黄河三角洲贝壳堤放线菌多样性及抑菌活性

研究旨在揭示黄河三角洲贝壳堤放线菌多样性,以便从中寻找新的具有生物防治功能的微生物资源。采用3种分离培养基,利用稀释平板涂布法对贝壳堤土壤样品进行分离;通过形态特征、生理生化实验及16S rDNA基因测序对放线菌进行了分类鉴定。结果表明分离到的174株放线菌分属于10个科,13个属,其中链霉菌属(42%)和拟诺卡氏菌(11%)为优势菌属。16S rDNA基因分析表明,61%的菌株与已知菌的相似性都在99%以下,其中菌株BK-17的16S rDNA序列与同源性最近的菌株相似率仅为95.2%。以2株细菌和5株植物病原真菌作为指示菌,进行了抑菌活性测定,有94株至少对1种指示菌有抑菌作用,有8株对7种指示菌都有很强的拮抗作用。以上研究结果表明黄河三角洲贝壳堤土壤中存在丰富的放线菌资源,存在很多潜在的新菌种和活性菌株,有进一步研究和开发的价值。     

广西沿海地区红树林根系土壤中放线菌的分离与鉴定

本研究通过分离纯培养,从广西北海及防城港红树林根系土壤中分离出放线菌并提取其总DNA,用放线菌通用引物对获得菌株的16SrDNA进行PCR扩增,对获得的扩增产物进行DNA序列测定及菌株鉴定。研究结果表明,从红树林根系土壤样品中分离出15株典型放线菌菌株。16SrDNA测序比对鉴定结果显示,15株典型放线菌菌株中有12株属于链霉菌属（Streptomyces）,是常见菌属;3株属于拟诺卡氏菌属（Nocardiopsis）,为稀有放线菌。本研究分离纯化获得15株典型放线菌,初步揭示了广西沿海地区红树林土壤中放线菌的多样性。     

苏云金芽孢杆菌S1478—1分离株的特性鉴定及cry1Ac同源基因克隆

本研究对从海南岛尖峰岭热带雨林自然保护区的土壤样品中分离出的＆菌株S1478～1进行了特性鉴定,研究表明S1478—1分离株菌落形态和生长特征和励参照菌株HD73极其相似。16SrDNA序列分析表明,S1478—1分离株与其它＆thuringiensis、B．cereus和B.anthracis的16S rDNA序列相似性达到99％。分离株能产菱形伴胞晶体,SDS-PAGE蛋白电泳分析表明,菌株在生长后期,形成芽孢同时分泌130kD大小的晶体蛋白。生物测定表明S1478—1分离株对小菜蛾具有很高的毒杀活性,LC50值高达5．159×10^8cfu／mL。初步显示S1478—1分离株可作为防治鳞翅目害虫的生物农药菌株。利用PCR—RFLP方法鉴定S1478—1分离株含有cry1Ac同源基因,以PCR粘性端克隆方法扩增全长基因,序列测定表明该基因ORF为3537bp,编码1178个氨基酸,推定的编码蛋白分子量为133．3kD,与其它cry1Ac基因序列最高达到99％同源,因此,该基因可作为杀虫工程菌及培育转基因抗虫作物的候选基因。     

盐穗木根际耐盐碱细菌的分离筛选及鉴定

将采集于新疆和田地区于田县盐碱地的2种盐穗木根际土壤样品制备成菌悬液,经适当稀释后,涂布于牛肉膏蛋白胨培养基中,经分离纯化获得20株菌株。经过分别或同时提高培养基的盐、碱浓度进行菌株耐盐碱性能的筛选,获得1株耐盐碱菌株D8,其在pH值12且盐(NaCl)浓度达到120 g/L的培养基中仍可生长,该菌株既属于极端嗜盐微生物又属于极端嗜碱微生物。通过对筛选出来的菌株进行革兰氏染色制片、生理生化检测、电镜观察、Biolog鉴定、16S rRNA基因测序及系统发育树的建立来鉴定种属关系。鉴定结果是该菌为太平洋盐单胞菌(Halomonas pacifica)。     

铁锰结核土壤锰氧化细菌多样性及新菌属分析

尽管细菌的锰氧化作用被认为是自然界中氧化锰矿物形成的主要成因,但目前国内外对陆地土壤环境中锰氧化细菌的种群组成与多样性方面的研究甚少。本研究对采集于山东崅峪一处含铁锰结核的棕壤进行了可培养锰氧化细菌分离、活性测定与多样性调查,结果发现表层土壤(A层:0～20 cm)的可培养锰氧化细菌是最丰富的,但是高锰氧化活性的细菌主要分布在心土层(B层:20～40 cm)和底土层(C层:>75 cm)。通过对具有高锰氧化活性的分离菌株16S rRNA基因的扩增、测序和序列BLAST比对分析,发现了7个此前未见报道的具有锰氧化活性的新菌属。此外,对5株具有高锰氧化活性的分离菌株和土壤样品的进行了16S rRNA基因V3产物的变性梯度凝胶电泳(DGGE)分析,结果显示此5株高锰氧化菌株并非都是土壤中的高丰度细菌。     

结晶纤维素降解细菌的筛选、分离与鉴定

采用以结晶纤维素（Avicel）为唯一碳源的平板活性筛选法,从60份森林腐殖土、腐术样品中分离得到2株在pH5．0、37℃条件下高效降解结晶纤维素的细菌菌株GXN152和GXN153。以菌株的总DNA基因为模板,用细菌的16S rRNA基因的通用引物扩增得到菌株GXN152和GXN153的16S rRNA基因序列。测序分析表明菌株GXN152的16S rRNA基因序列和洋葱假单胞菌（Burkholderia cepacia）菌株CNR22的16S rRNA基因序列的同源性最高,具有98％的同源性,生理生化特性检测表明菌株GXN152具有洋葱假单胞菌的鉴别特征,将结晶纤维素降解菌GXN152鉴定为洋葱假单胞菌。菌株GXN153的16S rRNA基因序列和类芽孢杆菌（Paenibacillus favisporus）菌株GMP01的16S rRNA基因序列的同源性最高,具有99％的同源性,生理生化特性检测表明菌株GXN153具有类芽孢杆菌的鉴别特征,将纤维素降解菌GXN153鉴定为类芽孢杆菌。     

广东省刺竹内生固氮菌多样性

采用好氧和厌氧培养方法对广东省刺竹(Bambusa blumeana)的根、茎和叶的内生细菌进行了分离和纯化,利用乙炔还原法检测了各菌株固氮酶活性,共获得40株具有较高固氮酶活性的刺竹内生固氮菌。对所获得的菌株进行SDS-PAGE全细胞蛋白电泳聚类分析,在80%相似性水平上,选取不同类群的代表菌株进行16S rRNA基因全序列测定。结果表明广东省刺竹内生固氮菌具有一定的多样性,它们分属Azospirillum(α-亚纲)、Escherichias和Pseudomonas(γ-亚纲)、Aquaspirillum(β-亚纲)。     

一株热带芽孢杆菌的分离鉴定及促生作用研究

为了筛选高效植物根际促生菌株,分析菌株促生应用潜力,为微生物肥料的研发提供宝贵的菌种资源。采集了海南省乐东黎族自治县的根际土壤,采用稀释涂布法分离筛选促生菌株,研究其产吲哚乙酸(IAA)、铁载体、蛋白酶、溶磷的能力,通过16S r RNA基因系统发育分析和全基因组特征对新分离菌株进行物种鉴定。分离得到一株产多种促生活性物质的菌株S03,基于16S rRNA基因和系统发育树分析结果表明,新分离菌株S03与NR157736热带芽孢杆菌(Bacillus tropicus)1A01406亲缘关系最近,结合全基因测序结果,确定菌株S03为Bacillus tropicus。Bacillus tropicus S03基因组全长5.42 Mb,碱基对G+C含量为36.02%,共编码5666个基因。通过GO数据库对比分析,S03具备促生物质(IAA、铁载体等)合成相关的基因。Bacillus tropicus S03的促生潜力良好,IAA产量为45.73 mg·L^-1,溶解无机磷能力为82.30 mg·L^-1,同时还具备产蛋白酶和铁载体的能力,Bacill...     

贵州市售婴儿配方乳粉中克罗诺杆菌的污染情况及特征分析

为揭示贵州省市售婴儿配方乳粉(PIF)中克罗诺杆菌的污染情况及种群特征,本研究以350份PIF样品为研究对象,对PIF中的克罗诺杆菌进行分离鉴定;利用API 20E生化鉴定法、16S rRNA测序法和多位点序列分型(MLST)法对分离株进行生化表型分型和基因分型,最后利用Kirby-Bauer纸片扩散法进行菌株的耐药性分析。结果表明,350份样品中共分离出21株克罗诺杆菌,污染率为6%。21株克罗诺杆菌被分为6个生化表型,3个类群和11个序列型,其中ST193和ST157为分离自贵州省市售PIF的克罗诺杆菌的优势序列型。耐药性分析结果表明,21株克罗诺杆菌对四环素和万古霉素具有高度的耐药性,而对氨苄西林、庆大霉素和氯霉素较为敏感。本研究结果为贵州省市售PIF中克罗诺杆菌的防控提供了理论依据。     

几株固氮芽孢杆菌的分离与鉴定

摘要： 从小麦（Triticum aestivum）、玉米（Zea mays）、黑麦草（Lolium sp.）和柳树（Salix sp.）的根际土壤中分离得到能在无氮培养基上生长的29株芽孢杆菌（Bacilli），通过固氮酶活的测定以及固氮酶结构基因 nifH 的PCR扩增得到7株固氮芽孢杆菌。对这7株固氮菌株进行了生理生化性状测定、16S rDNA序列分析(GenBank accession No. AY373358, AY373360,~AY373364和AY376876)、G+C mol%含量的测定及DNA-DNA杂交实验，结果表明，其中5株菌属于芽孢杆菌，另外2株菌属于类芽孢杆菌。在这7株被鉴定的菌株中，菌株T1被鉴定为Bacillus cereus；菌株G1、C4和C5被鉴定为B. megaterium；菌株W5的生理生化性状、16S rDNA和G+C mol%与Bacillus marisflavi 相近；G2的生理生化性状和16S rDNA 与Paenibacillus polymyxa 接近，但在基于16S rDNA的系统发育树中却与P. durus 聚在最小的分支内；T7可能是Paenibacillus属中的一个新种。     

铅锌矿区重金属污染对微生物数量及放线菌群落结构的影响

采集四川省汉源县富泉乡万顺铅锌矿区5个不同重金属浓度的土壤样品,进行了微生物数量及放线菌多样性的研究。经分离、纯化得到43株不同的放线菌,然后对其进行BOXAIR-PCR和16SrDNAPCR-RFLP分析。结果表明,铅锌矿区重金属复合污染对土壤微生物数量有较大的影响,随着铅锌矿区重金属污染程度的加剧,土壤微生物的总数下降。相关性分析表明,重金属含量与细菌数量呈极显著负相关（P〈0.01）,与放线菌数量、真菌数量呈显著负相关（P〈0.05）。供试菌株的16SrDNA用HaeⅢ、HinfⅠ和TaqⅠ酶切后具有32种遗传图谱类型。BOXAIR-PCR的聚类结果表明在86%的水平上,所有菌株分为10个遗传类型,结果基本与16SrDNAPCR-RFLP聚类差异不大。来源于高重金属的含量样品的菌株基本聚在一起,可能是重金属含量影响了放线菌的分布。同时,16SrDNA序列聚类分析结合系统发育树分析表明链霉菌属是汉源铅锌矿区主要的放线菌属并且具有遗传多样性。     

西北半干旱荒漠草原与耕地土壤可培养微生物多样性及分布特征比较

采用稀释涂布平板、分离培养和16S r DNA序列分析法对我国甘肃白银地区半干旱荒漠草原土壤可培养细菌、放线菌、真菌数量及群落分布特征进行了分析,比较了荒漠草原和耕地土壤微生物多样性。发现荒漠草原土壤可培养细菌、放线菌、真菌数量分别为1.23×106、0.19×106、0.18×106cfu·g-1,耕地三类微生物数量分别是3.03×106、0.53×106、0.05×106cfu·g-1。荒漠草原可培养细菌、放线菌数量明显低于耕地,而真菌数量高于耕地。从荒漠草原分离出14株细菌,分别属于γ-变形菌纲(γ-Proteobacteria)噬冷杆菌属(Psychrobacter),放线菌门(Actinobacteria)皮球菌属(Kytococcus),厚壁菌门(Firmicutes)芽孢杆菌属(Bacillus)、亮氨酸芽孢杆菌属(Lysinibacillus)、土壤芽孢杆菌属(Solibacillus)、气球菌属(Aerococcus),优势菌为芽孢杆菌属和噬冷杆菌属。耕地分离出可培养细菌19株,分别属于ɑ-变形菌纲(ɑ-Proteobacteri)根瘤菌属(Rhizobium)、中华根瘤菌属(Sinorhizobium),γ-变形菌纲(γ-Proteobacteria)假单胞菌属(Pseudomonas),厚壁菌门(Firmicutes)芽孢杆菌属(Bacillus),放线菌门(Actinobacteria)微杆菌属(Microbacterium)、节杆菌属(Arthrobacter)、微球菌属(Micrococcus)、考克氏菌属(Kocuria),以放线菌门细菌为主(占57.9%)。从荒漠草原分离放线菌共8株,分别属于链霉菌属(Atreptomyces)、小单孢菌属(Micromonspora)、间孢囊菌属(Intrasporangium),而耕地主要为链霉菌属(Atreptomyces)、小单孢菌属(Micromonspora)。荒漠草原真菌主要是交链孢霉属(Alternaria)、芽枝霉属(Cladosporium),耕地土壤真菌包括青霉属(Penicillium)、交链孢霉属(Alternaria)、曲霉属(Aspergillus)、毛霉属(Mucor)、链孢霉属(Coniothecium)。试验结果表明,荒漠草原与耕地土壤微生物都具有较丰富的多样性,但微生物群落结构存在一定差异,同一区域不同深度土壤中微生物数量和种类也存在差异,耕地土壤微生物多样性明显高于荒漠草原。     

A simple method for detection of lipolytic microorganisms in soils

Media selective for the isolation of bacteria, actinomycetes and fungi were amended with 0.1% sunflower oil emulsified with 0.01% Tween 80. Lipase-producing microorganisms produced clear zones on these media. When lipase-producing bacteria were cultured on a polycarbonate membrane laid on the selective medium for bacteria, clear zones were produced on the medium when the membrane along with bacteria was removed. The agar disc cut from the clear zone also produced a clear zone when placed on the fresh medium, indicating that clear zone formation is the result of the activity of extracellular lipases. The largest population of lipase-producing microorganisms in an agricultural soil was actinomycetes followed by bacteria and fungi. Ranging from 12 to 75% of bacteria, actinomycetes and fungi isolates from soils collected from three different locations were capable of producing lipases. In general, relatively small percentages of soil bacteria were lipase producers, and lipase producers were more common among soil actinomycetes and fungi. These three groups of microorganisms appear to be all important in decomposition of oils in organic matters in soils.     

Non-streptomycete actinomycetes as biocontrol agents of soil-borne fungal plant pathogens and as plant growth promoters

Among soil microorganisms, bacteria and fungi and to a lesser extent actinomycetes, have received considerable attention as biocontrol agents of soil-borne fungal plant pathogens and as plant growth promoters. Within actinomycetes, Streptomyces spp. have been investigated predominantly, mainly because of their dominance on, and the ease of isolation from, dilution plates and because of the commercial interest shown on the antibiotics produced by certain Streptomyces spp. Many of non-streptomycete actinomycetes (NSA) taxa are therefore rarely reported in literature dealing with routine isolations of biocontrol agents and/or plant growth promoters from plant and soil. It is clear that special isolation methods need to be employed in routine isolations to selectively isolate NSA. Some interesting information exists, albeit in relatively few reports compared to that on other microorganisms, on the biological activities of NSA, especially in relation to their mechanisms of action in the biological control of soil-borne fungal plant pathogens and plant growth promotion. This review presents an overview of this information and seeks to encourage further investigations into what may be considered a relatively unexplored area of research. Certain soil environmental factors, especially in horticultural systems, could be manipulated to render the soil conducive for the biological activities of NSA. A variety of NSA isolated by selective methods have not only shown to be rhizosphere competent but also adapted for an endophytic life in root cortices. Some of the NSA, including endophytic strains that have shown potential to suppress soil-borne fungal plant pathogens, are able to employ one or more mechanisms of antagonism including antibiosis, hyperparasitism and the production of cell-wall degrading enzymes. Strains of NSA promote plant growth by producing plant growth regulators. Enhancement of plant growth by the antagonists are considered to help the host by producing compensatory roots that mask the impact of root diseases.     

Soil microorganisms antagonistic towards Rhizobium japonicum

Success in introducing Rhizobium japonicum strains into soil is related to their interaction with native microorganisms including some that are antagonistic. Actinomycetes, bacteria, fungi and rhizobiophages antagonistic towards strains of R. japonicum were counted directly using soil samples from field plots under different crop and soil management systems. The antagonistic actinomycete population varied from 1.3 × 10³ to 4.5 × 10⁵ g^?1 dry soil and ranged up to 90% of total actinomycetes. Soybean rhizosphere soil samples included antagonistic actinomycetes ranging up to 70% of total actinomycetes. The antagonistic bacterial population was less than 10% of total bacteria and the proportion did not vary significantly with crop or soil management practices. Antagonistic fungi were observed for many of the soils examined but they could not be counted. There were few rhizobiophages and they were found most frequently in soybean rhizospheres. Occasional bacterial and actinomycete colonies that stimulated growth of R. japonicum were randomly observed among the soil samples tested.     

Melanogenic actinomycetes (Streptomyces spp.) from Brazilian soils

Summary Melanogenic actinomycetes were isolated from cerrado soils. Starch agar with a neutral pH was the best medium for selecting pigment-producing colonies. A pigmentation screening test selected 52% of these as possible melanin producers. Tests on liquid (organic and inorganic) and solid (peptone and tyrosine) media, and enzymatic tests, confirmed about 90% as melanin producers, 68% as dihydroxyphenylalanine (DOPA)-melanin and 32% as possible other kinds of melanin producers. Melanin production occurred mostly with an organic N, or an inorganic N with an additional organic N source. An exception was observed with three strains, which were able to produce melanins with an entirely inorganic N source in the medium. Instability of melanin production was a common feature in many strains. Further characterization of melanins produced by actinomycetes compared with soil humic acids may clarify the possible role of melanogenic actinomycetes in soil organic matter formation.     

Moderately haloalkaliphilic actinomycetes in salt-affected soils

It was found that the population density of actinomycetes in solonchaks and saline desert soils varied from hundreds to tens of thousands of colony-forming units (CFUs) per 1 g of soil depending on soil type and was by 1–3 orders of magnitude lower than the number of mycelial bacteria in main soil types. Actinomycetes grow actively in saline soils, and the length of their mycelium reaches 140 m per 1 g of soil. Domination of moderately halophilic, alkaliphilic, and haloalkaliphilic actinomycetes, which grow well under 5% NaCl and pH 8–9, is a specific feature of actinomycetal complexes in saline soils. Representatives of Streptomyces and Micromonospora genera were found among the haloalkaliphilic actinomycetes. Micromonospores demonstrated lower (than streptomycetes) adaptability to high salt concentrations. Investigation of the phylogenetic position of isolated dominant haloalkaliphilic strains of streptomycetes performed on the basis of sequencing of the gene 16S rRNA enabled identifying these strains as Streptomyces pluricolorescens and S. prunicolor.     

Diversity and antibiotic-producing potential of cultivable marine-derived actinomycetes from coastal sediments of Turkey

Purpose

Marine environments, especially sediments, are rich sources of actinomycetes that provide many bioactive compounds, primarily antibiotics. The goal of this study was to investigate the diversity of cultivable actinomycetes and their potential to produce antibiotics from sediments collected from the coastal zones of Turkey.

Materials and methods

Thirty sediment samples were collected from nine different coastal sites in three seas surrounding the Anatolian Peninsula of Turkey. Of the samples, 6 were collected from one site in the Black Sea, 18 from seven sites in the Aegean Sea, and 6 from one site in the Mediterranean Sea. Strains of pure actinomycetes were isolated by modified actinomycetes isolation agar (MAIA), M1 agar, M6 agar, and modified R2A agar. Ethyl acetate extracts and fermentation broths were used for the evaluation of antimicrobial activity against antibiotic resistant test microorganisms. The identification of the isolates was undertaken by 16S rRNA gene sequencing.

Results and discussion

A total of 261 strains of actinomycetes were isolated, of which 66 (25 %) were active against at least one antibiotic-resistant microorganism. Sixty-five of the actinomycetes isolates with antimicrobial activity were Streptomyces spp. and one was Nocardia sp., which implied that genus Streptomyces was predominant. Whereas MAIA agar was the best medium to recover actinomycetes, M6 agar was superior to others for the isolation of antibiotic-producing strains.

Conclusions

Extensive screening of the extracts from the 261 isolates for antimicrobial activities revealed considerable potential to produce antibiotics. These findings imply that actinomycetes from marine sediments of the Anatolian Peninsula coasts have potential for the discovery of novel bioactive compounds.     

Studies on the ecology of actinomycetes in soil—VIII: Distribution and characteristics of acidophilic actinomycetes

Acidophilic actinomycetes, growing between pH 3.5 and 6.5, were isolated from 17 natural soils and mine wastes. Most isolates were Streptomyces spp. and selected ones were compared with some established neutrophilic species by assessment of overall similarity. Acidophilic and neutrophilic strains clustered separately, differing in a number of cultural and physiological characters, in addition to pH requirements. The possible importance of acidophilic actinomycetes in soil is discussed.     

广西大王岭和大明山自然保护区苏云金芽孢杆菌收集与鉴定

从广西大王岭和大明山两个自然保护区共采集到土样264份，共分离出597株芽孢杆菌，通过光学和电子显微镜检观察，16株分离株观察到伴胞晶体蛋白，初步确定为苏云金芽胞杆菌（Bacillus thuringiensis，简称Bt），出菌率为6．06％。在16株肪分离株中，有4株在芽孢形成过程中能产菱形晶体蛋白，其余12株能产圆形和其他形状的晶体蛋白。利用PCR—RFLP方法和SDS．PAGE方法对16株助分离菌进行了蛋白和基因型的鉴定，结果表明，16株分离株中含有4株crylAc基因，表达约130kD的晶体蛋白，其中含有cry30基因和cry40基因的菌株分别1株和3株，表达大约75kD的晶体蛋白；另外8株戤菌株表达蛋白大小不一，其基因型尚不能确定，有待进一步分析。生物测定表明，产菱形晶体含有crylAc基因的4株励分离株对鳞翅目小菜夜蛾幼虫有很强的毒杀活性，而其它分离株对小菜夜蛾没有毒杀活性。