甘肃地区道地药材根际固氮微生物分离鉴定

为调研甘肃中药材种植区的固氮微生物资源,从党参、黄芪和当归3种药材种植地的根际土壤中分离具有固氮酶活性的微生物,单菌株纯化并进行分子生物学鉴定,最后通过乙炔还原法检测各菌株的固氮酶活性。结果表明:这3种作物的根际土壤中固氮微生物资源丰富,共分离得到28株具有固氮酶活性的菌株,经鉴定主要为根瘤菌属、芽孢菌属、单孢菌属、无色杆菌属和粘着箭菌属等,固氮酶活性为53.5 C_2H_2nmol·(h·ml)-1~295.3 C_2H_2nmol·(h·ml)~(-1),无色杆菌属的固氮酶活性较高。此研究为下一步筛选适合中药材生长的微生物固氮菌肥的菌种提供一定的理论依据。     

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。     

固氮芽孢杆菌GD272的筛选鉴定及其固氮性能研究

对所选育出的一株固氮芽孢杆菌（编号为GD272）进行了形态、生理生化测定和接种效果研究。通过16S rDNA基因比对以及生理生化鉴定表明,该菌株属于芽孢杆菌Bacillus sp.。乙炔还原法测定显示该菌株具有较高的固氮酶活性;小白菜盆栽试验看出,接种GD272菌达到了施用化学氮肥的同等效果。菌株GD272在固氮微生物肥料生产中具有较好的开发应用前景。     

7株解有机磷细菌的分离和鉴定

从土壤中分离筛选出7株解有机磷的微生物。对这7株解磷细菌进行了形态、生理生化性状测定及16SrDNA序列分析(GenBankaccessionNo:S2,AY651922;S3,AY661923;X1,AY651925;Y1,AY651924;H1,AY663435;H2,AY663436andHe,AY663436)。其中S2、S3、X1和He属于假单胞菌属(Pseudomonas),Y1属于芽孢杆菌属(Bacillus),H1属于不动杆菌属(Acinetobacter),H2属于寡养单胞菌属(Stenotrophomonas)。进一步通过G C含量和DNA-DNA杂交研究,结果表明,S2、S3和X1为产碱假单胞菌(Pseudomonasalcaligenes),Y1为蜡状芽孢杆菌(Bacilluscereus)。     

几株解有机磷细菌的分离和鉴定*

从土壤中分离筛选出7株解有机磷的微生物。对这7株解磷细菌进行了形态、生理生化性状测定及16S rDNA序列分析(GenBank accession No: S2, AY651922; S3, AY661923; X1, AY651925; Y1, AY651924; H1, AY663435; H2,AY663436 and He, AY663436)。其中S2、S3、X1和He属于假单胞菌属(Pseudomonas)，Y1属于芽孢杆菌属(Bacillus)，H1属于不动杆菌属(Acinetobacter)，H2属于寡养单胞菌属(Stenotrophomonas)。进一步通过G+C含量和DNA-DNA杂交研究, 结果表明，S2、S3和X1为产碱假单胞菌(Pseudomonas alcaligenes),Y1为蜡状芽孢杆菌(Bacillus cereus)。     

马铃薯内生固氮菌的分离及其促生特性研究

内生固氮菌是一类重要的植物促生菌,占据着植物组织内有利于营养供应和微环境适宜的生态位,能更好的发挥促生功能,促生机制包括固氮、解磷、产植物生长素、产ACC脱氨酶、产铁载体及拮抗病害等。本文从连作3年马铃薯根筛选获得8株内生固氮菌,其中短芽孢杆菌属(Brevibacterium)3株,芽孢杆菌属(Bacillus)3株和泛菌属(Pantoea)2株。促生特性研究发现:菌株Brevibacterium sp.GWR4具有较高固氮酶活性[16.206nmol C2H4/(mg蛋白质·h)],与圆褐固氮菌的固氮酶活性具有极显著性差异(P0.01);菌株Pantoea spp.GWR2、GWR3具有极高的产IAA能力,分别为186.07、162.21μg/m L,GWR2兼具产ACC脱氨酶活性[3.74μmol/(mg蛋白质·h)],GWR3兼具溶解无机磷的能力;菌株Brevibacterium sp.GWR5可以拮抗尖孢镰刀菌MLSOF(Fusarium xysporum)和茄病镰刀菌MLS-QB(Fusarium solani),兼具溶解有机磷和产IAA能力;Bacillus spp.GWR7、GWR8兼具固氮和产IAA能力。盆栽试验显示,分别接种Pantoea sp.GWR3,Brevibacterium spp.GWR4、GWR5和Bacillus spp.GWR7、GWR8后小白菜鲜重显著高于未接菌对照处理。可见,这5株菌兼具多种促生特性且对小白菜具有较好的促生效果,有望进一步研究开发成为微生物肥料生产菌种。     

烟草根表可培养细菌群落组成及典型菌群的促生特性研究

植物微生物组是维护植物生长发育、提升抗逆防病的重要调控因素。为发挥植物微生物促进烟草生长、改善烟草根区微生态功能作用，本研究从烟草根表分离筛选可培养细菌组，并对不同菌株的促生能力进行测定。结果表明：①从烟草根表分离并鉴定出可培养菌株310株，隶属于31个属，其中主要为芽孢杆菌属(Bacillus)、假单胞菌属(Pseudomonas)；②对比分析发现假单胞菌属、芽孢杆菌属、寡养单胞菌属(Stenotrophomonas)和成对杆菌属(Dyadobacter)为4种供试土壤烟草根表共有的细菌类群；③对进一步筛选得到的16株菌株进行促生能力的测定，发现6株菌具有固氮能力，5株菌产铁载体，4株菌可溶解无机磷，4株菌产IAA；④盆栽试验验证16株菌株的促生效果，其中37.5% 的菌株对烟草生长具有显著促进作用，烟草株高、总鲜物质量和地下部干物质量分别比对照提高35.1%、27.9% 和30.7%。总之，从烟草根表分离获得多种具有促生能力的菌株，为未来构建促进烟草健康生长的复合菌剂提供了理论基础。     

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。     

库姆塔格沙漠北界阿奇克谷地土壤可培养细菌多样性

为明确库姆塔格沙漠北界阿奇克谷地的土壤细菌多样性,探索该地区土壤微生物与植物群落的相互作用关系,采用NA、R2A培养基分离培养土壤细菌,通过16S rDNA序列系统发育分析鉴定菌株。结果表明,R2A平板菌落数量和种类较多;发现13株潜在新种菌株,已知菌种分属3大类群、4属、20种,厚壁菌门(Firmicutes)为优势类群,芽胞杆菌属(Bacillus)为优势属,枯草芽胞杆菌(Bacillus subtilis subsp.inaquosorum)为优势菌种。与其他类似环境相比,阿奇克谷地土壤中可培养细菌生物量偏小,可培养的细菌多为抗性、耐性强的极端微生物。该地区特有的微生物资源、功能微生物和新菌资源丰富,为防沙治沙、微生物菌剂开发等研究提供物质基础。本研究结果对库姆塔格沙漠的自然生态保护以及极端环境微生物资源的应用具有重要意义。     

嗜碱菌碱性蛋白酶菌株的筛选

自新疆，宁夏，陕西采集了４５份盐碱地区土壤，分离筛选到一株碱８菌株，经初步鉴定属于蜡状芽孢杆菌。该菌在ｐＨ９以上的培养基上生长，产酶；于３７℃，ｐＨ１０，种龄１４ｈ，接种量１％，发酵４８ｈ的条件下，酶活稳定，酶活力在１０８９ｕ／ｍｌ。     

Diversity of Paenibacillus durus strains isolated from soil and different plant rhizospheres evaluated by ARDRA and gyrB-RFLP analysis

Strains belonging to Paenibacillus durus isolated from the rhizosphere of various grasses and from bulk soil were previously divided into five phenotypic groups (A1–A5) based on the fermentation pattern of six carbohydrates (A1: sorbitol (+), A2: dulcitol and tagatose (+), A3: starch and glycogen (+), A4: starch, glycogen and d-arabitol (+) and A5: negative for these carbohydrates). This study aimed to assess whether plant types select for specific P. durus phenotypic groups. For that purpose, polymerase chain reaction-restriction fragment length polymorphism analysis of part of genes encoding 16S rRNA (ARDRA) and DNA gyrase subunit B (gyrB-RFLP) were used to produce genetic fingerprints. ARDRA and gyrB-RFLP data were clustered together to generate a dendrogram and two main clusters were observed. Cluster I showed a predominance of strains isolated from wheat, maize and sugarcane rhizospheres. Strains isolated from maize were distributed among the five patterns of carbohydrate metabolism, while strains isolated from sugarcane showed to be predominantly able to metabolize starch and glycogen. Neither sorbitol- nor arabitol-metabolizing strains were found in cluster II, which consisted of strains isolated from soil and from all plant species used. Our results suggest that the plants influenced the diversity of P. durus in their rhizospheres.     

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。     

烟草根际土壤中解钾细菌的分离与多样性分析

土壤中含有丰富的钾元素,但主要以缓效态形式存在于钾长石或云母等硅酸盐矿物中,不能被作物直接吸收利用。解钾微生物能溶解硅酸盐矿物中的钾,提高土壤中作物可利用钾的含量,有望缓解我国钾肥短缺的现状。本研究利用选择性培养基,从烟草根际筛选钾细菌,基于16S rDNA序列分析烟草根际土壤解钾细菌的多样性,通过测定解钾细菌的解钾效能及对烟草的促生作用,筛选有应用潜力的优良解钾细菌菌株。结果表明,从四川、湖北和山东烟区烟草根际土壤分离获得的27株解钾细菌,在解钾固体培养上溶钾圈直径为0.11~0.30 cm。16S rDNA序列分析表明,烟草根际土壤解钾细菌主要包括变形菌门γ亚群(Gammaproteobacteria,85.18%)、变形菌门α亚群(Alphaproteobacteria,3.70%)、变形菌门β亚群(Betaproteobacteria,3.70%)、放线菌门(Actinobacteria,3.70%)和拟杆菌门(Bacteroidetes,3.70%),其中克雷伯菌属(Klebsiella)为优势菌属(66.67%)。27个菌株均有一定的解钾能力,解钾活性为0.59~4.40 mg.L–1。参试菌株均对烟草有一定的促生作用,利用解钾细菌菌液处理烟株20 d后,与对照相比,株高增加0.97%~38.64%,最大叶长增加4.40%~31.02%。本研究筛选出的菌株XF11、GM2、JM19和GL7具有较高的解钾活性和促进植物生长的能力,展现了良好的应用潜力。     

Diversity of Paenibacillus polymyxa populations in the rhizosphereof wheat (Triticum durum) in Algerian soils

The diversity of Paenibacillus polymyxa populations associated with the rhizosphere of durum wheat was investigated in Algerian soils sampled in regions where wheat had been cultivated for 5 and 26 years (Hamiz, H5 and Z26), 70 years (Algiers, D70), and more than 2 000 years (Tiaret, T2000 and K2000). A total of 111 strains were isolated by immunotrapping and identified as P. polymyxa using an API50CHB kit and restriction analysis of the amplified 16S rDNA gene. The phenotypic characteristics of the P. polymyxa populations were compared and the strains found not to cluster according to their origin. The longer the history of wheat cultivation, the lower the phenotypic diversity and the higher the frequency of nitrogen-fixing strains. Population genetic diversity, evaluated by ERIC-PCR, showed the same trends as phenotypic diversity. The distribution of ERIC genotypes among the different populations studied were compared using Pearson’s Chi-squared test. The strains isolated in D70, Z26, and H5 derived from soil populations sharing the same genetic structure, but those isolated in T2000 and K2000 each stemmed from a population with a specific genetic structure. These data suggest that the genetic structure of P. polymyxa populations has been affected by long-term wheat cultivation.     

Diversity of Paenibacillus polymyxa populations in the rhizosphereof wheat (Triticum durum) in Algerian soils

The diversity of Paenibacillus polymyxa populations associated with the rhizosphere of durum wheat was investigated in Algerian soils sampled in regions where wheat had been cultivated for 5 and 26 years (Hamiz, H5 and Z26), 70 years (Algiers, D70), and more than 2 000 years (Tiaret, T2000 and K2000). A total of 111 strains were isolated by immunotrapping and identified as P. polymyxa using an API50CHB kit and restriction analysis of the amplified 16S rDNA gene. The phenotypic characteristics of the P. polymyxa populations were compared and the strains found not to cluster according to their origin. The longer the history of wheat cultivation, the lower the phenotypic diversity and the higher the frequency of nitrogen-fixing strains. Population genetic diversity, evaluated by ERIC-PCR, showed the same trends as phenotypic diversity. The distribution of ERIC genotypes among the different populations studied were compared using Pearson’s Chi-squared test. The strains isolated in D70, Z26, and H5 derived from soil populations sharing the same genetic structure, but those isolated in T2000 and K2000 each stemmed from a population with a specific genetic structure. These data suggest that the genetic structure of P. polymyxa populations has been affected by long-term wheat cultivation.     

甘肃省白龙江流域豆科植物根瘤菌共生固氮研究

对甘肃省白龙江流域甘南、陇南的大部分地区豆科植物——根瘤菌资源进行了调查 ,共采集 2 1属45种豆科植物 6 9份根瘤样品。从 6 9份样品中获 6 3株根瘤菌 ,对其中 44株根瘤以 13种寄主进行了回接试验 ,31株结瘤 ,结瘤率 70 .5 %。该区根瘤一般带有粉红色 ,形态比较规则。乙炔还原活力测定结果表明 ,88.5 %为有效根瘤。不同种根瘤固氮活性相差不大 ,但高于甘肃省其它地区。对豆科植物在蓄水保土 ,改良土壤状况中的地位进行了讨论。     

甘肃省白龙江流域豆科植物根瘤菌共生固氮研究

对甘肃省白龙江流域甘南、陇南的大部分地区豆科植物——根瘤菌资源进行了调查,共采集21属45种豆科植物69份根瘤样品。从69份样品中获63株根瘤菌,对其中44株根瘤以13种寄主进行了回接试验,31株结瘤,结瘤率70.5%。该区根瘤一般带有粉红色,形态比较规则。乙炔还原活力测定结果表明,88.5%为有效根瘤。不同种根瘤固氮活性相差不大,但高于甘肃省其它地区。对豆科植物在蓄水保土,改良土壤状况中的地位进行了讨论。     

Polyphasic characteristics of bradyrhizobia isolated from nodules of peanut (Arachis hypogaea) in China

Peanuts (Arachis hypogaea L.) were introduced to China about 500 years ago. However, the diversity of Rhizobial strains in China that can nodulate peanut was poorly understand. Diversity and phylogeny of 50 slow-growing strains, isolated from root nodules of peanut in different geographical regions of China, were studied using polyphasic techniques. All stains were clustered by phenotypic tests into two distinct groups: Group I: 16S rRNA RFLP genotype 3, and Group II, which divided into 16S rRNA RFLP genotypes 1 and 2. Genotype 1 shares the same genotype with USDA110, USDA122 and USDA127 of Bradyrhizobium japonicum, and genotype 2 solely consisted of extra-slow growing bradyrhizobia isolated from Hongan, China. Results of 16S rRNA sequencing revealed that peanut bradyrhizobia were phylogenetically related to B. japonicum and their sequence divergence was less than 1.1%. Based upon the size of the internally transcribed spacer (ITS) between the16S and 23S RNA genes, strains were classified into ITS-I, ITS-II and ITS-III genotypes. Strains could be further divided into sub-clusters IA, IB, IIa, IIb and IIc five sub-clusters through ITS PCR-RFLP and repetitive extragenic palindromic PCR (REP-PCR) analysis. Host specificity test revealed that all peanut bradyrhizobia tested nodulated Phaseolus vulgaris and strains of clusters IIb and IIc nodulated Glycine soja efficiently. Bradyrhizobia isolated from peanut were related, but still exhibited phylogenetical divergence with B. japonicum.     

Indigenous rhizobia associated with native shrubby legumes in Taiwan

Root-nodulating bacteria were isolated and characterized from seven native shrubby legumes growing in Taiwan. Phenotypic characteristics measured included growth rates in various media, colony morphology, and tolerances to extremes of temperature, salt and pH. The isolates were very diverse phenotypically. Among the 83 isolates that were screened, the majority were fast-growing rhizobia. Twenty eight strains tolerated high concentration of salt (4.5% NaCl) and grew well between temperatures of 37 and 45 °C. The majority of the strains also tolerated extreme pH in their medium from 3.5 to 12. All strains formed nitrogen fixing nodules, and the highest activity was detected in the legume Hedysarum crinita L. PCR restriction fragment length polymorphism (PCR-RFLP) and sequencing of the small subunit ribosomal RNAs revealed that the majority of the isolates belonged to the genera Rhizobium, Bradyrhizobium and Agrobacterium. Only a single strain represented the genus Sinorhizobium. In addition, a strain related to Burkholderia from the β-class of the Proteobacteria (CC-CC-5) was found within nodules of the legume Catenaria caudatum. The study contributes to the understanding of symbiotic nitrogen fixation in selected wild legumes that are native to Taiwan and provides insights into the distribution of nodulating and nitrogen-fixing bacteria from other distinct lineages.     

Isolation, enumeration, and characterization of diazotrophic bacteria from paddy soil sample under long-term fertilizer management experiment

A study was undertaken to determine the free-living culturable diazotrophic bacteria of paddy soils from a long-term fertilizer management experiment. Long-term application of different fertilizers significantly affected the population of free-living diazotrophs. Out of 165 distinct bacterial morphotypes observed during the isolation process, only 32 were positive for both acetylene reduction assay (ARA), and nifH gene screening. The ARA activity of the isolates ranged from 1.8 to 2,844.7 nmol ethylene h^?1 mg protein^?1. The 16S rRNA analysis identified the isolates to be members of 13 different genera viz. Bacillus, Pseudomonas, Paenibacillus, Serratia, Ochrobactrum, Lysinibacillus, Burkholderia, Brevundimonas, Herbaspirillum, Novosphingobium, Sphingomonas, Xanthomonas, and Azorhizobium. Though partial nifH gene sequencing of diazotrophic isolates showed good consistency with that of 16S rRNA-based identification, some nifH sequences were similar to a variety of uncultured nitrogen-fixing bacteria. The diversity of free-living diazotrophic bacteria and the wide distribution of nifH sequences indicate the potential contribution of these microorganisms to nitrogen input to paddy fields.