海南岛热带雨林区芽孢杆菌收集及Bt菌鉴定

本研究系统地对海南岛热带雨林自然保护区进行了土壤样品的采集、芽孢杆菌的分离收集和Bt菌株的鉴定.从尖峰岭热带雨林区、五指山热带雨林区、吊罗山热带雨林区、霸王岭热带雨林区总共采集了土壤样品1 882份,采用醋酸钠培养基结合高温方法分离出芽孢杆菌3 924份,鉴定出Bt分离株158份,Bt菌株的分离率和出菌率分别为4.03%和8.40%.结果分析表明,海南岛热带雨林区芽孢杆菌及Bt菌株分布对环境和生态表现出一定的规律性,一般海拔900 m至1 400 m的Bt菌株含量高、植被覆盖率高,土壤腐殖质含量高的热带沟谷雨林带Bt菌株含量最高.显微观察发现,获得的Bt菌株其伴胞晶体有菱形、球形、方形、椭球形、不定性等多种形状.利用SDS-PAGE方法对获得的Bt分离株进行了伴胞晶体进行分析,发现伴胞晶体的分子量有20 kD到150 kD不等.进一步利用PCR-RFLP技术对Bt分离株进行了cry基因型的分析,初步发现这些Bt菌株含有cry1、cry3、cry4、cry6、cry30、cry40等基因型.我们还利用鳞翅目昆虫小菜蛾和鞘翅目昆虫椰心叶甲进行部分Bt分离株的生物测定,初步结果显示本研究鉴定出的Bt分离株具有不同的抗虫靶标,对同一靶标昆虫也表现出不同的杀虫活性.整体而言,本研究结果显示出海南岛热带热带雨林自然保护区因其独特的热带地理生境、自然的生物演化系统,使得热带雨林区蕴藏了Bt菌株资源多样化,值得期待挖掘出一些新的菌株和新的基因资源.     

我国部分地区土壤中的苏芸金芽孢杆菌和球形芽孢杆菌

从云南、贵州、四川和陕西4省的土壤中分离到大量苏芸金芽孢杆菌(Bacillus thuringie—nsis)和球形芽孢杆菌(Bacillus sphaeticus)菌株。血清型分析表明,苏芸金芽孢杆菌分离株分属于23个血清型中的13个血清型,另有近20％的自凝型菌株及部分与所有标准菌抗血清无反应的菌株。对该两种昆虫病原细菌的生态分布规律进行了分析。研究了全部苏芸金芽孢杆菌分离株对鳞翅目、鞘翅目及双翅目的6种昆虫的毒力特性、伴孢晶体与芽孢的形态,以及晶体蛋白质成分。观察和测定了球形芽孢杆菌分离株的形态和毒力,并分析了部分菌株的晶体蛋白质成分。得到22株高效苏芸金芽孢杆菌和2株高效球形芽孢杆菌。证明苏芸金芽孢杆菌是典型的土壤微生物类群,我国西南地区土壤中的苏芸金芽孢杆菌资源十分丰富。     

湖南、江西铀矿床区土壤中(B.cereus)分离及形态学比较

从湖南、江西两地铀矿床区采集土壤样品 13份 ,用选择性培养基从 10 -3 稀释的平板中分离到卵黄反应阳性菌 2 38株 (其中xw1,xw2 无该菌 )。从中筛选 8株 ,经纯化培养 ,对 8株进行了显微镜和电镜的形态观察 ,其形态为典型的杆菌 ,大小为 1.2～ 1.4μm× 2 .6～ 3.4μm ,芽孢为椭圆或橄榄果实形。没有孢囊和伴孢晶体 ,周生鞭毛。生理生化分析结果证明 ,8株均为典型的蜡状芽孢杆菌 (Bacilluscereus)。     

枯草芽孢杆菌(Bacillus subtilis)Tpb55灌根与烟草根围细菌多样性变化的相关性北大核心CSCD

【目的】利用454高通量测序技术分析生防菌株枯草芽孢杆菌(Bacillus subtilis)Tpb55对烟草根围土壤细菌群落的影响;同时测定施加Tpb55处理对烟草黑胫病的大田防效。【方法】试验设置Tpb55菌剂108 CFU/m L灌根和空白对照2个处理,分别在处理0、10、22 d采集烟草根围土壤,提取土壤细菌总DNA,扩增细菌16S r DNA V1-V3区,对扩增产物进行454高通量测序,使用Qiime软件分析不同处理的细菌群落结构。【结果】对照和处理样品测序后共获得了41207个优质序列,鉴定属于细菌的25个门。所有样品中优势菌群均为放线菌门(Actinobacteria)、变形菌门(Proteobacteria)和酸杆菌门(Acidobacteria)。在病情发展过程中,放线菌门的细菌丰度逐渐下降,变形菌门含量呈上升趋势。施用Tpb55后,酸杆菌门含量明显上升并高于对照。对照中芽孢杆菌科(Bacillaceae)和多样性指示菌草酸杆菌科(Oxalobacteraceae)的细菌丰度均明显下降,Tpb55处理的样品中芽孢杆菌科含量不断上升,草酸杆菌科含量相对稳定。Chao1、ACE和Shannon指数分析表明,Tpb55处理的样品中细菌多样性和丰富度不断提高且高于对照。Tpb55处理后10 d和22 d,OTU序列数据库中与Tpb55 16S r DNA V1-V3区PCR产物高度同源OTU数目分别为31和45。Tpb55处理的烟草黑胫病病情指数(5.29)显著低于对照(38.52)。【结论】施用Tpb55可以提高根围土壤细菌多样性和群落结构稳定性,这可能是其发挥良好生防作用的重要机制之一。     

土壤中产α-淀粉酶菌的初步筛选研究

本文研究了从土壤中筛选淀粉酶产生菌,并对其产酶条件进行了探讨。共分离得到3株活性较强的产淀粉酶菌;其中活性最强的为A1株,其圈菌比可达3.75,经过生理生化检测实验,初步鉴定为腊状芽孢杆菌杆菌。     

花生侵脉新赤壳菌果腐病生防芽孢杆菌的分离鉴定及防病效果

【目的】为了分离鉴定对花生侵脉新赤壳菌果腐病病原菌Neocosmospora vasinfecta具有抑制作用的根际芽孢杆菌。【方法】利用平板稀释法从花生的根际土壤分离芽孢杆菌,再采用平板对峙法筛选出对N.vasinfecta具有抑制作用的根际芽孢杆菌,通过形态观察、生理生化特性和分子生物学相结合的多相分类方法对生防根际芽孢杆菌进行分类鉴定,检测脂肽类抗生素合成基因类型,并进行花生侵脉新赤壳菌果腐病的田间防治试验。【结果】从花生根际土壤中分离到28株芽孢杆菌,其中对花生果腐病病原菌具有明显抑制作用有8株。多相分类法结果显示2株为枯草芽孢杆菌(Bacillus subtilis),6株为解淀粉芽孢杆菌(B.amyloliquefaciens)。脂肽类抗生素合成基因检测显示,8株生防芽孢杆菌含有至少1种脂肽类抗生素,其中所有生防菌均含有丰原素B合成基因,推测这些芽孢杆菌对N.vasinfecta的抑制机制可能与脂肽类抗生素的合成相关。田间防病实验结果显示,B.amyloliquefaciens GF-3和GF-22制备的生物有机肥均能有效降低NPRP的发病指数,其防治效率分别为32.35%和79.41%,增产率分别为19.12%和25.85%。【结论】分离鉴定了2株对花生侵脉新赤壳菌果腐病具有明显防治效果的根际芽孢杆菌,这不仅为花生侵脉新赤壳菌果腐病的生防制剂研制提供了菌株,还为研究防治机理奠定了基础。     

拮抗枯草芽孢杆菌KC-5的分离鉴定及其发酵优化

为了筛选出对植物病原菌具有拮抗作用的生物防治细菌,采用平板对峙法从蔬菜根际土壤中分离获得一株对多种病原真菌具有抑制作用的枯草芽孢杆菌,并对抑制后的病原菌菌丝进行了观察,结果表明该菌株对尖孢镰刀菌、串珠镰刀菌、层出镰刀菌以及腐皮镰刀菌均具有抑菌活性。通过形态特征、生理生化特征及16S rRNA序列分析,将该菌株鉴定为枯草芽孢杆菌(Bacillus subtilis),并对其发酵培养基进行了优化。     

一株养殖大菱鲆体表溶血性蜡状芽胞杆菌的分离鉴定及基因组分析

蜡状芽孢杆菌是能产生孢子的革兰氏阳性菌,广泛分布于水体和土壤中,因能引起食物中毒而广为人知。本研究中,从养殖大菱鲆体表分离到一株溶血性细菌,经形态学鉴定和系统发育学分析,鉴定为蜡状芽孢杆菌。根据分离的时间和地点,这株溶血性的菌株被命名为蜡状芽孢杆菌WH2015。溶血试验证实WH2015菌株能够裂解羊红细胞。对蜡状芽孢杆菌WH2015基因组测序和分析的结果显示其基因组大小为5.8 Mb,平均G+C含量为35%,有6 568个基因。与其它蜡状芽孢杆菌株系进行比较基因组分析显示,WH2015菌株基因组几乎拥有所有的核心溶血性基因,提示其溶血机理与其它报导过蜡状芽孢杆菌株系的溶血机理相似。本研究在分离鉴定养殖大菱鲆体表溶血性蜡状芽胞杆菌WH2015的基础上,对其基因组进行了测序和分析并鉴定了其所具有的致病性基因和毒力因子,帮助我们理解由蜡状芽孢杆菌引起的养殖大菱鲆疾病,以及为其防治措施的实施和疾病的治疗提供可靠的理论基础。     

作物根际联合固氮芽孢杆菌的分离鉴定及生态分布

本文报导一种富集和分离根际联合固氮芽孢杆菌的方法,该方法以果胶为唯一碳源的培养基进行富集,中性红为指示剂的培养基进行分离,根据固氮芽孢杆菌能产气以及在分离培养基平板上菌落为红色两个重要的鉴别性特征即可检出,准确性高,简便快速;应用此法从１５个地点６种作物共８８个根系样品中分离出５１株固氮芽孢杆菌,经鉴定均为多粘芽孢杆菌（Ｂａｃｉｌｕｓｐｏｌｙｍｙｘａ）;还初步讨论了多粘芽孢杆菌在几种作物根际的分布情况。     

钾长石矿区土壤解钾菌的分离与多样性

目的获得高效钾长石分解细菌及分析钾长石矿区土壤中解钾菌的多样性。方法采集湖南省吉首市钾长石矿区土壤,用钾长石为唯一钾源的选择性培养基筛选分离解钾细菌,采用形态特征观察、生理生化特性检测和基于16S rRNA基因序列的系统发育分析初步鉴定解钾菌株,并测定菌株摇瓶条件下的解钾能力。结果分离获得38株钾长石分解细菌,分别属于13个属,其中13株菌为剑菌属,4株菌为芽胞杆菌属。菌株L17的发酵液中有效钾含量最高,为87.66 mg/L,是对照组的15.8倍。结论钾长石矿区土壤细菌种类有较好的多样性,其中剑菌属和芽孢杆菌属为优势种群,Mitsuaria属的L17解钾能力最强。     

金矿床区蜡状芽孢杆菌孢壁蛋白SDS-PAGE图谱及聚类分析

用SDS PAGE方法对 5株从我国金矿床区采集到的典型的蜡状芽孢杆菌 (Bacilluscereus)C6,C14 ,B2 ,JY I T1,JY X T9的孢壁蛋白同标准株AS1.12 6的孢壁蛋白共同进行比较分析 ,计算出了各蛋白电泳带的近似分子量 ,又以它们的迁移距离为标准同苏云金芽孢杆菌 (Bacillusthuringiensis)的孢壁蛋白相比较 ,得到聚类分析树状图谱 ,表明具有聚金作用的蜡状芽孢杆菌在分类学上具有相似性 ,而与具有杀虫作用的Bt菌株在分类学上则相差较远。     

Quantification of magnetic susceptibility in several strains of Bacillus spores: implications for separation and detection

Three strains of Bacillus: Bacillus atrophaeus (formally Bacillus globigii), Bacillus thuringiensis, and Bacillus cereus were tested for their intrinsic magnetic susceptibility. All three strains when sporulated demonstrated significant magnetic susceptibility using an instrument referred to as Cell Tracking Velocimetry. Energy dispersive spectroscopy also confirmed the presence of paramagnetic elements, Fe and Mn, in the spore form of the bacteria. It was demonstrated that this magnetic susceptibility is sufficient to separate and deposit these spores on glass slides in a magnetic deposition system. These results indicate the potential to separate spores with intrinsic magnetic susceptibility directly out of water or air samples.     

High-resolution solid-state 13C nuclear magnetic resonance of bacterial spores: identification of the alpha-carbon signal of dipicolinic acid.

Natural-abundance solid-state 13C nuclear magnetic resonance spectra were obtained for bacterial spores for the first time by using the technique of cross-polarization magic-angle-spinning nuclear magnetic resonance spectroscopy. A resonance at about 150 ppm, detectable in spore samples having a Mn content of less than 0.05%, was consistent with an identification as the alpha-carbon signal of calcium dipicolinate; this signal was missing from a spore sample treated with acid to release dipicolinate and from a spore coat preparation. Carbohydrate peaks were particularly intense in spores and coat preparations of Bacillus macerans. Signals ascribable to beta-hydroxybutyrate were prominent in a B. cereus sample.     

风化程度不同的黄金矿石中微生物的调查

对我国广西某地尚未开采的黄金矿床中风化程度不同的黄金矿石进行微生物分离,结果分离出6株霉菌和6株细菌,未发现酵母菌、放线菌和专性厌氧菌。三种风化程度不同的矿石中均分布有蜡状芽抱杆菌和氧化亚铁硫杆菌。     

含金地质体表生带中主要微生物类群的研究

通过对16个含金矿区主要微生物类群的分布、生态结构的研究,发现不同类型的金矿具有不同的微生物类群.金矿矿石与金矿酸水中的自养硫细菌和异养菌分布具有明显差异,酸性水中含自养硫细菌较多,含异养菌极少;金矿氧化带中的异氧菌一般多于自氧硫细菌,其优势菌以芽孢杆菌属中蜡状芽孢杆菌为主.研究结果揭示了金矿中存在的主要微生物类群,对阐明表生金的形成、成岩和蚀变作用具有一定的意义,对发掘生物湿法冶金菌种资源具有重要作用.     

A method for the determination of bacterial spore DNA content based on isotopic labelling, spore germination and diphenylamine assay; ploidy of spores of several Bacillus species.

A reliable method for measuring the spore DNA content, based on radioactive DNA labelling, spore germination in absence of DNA replication and diphenylamine assay, was developed. The accuracy of the method, within 10-15%, is adequate for determining the number of chromosomes per spore, provided that the genome size is known. B subtilis spores were shown to be invariably monogenomic, while those of larger bacilli Bacillus megaterium, Bacillus cereus and Bacillus thuringiensis, often, if not invariably, contain two genomes. Attempts to modify the spore DNA content of B subtilis by altering the richness of the sporulation medium, the sporulation conditions (liquid or solid medium), or by mutation, were apparently unsuccessful. An increase of spore size with medium richness, not accompanied by an increase in DNA content, was observed. The implication of the apparently species-specific spore ploidy and the influence of the sporulation conditions on spore size and shape are discussed.     

Bacillus cereus is common in the environment but emetic toxin producing isolates are rare

AIMS: To determine the incidence of emetic toxin producing Bacillus cereus in soil, animal faeces and selected vegetable produce to compare the results with the previously reported high incidence in rice paddy fields. To examine whether the emetic toxin has antibiotic activity. METHODS AND RESULTS: The incidence of emetic toxin producing B. cereus was evaluated by plating on selective agar 271 samples of soils, animal faeces, raw and processed vegetables. Overall, 45.8% of samples were positive for B. cereus. One hundred and seventy-seven B. cereus isolates were recovered at 30 degrees C with the grand mean spore count being 2.6 +/- 1.7 log(10) CFU g(-1) and 148 B. cereus isolates were recovered at 7 degrees C with the grand mean spore count being 2.2 +/- 1.2 log(10) CFU g(-1) of the 177 B. cereus isolated at 30 degrees C, only 3 were positive for emetic toxin production at a titre of 1/64, 1/32, 1/16, respectively. Also, 1 of 148 B. cereus isolated at 7 degrees C was positive for emetic toxin production to a titre of 1/128. All positive isolates came from washed or unwashed potato skins, one was psychrotrophic as determined by PCR and growth at 7 degrees C on subculture. The emetic toxin was not shown to have any antibiotic effects in growth inhibition studies. CONCLUSIONS: While B. cereus was a common isolate, the incidence of the emetic strain was rare. This is in contrast to previous findings of the high incidence in rice paddy fields and the processing environment, which may suggest rice is a selective area for growth of the emetic strain of B. cereus. SIGNIFICANCE AND IMPACT OF STUDY: The finding that a psychrotrophic isolate of B. cereus can produce emetic toxin is the first ever such observation and suggests the possibility that psychrotrophic isolates could grow in refrigerated fresh foods and cause emesis. The incidence of emetic B. cereus strains in rice paddy fields now requires further study for comparison with the low incidence found in other soils. The emetic toxin failed to inhibit the growth of other bacterial, fungal and yeast species. Whether the toxin (which is similar in structure to the antibiotic valinomycin) plays a competitive role in the environment therefore remains unclear.     

Development of a Quantitative Immunological Assay for the Study of Spore Coat Synthesis and Morphogenesis

A quantitative assay employing (125)I-labeled antibody has been developed for Bacillus cereus T spore coat protein. Populations of antibody molecules with various affinities for inner or outer coat can be prepared by selective adsorption to and elution from different coat preparations. Adsorption to and elution from intact spores results in an antibody preparation at least 15 times more reactive to outer coat. This antibody is useful for measuring the time and extent of spore coat maturation, i.e., outer spore coat formation. Rifampin inhibits the increase in content of this coat antigen.     

北衙金矿矿石内部可培养细菌多样性初步研究

目的对北衙金矿矿石样品内部的可培养细菌进行分离并对其多样性进行研究。方法采集云南北衙金矿矿石样品,采用固体肉汤培养基、卵黄培养基及厌氧琼脂培养基分离矿石内部的可培养细菌,并利用16SrRNA基因序列构建系统发育树,初步评估细菌多样性。结果北衙金矿矿石内部细菌的主要种群包括厚壁菌门和放线菌门的不同菌属,包括芽孢杆菌属、类芽孢杆菌属、考克菌属及节杆菌属的菌株,其中抗逆性较强的优势菌群为放线菌门的细菌。结论本研究证实北衙金矿矿石内部的确存在大量可培羔±田萧．并且右种群名样件．     

Characterization of mesophilic bacilli in faeces of feedlot cattle

AIMS: To determine the identity and composition of mesophilic Bacillus spp. in faeces sampled from feedlot cattle. METHODS AND RESULTS: Faecal samples from 10 feedlot cattle were analysed. The total aerobic spore count increased from 4.6 x 10(4) CFU g(-1) (before feedlotting, day 0) to 1.6 x 10(6) CFU g(-1) (feedlot for day 76). A total of 150 randomly selected spore isolates (60 each from days 0 and 76 cattle, 30 from feed) were speciated using a Bacillus group-specific PCR-amplified ribosomal DNA restriction analysis technique (Wu et al. 2006). At day 0, Bacillus subtilis and Bacillus cereus predominated with a prevalence of 58.3% and 26.7%, respectively, whereas three species, B. subtilis (50.0%), Bacillus licheniformis (27.6%) and Bacillus clausii (20.0%) predominated in day 76 faecal samples. Of these, only the first two species were present in feed samples at a frequency of 70% and 30% respectively. All B. cereus isolates on day 0, possessed at least one of three enterotoxin genes (nheA, nheB and nheC) but these were completely eliminated after a period of feedlotting. All isolates of B. licheniformis were genotypically heterogeneous according to pulsed-field gel electrophoresis analysis. CONCLUSIONS: Cattle faeces contain large numbers of Bacillus spores representing different mesophilic species. Stable faecal populations of particular Bacillus spp. mimicking those found in feed, were subsequently established by feedlotting. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained and methods used in this study will help to investigate the indigenous Bacillus composition in the gastrointestinal tract of cattle and will further guide the administration of Bacillus probiotics.