暗黑鳃金龟甲幼虫肠道细菌分离及鉴定

目的从微生态学角度研究暗黑鳃金龟甲幼虫营养生理活动,探讨其肠道菌群的构成,为其资源开发及生物防治提供理论依据。方法按传统分离方法,从暗黑鳃金龟甲幼虫肠道环境中分离纯化获得10个细菌菌株,对其菌体形态、染色反应、培养性状、生理生化反应进行了系统研究。结果研究结果表明,上述10个细菌菌株分别属于鲁氏耶尔森菌（Yersinia．ruckeri）、侧胞芽胞杆菌（Bacilluslaterosporus）、坚强芽胞杆菌（Bacillus．firmus）、放线杆菌属（Actinobacillus）、飞虫杀雄菌（Arsenophonusllasoniae）、不动杆菌属（Acinetobacter）、气单胞菌Aeromonas）、沙门菌属（Sal—moneUa）、短芽胞杆菌（Bacillusbrevis）、变形菌属（Proteus）。结论通过对暗黑鳃金龟甲幼虫肠道细菌的鉴定,其肠道细菌在培养性状、生理性状、生理生化测定等方面存在较多差异。     

龟纹瓢虫四虫态肠道细菌分离及鉴定

目的通过对龟纹瓢虫四个虫态肠道菌群研究,探讨其营养生理,为改良龟纹瓢虫人工饲料,大量扩繁天敌昆虫奠定基础。方法按照传统的分离方法从龟纹瓢虫瓢卵、幼虫、蛹及雌成虫或肠道内分离15个不同菌株,对其染色反应、培养性状,菌体形态和生理生化性状进行系统研究。结果从龟纹瓢虫卵内分离鉴定出短芽胞杆菌（Bacillus brevis）、球性芽胞杆菌（Bacillus sphaericus）、棍状杆菌属（Clavibacter）和一种未知分类地位的菌;幼虫肠道内分离鉴定柠檬酸杆菌属（Citrob acter）、短状杆菌属（Brachybacterium）、微杆菌属（Microbacterium）、皮杆菌属（Dermabacter）、浸麻芽胞杆菌属（Bacillus macerans）、纤维单胞菌属（Cellulomonas）;蛹肠道分离鉴定出短芽胞杆菌（Bacillus brevis）、蜡状芽胞杆菌（Bacillus cereus）、微杆菌属（Microbacterium）、不动杆菌属（Acinetobacter）;成虫肠道分离鉴定出短芽胞杆菌（Bacillus brevis）、球性芽胞杆菌（Bacillus sphaericus）、柠檬酸杆菌属（Citrob acter）、不动杆菌属（Acinetobacter）、地衣芽胞杆菌（Bacillus licheniformis）、凝结芽胞杆菌（Bacillus coagulans）和巨大芽胞杆菌（Bacillusmegaterium）。结论不同虫态龟纹瓢虫肠道中细菌种类不同,为进一步研究适合龟纹瓢虫不同时期生长和繁殖的人工饲料的配比提供了依据。     

斜纹夜蛾幼虫肠道细菌分离鉴定及其功能初步分析

昆虫肠道微生物对其寄主的生长发育、营养代谢、免疫以及农药抗性等方面都发挥着重要作用。为研究斜纹夜蛾Spodoptera litura幼虫肠道细菌的多样性,并为其功能验证做准备,本文利用传统微生物分离纯培养方法从斜纹夜蛾4龄幼虫肠道中共分离鉴定得到10株细菌,分别为属于变形菌门(Proteobacteria)的脱氮假单胞菌(Pseudomonas denitrificans),不动细菌(Acinetobacter sp.),肺炎克雷伯氏菌(Klebsiella pneumoniae)和肠杆菌(Enterobacter sp.);属于厚壁菌门(Firmicutes)的鸡葡萄球菌(Staphylococcus gallinarum),蒙氏肠球菌(Enterococcus mundtii),蜡样芽胞杆菌(Bacillus cereus)和枯草芽胞杆菌(Bacillus subtilis)以及放线菌门(Actinobacteria)的微杆菌(Microbacteriums sp.)和乳酪棒杆菌(Corynebacterium casei)。变形菌门和厚壁菌门是斜纹夜蛾肠道可培养细菌中的优势菌群。功能验证实验表明肠杆菌具备纤维素降解能力,微杆菌具备很强的苯酚降解能力。本研究为未来深入研究斜纹夜蛾肠道微生物的功能提供了方向和菌株材料。     

东北地区耐盐芽胞杆菌筛选统计及初步鉴定

采用温度筛选与表面定向培养相结合的方法对东北地区土壤中可培养耐盐芽胞杆菌进行分离和筛选,得到137株芽胞杆菌,其中耐盐芽胞杆菌74株,占总芽胞杆菌数量的54%,最适盐浓度均为1%,耐盐能力在4%-14%之间。通过扩增耐盐菌株的16S rRNA基因序列,对其进行分子鉴定和分类,获得东北地区土壤中可培养的耐盐芽胞杆菌的多样性信息。通过同源性比对和耐盐性差异,确定36株差异耐盐芽胞杆菌,分属于芽胞杆菌属中的7个种。其中多数菌株为苏云金芽胞杆菌(Bacillus thuringiensis)(14株,占总数38.9%,最高耐盐性在4%-9%NaCl之间)。其次依次为蜡样芽胞杆菌(Bacillus cereus)(7株,19.4%,4%-8%NaCl),枯草芽胞杆菌(Bacillus subtilis)(7株,19.4%,8%-11%NaCl),炭疽芽胞杆菌(Bacillus anthracis)(4株,11.1%,5%-7%NaCl),弯曲芽胞杆菌(Bacillus flexus)(2株,5.6%,9%-14%NaCl),球形芽胞杆菌(Bacillus sphaericus)(1株,2.8%,5%NaCl)和阿氏芽胞杆菌(Bacillus aryabhattai)(1株,2.8%,6%NaCl)。弯曲芽胞杆菌和枯草芽胞杆菌的耐盐能力较好。从中选取3株代表菌株,明确了其培养特性、形态特征及生理生化特性,并对其分别进行了系统发育分析。     

四川海螺沟冰川土样芽胞杆菌资源分析

【背景】芽胞杆菌(Bacillus-like)是一类能形成具有强抗性芽胞且可在多种极端环境下存活的细菌,其产生的多种功能代谢产物在多种领域具有重要研究价值。由于冰川低温、寡营养的独特生态环境,其存在的芽胞杆菌可能具有特殊性,因此研究冰川芽胞杆菌有利于发掘新基因、丰富芽胞杆菌多样性。【目的】了解四川海螺沟冰川土壤芽胞杆菌资源,为挖掘芽胞杆菌新资源提供基础。【方法】采用纯培养法分离获得冰川土壤芽胞杆菌资源,利用16S r RNA基因进行系统发育分析,测定代表性菌株的生理生化特性并采用类平均法和欧氏距离模型进行聚类分析。【结果】共筛选到可培养细菌44株,经16S r RNA基因鉴定确定其中36株为芽胞杆菌,隶属于4个属的19个种,分别为芽胞杆菌属(Bacillus)11个种24株、类芽胞杆菌属(Paenibacillus)2个种3株、短芽胞杆菌属(Brevibacillus)4个种5株和赖氨酸芽胞杆菌属(Lysinibacillus)2个种4株,其中以芽胞杆菌属(Bacillus)为优势属。分离菌中仅3株芽胞杆菌可在4°C生存,7株能在50°C生长,大部分菌株在30°C下生长良好;有74%菌株能耐碱,有37%菌株能在无盐条件下生长。根据生理生化结果,采用类平均法和欧氏距离模型进行聚类分析,可分为3组,分别包含7种、4种和6种芽胞杆菌。第1组均可以水解七叶灵和利用葡萄糖,第2组均不能水解七叶灵和利用葡萄糖,第3组仅能共同利用葡萄糖。【结论】四川海螺沟冰川土壤蕴藏着较为丰富的芽胞杆菌资源,为芽胞杆菌新资源挖掘提供了资源保障。     

黄粉虫不同虫态肠道细菌分离及鉴定

目的研究黄粉虫肠道细菌的类群与其取食的关系,为进一步开发利用提供理论依据。方法从人工饲养的黄粉虫幼虫、蛹和成虫肠道环境中分离纯化获得9个细菌菌株,对其菌体形态、染色反应、培养性状、生理生化反应进行系统研究。结果研究结果表明,上述9个细菌菌株分别属于放线杆菌属（Actunobacillus）、丙酸杆菌属（Propionibacterium）、柠檬酸杆菌属（Citrobacter）、沙雷菌属（Serratia）、芽胞杆菌属（Bacillus）、皮杆菌属（Dermabacter）、短状杆菌属（Brachybacterium）、棍状杆菌属（Clavibacter）和微小杆菌属（Exiguobcacterium）。结论通过对黄粉虫不同虫态即幼虫、蛹和成虫肠道细菌分离,其细菌种类存在一定差别。     

海洋中分离的抗白念珠菌芽胞杆菌性状特征及系统鉴定

对筛选自中国南海、黄海、渤海4个地区近海海水样品的7株有抗白念珠菌活性,且稳定性较强的芽胞杆菌的形态特征、培养特征及生理生化试验等进行系统分析比较,结果表明,LU-B02为凝结芽胞杆菌(Bacillus coagulans),LU-B13为蜡样芽胞杆菌(Bacillus cereus),其他各菌株为短小芽胞杆菌(Bacillius pumilus),除LU-B02耐盐度5%外,其他耐盐度达10%以上。它们均属于芽胞杆菌的第一群。16S r DNA基因同源性序列比较进一步证实LU-B02为凝结芽胞杆菌,它与凝结芽胞杆菌标准菌株ATCC15950在鉴定特征上虽然相同,但与后者相比,最高生长温度较低,耐盐性较强,可以利用阿拉伯糖、木糖、甘露醇等碳源,可水解酪素,并可稳定地产生抗白念珠菌活性物质,将其命名为Bacillus coagulans subsp.heishijiaosis。尚未见抗白念珠菌的凝结芽胞杆菌菌株的有关报道。     

中华通草蛉越冬成虫肠道可培养好氧细菌多样性分析

本研究以昆虫微生态学原理为指导探究中华通草蛉Chrysoperla sinica(Tjeder)的肠道菌群构成,为中华通草蛉的营养生理研究及人工繁殖利用提供理论依据和技术基础。采用无菌解剖及纯培养分离检测技术,从雌、雄成虫肠道环境中分离、纯化、培养,获得10个细菌菌株,对菌体形态、染色反应、培养形状、生理生化反应进行系统研究。结果表明,上述10个菌株分别属于摩根氏菌属(Morganella)、短小芽孢杆菌(Bacillus pumilus)、地杆菌属(Terrabacter)、爱德华氏菌属(Edwardsiella)、色杆菌属(Chromobacterium)、热黄杆菌(Flavobacterium thoupophilum)、放线杆菌属(Actinobacillus)、棍状杆菌属(Clavibacter)、邻单胞菌属(Plesiomonas)、预研菌属(Yokenella);越冬期雌、雄成虫肠道细菌的种类和数量存在较大差异,可能与雌、雄成虫不同的营养和生理需求有关,两者关系还需进一步研究。     

小菌虫肠道可培养细菌的分离鉴定 及产消化酶活性分析

摘要：目的 分离小菌虫肠道可培养细菌,并研究其产消化酶活性,探讨肠道细菌对小菌虫消化食物的影响。方法 采用传统细菌分离培养方法分离小菌虫肠道细菌,利用16S rDNA序列进行细菌分子鉴定;利用筛选培养基鉴别各细菌的产蛋白酶、脂肪酶、淀粉酶和纤维素酶活性。结果 在小菌虫肠道中分离到4种可培养细菌,分别是枯草芽胞杆菌（Bacillus subtilis）、肺炎克雷伯菌（Klebsiella pneumoniae）、Pseudocitrobacter faecalis和芽胞杆菌（Bacillus sp.）。其中,2种芽胞杆菌属细菌有产消化酶活性。枯草芽胞杆菌有产纤维素酶、淀粉酶和蛋白酶活性;芽胞杆菌仅有产蛋白酶活性,但产酶能力低于枯草芽胞杆菌。结论 小菌虫肠道细菌中可培养细菌结构简单,但其中的芽胞杆菌属细菌有产纤维素酶、淀粉酶和蛋白酶能力,说明小菌虫肠道中的2种芽胞杆菌属细菌可能有协助小菌虫进行食物消化的功能。     

鸡源益生芽胞杆菌的筛选

根据菌体对低pH和高胆盐的耐受性、产酶特性和对病原菌的抑菌效果,对家鸡肠道中的芽胞杆菌进行了筛选。结果表明,有4株芽胞杆菌(A2、C5、C19和D8)能抑制金黄色葡萄球菌(Staphylococcus aureus)的生长。这4株菌在pH 3.0和高胆盐条件下的存活率都达到60%以上,产淀粉酶和蛋白酶的能力也较好,表明这些菌株具有作为益生菌的开发潜力。经形态学和生理生化反应鉴定,初步确定A2和D8为地衣芽胞杆菌(Bacillus licheniform is),C5和C19为蜡样芽胞杆菌(Bacillus cereus)。     

小碎斑鱼蛉幼虫资源成分分析及价值评价

针对小碎斑鱼蛉Neochauliodes sparsus(Liu et Yang)幼虫,进行了资源成分测定,分析。结果表明:小碎斑鱼蛉幼虫干物质中蛋白质、氨基酸、脂肪、糖类及灰分含量分别为67.69%、56.02%、10.4%、1.59%、7.05%,含有18种氨基酸,包括8种人体必需氨基酸,其必需氨基酸占总氨基酸含量的45.32%,必需氨基酸与非必需氨基酸含量的比值为82.89%,第一限制性氨基酸为色氨酸;此外,还含有Ca、P、Fe、Zn等多种矿物质和微量元素。在分析了小碎斑鱼蛉幼虫资源成分的基础上,对其开发利用价值进行了评价。     

稻瘟病拮抗菌株的分离、筛选及鉴定

从水稻病健叶、茎和根组织以及稻田土壤中共分离得到细菌菌株321株。经发酵法初筛,对稻瘟病菌丝生长有抑制作用的有57株,再通过平板对峙法复筛,具有强烈拮抗作用的菌株有5株,其抑菌距离达16mm以上。分别对5个菌株进行形态学观察、生理生化指标进行鉴定,结果有1株(No.156)为Bacillus subtilis,2株(No.171和No.177)为Bacillus pumillus,2株(No.192和No.279)为Bacillus ploymyxa。     

窗胸萤肠道细菌分离及鉴定研究

从窗胸萤的5龄幼虫中分离纯化获得10个细菌菌株,并对其菌体形态、染色反应、培养性状、生理生化性状进行了系统研究。鉴定结果表明,上述10个细菌菌株分别属于埃希氏菌属(Escherichia)、金色单胞菌属(Chryseomonas)、放线杆菌属(Actinobacillus)、摩根氏菌属(Morganella)、李斯特菌属(Listeria)、金杆菌属(Aureobacterium)、微杆菌属(Microbacterium)、地杆菌属(Terrabacter)、芽孢杆菌属(Bacillus)和黄杆菌属(Flavibacterium)。     

塑料饲养大蜡螟幼虫肠道可培养细菌多样性

【背景】昆虫肠道中存在大量的微生物,是昆虫正常生命活动所必需的。它们能够促进维生素的合成、脂肪和碳水化合物的吸收及利用,同时还可以保护宿主抵御天敌,忍受高温以及促进毒素或异生素的代谢,间接促进资源开发。【目的】研究PE塑料饲喂的大蜡螟幼虫肠道可培养细菌的多样性。【方法】利用16S rRNA基因序列分析技术,结合菌落形态和细胞形态及相关生理生化特征鉴定细菌种类。【结果】从大蜡螟幼虫肠道分离纯化的40株可培养细菌得到16种不同细菌遗传型,分别属于芽孢杆菌科(Bacillaceae)、肠球菌科(Enterococcaceae)、葡萄球菌科(Staphylococcaceae)、莫拉菌科(Moraxellaceae)4个科。其中芽孢杆菌科是肠道可培养细菌的优势细菌种类。结合菌落和细胞形态及生理生化特征,确定肠道可培养细菌为芽孢杆菌属9株、肠球菌属4株、葡萄球菌属2株以及不动杆菌属1株。【结论】通过研究大蜡螟幼虫肠道可培养细菌群落结构组成,可为开展大蜡螟肠道的微生态研究提供相关理论基础。     

特异性杀虫基因Bt cry3A在桑粒肩天牛幼虫两种肠道常驻内生菌中的转化和表达

[目的]将特异性杀虫毒蛋白基因Bt cry3A转入桑粒肩天牛(Apriona germari Hope,Ag)幼虫肠道常驻内生菌中,构建能在天牛幼虫肠道中定殖并表达特异性杀虫基因Bt cry3A的工程菌.[方法]以传统方法和16S rDNA分子生物学分析等方法分离、鉴定Ag幼虫肠道优势的常驻内生菌,从中筛选出适合转化的候选菌株.利用电转化技术将含有对鞘翅目昆虫具专一性毒力Bt cry3A基因的Escherichia coli-Bacillus thuringiensis穿梭表达质粒pHT305a和pHT7911分别转入Ag幼虫肠道常驻内生菌短短芽孢杆菌(Brevibacillus brevis Ag12,Ag12)和苏云金芽孢杆菌(Bacillus thuringiensis Ag13,Ag13)中.[结果]从Ag幼虫肠道共分离获得18个不同种的可培养细菌菌株,并从中选取菌株Ag12和Ag13作为出发菌株转入Bt cry3A基因.经质粒稳定性试验、转化子生长特性测试、伴胞晶体电镜检测、毒蛋白SDS-PAGE分析、工程菌定殖性分析以及生物毒力测试,结果显示cry3A基因已经成功转入Ag幼虫的常驻内生菌短短芽孢杆菌和苏云金芽孢杆菌中,并且工程菌Ag12-305a、Ag13-305a、Ag 12-7911和Ag13-7911都能在天牛幼虫肠道内稳定生长、繁殖并表达分子量约65kDa的伴孢晶体杀虫蛋白Cry3A.[结论]Bt cry3A基因已成功转入桑粒肩天牛幼虫肠道优势常驻内生菌中,获得了四株能在桑粒肩天牛幼虫肠道内定殖,并能表达目的杀虫基因Btcry3A的转基因工程菌.     

Expression of mosquito active toxin genes by a Colombian native strain of the gram-negative bacterium Asticcacaulis excentricus

Mosquito control with biological insecticides, such as Bacillus sp. toxins, has been used widely in many countries. However, rapid sedimentation away from the mosquito larvae feeding zone causes a low residual effect. In order to overcome this problem, it has been proposed to clone the Bacillus toxin genes in aquatic bacteria which are able to live in the upper part of the water column. Two strains of Asticcacaulis excentricus were chosen to introduce the B. sphaericus binary toxin gene and B. thuringiensis subsp. medellin cry11Bb gene cloned in suitable vectors. In feeding experiments with these aquatic bacteria, it was shown that Culex quinquefasciatus, Aedes aegypti, and Anopheles albimanus larvae were able to survive on a diet based on this wild bacterium. A. excentricus recombinant strains were able to express both genes, but the recombinant strain expressing the B. sphaericus binary toxin was toxic to mosquito larvae. Crude protease A. excentricus extracts did not degrade the Cry11Bb toxin. The flotability studies indicated that the recombinant A. excentricus strains remained in the upper part of the water column longer than the wild type Bacillus strains.     

Isolation and characterization of two hydrocarbon-degrading Bacillus subtilis strains from oil contaminated soil of Kuwait

Two strains of hydrocarbon-utilizing bacteria were isolated from soil samples of the Kuwait Burqan oil field at a temperature of 37 degrees C. The bacteria were motile endospore-forming rods with slight differences in their metabolic patterns and 16S rRNA sequence. Vegetative cells of the strains designated as AHI and AHII had an ultrastructure typical of gram-positive bacteria and showed gram-positive staining. The bacteria did not show pigmentation. Best growth was observed at 37 degrees C at neutral pH and NaCl concentrations in the range of 5-10 g per l. Both strains were obligatory aerobic and developed on synthetic media with either Diesel fuel, n-decan or naphthalene as the sole carbon and energy source. No specific growth factors were required. On the basis of their morphological, physiological and biochemical features, as well as their 16S rRNA analysis and electron microscope study, both strains were assigned to the species of Bacillus subtilis.     

A study on the possible utilization of immunodiffusion and immunofluorescence techniques as the diagnostic methods for American foulbrood of honeybees (Apis mellifera)

Four types of antisera were obtained from rabbits hyperimmunized with either spores or vegetative rods from two strains of the American foulbrood pathogen, Bacillus larvae. The specificity and sensitivity of these antisera were tested with immunofluorescence and immunodiffusion methods. No cross-reactions were observed between the antisera and other different species of Bacillus or different genera of bacteria. The specificity was not found between the antisera and two strains of B. larvae although stronger fluorescent intensity was observed between the antiserum and its corresponding strain of antigen in the immunofluorescence tests. Eight samples of 1- to 2-day-old larvae, 3- to 4-day-old larvae, decayed tissue, and dry remain, collected from eight infected colonies, were tested against antisera by the immunofluorescence and the immunodiffusion methods. The results indicated that both methods are sensitive and specific for making diagnosis of field samples of American foulbrood of honey bees.     

Identification of entomopathogenic Bacillus isolated from Simulium (Diptera, Simuliidae) larvae and adults.

Entomopathogenic bacteria isolated from Simulium larvae and adults from breeding sites in the states of S?o Paulo and Rio de Janeiro, Brazil, were identified as 18 strains of Bacillus thuringiensis and one of B. sphaericus. Most of these strains were serotyped according to their flagellar antigens. However, nine of the B. thuringiensis samples, could not be serotyped and were designated as "autoagglutinating"; they were also shown to be toxic in preliminary tests against Aedes aegypti larvae. Additionally, B. sphaericus was also shown to be toxic towards Culex quinquefasciatus larvae.     

Plasmid transfer between strains of Bacillus thuringiensis infecting Galleria mellonella and Spodoptera littoralis

To determine the possibility of plasmid transfer occurring between strains of Bacillus thuringiensis in infected lepidopterous larvae, Galleria mellonella and Spodoptera littoralis were infected with two or more strains of B. thuringiensis and the resulting bacteria from the dead insects were examined for plasmid transfer. Transfer rates of plasmids coding for crystal production and tetracycline resistance were high, reaching levels similar to those obtained in laboratory broth cultures. Transfer was higher in G. mellonella than S. littoralis, probably due to the greater ability of B. thuringiensis to colonize the larvae. In broth cultures, B. thuringiensis was also able to transfer plasmids into sporeforming bacteria present in soil samples. The results suggest that plasmid transfer between strains of B. thuringiensis occurs in nature, resulting in the production of new combinations of delta-endotoxins within populations of the bacteria.