phenomenon was supported by our observation where we found significant amount of iturin A from the root zone soil of the seed treated plants.

Study on Mutagenic Breeding of Bacillus Subtilis and Properties of Its Antifungal Substances

Bacillus subtitles JA isolated by our laboratory produced a large amount of anti-fungal substances, which had strong inhibitory activity against various plant pathogenic fungi, such as Rhizoctonia solani, Fusarium graminearum and so on. Ion beam implantation as a new mutagenic methods was applied in our studay. After B. subtitles JA was implanted by N+ ions, a strain designated as B. subtitles JA-026 was screened and obtained, which had a higher ability to produce those antifungal substances. A series of experiments indicated that the antifungal substances were thermostable and partially sensitive to proteinases K and tryproteinase. When the fermentating broth was fractionated with ammonium sulphate of a final saturation of 70%, the precipitate-enhanced inhibitory activity while the supernatant lost this activity. It appeared that the antifungal substances were likely to be protein.

Expression of a Lysine-rich Gene in Bacillus subtilis 168

Lysine-rich protein gene （lys） was cloned from winged bean （Psophocarpus tetragonolobus （L.） DC）, and cloned into prokaryotie expression vector pHT43, the recombinant plasmid pHT43/lys were constructed and then transferred into Bacillus subtilis168, upon IPTG induction, the recombinant protein was expressed, and the content of lysine was detected by HPLC. The result showed that lysine content increased by 9.85%. It was suggested that introducing lys gene into Bacillus subtilis 168 was an effective way to improve its nutrition quality.

Purification of Two Antimicrobial Substances Produced by Bacillus subtilis Strain B11 and Their Properties

Two antimicrobial substances produced by Bacillus subtilis strain B 11 were purified by boiling, DEAE 52 anion exchange chromatography and aluminum oxide adsorption chromatography, These purified substances showed only one spot on silica gel thin layer chromatography （TLC）. Antimicrobial assays showed that antimicrobial substances A and B inhibited the growth of plant pathogens such as Fusarium oxysporum Schl f.sp. niveum, Rhizoctonia solani, Ralstonia solanacearum and Xanthomonas oryzae pv. oryzae. In addition, antimicrobial substance B could also inhibit the growth of Magnaporthe grisea. These two antimicrobial substances were resistant to proteolytic enzymes and temperature as high as 121℃.

Colonization Ability of Bacillus subtilis HAS in Sugarcane

Bacillus subtilis HAS had good control effects on sugarcane smut in the field. Colonization dynamics of B. subtilis HAS were studied through biotics marker and pot culture, to understand the bio-control mechanism of the strain and provide experimental basis for commercialization. The results showed that B.subtilis HAS had strong colonization ability, which could be colonized in sugarcane root, stem, leaf and rhizosphere for long time, but the colonization levels were different. The colonization density of B.sutili HAS remained 103 CFU/g soil in the rhizosphere and 102 CFU/g （fresh weight） in the root after inoculation of 60 d. The colonization in the rhizosphere decreased first, then increased and decreased again, and finally stabilized. The colonization in the root first increased then decreased. Meantime, colonization status in stems and leaves showed that the colonization density in stems and leaves was 10 CFU/g （fresh weight）, indicating that the colonization level was not high in stem and leaf.