台湾地区芽胞杆菌物种多样性

了解芽胞杆菌资源多样性, 可为芽胞杆菌功能资源挖掘和菌剂开发提供基础。从台湾地区8个市(县)采集土壤样本, 从20份土壤样品中分离获得了136株芽胞杆菌, 采用16S rRNA基因同源性将其鉴定为芽胞杆菌科的2个属、20个种。分别属于芽胞杆菌属(Bacillus)的16个种和赖氨酸芽胞杆菌属(Lysinibacillus)的4个种。根据分离频度分析得知, 台湾地区土壤中的芽胞杆菌优势菌群为阿氏芽胞杆菌(Bacillus aryabhattai)、苏云金芽胞杆菌(B. thuringiensis)和蜡样芽胞杆菌(B. cereus), 其他种类分布极其不均匀。芽胞杆菌Shannon多样性指数为1.2925-2.5850, 最高的为台中市和嘉义市(2.5850), 最低的为桃园县(1.2925)。根据分离频度对芽胞杆菌种类的聚类分析显示, 当欧式距离λ = 20时, 芽胞杆菌种类可分为高频度分布类型如阿氏芽胞杆菌(B. aryabhattai), 低频度分布类型如简单芽胞杆菌(B. simplex)。依据分离频度对8个采样点间的聚类分析未发现采样点间的芽胞杆菌种类分布的相关性。本研究认为台湾地区土壤中蕴藏着丰富芽胞杆菌种类多样性高, 具有很大的开发潜力。     

大小兴安岭可培养细菌的资源多样性

细菌作为微生物中的重要组成部分, 在有机质的形成与分解、维持生态系统平衡、促进动植物发育等多方面都有着重要作用。2014-2017年, 我们采用常见培养基分离纯培养法及16S rRNA基因序列分析方法对大小兴安岭地区土壤可培养细菌的群落结构和多样性进行了调查研究。结果表明: 从大小兴安岭地区的17个自然保护区内不同生境的土壤中分离获得3,180个菌株, 隶属于24属120种。其中, 芽孢杆菌属(Bacillus)的种数和株数最多, 分别为38种和2,419株, 是大小兴安岭地区可培养细菌的绝对优势类群(占总株数的76.1%); 其次是短杆菌属(Brevibacterium)(13.0%)。大兴安岭地区的物种数、Simpson多样性指数和Shannon-Wiener指数高于小兴安岭地区。优势类群芽孢杆菌属的枯草芽孢杆菌(B. subtilis)、苏云金芽孢杆菌(B. thuringiensis)、巨大芽孢杆菌(B. megaterium)等有重要的生产及科学研究价值。     

新疆伊犁马铃薯根际芽胞杆菌纯培养多样性

了解马铃薯根际土壤中芽胞杆菌种类多样性,可为挖掘芽胞杆菌新资源提供基础。从新疆伊犁州9个地点采集了30份马铃薯根际土壤样品,采用可培养法从中分离芽胞杆菌,通过16S rRNA基因同源性鉴定了分离菌株的分类地位。共获得芽胞杆菌349株,基于菌落形态特征和16S rRNA基因序列确定了66个代表菌株,其中14株与近缘种模式菌株的16S rRNA基因序列相似性介于93.0–98.5%之间,为潜在芽胞杆菌新种。349株芽胞杆菌属于9属66种,分别为芽胞杆菌属(Bacillus)的41个种(41/66,62.1%),为最优势属;类芽胞杆菌属(Paenibacillus)有9种(9/66,13.6%),赖氨酸芽胞杆菌属(Lysinibacillus)共6种(6/66,9.1%),嗜冷芽胞杆菌属(Pscychrobacillus)共4种(4/66,6.1%),虚构芽胞杆菌属(Fictibacillus)有2种(2/66,3.0%),短芽胞杆菌属(Brevibacillus)、大洋芽胞杆菌属(Oceanobacillus)、鲁梅尔芽胞杆菌属(Rummelibacillus)和解硫胺素芽胞杆菌属(Aneurinibacillus)皆为1种。新疆9个地点马铃薯根际土壤中芽胞杆菌含量为2.20–8.86×104 cfu/g,其中特克斯蒙古乡马铃薯根际土壤中芽胞杆菌的菌落含量和种类最多,分别为8.86×104 cfu/g和37种;尼勒克县马场马铃薯根际土壤中的芽胞杆菌含量最少,特克斯去昭苏路上和阜康西沟村的芽胞杆菌种类最少,仅9种。但每个地点的优势种均相同,均为简单芽胞杆菌(Bacillus simplex)和阿氏芽胞杆菌(B.aryabhattai)。芽胞杆菌种类可划分为高频度分布型(简单芽胞杆菌等4种)、中频度分布型(植物内生芽胞杆菌等12种)和低频度分布型(其余51种芽胞杆菌,如蕈状芽胞杆菌)。新疆9个地点芽胞杆菌种类分布可分为高含量高丰度型(特克斯蒙古乡和农技推广园)和低含量低丰度型(如尼勒克县马场等地)。相关性分析发现,马铃薯根际芽胞杆菌种类分布与海拔高度无显著相关性。新疆马铃薯根际土壤蕴含种类丰富的芽胞杆菌资源,可为芽胞杆菌功能菌株开发提供丰富的菌种来源。     

大莲湖池杉林土壤细菌的季节性变化

应用稀释平板法对大莲湖池杉林湿地土壤细菌进行分离,采用16S rDNA序列分析法对所分离细菌进行鉴定。结果表明,池杉林不同季节土壤细菌种类和数量有差异。其中夏季土壤细菌数量和种类最多,春、秋季次之,冬季最少。四个季度共分离得到60株菌株,分属15个细菌种属,分别为芽胞杆菌(Bacillus),假单胞菌(Pseudomonas),黄杆菌(Flavobacterium),红球菌(Rhodococcus),北里孢菌(Kitasatosporia),金黄杆菌(Chryseobacterium),不动杆菌(Acinetobacter),鞘氨醇杆菌(Sphingobacterium),丛毛单胞菌(Comamonas),伯克霍尔德氏菌(Burkholderia),链霉菌(Streptomyces),沙雷氏菌(Serratia),肠杆菌(Enterobacter),窄食单胞菌(Stenotrophomonas)和节杆菌(Arthrobacter)。     

基于可培养法分析亚热带植物内生与根际芽胞杆菌种类分布异质性

【背景】芽胞杆菌是农业上重要的微生物菌剂,对植物具有促生、防病、防虫等作用。【目的】了解亚热带植物内生和根际芽胞杆菌的种群分布,为其功能挖掘提供科学依据。【方法】采用可培养手段对甘蔗(Saccharum officinarum)、红麻(Hibiscus cannabinus L.)、黄麻(Corchorus capsularis L.)、黄秋葵(Abelmoschus esculentus)和玫瑰茄(Hibiscus sabdariffa)根际土壤和根内生芽胞杆菌进行分离,利用16S rRNA基因测序对分离菌株进行分类鉴定,并分析系统发育地位。【结果】共获得了可培养芽胞杆菌菌株144株,其中根内生芽胞杆菌82株,根际土壤62株。经16S rRNA基因测序鉴定为芽胞杆菌4个属37个种,分别为芽胞杆菌属、短芽胞杆菌属、类芽胞杆菌属和赖氨酸芽胞杆菌属。芽胞杆菌菌落数量和种类在根部及其根际土壤中差异较大,根际土壤中芽胞杆菌菌落数量远远大于根部,根际土壤中芽胞杆菌菌落含量范围为(0.2-370.0)×10~5CFU/g,而根部为(0.1-81.0)×10~3 CFU/g。根部分离获得的芽胞杆菌种类远大于根际土壤中,从作物根际土壤中共获得了芽胞杆菌19个种,从根部获得内生芽胞杆菌共32个种。阿氏芽胞杆菌(Bacillus aryabhattai)、仙草芽胞杆菌(Bacillus mesonae)和假蕈状芽胞杆菌(Bacillus pseudomycoides)同时存在于4种作物的根际土壤和根部,其他芽胞杆菌种类仅存在于1种作物的根际或者根部。【结论】亚热带作物根际土壤和根部芽胞杆菌种类和数量极为丰富,而且还存在可分离培养的芽胞杆菌潜在新物种,这为了解植物与根际微生物相互作用、根际环境生态平衡提供了理论基础和科学依据。     

苏云金芽孢杆菌及其杀虫晶体蛋白 作用机制的研究进展

综合叙述了苏云金芽胞杆菌Bacillus thuringiensis和杀虫晶体蛋白的作用机制及在不同水平上解释这些机制的一些流行模型和有关亚分子结构的作用。     

基于可培养方法分析云南腾冲小空山火山谷芽胞杆菌分布特征

【目的】为了解云南腾冲小空山火山谷土壤中可培养芽胞杆菌种类分布特征。【方法】采用可培养手段对小空山火山谷阳坡、谷底和阴坡土壤中的芽胞杆菌进行分离培养,根据16S rRNA基因序列同源性对分离菌株进行鉴定,并分析系统发育地位。利用Canoco5软件分析采样点芽胞杆菌种类分布特征与土壤样品理化性质的相关性。【结果】从火山谷土壤样品中共分离获得180株芽胞杆菌,16S rRNA基因测序鉴定结果表明分离菌株隶属于芽胞杆菌纲2个科(芽胞杆菌科和类芽胞杆菌科)、6个属、34个种,其中芽胞杆菌属(Bacillus) 11个种,类芽胞杆菌属(Paenibacillus) 14个种,短芽胞杆菌属(Brevibacillus)3个种,赖氨酸芽胞杆菌属(Lysinibacillus)4个种,嗜冷芽胞杆菌属(Psychrobacillus)1个种和绿芽胞杆菌属(Viridibacillus)2个种,其中7个菌株与其最近模式菌株16SrRNA相似性低于种的界定阈值(98.65%),为芽胞杆菌潜在新物种。优势属为芽胞杆菌属和类芽胞杆菌属,优势种为蕈状芽胞杆菌(Bacillusmycoides),图瓦永芽胞杆菌(Bacillustoyonensis),蜡状芽胞杆菌(Bacilluscereus),解木糖赖氨酸芽胞杆菌(Lysinibacillusxylanilyticus),蜂房类芽胞杆菌(Paenibacillusalvei)和沙地绿芽胞杆菌(Viridibacillus arenosi)。其中16个种分离自阳坡,29个种分离自阴坡,9个种分离自谷底,三者共同种类为6种。阳坡、谷底和阴坡的芽胞杆菌种群分布Bray-Curtis相似性为62.4%,多样性分析结果表明,Shannon指数(H′)大小次序为阴坡阳坡谷底。环境因子分析发现,芽胞杆菌种群分布多样性特征与其土壤的海拔高度、碳氮比和硫含量呈负相关,而和碳源和氮源含量呈正相关。【结论】从以上结果得出,云南腾冲火山谷有着较为丰富的芽胞杆菌资源,且还存在可分离培养的芽胞杆菌的潜在新物种,为利用火山微生物资源提供了保障。     

养猪微生物发酵床芽胞杆菌空间分布多样性

了解微生物发酵床大栏养猪垫料中的芽胞杆菌多样性和空间分布规律,为微生物发酵床管理、芽胞杆菌新资源挖掘及菌剂开发奠定基础。将发酵床划分为32个方格(4行×8列),采用五点取样法获得每个方格的样品。采用可培养法从32份样品中分离芽胞杆菌菌株,利用16S rRNA基因序列初步鉴定所分离获得的芽胞杆菌种类。利用聚集度指标和回归分析法,分析芽胞杆菌的样方空间分布型。通过Shannon-Wiener多样性指数、Simpson优势度指数、Hill指数及丰富度指数分析,揭示微生物发酵床中芽胞杆菌的空间分布多样性。从32份样品中共获得芽胞杆菌452株,16S rRNA基因鉴定结果表明它们分别隶属于芽胞杆菌纲的2个科、8个属、48个种。其中,种类最多的为芽胞杆菌属(Bacillus),30种;赖氨酸芽胞杆菌属(Lysinibacillus),6种;类芽胞杆菌属(Paenibacillus),5种;短芽胞杆菌属(Brevibacillus),3种;鸟氨酸芽胞杆菌属(Ornithinibacillus)、大洋芽胞杆菌属(Oceanibacillus)、少盐芽胞杆菌属(Paucisalibacillus)和纤细芽胞杆菌属(Gracilibacillus)各1个种。芽胞杆菌种类在发酵床空间分布差异很大,根据其空间出现频次,可分为广分布种类,如地衣芽胞杆菌(Bacillus licheniformis);寡分布种类,如根际芽胞杆菌(B.rhizosphaerae);少分布种类,如弯曲芽胞杆菌(B.flexus)。依据其数量,可分为高含量组优势种群,如地衣芽胞杆菌(B.licheniformis);中含量组常见种群,耐盐赖氨酸芽胞杆菌(Lysinibacillus halotolerans);寡含量组寡见种群,如根际芽胞杆菌(B.rhizosphaerae);低含量组偶见种群,如土地芽胞杆菌(B.humi)。空间分布型聚集度和回归分析测定表明,芽胞杆菌在微生物发酵床的分布类型为聚集分布。微生物发酵床垫料中芽胞杆菌种类总含量高达4.41×108个/g,其种类含量范围为0.01—94.1×106个/g(均值为8.96×106个/g),丰富度指数(D)、优势度指数(λ)、Shannon-Wiener指数(H')和均匀度指数(J')分别为0.4928、0.2634、1.3589和0.9803,其中香农指数最大的单个芽胞杆菌种类为地衣芽胞杆菌(B.licheniformis)。根据芽胞杆菌种类多样性指数聚类分析,当欧式距离λ=17时,可分为高丰富度高含量和低丰富度低含量类型。微生物发酵床的芽胞杆菌种类丰富、数量高,是一个天然的菌剂"发酵罐",有望直接作为微生物菌剂,应用于土壤改良、作物病害防控、污染治理等领域。     

农林生态区土壤中的可培养芽胞杆菌资源调查——以四川省和重庆市部分地区为例

调查了四川和重庆农林生态区土壤中的芽胞杆菌资源种群分布,为芽胞杆菌功能资源挖掘和菌剂开发提供基础。采用可培养法从农林生态区土壤中分离获得芽胞杆菌,利用细菌16S rRNA基因初步确定其分类地位,并通过多样性指数分析芽胞杆菌种群结构。结果表明,共分离获得芽胞杆菌95株,经16S rRNA基因鉴定,7个属38个种,其中芽胞杆菌属(Bacillus)22个种、赖氨酸芽胞杆菌属(Lysinbacillus)4个种、类芽胞杆菌属(Paenibacillus)7个种、短芽胞杆菌属(Brevibacillus)2个种、嗜冷芽胞杆菌属(Psychrobacillus)1个种、鲁梅尔芽胞杆菌属(Rummeliibacillus)1个种和虚构芽胞杆菌属(Fictibacillus)1个种。其中,6株芽胞杆菌与其最相近模式菌株的16S rRNA相似性低于98.65%,为潜在新种。农林生态区土壤芽胞杆菌多样性指数香农指数大小依次为乐山林地盐亭县农地重庆林地盐亭县林地。以芽胞杆菌种类为样本,含量为指标,当欧式距离λ=15时,芽胞杆菌种类可分为高含量分布广型和低含量分布不均匀型。综上所述,农林生态区土壤蕴藏着较丰富的芽胞杆菌种类,为芽胞杆菌资源开发和利用提供了资源保障。     

永兴岛土壤芽胞杆菌多样性与抗线虫活性分析

从永兴岛的土壤中分离到187株芽胞杆菌,利用16S r DNA序列分析与RFLP技术对芽胞杆菌进行多样性分析,并初步探索了其抗南方根结线虫的活性。16S r DNA PCR-RFLP酶切图谱和代表菌株系统发育分析表明,永兴岛土壤中分离的187株芽胞杆菌分属于16个遗传类群,这些芽胞杆菌主要分布在Bacillus、Lysinibacillus、Paenibacillus、Brevibacillus和Sporosarcina 5个属。抗线虫的筛选结果显示,有5株芽胞杆菌表现出很好的抗线虫活性,其中活性最高的DB14381,可能是Lysinibacillus属的新资源。本研究揭示了永兴岛土壤蕴含丰富的芽胞杆菌物种多样性及潜在的新分类单元,并且存在拮抗南方根结线虫的芽胞杆菌资源。     

Natural occurrence of Bacillus thuringiensis and Bacillus cereus in eukaryotic organisms: a case for symbiosis

Bacillus thuringiensis and Bacillus cereus, two members of the Bacillus cereus sensu lato group, are most noted for their virulence in, respectively, arthropods and mammals including humans. Because of their pathogenicity to insects in particular, and their narrow host range, strains of B. thuringiensis have been utilised successfully as biocontrol agents of insect pests. Whereas B. cereus is not an established entomopathogen, certain strains of this species are well known to be etiological agents of gastrointestinal and emetic syndromes in humans. While much is known about the taxonomic properties and molecular basis for virulence of B. thuringiensis and B. cereus, comparatively less is known about their ecology in natural environments. Thus, there are limited data regarding their resilience, i.e. recycling of vegetative and sporulated phases of growth in soil, ecolgical niches including symbiotic interactions with other organisms, and the impact on ecosystems in which they proliferate. Nevertheless, based on recent data, a picture is beginning to emerge that B. thuringiensis and B. cereus are capable of establishing mutual and commensal relationships with both animals and plants. In this regard, these bacilli can proliferate in the digestive tracts of animals, where upon defecation they form dormant spores in the soil, and to a lesser extent on the phylloplane and rhizospheres of plants. Altogether, current evidence strongly suggests that B. thuringiensis and B. cereus are capable of completing their life cycles and recycling through various reservoirs, including animals, plants, and soil. This review focuses on the current knowledge pertaining to the ecologic interactions between B. thuringiensis and B. cereus and eukaryotic hosts, with special emphasis on symbiosis.     

Hydrogen and polyhydroxybutyrate producing abilities of microbes from diverse habitats by dark fermentative process

Thirty five bacterial isolates from diverse environmental sources such as contaminated food, nitrogen rich soil, activated sludges from pesticide and oil refineries effluent treatment plants were found to belong to Bacillus, Bordetella, Enterobacter, Proteus, and Pseudomonas sp. on the basis of 16S rRNA gene sequence analysis. Under dark fermentative conditions, maximum hydrogen (H₂) yields (mol/mol of glucose added) were recorded to be 0.68 with Enterobacter aerogenes EGU16 followed by 0.63 with Bacillus cereus EGU43 and Bacillus thuringiensis EGU45. H₂ constituted 63–69% of the total biogas evolved. Out of these 35 microbes, 18 isolates had the ability to produce polyhydroxybutyrate (PHB), which varied up to 500 mg/l of medium, equivalent to a yield of 66.6%. The highest PHB yield was recorded with B. cereus strain EGU3. Nine strains had high hydrolytic activities (zone of hydrolysis): lipase (34–38 mm) – Bacillus sphaericus strains EGU385, EGU399 and EGU542; protease (56–62 mm) – Bacillus sp. strains EGU444, EGU447 and EGU445; amylase (23 mm) – B. thuringiensis EGU378, marine bacterium strain EGU409 and Pseudomonas sp. strain EGU448. These strains with high hydrolytic activities had relatively low H₂ producing abilities in the range of 0.26–0.42 mol/mol of glucose added and only B. thuringiensis strain EGU378 had the ability to produce PHB. This is the first report among the non-photosynthetic microbes, where the same organism(s) – B. cereus strain EGU43 and B. thuringiensis strain EGU45, have been shown to produce H₂ – 0.63 mol/mol of glucose added and PHB – 420–435 mg/l medium.     

Microbial combinatorics: a simplified approach for isolating insecticidal bacteria

Bacteria can control pest insects that damage food crops, vector diseases and defoliate trees. Conventionally, isolation of these bacteria has been from soil and sporadically from dead insects. A simplified approach for isolating insecticidal bacteria from soil using the target insect as the selective agent was employed in this study. Instead of isolating single strains of bacteria from soil and testing each individual strain for insect toxicity, mixtures of bacteria present in each soil sample were tested together directly for toxicity using Manduca sexta (Linnaeus) (Lepidoptera: Sphingidae) as a model insect. Thirty-five soil suspensions or bacterial suspensions of the 40 suspensions tested killed at least one M. sexta larva. All but one bacterial culture isolated from dead larvae and retested for toxicity, killed at least one M. sexta larva. Nineteen bacterial strains isolated from larvae killed in the first test, were identical to the bacteria fed to the retested larvae. Of the 19 strains isolated, 14 were identified by 16S rDNA sequencing as belonging to the Bacillus cereus group including three strains that formed crystals that were identified as B. thuringiensis. Of the three other spore-forming strains, two were identified as psychrotrophic B. weihenstephanensis and the third as Lysinibacillus fusiformis. Two others were identified as Enterococcus faecalis. This approach, microbial combinatorics, reduces the number of insects necessary for toxicity screening and associated time and resources compared to conventional methods that first isolate bacteria and then individually test for toxicity as well as a means of discovery of new pathogens using the insect as the selective agent.     

WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line

Bacillus cereus continues to be one of the important foodborne pathogens due to its ability to produce various heat-labile and -stable toxins. Several methods have been developed to assess the pathogenicity of the B. cereus strains; however, most of these take more than 2–3 days to provide confirmatory results. In this study we standardized a one-step cytotoxicity assay using WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) and compared with the traditional MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)-based assay for rapid detection of cytotoxic Bacillus spp. using Chinese hamster ovary (CHO) cell line. Crude toxin preparations from 50 isolates of Bacillus spp. were exposed to CHO cell line for 1 h or 24 h and the cytotoxicity was determined by using WST-1 and MTT-based methods. Most B. cereus strains and some strains of other Bacillus species from our collection or from food sources showed comparably high cytotoxicity using either of the methods (P = 0.81); however, WST-1 assay provided results in only 3 h while MTT assay in 44–52 h. A positive correlation (R² = 0.93) between WST-1 and MTT assays strongly suggests that the WST-1-based cytotoxicity assay could be used as an alternative method to MTT assay for rapid (3 h) confirmation of toxigenic Bacillus species in foods prior to their retail distribution or consumption.     

Overexpression of serine alkaline protease encoding gene in Bacillus species: performance analyses

Bacillus species carrying subC gene encoding serine alkaline protease (SAP) enzyme were developed in order to increase the yield and selectivity in the bioprocess for SAP production. For this aim, subC gene was cloned into pHV1431 Escherichia coli–Bacillus shuttle vector, and transferred into nine host Bacillus species, i.e. B. alvei, B. amyloliquefaciens, B. badius, B. cereus, B. coagulans, B. firmus, B. licheniformis, B. sphaericus and B. subtilis. The influence of the host Bacillus species on SAP production on a defined medium with glucose was investigated in bioreactor systems. For each of the recombinant (r-) Bacillus species, effects of initial glucose concentration on cell growth and SAP production were investigated; and, physiological differences and similarities between the wild-type and r-Bacillus species are discussed. The highest biomass concentration was obtained with r-B. coagulans as 3.8 kg m⁻³ at the initial glucose concentration of C_Go=20 kg m⁻³ and the highest volumetric SAP activity was obtained with r-B. amyloliquefaciens as 1650 U cm⁻³ at C_Go=20 kg m⁻³. Overall SAP activity per amount of substrate consumed was the highest for r-B. sphaericus (137 U g⁻¹ cm⁻³) and r-B. licheniformis (130 U g⁻¹ cm⁻³). Among the r-Bacillus species the highest activity increase compared to the wild types was obtained with r-B. sphaericus while the lowest increase was obtained with r-B. amyloliquefaciens and r-B. licheniformis due to high SAP production potential of the wild-type strains. During storage of the host microorganisms, r-B. alvei and r-B. amyloliquefaciens were not able to bear the recombinant plasmid, probably, due to the restriction enzymes synthesized. Due to the highest stable volumetric activities r-B. licheniformis (950 U cm⁻³) and r-B. sphaericus (820 U cm⁻³) appear to be the favorable hosts for the production of SAP. All the r-Bacillus species excreted organic acids oxaloacetic and succinic acids, but, none excreted the amino acid valine. The variations in by-product distributions with each recombinant organism were also discussed.     

燕麦孢囊线虫孢囊定殖菌物的多样性

为揭示燕麦孢囊线虫孢囊定殖菌物的多样性,从青岛市选择3个罹患燕麦孢囊线虫病的冬小麦地,在返青期至成熟期,定时采集小麦根际燕麦孢囊线虫病土样,采用孢囊刺破法、单孢分离法或单菌丝分离法对定殖菌物进行分离纯化,并对定殖菌物进行形态鉴定和分子鉴定以及多样性分析。结果表明,从3个地点分离得到1 072个菌株,这些菌株归属于19目（含3个未定目）28科（含10个未定科）35属（含3个未定属）,其中轮枝孢属Verticillium的相对多度最高,为25.2%,是优势属;镰孢菌属Fusarium、黑团孢属Periconia及链格孢属Alternaria的相对多度分别为14.9%、13.5%及13.4%。示范园样品的Margalef’s丰富度指数,Shannon-Wiener多样性指数及Pielou均匀度指数均最高,分别为4.30、2.516及0.755;家属区样品的Margalef’s丰富度指数最低,为3.18;而葛家屯样品的Shannon-Wiener多样性指数和Pielou均匀度指数最低,分别为2.077和0.654。3个地点定殖菌物群落组成的Jaccard相似性指数在0.486-0.607之间。     

Development of a rapid and sensitive immunoassay for detection and subsequent recovery of Bacillus anthracis spores in environmental samples

Bacillus anthracis is considered a major threat as an agent of bioterrorism. B. anthracis spores are readily dispersed as aerosols, are very persistent, and are resistant to normal disinfection treatments. Immunoassays have been developed to rapidly detect B. anthracis spores at high concentrations. However, detection of B. anthracis spores at lower concentrations is problematic due to the fact that closely related Bacillus species (e.g., B. thuringiensis) can cross-react with anti-B. anthracis antibodies, resulting in false positive detections. Subsequent polymerase chain reaction (PCR) analysis is required to differentiate virulent strains. We report here on a protocol for the rapid, sensitive detection of B. anthracis spore using the Integrating Waveguide Biosensor followed by a method for the rapid release and germination of immunocaptured spores. A detection limit of ca. 10³ spores was achieved by incubating spores simultaneously with capture and detection antibodies (“liquid-phase” assay) prior to capture on capillary tubes/waveguides. Subsequent incubation with BHI broth directly in capillary tubes allowed for rapid germination, outgrowth, and release of spores, resulting in vegetative cells for PCR analysis.     

Re-identification of the halotolerant, biosurfactant-producing Bacillus licheniformis strain JF-2 as Bacillus mojavensis strain JF-2

Bacillus strain JF-2 (ATCC 39307) is a halotolerant, biosurfactant-producing bacterium that was initially described as a member of the species Bacillus licheniformis based on a limited set of phenotypic characteristics. Here, genetic and phenotypic analyses were employed to determine the relationship of Bacillus strain JF-2 to other Bacillus strains. The restriction patterns with AluI and analysis of gyrA and 16S rRNA gene sequences grouped Bacillus strain JF-2 with B. mojavensis^T and not with B. licheniformis^T. DNA–DNA similarity showed JF-2 was 75% similar to B. mojavensis^T and only 11% similar to B. licheniformis^T. Both strain JF-2 and B. mojavensis^T required DNA for anaerobic growth, but B. licheniformis^T did not. B. mojavensis^T and strain JF-2 did not grow anaerobically in thioglycollate medium or aerobically with propionate while B. licheniformis^T grew under these conditions. DNA–DNA similarity, gene sequence data and phenotypic characteristics all support the assignment of JF-2 as a member of the species B. mojavensis.