西北部分地区苦马豆根瘤菌的表型多样性研究

将采集自甘肃、新疆、宁夏等地区的苦马豆根瘤,经分离、纯化获得48株未知菌株,并选取7株参比菌株,进行唯一碳、氮源利用、对抗生素及染料抗性、耐盐性、初始pH生长、生长温度范围及酶活性等共113项生理生化测定;采用数值分类方法对未知根瘤菌进行表型多样性分析。结果表明：供试菌株在碳氮源利用、抗生素敏感性、抗逆性等方面存在着差异。该地区苦马豆根瘤菌具有较强的耐盐、耐碱能力,所有菌株均能在初始pH9~12的YMA培养基上生长,35%菌株可耐受6%的NaCl。从数值分类树状图可见,未知供试菌株在56%的相似水平上聚在一起,在72%的相似水平上分为5个表观群。群Ⅰ有39株菌,在74.6%相似水平聚合,中心菌株为CCNWGS0215;群Ⅱ有5株菌,在76%的相似水平聚合,中心菌株为CCNWGS0228;群Ⅳ有2株菌,在81.5%的相似水平聚合,群Ⅲ和群Ⅴ分别只有1株菌,它们与模式株分离,可能为潜在的新种。     

山东省根瘤菌表型多样性及数值分析

在山东省根瘤菌资源调查的基础上,选取31株快生根瘤菌和34株参比菌株,进行了102项表型性状测定和聚类分析。结果表明：不同地理来源、甚至同一地区来源或同种寄主的不同菌株在碳氮源利用、抗生素抗性、耐逆性等方面存在着较高的多样性。32% 的菌株可耐受300 μg/mL的新霉素,39%的菌株可耐受300 μg/mL的青霉素和洁霉素。2株菌能耐受4.0% 的NaCl。13株菌可以在pH11的条件下生长。在87％的相似水平上,未知菌株形成2个独立的亚群,第1亚群有3株菌,中心菌株为SD109;第2亚群有4株菌,中心菌株为SD083。     

分离自补骨脂等宿主根瘤的24株菌的数值分类和16S rDNA PCR-RFLP 研究

采用数值分类和16S rDNA PCR-RFLP对分离自云南省豆科植物补骨脂(Psoralea corylifolia)、葛藤(Pueraria lobata)、杭子梢(Campylotropis macrocarpa)等宿主的24株菌及10株根瘤菌参比菌株进行了研究。数值分类结果表明, 在84%相似性水平上, 所有的菌株可分为3群：群Ⅲ为未知菌群, 群Ⅰ为慢生菌群, 群Ⅱ为快生和中慢生菌群。从依据16S rDNA PCR-RFLP分析建立的树状图来看, 在70%相似性水平上, 所有的菌株可分为5个系统发育分支：分支Ⅰ和Ⅴ没有参比菌株, 为未知分支; 分支Ⅱ为Agrobacterium-Sinorhizobium-Rhizobium, 分支Ⅲ为Mesorhizo- bium, 分支Ⅳ为Bradyrhizobium。数值分类和16S rDNA PCR-RFLP的结果部分一致, 有2株菌与A. tumefaciens IAM13129T聚在一起。     

刺槐根瘤菌的表型多样性研究

从杨陵地区采集豆科树种刺槐的根瘤,经分离、纯化,获得40株未知菌株,并选取35株参比菌株,进行唯一碳源、氮源利用、对抗生素和染料的抗性、耐盐性、初始pH生长、生长温度范围及石蕊牛奶反应等共105项生理生化测定。结果表明：供试的刺槐根瘤菌在碳、氮源利用、抗生素敏感性、对染料的抗性程度等方面存在着差异。部分菌株具有较强的耐盐碱能力,其中42．5％的菌株能耐受3.0％的NaCl,17．5％的菌株可在初始pH12的YMA培养基上生长。从数值分类树状图可见,在86％的相似水平上未知菌株构成了3个新的类群,其中第1、2类群各有10株菌,中心菌株分别为NWYC113和NWYCl29,第3类群有7株菌,中心菌株为NWYC147。其分类地位需进一步研究和确定。     

分离自补骨脂等宿主根瘤的24株菌的数值分类和16S rDNA PCR-RFLP 研究

采用数值分类和16S rDNA PCR-RFLP对分离自云南省豆科植物补骨脂(Psoralea corylifolia)、葛藤(Pueraria lobata)、杭子梢(Campylotropis macrocarpa)等宿主的24株菌及10株根瘤菌参比菌株进行了研究.数值分类结果表明,在84%相似性水平上,所有的菌株可分为3群:群Ⅲ为未知菌群,群Ⅰ为慢生菌群,群Ⅱ为快生和中慢生菌群.从依据16S rDNA PCR-RFLP分析建立的树状图来看,在70%相似性水平上,所有的菌株可分为5个系统发育分支:分支Ⅰ和Ⅴ没有参比菌株,为未知分支;分支Ⅱ为Agrobacterium-Sinorhizobium-Rhizobium,分支Ⅲ为Mesorhizobium,分支Ⅳ为Bradyrhizobium.数值分类和16S rDNA PCR-RFLP的结果部分一致,有2株茵与A.tumefaciens IAM13129T聚在一起.     

链孢囊放线菌及其相关菌的数值分类研究

对链孢囊菌属的28株放线菌和4株气丝可形成孢囊的放线菌进行了形态、生理生化特性、生长条件、抗生素敏感性、抗菌谱等107项试验测定。根据Ssm相似性系数及平均链锁聚类方式,借助于电子计算机对这些菌株进行了比较。结果表明,11株国际公认的链孢囊菌属的标准菌和一株已知的链孢囊菌与供试未知菌,在59％的水平上归为一群。通过数值分类把所比较的菌株在77％水平上区分为不同的表观群,为进一步的分子分类学研究和种的鉴定提供了依据。     

我国蚕豆根瘤菌的数值分类及其16SrDNA PCR-RFLP研究

对分离自我国11个省24个地区49株蚕豆根瘤菌及11株参比菌株进行了唯一碳源、氮源、抗生素、耐逆性和酶活性等138个表型性状测定,并用MINTS软件进行聚类分析。结果表明,全部供试菌株在59％的相似水平上聚在一起,在80％的相似水平上可分为6个群。其中群4与参比菌株聚在一起,而其他5个群均由未知菌组成。进一步对36株菌进行了16S rDNA PCR—RFLP分析,在85％相似水平上供试菌可分为4个群和1个独立的分支,其聚群结果与数值分类结果有较好的一致性。表型及遗传型分析结果表明,我国蚕豆根瘤菌具有极大的多样性。     

PCR结合酶切-序列比对法鉴定B群脑膜炎奈瑟菌菌株

目的用PCR结合酶切-序列比对法对B群脑膜炎奈瑟菌菌株进行鉴定。方法用玻片凝集法对不同来源的15株B群脑膜炎奈瑟菌菌株进行初步检定,再用PCR结合酶切-序列比对法对上述15株菌株进行进一步鉴定,即用PCR结合酶切法扩增菌株的唾液酸转移酶sia D基因并对PCR产物进行酶切后,用BLAST软件将PCR产物测序结果与Gene Bank中原始sia D序列比对。结果 15株菌株玻片凝集结果均为阳性;15株菌株的PCR产物片段大小均为460 bp;TaqⅠ酶切后,13株菌株的酶切产物片段大小仍为460 bp,其PCR产物测序比对结果与B群脑膜炎奈瑟菌原始sia D序列同源性均达到99%;其余2株酶切产物片段大小约200 bp,与C群脑膜炎奈瑟菌sia D原始基因序列同源性分别为98%和99%。结论 15株菌株经PCR结合酶切-序列比对法鉴定,13株为B群脑膜炎奈瑟菌菌株,2株为C群脑膜炎奈瑟菌菌株;该方法可准确鉴定B群脑膜炎奈瑟菌菌株。     

我国蚕豆根瘤菌的数值分类及其16S rDNA PCR-RFLP研究*

对分离自我国11个省24个地区49株蚕豆根瘤菌及11株参比菌株进行了唯一碳源、氮源、抗生素、耐逆性和酶活性等138个表型性状测定,并用M INTS软件进行聚类分析。结果表明,全部供试菌株在59%的相似水平上聚在一起,在80%的相似水平上可分为6个群。其中群4与参比菌株聚在一起,而其他5个群均由未知菌组成。进一步对36株菌进行了16S rDNA PCR-RFLP分析,在85%相似水平上供试菌可分为4个群和1个独立的分支,其聚群结果与数值分类结果有较好的一致性。表型及遗传型分析结果表明,我国蚕豆根瘤菌具有极大的多样性。     

西北地区甘草根瘤菌的表型多样性研究*

选用分离自西北干旱、半干旱地区的甘草根瘤菌68株和34株参比菌株,进行了113项表型性状测定,结果表明：不同地理来源、同一地理来源、甚至同一植株不同根瘤菌株在碳氮源利用、抗生素敏感性、抗逆性等方面存在着差异。部分菌株具有较强的耐盐耐碱能力,其中有5株和1株菌分别能耐受50g／L、60g／L的NaCl2有43％的菌株能在初始DH12的YMA培养基上生长。从数值分类树状图谱发现,在85．5％的相似水平上供试菌株构成了3个表观群。群I有27株菌,除4株来自陕西外,其余均来自新疆。群Ⅱ有4株菌,皆来自陕西。群Ⅲ有8株菌,6株来自陕西,2株来自宁夏,它们与R．hainanense聚在一起。而群I、群Ⅱ没有与参比菌株聚在一起,可能是新的表观群,其分类地位需进一步确定。     

新疆苦豆子根瘤菌的数值分类研究

苦豆子(Sophora alopecuroides)对于干旱荒漠地区的畜牧业发展有着非常重要的意义,其生长特性与根瘤菌有密切关系。我们对分离自新疆苦豆子根瘤的67株根瘤菌及36个模式菌株进行了118项表型性状的测定,包括唯一碳源利用、唯一氮源利用、对抗生素和染料的抗性、耐盐性、初始pH值生长范围、生长温度范围及石蕊牛奶反应、氧化酶、过氧化氢酶和脲酶。对测定结果用聚类分析方法进行了分析,获得数值分类树状图。结果表明：新疆苦豆子根瘤菌在碳氮源利用、抗生素敏感性以及对染料的抗性程度等方面存在着差异。新疆苦豆子根瘤菌能耐受低温,并具有较强的耐盐、碱能力,所有供试菌株均能在初始pH值为9－12的YMA培养基上生长,92.5％的菌株能耐受3.0％的NaCl,91.0％的菌株能耐受4.0％的NaCl,有18株菌甚至能耐受5.0％和6.0％的NaCl。聚类结果表明, 在84.8%的相似性水平上,67个供试菌株构成了4个新的表观群,第Ⅰ、Ⅱ、Ⅲ、Ⅳ类群分别有21、7、4、3个菌株,中心菌株分别为NWBC152、NWTKX101、NWYJS12、NWLP112。此外,数值分类结果还表明,苦豆子根瘤菌与模式菌株的相似性较低,它们所形成的4个独立群可能有新种出现。     

A numerical taxonomic study of lactic acid bacteria isolated from irradiated pork and chicken packaged under various gas atmospheres

Ninety-four Gram-positive, catalase-negative bacteria, isolated from pork and chicken that had been packed in modified atmospheres and irradiated to 1·75 and 2·5 kGy respectively, were studied. The majority of the strains were Lactobacillus saké . Numerical taxonomy, with the group average clustering strategy, revealed the existence of six clusters at the 85% similarity level. The largest, Cluster 1, contained 78 (83%) of the test strains along with three Lact. saké strains. Cluster 2 contained three test strains and the type strains of Carnobacterium piscicola and Carn. divergens . Cluster 3 contained two chicken strains and Lact. curvatus . Cluster 4 contained a pork strain and Leuconostoc dextranicum . Clusters 5 and 6 contained four pork and two chicken strains respectively and no type strains. Four test strains remained unclustered as did the other reference strains included in the study.     

A numerical taxonomic study of lactic acid bacteria isolated from irradiated pork and chicken packaged under various gas atmospheres

Ninety-four Gram-positive, catalase-negative bacteria, isolated from pork and chicken that had been packed in modified atmospheres and irradiated to 1.75 and 2.5 kGy respectively, were studied. The majority of the strains were Lactobacillus saké. Numerical taxonomy, with the group average clustering strategy, revealed the existence of six clusters at the 85% similarity level. The largest, Cluster 1, contained 78 (83%) of the test strains along with three Lact. sak'e strains. Cluster 2 contained three test strains and the type strains of Carnobacterium piscicola and Carn. divergens. Cluster 3 contained two chicken strains and Lact. curvatus. Cluster 4 contained a pork strain and Leuconostoc dextranicum. Clusters 5 and 6 contained four pork and two chicken strains respectively and no type strains. Four test strains remained unclustered as did the other reference strains included in the study.     

广西不同时期IBV分离株S1基因高变区Ⅰ的遗传变异分析

对广西1985-2007年间分离到的22株传染性支气管炎病毒（IBV）的S1基因高变区I（HVRI）进行序列测定,并与发表的其他IBV参考株及鸽子分离的冠状病毒株的基因序列进行比较和分析。系统进化关系显示毒株可分为5个基因群,其中有16个广西分离株属第1群,它们与鸽子冠状病毒分离株的氨基酸序列同源性较高,与Massachusetts（Mass）型疫苗株的同源性较低。有15个分离株在33-34位和34～35之间分别有4个和3个氨基酸残基的插入,GX-NN6在33～34位和34～35位之间则均有4个氨基酸残基的插入;GX-YL1、GX-NN2与常用的Mass型疫苗株的亲缘关系最近,同属于第Ⅱ群;GX-G、GX-XD与日本同一时期分离的毒株JP Miyazaki 89亲缘关系最近,属于第Ⅲ群;GX-YL6、GX-NN7与欧洲毒株4／91亲缘关系较近,属于第V群。结果表明广西存在着多种类型IBV毒株的流行,毒株S1基因HVRI碱基的突变或插入比较普遍,可导致其氨基酸序列的变化,绝大部分毒株与目前常用的Mass型疫苗株的亲缘关系较低。同一时期的分离株同源性较高,但无明显的地域性差异。     

Phylogenetic relationship of street rabies virus strains and their antigenic reactivity with antibodies induced by vaccine strains. II. Correlation between genetic distance and antibody reactivity

The aim of these studies was the estimation of the influence of genetic divergence of reactivity with sera of people vaccinated against rabies of Polish rabies strains. Genetic similarity between CVS strain and street rabies strains of genotype 1 is relatively high. However, CVS strain showed the highest reactivity with standard immunoglobulin and sera of antirabies vaccinated people (measured by western blot method). It was completely different from street viruses. Cluster method based on genetic and serologic features indicated high difference between CVS strain and street rabies strains belonging to genotype 1 and genotype 5. On this basis CVS strain was classified as a separate cluster. The genetic divergence of rabies strains circulating in Poland suggests the need of permanent epidemiological and virological surveillance. Strains different in their genotypic and biotypic characteristics should be estimated according to their antigenic similarity to vaccine strain. In practice neutralisation test using mono- and polyclonal sera should be performed. Strains isolated from new or atypical animal species should be studied first of all.     

Identification of proteolytic rumen bacteria isolated from New Zealand cattle

The protease activities of 212 strains of rumen bacteria isolated from New Zealand cattle grazing pasture were measured. Thirty-seven per cent of strains had activity greater than or equal to the proteolytic rumen bacterium Prevotella ruminicola and 43 of these isolates were identified by morphology, carbon source utilization, Gram stain, biochemical tests and fermentation end-product analysis. Hierarchical Cluster Analysis showed that the strains formed four clusters: cluster A contained 26 strains and clustered with a reference strain of Streptococcus bovis; cluster C contained three strains and clustered with a reference strain of Butyrivibrio fibrisolvens , while clusters B (10 strains) and D (three strains) did not cluster with any of the remaining rumen bacterial type strains. Further tests identified strains of cluster B as Eubacterium budayi , while cluster D strains most closely resembled B. fibrisolvens and were described as B. fibrisolvens -like. An unclustered strain, C21a, was identified as P. ruminicola. The significance of these proteolytic bacterial populations is discussed in relation to protein breakdown in New Zealand ruminants.     

Recharacterization of Pseudomonas fulva Iizuka and Komagata 1963, and proposals of Pseudomonas parafulva sp. nov. and Pseudomonas cremoricolorata sp. nov

Seven Pseudomonas fulva strains obtained from culture collections were taxonomically studied. The seven strains were separated into three clusters (Clusters I to III) on the basis of 16S rRNA gene sequences, and located phylogenetically in the genus Pseudomonas sensu stricto. Further, the strains were classified into 4 groups (Groups I to IV) on the basis of DNA-DNA similarity. As a result, Cluster I was split into Groups I and II. Group I included the type strain of P. fulva and two strains, and levels of DNA-DNA similarity ranged from 88 to 100% among the strains. Group II contained two strains, and the level between the two strains ranged from 91 to 100%. Group III consisted of one strain. Group IV included one strain, and this strain showed a high level of DNA-DNA similarity with the type strain of Pseudomonas straminea NRIC 0164(T). Clusters II and III corresponded to Groups III and IV, respectively. The four groups were separated from one another and from related Pseudomonas species at the level from 3 to 45% of DNA-DNA similarity. The strains of Groups I, II, and III had ubiquinone 9 as the major quinone. According to numerical analysis by the use of 133 phenotypic characteristics, the seven P. fulva strains were split into four phenons (Phenons I to IV). The groups by DNA-DNA similarity corresponded well with the phenons produced by numerical taxonomy, and differential characteristics were recognized. Consequently, Group I was regarded as P. fulva because the type strain (NRIC 0180(T)) of this species was included in this group. Strains in Group II were identified as a new species, Pseudomonas parafulva sp. nov., and the type strain is AJ 2129 (=IFO 16636=JCM 11244=NRIC 0501). NRIC 0181 in Group III was identified as a new species, Pseudomonas cremoricolorata sp. nov., and the type strain is NRIC 0181 (=IFO 16634=JCM 11246). NRIC 0182 in Group IV was identified as P. straminea on the basis of the high level of DNA-DNA similarity with the type strain of this species.     

Sequence analysis of rpoB and rpoD gene fragments reveals the phylogenetic diversity of actinobacteria of genus Frankia

Partial rpoD, rpoB, and 16S rRNA gene sequences were obtained from databases and (or) amplified from 12 strains of Frankia. These strains belonged to either Cluster 1 (Alnus-, Myrica-, Comptonia-, and Casuarina-infective strains) or Cluster 3 (Elaeagnus-infective strain). An rpoD gene-based PCR approach was designed to allow the detection of frankiae in complex samples. Additionally, partial gene sequences obtained using 2 rpoB gene primer sets (named rpoB-1 and rpoB-2) were used to generate phylogenetic eurograms to find a molecular tool able to assess biodiversity among Frankia strains. The rpoB-2 primer set allowed separation of closely related strains and groupings representative of host plant compatibility groups. One exception to this was for strains ACN10a and ACN14a, isolated from the same geographical location. Results obtained showed that rpoB-2 is a tool of great interest to evaluate relatedness of Frankia strains, and assess biodiversity in this genus. Additionally, since rpoB-2 phylogenetic profiles of the Frankia strains studied reflected the species of host plants they were isolated from, the study of rpoB (a house-keeping gene) shows promise for future ecological studies on these symbioses.     

杏鲍菇菌种遗传多态性的ISSR分析

采用ISSR(Inter-simple Sequence Repeat)分子标记技术并应用NTSYSpc 2.10e生物软件对20株杏鲍菇菌种进行遗传聚类分析。结果表明,从22条引物中筛选出11条扩增条带清晰、多态性好的ISSR引物,共获得74个ISSR标记位点,51个多态性位点,多态比率为68.92%,产物片段大小在100~5 000 bp之间。聚类分析发现,遗传相似系数在0.828时,20株杏鲍菇菌种分为4个类群,当遗传相似系数达到0.896时,分为12个类群,杏528寿与其他菌种的亲缘系数最远,具有独立的遗传体系。