枯草芽孢杆菌W-1 α-淀粉酶的热稳定性研究

本文研究了温度对枯草芽孢杆菌W-1 a-淀粉酶的活力的影响。试验表明,该酶在60℃以下是稳定的;75℃加热10分钟全部失活,在0.02M CaCl_2和0.02M NaCl并存下,80℃加热10分钟,活力保持不变。

枯草芽孢杆菌对产蛋后期蛋鸡生产性能和蛋壳品质的影响

本试验旨在研究饲粮中添加枯草芽孢杆菌对产蛋后期蛋鸡生产性能和蛋壳品质的影响。选取420只产蛋率和体重相近的健康52周龄海兰褐蛋鸡,随机分为4个组,每组7个重复,每个重复15只鸡。对照组饲喂玉米-豆粕型基础饲粮,试验组分别在基础饲粮中添加200、400和800 mg/kg枯草芽孢杆菌。预试期1周,正试期12周。结果表明,与对照组相比:1)饲粮中添加200、400和800 mg/kg枯草芽孢杆菌对产蛋率和平均蛋重无显著影响(P>0.05),但显著降低了平均日采食量(P<0.05);饲粮中添加400和800 mg/kg枯草芽孢杆菌显著降低了料蛋比(P<0.05)。2)饲粮中添加200、400和800 mg/kg枯草芽孢杆菌显著增加了蛋壳厚度(P<0.05),显著降低了破软蛋率(P<0.05);饲粮中添加800 mg/kg枯草芽孢杆菌显著提高了蛋壳强度和壳重比例(P<0.05)。3)饲粮中添加800 mg/kg枯草芽孢杆菌改善了蛋壳的超微结构,显著增加了蛋壳中钙含量(P<0.05)。由此可见,饲粮中添加枯草芽孢杆菌可以改善产蛋后期蛋鸡生产性能,提高蛋壳品质,降低破软蛋率。本试验条件下,饲粮中添加800 mg/kg枯草芽孢杆菌为宜。

紫苏籽油抗菌活性研究

以革兰氏阴性菌大肠杆菌和革兰氏阳性菌枯草芽孢杆菌作为供试菌株,采用滤纸片法测定评估了紫苏籽油经过皂化提取之后其提取液抑制大肠杆菌和枯草芽孢杆菌的能力,并将所得的结果与大豆油的抑菌作用进行比较。结果表明,紫苏籽油提取液对大肠杆菌和枯草芽孢杆菌两个供试菌株的抑制效果较强,且对于枯草芽孢杆菌的抑制效果要优于大肠杆菌,且紫苏籽油对大肠杆菌和枯草芽孢杆菌的抑制效果要优于大豆油。

来自鳗鲡的二株益生菌适宜培养基的研究

用正交试验法,通过研究培养基基础成分、主要添加成分和无机盐对养殖鳗鲡(Anguilla japonica)肠道益生菌枯草芽孢杆菌(A40209CDC4)和少动鞘氨醇单胞菌(A31009NA)的生长影响,确定了优化的培养基。菌株A31009NA的优化配方为:蛋白胨1.0%,牛肉膏0.1%,食盐0.5%,酵母膏0.5%,葡萄糖1.0%,硫酸铵0.05%,MgSO4.7H2O 0.1 g/L,CuSO4.5 H2O 0.15 mg/L,MnSO4.H2O 1.5 mg/L,CoCl.H2O 0.15 mg/L;A40209CDC4优化培养基配方为:蛋白胨1.0%,牛肉膏0.1%,食盐1.0%,酵母膏0.5%,硫酸铵0.05%,MgSO4.7 H2O 0.1 g/L,MnSO4.H2O 1.5 mg/L,CoCl.H2O 0.15 mg/L,CaCl20.02 g/L。优化培养基与普通营养肉汤(蛋白胨1.0%,牛肉膏0.3%,食盐0.5%)培养对照试验结果表明,优化培养基有效地提高了细菌产量。

Screening of Biocontrol Strain Bacillus subtilis by N^+ Ion Beam Implantation

[Objective] This study was to investigate the effect of N＋ ion beam implantation on the survival rate and mutation rate of biocontrol strain Bacillus subtilis. [Method] The factors influencing B. subtilis ion beam implantation, including culture time, dilution concentration, solvent, drying time of mycoderm were optimized. B. subtilis cells were implanted by using ion beam at dose of 2.0×10^14～4.0×10^14 ions/cm2 and the energy of 30 kev. Then the methods of culturing colonies confronting each other on plate and Oxford cup diffusion were used to screening strains. [Result] The optimal parameters were found as follows： culture in liquid for 20-24 h, dilution with sterile water to 106 cells/ml and drying time of 60 min for sample preparation; the optimal N＋ ion beam implantation dose of 2.0×10^14～4.0×10^14 ions/cm2 at the energy of 30 kev, the survival rate of 8.43%-26.71% and the mutation rate of 3.50%-5.43%. [Conclusion] This study provided reference for ion beam implantation mutation of B. subtilis.

Effects of Heavy Metal Stress on the Growth Curves of Two Sepcies of Bacteria

[Objective] The aim was to study on the effects of heavy metal stress on the growth curves of Escherichia coli and Bacillus subtili. [Method] Using traditional culture method,Escherichia coli and Bacillus subtili were cultured under heavy metal stress including Cu2＋,Hg2＋,Pb2＋,Cd2＋ and Cr6＋ in different concentrations. Then the growth curves of the bacteria were determined to investigate the effects of exogenous heavy metals on the growth of the two kinds of bacteria. [Result] The proliferation of the two bacteria was inhibited at high concentrations of Hg2＋ and Cd2＋ respectively,and G＋ is more sensitive to them than G-; when the heavy metal concentration was 50 mg/L,the toxicity of the five kinds of heavy metals on the two bacteria was Hg2＋Cd2＋Cu2＋Cr6＋≈Pb2＋. [Conclusion] The research will provide a basis to explore the effects of heavy metal on environment and ecological system.

Preliminary Study on Garlic Root Exudates Influences to the Growth of Pomegranate Wilt Pathogen(Ceratocystis fimbriata)and Bacillus subtilis

[Objectives] This paper aims to explore the possibility to intercrop garlic with pomegranate tree to control pomegranate wilt.[Methods] Root exudates of garlic is cultivated in 1/5 concentration of MS solution and distilled water is examined in lab to test their effect to growth of mycelia of pomegranate wilt pathogen（Ceratocystis fimbriata）and multiplication of Bacillus subtilis.[Results] The result shows that garlic root exudates whatever cultivated in MS solution or distilled water could not inhibit or promote mycelia growth of C.fimbriata.However,garlic root exudates cultivated in both methods effectively promote multiplication of B.subtilis.[Conclusions] It is suggested that intercropping garlic with pomegranate tree by combining application B.subtilis could be a promising way to prevent pomegranate wilt spread in practice.

Research on the Fermentation Conditions of Bacillus subtillus B-332

[Objective] The paper was to ascertain the fermentation conditions and medium formula of Bacillus subtilis B-332. [Method] Using single factor and orthogo- nal experimental designs, the fermentation medium and culture conditions for B. sub- i tills B-332 were optimized through shaking flask culture. [Result] The optimal culture and inoculation time was 18 h, and the optimized medium formula was as follows： soybean powder 0.60 g/L, sucrose 0.25 g/L, ammonium sulfate 0.07 g/L, trisodium citrate 0.03 g/L, dipotassium hydrogen phosphate 0.003 g/L, magnesium sulfate 0.005 g/L, ferrous sulfate 0.000 5%; the fermentation condition was as follows： tem- perature 30 ℃, shaking bed speed 180 r/min, shaking bottle volume 80 ml/500 ml. In the optimal fermentation conditions, the fermented spore quantity was 1.43×10^11 cfu/ml, which was 34.48 times higher than the spore quantity of 4.03×10^9 cfu/ml in initial condition. [Result] The study laid the foundation for factory production of the strain.

On the Bacteriostatic Activity of Bacillus subtilis and Pyraclostrobin as Well as Their Mixtures to Grape Anthracnose and the Field Disease Control Efficiency

[Objective] To screen out the biological compound bactericides for grape anthracnose, reduce and replace the use of chemical pesticide. [Methods] The de- termination on the indoor bacteriostatic activity of different proportions of Bacillus subtilis and pyraclostrobin to grape anthracnose was carried out, and mycelial growth rate method was adopted to determine the toxicity of Bacillus subtilis and pyraclostrobin as well as their 5 mixtures to grape anthracnose. [Results] The EC50 of Bacillus subtilis and pyraclostrobin as well as their mixture combinations of 1：1, 1：2, 1：3, 1：4 and 1：5 to grape anthracnose were respectively 1.969 8, 1.527 4, 1.373 2, 1.294 8 and 1.247 3 μg/ml; the synergistic coefficients （SR） of the 5 mix- ture combinations to grape anthracnose were 1.70, 1.25, 1.13, 1.12 and 1.12, re- spectively, in which the synergistic effect of 1：1 was the largest. The indoor biologi- cal activity of pyraclostrobin（EC50 was 1.054 0μg/ml） was higher than that of Bacil- lus subtilis（EC50 was 15.017 5 μg/ml）. 50 d after the agentia（before the harvesting）, the investigation results showed that 1 000-fold dilution, 1 500-fold dilution and 2 000- fold dilution as well as each single dosage of 20% pyraclostrobin .200×10^8 cfu/g Bacillus subtili wettable powder all had better control efficiency to grape anthracnose after fruit setting and before bagging, in which the treatments of high concentration and middle concentration were higher than the treatments of low concentration and two single dosages： the highest control efficiency of high concentration was 90.03%, which was higher than all other treatments; the control efficiency of middle concen- tration was 87.01%, which was higher than that of low concentration and each sin- gle dosage; the control efficiency of low concentration was 84.11%, which was high- er than 1 000-fold dilution of 1 000×10^8 cfu/g Bacillus subti/i wettable powder （the control efficiency was 64.60%） and 2 000-fold dilution of 250 g/L Bacillus subti/i wettable powder （the control efficiency was 81.07%）. In addition, each treatment al- so had better control efficiency to other cluster diseases, such as white rot, etc., and the control efficiency was almost the same as that of anthracnose. [Conclusion] It was suggested that the prevention concentration of 20% pyraclostrobin .200×10^8 cfu/g Bacillus subtili wettable powder to grape anthracnose after fruit setting and before bagging was 1 000-fold - 2 000-fold dilution.

Toxicity Test and Field Control Effects of 4 Different Fungicides on Grape Downy Mildew

[Objective] This study was conducted to investigate the biocontrol activity of Streptomyces corchorusii strain NF0919 and Bacillus subtilis D J-6 WP to grape downy mildew. [Methed] We determined the indoor toxicity of the supernatant of S. corchorusii strain NF0919, 1.0×1011 cfu/g B. subtilis D J-6 WP, mancozeb and dimethomorph on Plasmopara viticola by the leaf disc method, respectively, and a field efficacy trial was conducted. [Result] The results showed that the ECso values of the supernatant of strain NF0919, 1.0×1011cfu/g B. subtilis D J-6 WP, mancozeb and dimethomorph were 96.285 9, 86.603 8, 69.947 2 and 7.263 6 μg/ml, respec- tively. The values of field efficacy in preventive experiments for grape downy mildew on the 7th day after 2 times of spraying 20 times diluent of the supernatant of strain NF0919 and 1 000 times diluent of 1.0×1011 cfu/g B. subtilis D J-6 WP were 71.55% and 70.71%, respectively, and the values of field efficacy on the 14th day after the 2 times of fungicide application were 67.54% and 68.19%, respectively. The values of field efficacy in curative experiments on the 7th day after 2 times of spraying 20 times diluent of the supematant of strain NF0919 and 1 000 times diluent of 1.0×1011 cfu/g B. subtilis D J-6 WP were 59.72% and 56.07%, respectively, and the val- ues of field efficacy on the 14th day after the 2 times of fungicide application were 56.88% and 57.46%, respectively. The field efficacy values of the 2 tested biocon- trol agents were equivalent. The protective effect showed no significant difference between each of tested biocontrol agents and 300 times diluent of 50% mancozeb WP, but there was a significant difference in the efficacy between each of tested biocontrol agents and 200 times diluent of 40% dimethomorph SC. [Conclusion] The S. corchorusii strain NF0919 and B. subtilis D J-6 WP had certain biocontrol poten- tial to grape downy mildew and development value.

Curing of the Bacillus subtilis Plasmid Using Sodium Dodecyl Sulfate

Curing of Bacillus subtilis plasmid using sodium dodecyl sulfate (SDS)was studied in order to obtain a host strain. An overnight culture of Bacillus subtilis 24/pMX45 was used to inoculate fresh LB containing SDS (0-0.008%). No growth of 24/pMX45 was observed when LB contained an SDS concentration of 0.006% or greater, and the sublethal concentration (w/v) of SDS was 0.005% with a killing rate of 99%. Samples were diluted and plated on LB agar, individual colonies were randomly picked to a selective agar medium by tooth to screen for loss of plasmid-encoded erythomycin resistance. CsCl-EtBr gradient centrifugation and plasmid DNA profile demonstrated that plasmid-cured derivative A7 has completely lost its plasmid. A7 had a shorter lag, and its cell concentration was consistently higher than that of the 24/pMX45. Elimination of the plasmid was first observed after 24/pMX45 had been treated with SDS for 8 h. The percent elimination then continued to increase until about 22 h, after which the fraction of cured cell in the population remained constant. Plasmid cured cell numbers were measured in a separate control culture of 24/pMX45 untreated by SDS. No spontaneous loss of pMX45 was observed after 24/pMX45 were incubated for 24 h and 48 h with shaking at 37 ℃.These results suggested that SDS can be used as curing agent to eliminate the plasmid of Bacillus subtilis.

Control Effects of Bacillus subtilis DJ-6 and Pyraclostrobin Alone and in Combination Against Fusarium oxysporum

[Objective] This study was conducted to screen a synergistic biological fungicide complex to control Fusarium wilt, reducing the use of chemical pesticides. [Method] The inhibitory effects of Bacil us subtilis DJ-6 and pyraclostrobin alone or in combination at five ratios against Fusarium oxysporum were detected by mea-suring mycelium growth rate in laboratory tests. The growth promotion and disease control effect of combined or single use of 20% pyraclostrobin and 2 ×1011 cfu/g B. subtilis DJ-6 WP at 1∶1 000, 1∶2 000 and 1∶3 000 dilutions were detected in field trials. [Result] The EC50 values of combined use of B. subtilis DJ-6 and pyra-clostrobin at ratios of 1∶1, 1∶2, 1∶3, 1∶4 and 1∶5 against F. oxysporum were 5.311 5, 4.008 6, 3.570 6, 3.350 9 and 3.218 9 μg/ml, with the synergistic ratios （SR） of 2.28, 1.77, 1.53, 1.64, 1.11, among which the synergetic effect at 1∶1 was the best. The fungicidal activity of pyraclostrobin was greater than that of B. subtilis DJ-6 in laboratory tests. Field trials revealed that al the 1∶1 000, 1∶2 000 and 1∶3 000 dilu-tions of 20% pyraclostrobin·2×1011 cfu/g B. subtilis DJ-6 WP in combination, 1∶1 000 dilution of 1 ×1012 cfu/g B. subtilis DJ-6 WP and 1∶2 000 dilution of 250 g/L pyra-clostrobin EC promoted the growth of strawberry by increasing plant height, leaf petiole, leaf blade area and stem diameter. Among them, the treatments with 1∶1 000 and 1∶2 000 of 20% pyraclostrobin · 2×1011 cfu/g B. subtilis DJ-6 WP in combina-tion had better effects than other treatments. The control effects of al the treat-ments were measured 30 and 80 d after fungicide application. The control effects of 1∶1 000 dilution of 20% pyraclostrobin and 2×1011 cfu/g B. subtilis DJ-6 in combina-tion were up to 100% and 93.11%, which were higher than those in al other treat-ments. The second highest control effects were found in the treatment with 1∶ 2 000 dilution of 20% pyraclostrobin and 2×1011 cfu/g B. subtilis DJ-6 in combination, they were 92.49% and 86.49%, higher than those in other treatments except the 1∶1 000 dilution of 20% pyraclostrobin and 2 ×1011 cfu/g B. subtilis DJ-6 in combination. The control effects of 1∶3 000 dilution of 20% pyraclostrobin and 2×1011 cfu/g B. subtilis DJ-6 in combination were 82.61% and 72.42%, higher than those in treatment with 1∶1 000 dilution of 1×1012 cfu/g B. subtilis DJ-6 WP, but lower than those in treat-ment with 1∶2 000 dilution of 25% pyraclostrobin EC. [Conclusion] Al the results re-vealed that the combination use of 20% pyraclostrobin and 2 ×1011 cfu/g B. subtilis DJ-6 WP at 1∶1 000 to 1∶2 000 dilution had better control effect against strawberry Fusarium wilt.

Screening and identification of an antagonistic bacterium strain LHB02 and the optimization of its fermentation conditions

A strain LHB02 with strong antibacterial activity against some aquatic pathogens was screened from the coastal marine sediment in Fujian province, China. LHB02 was identified as Bacillus subtilis, based on its 16S rRNA sequence, together with the morphological, physiological and biochemical characteristics. The antagonistic activity of strain LHB02 and its optimal fermentation conditions were also investigated. The results showed that LHB02 had strong antagonistic activity against 3 species of vibrios： Vibrio harveyi,Vibrio alginolyticus and Vibrio anguillarum, and the optimum fermentation conditions for the strain LHB02 were as follows： KB culture medium （peptone 20 g, glycerol 10 mL, K2HPO4 1.5 g, MgSO 4 .7H2O 1.5 g, H2O 1 000 mL）; temperature, 28 ℃; pH ,7.0; culture time, 36 h; and inoculation amount, 1.5% （v/v）.

Antagonistic Endophytic Bacteria Isolated from Different Growth Stages of Cotton for the Control of Cotton Verticillium wilt

[Objective] This study aimed to investigate the combined control effects of endophytic bacteria at different growth stages against cotton Verticfllium wilt and pro- vide a new strategy for the biocontrol of other soil-borne diseases. [Method] Endophytic bacteria with high resistance against Verticillium wilt were isolated from seedling, squaring and boll-setting cotton vascular, respectively. Their 16S rDNA se- quences were detected for comparative analysis. Three biocontrol strains were se- lected and identified, whose colonization roles in cotton plants were explored. The control efficiency was determined with indoor and field experiments. [Result] Accord- ing to the 16S rDNA sequence homology, the three strains were identified as Paeni- bacillus polyrnyxa YUPP-8, Paenibacillus xylanilyticus YUPP-1 and Bacillus subtilis YUPP-2, respectively. Results of colonization assessment showed that three strains all could be successfully colonized in cotton vascular. However, application amount had a positive effect on the number of colonized biocontrol bacteria in cotton, strain YUPP-8 had the largest number of colonized biocontrol bacteria in seedling period, strain YUPP-1 had the largest number of colonized biocontrol bacteria in squaring period, and strain YUPP-2 had the largest number of colonized biocontrol bacteria in boll-setting period. Indoor pot experiment showed that cotton plants in combined bio- control bacteria treatment group were not infected in flowing period, while Verticillium wilt morbidity rate of cotton treated with single strain in seedling period were 6.7% （YUPP-8）, 6.7% （YUPP-1） and 13.3% （YUPP-2）; however, Verticillium wilt morbidity rate wilt of the control reached 80%. Field experiment conducted during 2010-2011 showed that the combined application of three strains had better effect than separate application; specifically, Verticillium wilt morbidity rate and disease index of cotton in boll-setting period with combined application of three strains in 2010 were 9.4% and 6.5, respectively, while those in control group were 47.5% and 32.8; results in 2011 were similar to 2010, with higher disease severity. These results indicate that com- bined application of endophytic bacteria at different growth stages has great applica- tion potential in control of cotton Verticillium wilt. [Conclusion] This study preliminarily overcomes the defects in the application of biocontrol bacteria and provided reference for the prevention and treatment of other soil-borne diseases.

Optimization of Sporulation Conditions of Biocontrol Bacteria B579 by Two-step Control Strategy

[Objective] For preparing the biopesticide of Bacillus subtilis B579 with high spore concentration,the sporulation conditions were optimized.[Method] Two-step fermentation control strategy was used,in which,the first phase（0-10 h）was to improve cell growth,and the second phase（10-30 h）was to promote spore formation.Four factors including initial glucose concentration,fermentation pH,temperature（in the second phase）and shaking speed（in the second phase）were optimized using the methods of single factor test and orthogonal experiment.[Result] The initial glucose concentration showed a significant effect on sporulation.The optimal conditions for the spore formation of B.subtilis B579 were as follows：initial glucose concentration 5 g/L,fermentation pH 7.0,the temperature for the first phase 37 ℃,and the shaking speed for the first phase 180 r/min,the temperature for the second phase 40 ℃,and the shaking speed for the second phase 200 r/min;in addition,the first phase was 10 h and the second phase of fermentation was conducted for 30 h.Under such conditions,the spore concentration and spore formation rate could reach 9.43×108 CFU/ml and 90.99%,respectively,which represented 6.70-fold and 2.43-fold increase compared with those before optimization.[Conclusion] The spores concentration of biocontrol agent was improved using two-step control strategy,which provided the basis for biopesticide production in large scale.

Inhibitory Effects of Bacillus subtilis on Staphylococcus aureus

[Objective] To produce drug resistance, seek non-toxic environmental so as to change the current biological drugs that did not excessive use of antibiotics. [Methods] A strain of Bacillus was purified and isolated from fresh and healthy in- testines of grass carps. Biochemical identification was carried out by conventional bacterial biochemical test method. Two pairs of primers were designed, 16S rRNA detection and sequencing analysis were carried out. Drug sensitive test was carried out by agar diffusion method. In vitro inhibition test on Staphylococcus aureus was carried out by Oxford cup method. [Results] The isolated bacterium had basically the same biochemical characters as Bacillus subtilis; and the homology reached 100%. Thus, the isolated bacterium was identified to be Bacillus subtilis. It was insensitive to amoxicillin, ampicillin, penicillin G and so on, but sensitive to amikacin, cefalexin, ciprofloxacin and cefradine. The inhibitory effects of Bacillus subtilis on Staphylococ- cus aureus were significant. The minimum inhibitory concentration （MIC） was 2.8×10^8×2^-5/ml and minimum bactericidal concentration （MBC） was 2.8×10^8×2^-2/ml. [Conclusions] The isolated Bacillus subtilis could be used to prevent and control diseases caused by Staphylococcus aureus, and reduce the abuse of antibiotics.

贝莱斯芽孢杆菌HN-1突变体文库的构建及Arp基因的鉴定

贝莱斯芽孢杆菌HN-1是一株对香蕉枯萎病4号生理小种尖孢镰刀菌(Fusarium oxysporum f.sp.cubense race 4)具有良好抑制活性的生防芽孢杆菌。本研究旨在构建生防菌HN-1的突变体库,在突变体库的基础上深入研究生防芽孢杆菌的抑菌机理,针对香蕉枯萎病的生物防治夯实理论基础。将携带转座子TnYLB-1的温敏型质粒载体pMarA质粒利用化学法转化进入贝莱斯芽孢杆菌HN-1中,通过50℃高温诱导后构建了由1 800个HN-1突变体组成的贝莱斯芽孢杆菌HN-1突变体文库,质粒消除率达99.7%。通过平板抑菌法筛选得到一株对尖孢镰刀菌Foc4的抑制活性降低的突变体,利用反向PCR技术确定插入破坏的基因与贝莱斯芽孢杆菌模式菌株FZB42 arsenic resistance protein (RBAM-550730)基因同源性为99%,在系统进化树中与贝莱斯芽孢杆菌模式菌株FZB42中的该基因属于同一分支。通过筛选鉴定HN-1突变体文库中抑菌活性丧失突变体发现HN-1中Arp基因与生防芽孢杆菌HN-1抑制尖孢镰刀菌的活性有密切的关系。

Isolation and Identification of Triazophos-degrading Bacillus subtilis str. C-Y106 and Its Degradation Characteristics

[Objective] The aim was to isolate the triazophos-degrading strain and study its degradation characteristics. [Method] A triazophos-degrading bacterium strain C-Y106 was isolated from sludge in an aeration tank of triazophos manufacture. Then the strain C-Y106 was identified according to the morphology,physiological and biochemical characteristics,and 16S rRNA sequence analysis. The effect of medium with different nutrients on triazophos-degrading rate by C-Y106 was studied. [Result] The strain C-Y106 was identified as Bacillus subtilis. The strain C-Y106 could grow in the mineral salt medium with 40 mg/L of triazophos as the sole sources of carbon,Nitrogen and Phosphorus. The triazophos-degrading rate was the highest as 76.8% in the mineral salt medium with 40 mg/L of triazophos as the sole source of Phosphorus,after being incubated at 31 ℃,pH 8.0 and 150 r/min for 60 h. [Conclusion] The research had provided theoretical basis for the identification and purification of enzymes for triazophos degradation.

Antibacterial Activity of Bacillus subtilis and Properties of Protein Crude Extract

In order to get biological drugs with no resistance or toxic side effects and to reduce the use of antibiotics, a strain of Baci//us subtilis was isolated from animal intestine, and the isolate was identified by molecular biological method; in vitro an- tibacterial test of the isolate was performed using agar diffusion method; the optimal fermentation condition of the isoJate was screened by conventional culture method; the antibacterial crude protein of the isolate was extracted by saturated ammonium sulfate method; the physicochemical properties of antibacterial crude protein was de- tected by comparison method; The results showed that the isolate was B. subti/is, which had antibacterial effects on Staphy/ococcus aureus, streptococcus and swine erysipelas. The fermentation effect of the isolate was the best under the condition of temperature 30 ~C, pH 7, liquid volume 75 ml/250 ml, inoculation volume 20% and culture time 48 h. The antibacterial effect of the isolate was the best when extract- ed by 80% saturated ammonium sulfate. The antibacterial crude protein had strong resistance to heat and acid. Organic solvent and UV irradiation had some influences on antibacterial crude protein. Proteases had hydrolytic effects on antibacterial crude protein. The isolated B. subti/is can be used to prevent and control the diseases caused by S. aureus, streptococcus and swine erysipelas, and can regulate intesti- nal microecology by adding into expanded feeds.

Purification of Antifungal Protein from Biocontrol Strain XG-1 against Fusarium Wilt of Watermelon

[Objective] This study aimed to isolate and purify the antifungal protein against Fusarium oxysporum f. sp niveum （FON） from Bacillus subtilis XG-I. [Method] The crude protein was obtained by using fractional precipitation with am- monium sulfate, then chromatography using both DEAE-Sepharose FF anion ex- change and Sephacryl S-100 gel filtration columns was adopted for further purifica- tion of the protein, followed by MALDI-TOF-MS analysis of the structure of the at- tained protein which was antagonistic to FON. [Result] The result from MALDI-TOF- MS analysis suggested that the antifungal protein extracted from strain XG-1 was highly homologous to the flagellin （gi114278900） deriving from B. subtilis, with a pro- tein score of 248 and a coverage rate of 63%. It was thus speculated that this an- tagonistic protein was a kind of flagellin with a molecular mass of 30.6 kD. [Conclusion] The results from this study provide theoretical basis for clarifying the mechanisms of the action of strain XG-1 as well as important references for the preven- tion and control of watermelon Fusarium wilt.