一株抗真菌解淀粉芽孢杆菌的分离鉴定及其发酵条件的初步研究

从堆肥中分离到一株对植物病原菌尖孢镰刀菌(Fusarium oxysporum)具有强烈抗菌活性并具有较广抗菌谱的细菌Q-12菌株。通过形态观察、生理生化实验1、6S rDNA同源性序列分析以及部分特异性基因序列分析,鉴定该菌为解淀粉芽孢杆菌。该菌的最适培养基组成为:葡萄糖5g/L,NH4Cl 1g/L,牛肉膏0.8g/L,氯化镁5g/L。最适培养温度为33℃,最适培养pH为6.0,最适培养时间为40h。     

解淀粉芽胞杆菌抗真菌活性研究进展

解淀粉芽胞杆菌(Bacillus amyloliquefaciens)具有很强的抑制植物病原真菌的能力。其菌体细胞能产生多种酶类、脂肽类抗生素、生物表面活性素、聚酮类化合物和抑菌蛋白,同时具有诱导植物产生系统抗性(ISR)的能力,因此在工农业、种植业、养殖业、食品加工业、果蔬的采后保鲜和饲料业等行业具有重要价值。本文对解淀粉芽胞杆菌抗真菌作用、抗真菌能力提高策略、抗菌化合物合成调节、抑制真菌机制及其引发的ISR等问题进行了深入探讨和综述。     

解淀粉芽孢杆菌NK10.BAhjaWT抑真菌作用的研究

芽孢杆菌属以多产抗菌素闻名。通过筛选几十株不同来源的芽孢杆菌, 获得1株具有强抑真菌活性的芽孢杆菌。经过16S rDNA检测与Biolog分析, 确定此株菌为解淀粉芽孢杆菌。本实验对摇瓶发酵的条件进行了优化, 经过对发酵上清液硫酸铵盐析、透析、真空冷冻干燥获得粗提蛋白。并对粗蛋白的热稳定性、pH稳定性、抗蛋白酶水解能力、离子稳定性以及抑真菌谱进行了研究, 最后使用扫描电镜对抑真菌机制进行了探索。     

解淀粉芽孢杆菌NK10.BAhjaWT抑真菌作用的研究

芽孢杆菌属以多产抗菌素闻名.通过筛选几十株不同来源的芽孢杆菌,获得1株具有强抑真菌活性的芽孢杆菌.经过16S rDNA检测与Biolog分析,确定此株菌为解淀粉芽孢杆菌.本实验对摇瓶发酵的条件进行了优化,经过对发酵上清液硫酸铵盐析、透析,真空冷冻干燥获得粗提蛋白.并对粗蛋白的热稳定性、pH稳定性、抗蛋白酶水解能力、离子稳定性以及抑真菌谱进行了研究,最后使用扫描电镜对抑真菌机制进行了探索.     

菜心炭疽病菌拮抗细菌的筛选及鉴定

从土壤中分离筛选获得2株对菜心炭疽病菌——希金斯炭疽菌(Colletotrichum higginsianum)具有强烈抑制作用的芽孢杆菌菌株19E2、13A1。通过形态特征观察、生理生化特性测定, 结合16S rDNA序列同源性分析以及部分基因特异性序列分析, 将2株菌鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。抑菌试验的结果表明: 当19E2和13A1的培养液在PDA培养基中的浓度为10%时, 抑菌率分别达到75.62%和85.35%, 当培养液浓度升高到20%时, 可完全抑制菜心炭疽病菌生长。抗菌谱测定结果显示: 2株菌对辣椒炭疽菌(C. capsici)、胶孢炭疽菌(C. gloeosporioides)、灰霉 (Botrytis cinerea)、苞叶芋柱帚霉(Cylindrocladium spathiphylli)和尖镰孢(Fusarium oxysorum)等重要病原菌具有明显的作用效果。     

Biocontrol potential of two novel grapevine associated Bacillus strains for management of anthracnose disease caused by Colletotrichum gloeosporioides

A study was conducted to identify bacterial antagonists for biological control of anthracnose which is one of the economically important diseases of grapes. In India, it is caused by Colletotrichum gloeosporioides. Two hundred and ninety-three bacteria were isolated from the grape ecosystem of 43 spatially distant vineyards in peninsular India. Of these, 25 bacteria substantially inhibited the radial growth of C. gloeosporioides in in vitro studies and 18 bacteria significantly reduced infections in vivo. Of these 18 bacteria, 5 and 3 bacteria also significantly reduced percent disease index (PDI) of downy and powdery mildew diseases, respectively. These bacteria were labelled as strains, DR-38, DR-39, TL-171, DRo-198, TS-204, TS-205, and DR-219, and were identified as Bacillus species based on morphological and molecular characterisation. Aqueous suspensions of all these strains applied as foliar sprays at 1×10⁸ cfu/ml on field grown vines significantly lowered the PDI and the AUDPC (area under disease progress curve) of anthracnose when compared with the untreated control, except DRo-198. Strains TS-204 and TL-171 recorded lower PDIs and AUDPC when compared with the other five strains, and TS-204 could effectively suppress ripe rot of berries, too, in vivo. Strains TS-204 and TL-171 are identified for biocontrol of anthracnose in grapes.     

Integrated control of Colletotrichum gloeosporioides on mango fruit in Taiwan by the combination of Bacillus subtilis and fruit bagging

Anthracnose disease caused by Colletotrichum gloeosporioides in Jingkwang mango grown in Taiwan was significantly reduced by the integration of fruit bagging with either B. subtilis strain LB5 or fungicides. The combined treatments were most effective in reducing early infection during the 2004 season, leading to 56.4 and 58.3% reduction, respectively, while in 2003 reduction accounted for 51 and 52.3%, respectively. Post-harvest application of B. subtilis strain LB5 cell suspensions on fruits already treated by bagging, bagging+LB5 and baggingfungicides in the field reduced anthracnose incidence significantly at all tested concentrations. These results indicate that biocontrol efficacy of B. subtilis LB5 may be due to the prevention of early fruit infection, thereby reducing significantly anthracnose incidence in ripening fruits to much lower levels than those obtained by using a conventional single post-harvest treatment.     

Rapid Detection of Colletotrichum lagenarium,Causal Agent of Anthracnose of Cucurbitaceous Crops,by PCR and Real‐time PCR

Rapid and accurate polymerase chain reaction (PCR) and real‐time PCR methods were developed for the detection of Colletotrichum lagenarium, the causal agent of anthracnose, in tissues of squash (Cucurbita moschata), watermelon (Citrullus lanatus), cucumber (Cucumis sativus) and muskmelon (Cucumis melo). PCR assays amplified different internal transcribed spacer sequences from C. lagenarium, so effectively detected this pathogen in infected tissues. PCR analysis with the primer co‐m‐337F1/R1 was able to differentiate C. lagenarium from other fungal pathogens, including Colletotrichum spp., Fusarium spp., Alternaria spp. and Didymella spp. An optimized real‐time PCR assay was developed to detect and monitor C. lagenarium in both infected plant tissues and soil samples. The sensitivity of real‐time PCR can detect down to 1 pg of DNA. Thus, PCR‐based analysis is a useful technique for rapid detection and diagnosis of C. lagenarium in infected plants or infested soils.     

Environmental dynamics of Bacillus amyloliquefaciens CCMI 1051 antifungal activity under different nitrogen patterns

Aims: The aim of this study was to evaluate the influence of environmental conditions on the antifungal activity of the Bacillus sp. CCMI 1053 cultures. Methods and Results: The electrospray ionization mass spectra (ESI‐MS) analysis was used to detect the active peptides produced by Bacillus amyloliquefaciens CCMI 1051 cultures in a glucose‐containing medium to which four different nitrogen sources were added. The cultures produced different patterns of Bacillus sporulation and distinct antifungal activity of the cell‐free culture broths. Conclusions: The highest sporulation obtained corresponds to higher antifungal activity when it is formed after 3 days of microbial growth. The antifungal activity against Trichoderma harzianum CCMI 783 is more influenced by the concentration on the nitrogen source than the culture time of incubation. The association of nitrogen concentration and the time of incubation is particularly relevant in the expression of the antifungal activity. Significance and Impact of the Study: The present findings allow the reduction of the use of chemical pesticides and to limit some plant diseases. The association of the nitrogen source and the time of incubation is a novelty, which would improve the production of secondary metabolites. Both economical and environmental benefits arise from the study.     

鲟源嗜水气单胞菌拮抗解淀粉芽孢杆菌微胶囊的制备工艺及其特性

【目的】优化鲟源嗜水气单胞菌拮抗解淀粉芽孢杆菌微胶囊的制备工艺,并观察其特性。【方法】以明胶为壁材,采用单因素法,考察了明胶浓度、进风温度、进料速度、空气流量等因素对解淀粉芽孢杆菌微胶囊有效含菌量的影响,并进一步通过正交试验设计优化制备解淀粉芽孢杆菌微胶囊的喷雾干燥工艺参数,观察其微胶囊颗粒形态以及对人工模拟胃液和肠液的耐受力。【结果】解淀粉芽孢杆菌微胶囊喷雾干燥的最佳制备工艺条件为:明胶浓度为3%,进风温度为155°C,进料速度为8 mL/min,空气流量为700 L/h,各因素对其喷雾干燥工艺的影响程度为:明胶浓度进料速度空气流量进风温度。此外,解淀粉芽孢杆菌微胶囊的颗粒呈球形,表面有凹陷,但没有孔和裂纹,颗粒粒径分布基本均匀,平均大小为9.22μm,对人工模拟胃液和肠液具有较好的耐受力,对鲟源嗜水气单胞菌具有良好的生长抑制效果。【结论】本研究结果为鲟源嗜水气单胞菌拮抗解淀粉芽孢杆菌微胶囊的工业化生产奠定了基础。     

Mulberry anthracnose antagonists (iturins) produced by Bacillus amyloliquefaciens RC-2

Bacillus amyloliquefaciens strain RC-2 produced seven antifungal compounds (1-7) secreted into the culture filtrate. These compounds inhibited the development of mulberry anthracnose caused by the fungus, Colletotrichum dematium. Chemical structural analyses by NMR and FAB-MS revealed that all these compounds were iturins (cyclic peptides with the following sequence: L-Asn --> D-Tyr --> D-Asn --> L-Gln --> L-Pro --> D-Asn --> L-Ser --> D-beta-amino acid -->) and compounds 1-6 are identical to iturins A-2-A-7, respectively. Compound 7 (iturin A-8) is a new iturin, which has a -(CH(2))(10)CH(CH(3))CH(2)CH(3) group as a side chain in the beta-amino acid in the molecule.     

Role of Bacillus amyloliquefaciens Y1 in the control of Fusarium wilt disease and growth promotion of tomato

The objective of this study was to examine the effects of Bacillus amyloliquefaciens Y1 on the control of Fusarium wilt disease and subsequent improvement in the growth of tomato plants. The Y1 strain strongly inhibited Fusarium oxysporum f. sp. lycopersici in vitro and also produced indole-3-acetic acid (IAA) in both the presence and absence of tryptophan. Over 96% of tomato seeds germinated when treated with either water, tryptone soy broth, or Y1 cultures, whereas root (5.40?cm) and shoot (5.15?cm) lengths were greatest in tomato seedlings treated with Y1 cultures that lacked tryptophan. Three experimental treatments – Black White medium (BW), BW medium with a commercial fungicide (BW?+?F), and Y1 culture inoculated in BW medium (Y1) – were applied to control Fusarium wilt disease under in vivo conditions. Application of Y1 culture and BW?+?F led to significantly lower disease incidence than did BW; moreover, shoot length and fresh and dry weight of both roots and shoots were greater in plants treated with Y1 than in plants treated with either BW or BW?+?F. A similar trend was observed for chitinase and β-1,3-glucanase activities in roots and leaves of tomato plants in all treatment groups over most of the experimental period. Finally, the presence of Y1 in the rhizospheric soils of Y1-treated plants resulted in a significant reduction in the populations of other bacteria. The results of our study demonstrated the effectiveness of Y1 not only in the control of Fusarium wilt disease but also for the enhancement of plant growth in cultivated tomato.     

内生解淀粉芽孢杆菌CC09产Iturin A摇瓶发酵条件优化

【目的】提高内生解淀粉芽孢杆菌CC09发酵产抗菌脂肽Iturin A的产量。【方法】首先采用单因子实验研究了碳源、氮源、NaCl浓度、pH、温度、转速和装液量等因子对CC09产Iturin A能力的影响,然后对其中显著性因子:氮源浓度、pH、温度及装液量4个因素进行正交实验,进一步优化发酵条件。【结果】优化培养基组成及发酵条件可以提高CC09菌株的生长速度及产Iturin A的量,其中可溶性淀粉以及一定比例的蛋白胨和酵母粉是CC09菌株产Iturin A的良好碳源和氮源;培养温度、装液量、培养液pH等也对CC09菌株产Iturin A有显著影响。优化后的培养基成分:可溶性淀粉(碳源)5 g/L、比例为3:1的胰蛋白胨酵母粉混合氮源15 g/L、NaCl 1 g/L;最佳培养条件:pH 6.0、28°C、摇床转速120 r/min、培养瓶装液量20%。【结论】在此条件下,Iturin A的产量可达到690 mg/L,较优化前的138 mg/L提高了4倍。     

纳豆芽孢杆菌Bna05菌株产抗霉脂肽的鉴定

【目的】分析枯草芽孢杆菌纳豆菌亚种Bna05菌株代谢产物中脂肽类物质的存在情况,并探讨它们在抗霉功能中所发挥的作用。【方法】利用特异性引物对Bna05菌株进行脂肽合成酶类基因片段扩增、测序和BLAST比对分析;通过平板抑菌圈区域取样法获得Bna05菌株的高抗霉活性代谢产物,对该产物进行反相高效液相色谱(RP-HPLC)分离;用琼脂微稀释法测定分离物的抗霉活性,并对活性分离物进行质谱鉴定。【结果】Bna05菌株含有sfp和srf AA基因,未检测到itu C、itu D、fen D、fen ACE、bym B、bym C基因;RP-HPLC分离得到3组抗霉活性物质F_2、F_3和F_4,F_2中未检测到脂肽类物质,从F_3和F_4中分别鉴定出两类Surfactin同系物:V_7-surfactin和I/L_7-surfactin。两类Surfactin分别与F_2组合使用时,均表现出抗霉协同作用;此外,与Surfactin单独使用相比,两类Surfactin混合物与F_2组合后的协同抗霉活性得到进一步增强。【结论】Bna05菌株所产脂肽类物质主要是V_7-surfactin和I/L_7-surfactin,Surfactin与Bna05菌株所产其它活性物质之间存在抗霉协同作用,而V_7-surfactin和I/L_7-surfactin的同时存在,对于增强这种协同抗霉作用是有利的。     

高产3-羟基丁酮解淀粉芽孢杆菌的选育及发酵优化

3-羟基丁酮(Acetoin)作为一种重要的食用香料和平台化合物被广泛应用于医药、食品等领域。为改善解淀粉芽孢杆菌Bacillus amyloliquefaciens的3-羟基丁酮生产能力,采用常压室温等离子体(ARTP)和~(60)Coγ射线进行复合诱变,以3-羟基丁酮产量为分析指标,筛选获得最优突变株B.amyloliquefaciens H-5,3-羟基丁酮产量为68.2 g/L。为进一步实现3-羟基丁酮的高效生产,对此突变株进行5 L发酵罐水平的培养条件优化,并于30 L发酵罐上进行放大培养,最终3-羟基丁酮产量达85.2 g/L,较出发菌株B.amyloliquefaciens FMME088提高了26.8%。上述研究结果表明,ARTP和~(60)Coγ射线复合诱变能够有效获得高产菌株,该突变株具有较高的工业化微生物发酵生产3-羟基丁酮的潜能。     

枯草芽孢杆菌抗菌蛋白X98Ⅲ的纯化与性质

枯草芽孢杆菌（Bacillussubtilis）BS－98是一株能强烈抑制苹果轮纹病菌（Physalosporapiricola）等植物病原真菌的拮抗菌株。BS－98菌株培养液经硫酸铵分级盐析、SephadexG－100柱层析和DEAE－纤维素（DE32）柱层析后分离纯化出一种抗菌蛋白,命名为X98Ⅲ。蛋白电泳分析结果表明,此蛋白分子量为59000,等电点为4．50．醋酸纤维膜电泳后经特异染色证明X98Ⅲ含糖及胀。用DNS法测其含糖量为6％。此蛋白对热稳定,对蛋白酶部分敏感。氨基酸组分分析表明,该蛋白含11种不同氨基酸,尤富含谷氨酸和半胱氨酸等,而缺少天冬氨酸等。纯化后的X98Ⅲ对苹果轮纹病菌、芦笋茎枯病菌等有很强的抑制作用。X98Ⅲ的抑菌机理主要是溶解细胞壁,造成菌丝畸形、孢子不发芽或发芽异常。     

pH与溶氧控制对解淀粉芽胞杆菌发酵粗甘油生产2,3-丁二醇的影响

首次利用一株安全菌株解淀粉芽胞杆菌发酵生物柴油副产物粗甘油生产2,3-丁二醇。溶氧和pH是影响微生物法生产2,3-丁二醇的最主要因素。结果表明,发酵过程中不控制pH更有利于2,3-丁二醇合成;采用三阶段控制搅拌转速策略,2,3-丁二醇产量最大值达到?38.1?g/L,生产强度达到1.06?g/(L·h),与恒定转速获得的最好结果相比较,分别提高了14.8%和63.1%。采用脉冲流加发酵时,2,3-丁二醇产量达到71.2 g/L,2,3-丁二醇生产强度达到0.99 g/(L·h),这是目前报道的利用粗甘油合成2,3-丁二醇的最高产量。