宁夏荒漠草原小叶锦鸡儿可培养内生细菌多样性及其分布特征

对宁夏荒漠草原小叶锦鸡儿各部分组织中可培养内生细菌的遗传多样性和分布特征进行了分析,并对菌株的耐盐性、耐酸碱性和生长温度范围进行了测定。结果表明:各部分组织中内生细菌的数量和群落组成存在明显差异,根部细菌种群密度最大,分离数量最多,叶部次之,茎部和种子数量最少。78株分离菌株经16S rDNA-RFLP分析共产生9种遗传图谱类型,16SrDNA序列分析表明供试菌株分别归属于芽孢杆菌属、类芽孢杆菌属、假单胞菌属、贪铜菌属、罗尔斯顿菌属和中华根瘤菌属,与其它植物相比多样性较为单一;芽孢杆菌属是小叶锦鸡儿的优势内生菌群(占87.2%),分布于所有组织中。菌株的抗逆能力较强,多数菌株可耐受6%的NaCl,能在pH值5.0—10.0及45℃的条件下生长,3株芽孢杆菌属细菌具有较强的耐盐、耐酸碱、耐高温性能。     

金沙江干热河谷区胡枝子（Lespedeza Michx）根瘤菌多样性及其系统发育

研究利用数值分类、BOXAIR-PCR指纹图谱、16SrDNA PCR-RFLP、16S rDNA和GSⅡ序列分析等方法,研究了分离自金沙江干热河谷区的86株胡枝子根瘤菌的多样性和系统发育,结果表明金沙江干热河谷区胡枝子根瘤菌蕴涵丰富的生物多样性。通过数值分类,供试未知菌株表现出极大的表型性状多样性,能耐高温（60℃）和低pH（4.0）,在低温（10℃）或者高pH（9.0）条件下生长很差,耐盐性也很差。供试未知菌株的16S rDNA用HaeⅢ、MspⅠ、HinfⅠ和TaqⅠ酶切后具有16种遗传图谱类型,其中10株供试未知菌的16S rDNA遗传图谱类型不同于所选用的已知参比菌株。BOXAIR-PCR的分群结果分散,很多在16S rDNA PCR-RFLP中具有相同遗传类型的菌株也表现较大差异,表明了供试菌株在基因组水平上差异很大。序列分析结果表明,6株代表菌株分布于Rhizobium、Sinorhizobium、Mesorhizobium、Bradyrhizobium4个属,16S rDNA序列与GSⅡ序列分别构建的系统发育树在属水平上基本一致,但16S rDNA序列的同源性比GSⅡ高,6株代表菌株间16S rDNA序列的同源性在87.5%～99.5%之间,GSⅡ序列的同源性在79.4%～89.8%之间;而代表菌株与亲缘关系最近的参比菌株间的16S rDNA序列的同源性为99.9%～100%,GSⅡ的同源性为88.9%～99.6%。     

毛乌素沙地中间锦鸡儿根瘤菌遗传多样性及16S rDNA全序列分析

野生豆科植物中间锦鸡儿是毛乌素沙地的优势种。从蛋白质和DNA水平分析与其共生的根瘤茵的遗传多样性。在蛋白质水平上,24株中间锦鸡儿根瘤菌和9株参比菌株分为2组,A组包含95．8％供试中间锦鸡儿根瘤菌,参比菌株聚为B组,菌株GH72不与其余供试根瘤菌聚类。应用16S　rDNA　PCR—RFLP方法将供试菌株分为22种基因型,中间锦鸡儿根瘤菌组成12种,其余10种由参比菌株构成。表明中间锦鸡儿根瘤菌具高水平遗传多样性。选取代表菌株GH2001进行16S　rDNA全序列测定。与已知相关根瘤菌菌株16S　rDNA进行同源性比较,构建系统发育树状图。GH2001位于Rhizobium分支,与且Agrobacterium radiobacter,Ag.rubi,Rhizobium giardinii,R.mongolense,R.yanglingense,R.galegae和R.huautlense的序列同源性分别达到99％、98．3％、96．3％、95．5％、95．6％、95．27％和95．7％。     

广西葛根内生细菌的分离鉴定及其促生特性

【背景】植物内生菌长期与宿主共生,对宿主生长发育产生影响。葛根作为重要的药食两用作物,葛根内生菌的研究具有重要实践意义。【目的】对广西葛根根部内生细菌进行分离、鉴定及促植物生长特性分析,旨在了解该药食同源植物内生细菌种群结构及其促生特性,为分析内生菌群体在药食同源植物产量和品质形成的作用及其内生细菌资源的开发利用提供参考。【方法】采用6种不同的培养基从广西葛根的根瘤、根系和根愈伤组织分离内生细菌,16S rRNA基因测序和系统发育分析内生细菌的分布特征和遗传多样性,采用生理生化方法测定分离菌株的固氮活性、溶磷特性、产生嗜铁素、分泌吲哚乙酸(indole-3-aceticacid,IAA)等促生特性。【结果】从葛根根瘤、根系和根部愈伤组织中共分离得到223个菌株,16S rRNA基因测序鉴定这些菌株隶属于2门4纲10科19属,其中芽孢杆菌属、假单胞菌属、土壤杆菌属、肠杆菌属为葛根优势菌群;内生细菌数量和群落组成存在明显的组织特异性,其数量表现为根瘤>根系>根愈伤组织,但其种群多样性表现为根愈伤组织>根系>根瘤。不同培养基分离出的细菌种群丰富度有差异。从供试菌株中筛...     

药用植物内生芽孢杆菌的多样性和系统发育研究

[目的]了解药用植物内生芽孢杆菌的生物多样性.[方法]采用数值分类、16S rDNA PCR RFLP、BOX-PCR指纹图谱和16S rDNA序列分析技术对分离于几种药用植物的内生芽孢杆菌和已知参比菌株进行表型、遗传多样性及系统发育研究.[结果]供试菌株在数值分类聚类分析中在84%的相似水平上产生13个表观群.16S rDNAPCR-RFLP分析表明供试菌株表现出丰富的遗传多样性.BOX-PCR指纹图谱分析进一步证明药用植物的内生芽孢杆菌的基因组也具有多样性,聚群的结果与数值分类有较好一致性.用软件在Genbank中进行所得序列的同源性检索,并构建系统发育树.由16S rDNA序列分析可知,供试的代表菌株SCAU11与球形芽孢杆菌(Bacillus sphaericus)亲缘关系最近,SCAU78和SCAU25为枯草芽孢杆菌(Bacillus subtilis)的两个亚种,代表菌株SCAU39与巨大芽孢杆菌(Bacillus megaterium)的亲缘关系最近.[结论]研究结果表明药用植物内生芽孢杆菌具有明显的表型和遗传多样性.     

陕西太白尾矿废弃地豆科植物根瘤菌16S rDNA PCR-RFLP及全序列分析

应用16S rDNA-RFLP和16S rDNA全序列测定方法,对分离自陕西太白金矿尾矿废弃地的55株根瘤菌和12株参比菌株进行了遗传多样性和系统发育地位研究。采用平均连锁法(UPMGA)对16S rDNA PCR-RFLP聚类,结果显示所有菌株在72%的水平上聚到一起。根据参比菌株的种属关系,将供试菌株初步分成6个遗传发育群。群Ⅰ为根瘤菌属,群Ⅱ为中华根瘤菌属,群Ⅲ是中慢生根瘤菌属,群Ⅳ为土壤杆菌属,群V为一未知群,群Ⅵ为慢生根瘤菌属。分离自天蓝苜蓿的根瘤菌主要分布在群Ⅱ,截叶胡枝子根瘤菌在各个群内均有分布,表现出丰富的遗传多样性。选取群Ⅰ、Ⅱ的代表菌株TB17-1、TB50-1进行16S rDNA全序列测定分析,结果显示TB17-1与Rhizonbium leguminosarumUSDA2370的同源性高达99.7%,TB50-1与Sinorhizobium melilotiLMG6133的同源性为100%。全序列测定结果与RFLP分析结果基本一致。     

若尔盖高原沼泽土和草甸风沙土细菌16S rDNA PCR-RFLP和系统发育分析

采用稀释平板法从若尔盖高原沼泽土和草甸风沙土分离获得66株细菌,在16S rDNA PCR-RFLP分析的基础上,测定了22株代表菌株的16S rDNA序列,构建了供试细菌的系统发育关系。16S rDNA PCR-RFLP分析中,在78%相似性水平处,除REG14,REG20,REG22和REG55单独成群外,其余菌株分为8个遗传群,其中,群Ⅰ和群Ⅳ最大,均有15个菌株,其次是群Ⅷ,由11个菌株组成,其余21个菌株分布于5个群内。对22个代表菌株16SrDNA全序列系统发育表明,这些菌株分布于不同的系统发育分支。其中,以芽孢杆菌属(Bacillus)、假单胞菌属(Pseudomonas)和分枝杆菌属(Mycobacterium)为主,其余菌株分布于Lysinibacillus属、中华根瘤菌属(Sinorhizobium)、不动杆菌(Acinetobacter)、固氮菌属(Azotobacter)、红球菌属(Rhodococcus)、微球菌属(Micrococcus)和醋酸杆菌属(Acetobacter)等7个属。     

几种经济作物根际拮抗细菌的多样性

采用抗菌谱测定以及BOXAIR-PCR、生理生化特征和16S rDNA序列分析等方法对分离自10种作物根际的55株拮抗细菌的多样性及主要拮抗菌类群进行了分析.结果表明： 根际拮抗细菌的拮抗作用具有丰富的多样性;BOXAIR-PCR分析中供试菌在721％相似性水平上可以聚为7个群,850%相似性水平上聚为25个群;所有供试根际拮抗菌分别属于芽孢杆菌属（Bacillus）芽孢杆菌属（Paenibacillus）、短芽孢杆菌属（Brevibacillus）、假单胞菌属（Pseudomonas）和产碱菌属（Alcaligenes）.作物根际拮抗菌具有丰富的遗传多样性和拮抗性能多样性.     

沙冬青根瘤菌遗传多样性和系统发育分析

利用16S rDNA RFLP、16S-23S rDNA RFLP和16S rDNA序列分析方法,对分离自宁夏和内蒙古阿拉善地区的沙冬青根瘤菌进行了遗传多样性和系统发育分析.结果表明,分离自不同地区沙冬青根瘤菌的44株测试菌株分别归属于中慢生根瘤菌属(Mesorhizobium)、叶杆菌属 (Phylobacterium)、中华根瘤菌属(Sinorhizobium)、根瘤菌属(Rhizobium)、土壤杆菌属(Agrobacterium)5个属种.受寄主和地理环境因素的影响,沙冬青根瘤菌具有丰富的遗传多样性.     

Ugan古河道胡杨可培养内生细菌的多样性

摘要：【目的】为了了解塔河废弃古河道胡杨可培养内生细菌的多样性。【方法】从2棵胡杨树干部抽出其内存液,采用三种不同的培养基对样品的内生细菌进行了分离纯化;对它们进行16S rDNA测定和系统进化分析。【结果】分离纯化不同表型的细菌62株,对它们的16S rDNA序列分析表明,62株菌分别属于四个大类群;厚壁菌门（Firmicutes）、放线菌门(Actinobacteria)、α-变形菌纲(Alpha Proteobacteria) 、γ-变形菌纲(Gamma Proteobacteria),18个属,32个种;芽孢杆菌属和假单胞菌属是胡杨可培养内生细菌的优势细菌种群,它们分别占已测种群的40.32%、16.13%。其中菌株KTH-63为葡萄球菌科的潜在的新属新种,它与最近源菌株的16S rDNA序列相似率为92.491%;9株菌KLH-21、KLH-1、KTH-8、KTH-14、KNA-26、KLH-18、KTH-20、KNA-3、KLH-25是潜在的新种（16S rDNA相似率为96.089 %-97.769 %）,胡杨树干内存液中潜在新种的发现率高达总分离检测菌株的16.13 % 。本研究获得的胡杨可培养内生细菌的群落结构数据给植物内生细菌新增了10个属,18个种。【结论】胡杨具有多样性极其丰富的可培养内生细菌菌种资源,土著新种的发现频率超出了预期,胡杨可培养内生细菌的群落结构极大地刷新了植物内生细菌的种群记录,极具进一步发掘的潜力。     

Isolation and Characterization of Endophytic Bacteria from the Nickel Hyperaccumulator Plant <Emphasis Type="Italic">Alyssum bertolonii</Emphasis>

We report the isolation and characterization of endophytic bacteria, endemic to serpentine outcrops of Central Italy, from a nickel hyperaccumulator plant, Alyssum bertolonii Desv. (Brassicaceae). Eighty-three endophytic bacteria were isolated from roots, stems, and leaves of A. bertolonii and classified by restriction analysis of 16S rDNA (ARDRA) and partial 16S rDNA sequencing in 23 different taxonomic groups. All isolates were then screened for siderophore production and for resistance to heavy metals. One isolate representative of each ARDRA group was then tested for plant tissue colonization ability in sterile culture.Obtained results pointed out that, despite the high concentration of heavy metals present in its tissues, A. bertolonii harbors an endophytic bacterial flora showing a high genetic diversity as well as a high level of resistance to heavy metals that could potentially help plant growth and Ni hyperaccumulation.     

Genetic diversity of rhizobia isolated from Caragana intermedia in Maowusu sandland,north of China

AIMS: Thirty-three rhizobial strains isolated from nodules of Caragana intermedia in Maowusu sandland were examined for their genetic diversity and putative phylogenetic position. METHODS AND RESULTS: Isolates from Caragana intermedia were classified into 12 genotypes by 16S rDNA polymerase chain reaction-restriction fragment length polymorphism (RFLP), which showed no distinct relationships with those of the reference strains. The genotypes of rhizobia were not related to geographical location. Thr 16S rDNA sequence of representative strain GH2001 from dominant genotype 2 shared high homologuey with some Rhizobium species: Rh. giardinii (96.4%), Rh. huautlense (95.3%), Rh. galegae (95.7%), Rh. yanglingense (95.2%), Rh. mongolense (95.6%), Rh. radiobacter (99%) and Rh. rubi (98.3%). CONCLUSIONS: A high degree of genetic diversity existed among rhizobia nodulating Caragana intermedia in Maowusu sandland. Most of the new isolates might belong to Rhizobium. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that the rich diversity of rhizobia might have contributed to the adaptation of the arid region. These strains could be valuable at the economic and ecosystem level.     

乌头产吲哚乙酸内生细菌遗传多样性、抗逆性及其对水稻幼苗生长的影响

[目的]探究乌头产吲咮乙酸(IAA)内生细菌的遗传多样性、溶磷解钾能力、抗逆能力及其对水稻幼苗生长的影响,为道地产区乌头产业可持续发展提供科技支撑.[方法]从健康乌头植株分离可培养内生细菌,采用Salkowski比色法测定内生细菌产IAA能力,16S rDNA限制性片段长度多样性(16S rDNA-RFLP)及16S ...     

川楝内生放线菌的分离及多样性研究

【目的】分离四川省各个地区川楝内生放线菌并研究其物种多样性。【方法】应用7种选择性分离培养基分离样品根、茎、叶、树皮和果实中的内生放线菌,采用16SrRNA基因RFLP分析代表菌株多样性。【结果】研究共获得403株内生放线菌。不同地点、不同植株部位、不同培养基分离得到的内生放线菌数目均有差异。广元采集的样品分离得到的数目最多,为86株;最少的是绵阳,仅有12株。从植物表皮中分离到148株放线菌,占获得菌株总数的36.7%;而从果中分离到31株,仅占获得菌株总数的7.6%;虽然从根部分离到的数量也很少,但是其出菌率却是最高的。5号和3号培养基的分离效果最为理想。16S rRNA基因RFLP分析结果显示所有供试菌株在68%的相似性上聚在一起,在84%的相似水平上分成了10个遗传类型。代表菌株的16SrRNA基因序列测定及系统发育分析结果表明:分离得到的放线菌包括4个属,分别是链霉菌属(Streptomyces)、北里孢菌属(Kitasatospora)、节杆菌属(Arthrobacter)、克里布所菌属(Kribbella)。其中,链霉菌是优势类群,占代表菌株数目的比例高达91%,而稀有放线菌的比例只有9%。【结论】研究发现的川楝内生放线菌主要属于链霉菌属(Streptomyces)、北里孢菌属(Kitasatospora)、节杆菌属(Arthrobacter)、克里布所菌属(Kribbella)。     

Isolation and identification of actinobacteria from surface-sterilized wheat roots

This is the first report of filamentous actinobacteria isolated from surface-sterilized root tissues of healthy wheat plants (Triticum aestivum L.). Wheat roots from a range of sites across South Australia were used as the source material for the isolation of the endophytic actinobacteria. Roots were surface-sterilized by using ethanol and sodium hypochlorite prior to the isolation of the actinobacteria. Forty-nine of these isolates were identified by using 16S ribosomal DNA (rDNA) sequencing and found to belong to a small group of actinobacterial genera including Streptomyces, Microbispora, Micromonospora, and Nocardiodes spp. Many of the Streptomyces spp. were found to be similar, on the basis of their 16S rDNA gene sequence, to Streptomyces spp. that had been isolated from potato scabs. In particular, several isolates exhibited high 16S rDNA gene sequence homology to Streptomyces caviscabies and S. setonii. None of these isolates, nor the S. caviscabies and S. setonii type strains, were found to carry the nec1 pathogenicity-associated gene or to produce the toxin thaxtomin, indicating that they were nonpathogenic. These isolates were recovered from healthy plants over a range of geographically and temporally isolated sampling events and constitute an important plant-microbe interaction.     

Genetic diversity of rhizobia isolated from Astragalus adsurgens growing in different geographical regions of China

The genetic diversity among 95 isolates from Astragalus adsurgens was investigated using molecular biological methods. All of the isolates and 24 reference strains could be differentiated by AFLP, REP-, ERIC- and BOX-PCR fingerprinting analysis. By cluster analysis of the data, 31 AFLP and 38 Rep-PCR genomic groups were delineated, indicating considerable genetic diversity among the isolates. Fifty-four representative strains were further analyzed by RFLP of PCR-amplified 16S and 23S rDNA, revealing 26 rDNA genotypes among the isolates. The phylogenetic relationship of the isolates was determined by partial sequencing of 16S rRNA genes of 16 strains. The results suggest that the A. adsurgens rhizobia belong to the genera Agrobacterium, Mesorhizobium, Rhizobium and Sinorhizobium.     

黄土高原地区大豆根瘤菌的遗传多样性和系统发育

【目的】研究黄土高原地区大豆根瘤菌的遗传多样性和系统发育。【方法】采用BOX-PCR、16S rDNAPCR-RFLP、16S-23S IGS PCR-RFLP和16S rRNA基因序列分析方法对分离自我国黄土高原地区4个省的15个地区的130株大豆根瘤菌及部分参比菌株进行了遗传多样性和系统发育分析。【结果】BOX-PCR反映的菌株多样性最丰富,形成的遗传群最多,16S rDNA PCR-RFLP方法在属、种水平上聚群较好,16S-23S IGSPCR RFLP反映的多样性介于BOX-PCR和16S rDNA PCR-RFLP之间,能够较好地反映出属、种和亲缘关系很近的菌株间的差异,3种方法聚类分析结果基本一致,可将所有供试菌株分为两大类群,中华根瘤菌属(Sinorhizobium)和慢生根瘤菌属(Bradyrhizobium)。从系统发育来看,供试的快生大豆根瘤菌为费氏中华根瘤菌(Sinorhizobium fredii),慢生大豆根瘤菌为日本慢生大豆根瘤菌(Bradyrhizobium japonicum)和辽宁慢生根瘤菌(Bradyrhizobium liaoningense)。【结论】我国黄土高原地区大豆根瘤菌具有较丰富的遗传多样性,S.fredii优势种,慢生大豆根瘤菌仅占10%,同时,分离到2株B.liaoningense。     

Genetic diversity and phylogeny of rhizobia isolated from Caragana microphylla growing in desert soil in Ningxia, China

Rhizobia are soil bacteria with the capacity to induce nitrogen-fixing nodules on the roots or stems of legume plants. A total of 40 bacterial isolates from the root nodules of Caragana microphylla growing in desert soil in Ningxia, China, were analyzed for genetic diversity and phylogenetic position. These isolates were classified into 7 types of 16S ribosomal DNA (rDNA) using polymerase chain reaction-restriction fragment length polymorphism analysis. They were grouped into 4 clades, Rhizobium-Agrobacterium, Sinorhizobium, Phyllobacterium, and Bradyrhizobium, when the phylogenies of 16S rDNA, recA, and atpD genes were applied. Phylogenetic analysis showed that the tree generated from the 16S rDNA sequencing agreed with that produced from the recA and atpD genes. By analyzing phylogenetic relationship using the 3 loci, the isolates in the branches of Phyllobacterium and Sinorhizobium could be identified as P. brassicacearum and S. meliloti. The isolates in the branch of Rhizobium-Agrobacterium were the most abundant microsymbiont of C. microphylla and were designated R. leguminosarum, R. galegae, R. alamii, and A. tumefaciens. Two isolates with low sequence similarity to the known species of Bradyrhizobium might be novel species in this genus.     

油菜内生细菌16S核糖体DNA的RFLP分析

植物内生细菌定殖在植物组织内部但不引起明显的病害症状。从健康油菜植株的不同器官中分离到大量内生细菌,这些细菌菌落形态存在明显的差异,表明油菜组织中存在大量内生细菌,且类群丰富。分离到的122株内生细菌根据菌落形态可以划分为35类;利用细菌通用引物对16S核糖体DNA进行扩增, 获得约15 kb片段,分别用内切酶HaeⅢ和MspⅠ对扩增产物进行限制性酶切,产生不同的酶切图谱,根据酶切图谱聚类分析结果,所有供试菌株被归为39类,这一结果从遗传上显示油菜内生细菌类群的多样性。两种方法归类结果比较发现菌落特征所反映的信息量有限,只能作为初步的参考指标,核糖体DNA的限制性片段长度多态性分析快速、准确,可以作为油菜内生细菌多样性分析的一种有效方法