自然环境胁迫对旱冬瓜Frankia菌基因多样性的影响

利用rep-PCR方法,研究云南鸡足山及无量山不同生境下旱冬瓜根瘤内Frankia菌基因多样性及其变化,以了解不同自然环境胁迫对Frankia菌基因多样性的影响。结果表明,多样性随地域、海拔和坡向不同而变化,鸡足山Frankia菌基因类型比无量山丰富。鸡足山旱冬瓜根瘤内的Frankia菌在山底2300m处,Shannon指数平均为0．90;山顶海拔2650m以上,Shannon指数随之上升到1．33。南坡Frankia菌多样性高于北坡,表明多样性指数与环境胁迫大小成正相关,自然环境胁迫是产生和保持Frankia菌基因多样性的重要因子之一。     

中国沙棘(Hippophae rhamnoides ssp. Sinensis)根瘤内Frankia菌的遗传多样性

为了研究中国沙棘亚种共生菌Frankia的遗传多样性,利用PCR-RFLP分子标记方法,对从青海西宁到内蒙古库伦17个地点采集的106个中国沙棘根瘤样品进行遗传多样性分析.供试样品nifD-nifK基因间隔区(IGS)扩增产物分别用3种内切酶(HinfⅠ、HaeⅢ和MboⅠ)酶切,共产生21条酶切谱带,其中17条为多态性条带,多态位点百分比(PPL)为80.99%,所有样品可被划分为9个基因型.结果表明中国沙棘根瘤内的Frankia菌有丰富的遗传多样性,土壤质量较好地点的丰富度高于土壤质量较差地点,海拔较高地区的丰富度高于海拔较低地区,多数地点至少有2种不同基因型的Frankia 菌.聚类分析显示Frankia 菌不同基因型间的遗传距离在4.88%～55.96%之间,它们在不同地点的分布是不均匀的,没有发现不同基因型菌间的亲缘关系与地点有相关性.中国沙棘根瘤中Frankia菌可分为两个基因型组,组内基因型分布比较一致,而组间有明显差异.     

长白山赤杨丛枝菌根真菌多样性的半巢式LP-PCR-SSCP检测分析

利用半巢式LP-PCR-SSCP技术,对中国吉林长白山不同海拔生境下3种赤杨共生丛枝菌根真菌的多样性进行检测分析。结果表明,该地区赤杨属东北赤杨、西伯利亚赤杨及色赤杨共生丛枝菌根真菌在科乃至种的水平上并未随宿主的变化表现出丰富的多样性;3个树种在自身属的水平上与共生的球囊霉科(Glomaceae)至少1种AMF,即G．intraradix,在种的水平上表现出不相关于宿主海拔高度的某种相互选择性。     

ARDRA对植物根瘤内共生放线菌Frankia多样性的研究

应用原核生物16SrDNA特异性引物rD1和fD1,通过ARDRA（amplified ribosomal DNA restriction analysis）法直接扩增自中国云南、东北地区赤杨属3种植物和沙棘属1种植物根瘤内FrankiaDNA,得到一长约1500bp的扩增产物,选用两种内切酶HaeⅢ、AfaⅠ联合对扩增产物进行酶切,得到稳定的酶切图谱,将所测48个感染赤杨的Frankia样本区分为3个不同的组,所测43个感染沙棘的Frankia样本区分为3个不同的组,显示根瘤内Frankia存在丰富的遗传多样性。     

色赤杨根瘤内生菌Frankia sp,At~4的分离和侵染能力的鉴定(简报)

放线菌与非豆科木本植物的共生体系具有强大的固氮能力,可将大气中氮素转变成化合态,以满足植物需要,并培肥土壤,在绿化造林中起着重要作用。近年来,国内外均在积极开展这方面的工作,使其成为固氮研究中最活跃的领域之一。最近,我们已从色赤杨根瘤中分离到了几株Frankia放线菌,并进行了形态学和宿主植物回接结瘤等方面的鉴定工作。已证实At4是一株能使色赤杨结瘤固氮的Frankia菌株。     

内蒙古沙棘共生菌Frankia的遗传多样性

利用RFLP分子标记方法,在自然条件下对内蒙古东、西部8个地点采集的24个沙棘根瘤样品进行沙棘共生菌Frankia的遗传多样性分析。结果表明,供试样品nifD-nifK基因间隔区(IGS)扩增片段的大小约1 100bp;不同样品的酶切图谱有明显差异;有些样品产生复合RFLP型,揭示在自然状态下不同基因型的Frankia菌株可共同侵染同一沙棘寄主。聚类分析显示,来源于相同地点和不同地点的根瘤样品内的Frankia菌株间均有遗传多样性;没有发现Frankia菌株遗传多样性的分布与采样地点有相关性。     

长白山四种赤杨丛枝菌根真菌侵染多样性的巢式PCR-RFLP分析

采集中国吉林长白山不同海拔的4种赤杨根须样本,利用巢式PCR-RFLP方法检测丛枝菌根真菌(AMF)对样品的侵染情况,PCR结果经限制性内切酶分析. 结果表明,赤杨根内AMF存在丰富的基因多样性.AMF的侵染有从宿主混乱性向宿主专一性发展的趋势.东北赤杨AMF的宿主专一性水平最强,球囊霉属已成为东北赤杨的优势侵染类群;其余3种赤杨的AMF则出现宿主混乱现象.宿主因素比海拔因素对AMF侵染有更重要的影响.     

Frankia菌同功酶图谱分析和分类

利用同功酶图谱的差异性对Frankia菌进行分类识别是一种非常有效的方法,不同的Frankia菌株具有不同的同功酶(过氧化物酶和酯酶)图谱。根据图谱之间的相似性,可以将20株Frankia菌分成三大组,即赤杨组、木麻黄组和胡颓子组,这与其它分类结果基本符合,只是细枝木麻黄菌株Cc01的过氧化物酶图谱与其它菌株完全不同。从同种植物根瘤中分离的内生菌具有不同的同功酶图谱,进一步证实了遗传结构不完全相同的菌株可以同时共生于同种根瘤内,同时根据二种同功酶的电泳分布率,说明了Frankia菌株遗传信息的广泛分布性。     

甘南高寒地区不同海拔西藏沙棘根瘤内生菌多样性

为了研究甘南高寒地区3个不同海拔西藏沙棘根瘤中内生菌的多样性,采用Illumina MiSeq高通量测序技术对3种不同海拔的西藏沙棘根瘤内生菌的菌群组成和多样性进行了分析。实验结果表明,3个不同海拔西藏沙棘根瘤中的内生菌主要包括6大门,分别为蓝藻门(Cyanobacteria),放线菌门(Actinobacteria),变形菌门(Proteobacteria),拟杆菌门(Bacteroidetes),梭杆菌门(Fusobacteria)和厚壁菌门(Firmicutes)。其中占主导地位的微生物是蓝藻门和放线菌门,蓝藻门在3个海拔的西藏沙棘根瘤中的丰度分别为68.1%,64.7%和66.0%,放线菌门在3个海拔的西藏沙棘根瘤中的丰度分别为28.6%,30.2%和29.5%。放线菌门弗兰克氏菌属(Frankia)为共同的优势菌属之一,在3个海拔的丰度分别为28.2%,29.8%和29.1%。3个不同海拔西藏沙棘根瘤中的内生菌除了能与沙棘共生固氮的弗兰克氏菌外,还存在其他的微生物群落,可能存在一些潜在的有价值的内生菌种资源。     

非豆科固氮树木根瘤内生菌(Frankia)的分离和纯培养简报

1981年9月,蒋建德、杨慧凡等用蔗糖离心法从四川桤木(Alnus cremastogyne Burk.)的根瘤中分得内生菌Frankia sp.Acc 13。从而在我国获得了第一个纯培养的菌株。以Acc13作为接菌体,在所回接的四川桤木幼苗根系上形成大量根瘤。根瘤具有固氮能力,乙炔还原活性为15.7μM乙烯/克鲜瘤重·小时。赵振英、钟玉华等由赤杨属的毛赤杨(Alnus hirsuta Turcz.)、欧洲赤杨(A.gluti-     

Correlations between the ages of Alnus host species and the genetic diversity of associated endosymbiotic Frankia strains from nodules

Nodule samples were collected from four alder species: Alnus nepalensis, A. sibirica, A. tinctoria and A. mandshurica growing in different environments on Gaoligong Mountains,Yunnan Province of Southwest China and on Changbai Mountains, Jilin Province of Northeast China. PCR-RFLP analysis of the IGS between nifD and nifK genes was directly applied to uncultured Frankia strains in the nodules. A total of 21 restriction patterns were obtained. The Frankia population in the nodules of A. nepalensis had the highest genetic diversity among all four Frankia populations; by contrast, the population in the nodules of A. mandshurica had the lowest degree of divergence; the ones in the nodules of A. sibirica and A. tinctoria were intermediate. A dendrogram, which was constructed based on the genetic distance between the restriction patterns, indicated that Frankia strains from A. sibirica and A. tinctoria had a close genetic relationship. Frankia strains from A. nepalensis might be the ancestor of Frankia strains infecting other Alnus species. From these results and the inference of the ages of Alnus host species, it is deduced that there was a co-evolution between Alnus and its microsymbiont Frankia in China.     

Correlations between the ages of Alnus host species and the genetic diversity of associated endosymbiotic Frankia strains from nodules

Nodule samples were collected from four alder species: Alnus nepalensis, A. si-birica, A. tinctoria and A. mandshurica growing in different environments on Gaoligong Mountains, Yunnan Province of Southwest China and on Changbai Mountains, Jilin Province of Northeast China. PCR-RFLP analysis of the IGS between nifD and nifK genes was directly applied to uncultured Frankia strains in the nodules. A total of 21 restriction patterns were obtained. The Frankia population in the nodules of A. nepalensis had the highest genetic diversity among all four Frankia populations; by contrast, the population in the nodules of A. mandshurica had the lowest degree of divergence; the ones in the nodules of A. sibirica and A. tinctoria were intermediate. A dendrogram, which was constructed based on the genetic distance between the restriction patterns, indicated that Frankia strains from A. sibirica and A. tinctoria had a close genetic relationship. Frankia strains from A. nepalensis might be the ancestor of Frankia strain     

福建省放线菌结瘤植物共生菌Frankia遗传多样性研究

[目的]了解福建省放线菌结瘤植物共生固氮菌Frankia的遗传多样性.[方法]利用16S-23SrDNA间隔区(rrn)和nifD-K基因间隔区的PCR扩增和RFLP技术,分析了福建省木麻黄、杨梅、桤木、胡颓子等共生Frankia纯培养菌株的遗传差异.[结果]17个菌株获得rrn扩增片段,2个杨梅菌株和1个胡颓子菌株扩增未成功,酶切图谱经聚类分析表明6个地点的细枝木麻黄、短枝木麻黄、粗枝木麻黄12个共生Frankia菌株同源性高,属于一个类群,2个地点的4个杨梅菌株和1个四川桤木菌株亲缘关系近,为另一类群.25个Frankia菌株的,nifD-K基因间隔区PCR-RFLP分析结果显示,7个地点的3种木麻黄14个菌株聚类为一个类群,4个地点的7个杨梅菌株、2个地点的2个四川桤木菌株以及1个台湾桤木菌株聚类为另一个类群,胡颓子菌株则为独立的类群.[结论]研究结果表明福建省共生Frankia遗传多样性丰富.     

Genetic diversity among Frankia strains nodulating members of the family Casuarinaceae in Australia revealed by PCR and restriction fragment length polymorphism analysis with crushed root nodules.

DNA extracted directly from nodules was used to assess the genetic diversity of Frankia strains symbiotically associated with two species of the genus Casuarina and two of the genus Allocasuarina naturally occurring in northeastern Australia. DNA from field-collected nodules or extracted from reference cultures of Casuarina-infective Frankia strains was used as the template in PCRs with primers targeting two DNA regions, one in the ribosomal operon and the other in the nif operon. PCR products were then analyzed by using a set of restriction endonucleases. Five distinct genetic groups were recognized on the basis of these restriction patterns. These groups were consistently associated with the host species from which the nodules originated. All isolated reference strains had similar patterns and were assigned to group 1 along with six of the eight unisolated Frankia strains from Casuarina equisetifolia in Australia. Group 2 consisted of two unisolated Frankia strains from C. equisetifolia, whereas groups 3 to 5 comprised all unisolated strains from Casuarina cunninghamiana, Allocasuarina torulosa, and Allocasuarina littoralis, respectively. These results demonstrate that, contrary to the results of previous molecular studies of isolated strains, there is genetic diversity among Frankia strains that infect members of the family Casuarinacaeae. The apparent high homogeneity of Frankia strains in these previous studies probably relates to the single host species from which the strains were obtained and the origin of these strains from areas outside the natural geographic range of members of the family Casuarinaceae, where genetic diversity could be lower than in Australia.     

Diversity of Frankia strains associated to Myrica gale in Western Europe: impact of host plant (Myrica vs. Alnus) and of edaphic factors

Myricaceae can be nodulated by a variety of Frankia strains isolated from other actinorhizal families. Consequently, the genus Myrica has been considered to have low specificity with respect to microsymbiont taxa. In contrast to controlled studies of Myrica infectious capacity, field studies in North America have indicated that M. gale symbionts belong to the genetic group of Alnus-infective strains. Myrica gale is the most widely distributed species in the genus so this study focused on describing the genetic diversity of M. gale-nodulating strains from 10 sites in Western Europe across a range of edaphic conditions. When possible, the specificity of M. gale-infective strains was compared with that of Alnus-infective strains from the same sites. Nodular strains from Belgium, France and Spain were characterized using PCR-RFLP of rrs gene and 16S-23S IGS. rrs-RFLP patterns showed a high level of homogeneity among European strains with one dominant genotype. IGS-RFLP patterns revealed the largest inter and intrasite diversity in France. In Belgium, Frankia strains were found to occur in two groups according to soil pH and organic matter characteristics of the sites. European M. gale-infective strains were genetically different from European Alnus and North American M. gale-infective strains indicating the possibility of different pathways of co-evolution among geographically isolated populations.     

应用ARDRA技术研究Frankia菌多样性

应用原核生物16SrDNA特异性引物rD1和fD1,对分自4个分类接种群的12株纯培养Frankia菌总DNA进行扩增,得到1条长约1500bp的扩增产物.选用2种内切酶HinfI,MspI对扩增产物进行酶切,得到稳定的酶切图谱.对图谱的分析结果表明,Frankia菌间存在极其丰富的遗传多样性.     

Diversity and specificity of Frankia strains in nodules of sympatric Myrica gale, Alnus incana, and Shepherdia canadensis determined by rrs gene polymorphism

The identity of Frankia strains from nodules of Myrica gale, Alnus incana subsp. rugosa, and Shepherdia canadensis was determined for a natural stand on a lake shore sand dune in Wisconsin, where the three actinorhizal plant species were growing in close proximity, and from two additional stands with M. gale as the sole actinorhizal component. Unisolated strains were compared by their 16S ribosomal DNA (rDNA) restriction patterns using a direct PCR amplification protocol on nodules. Phylogenetic relationships among nodular Frankia strains were analyzed by comparing complete 16S rDNA sequences of study and reference strains. Where the three actinorhizal species occurred together, each host species was nodulated by a different phylogenetic group of Frankia strains. M. gale strains from all three sites belonged to an Alnus-Casuarina group, closely related to Frankia alni representative strains, and were low in diversity for a host genus considered promiscuous with respect to Frankia microsymbiont genotype. Frankia strains from A. incana nodules were also within the Alnus-Casuarina cluster, distinct from Frankia strains of M. gale nodules at the mixed actinorhizal site but not from Frankia strains from two M. gale nodules at a second site in Wisconsin. Frankia strains from nodules of S. canadensis belonged to a divergent subset of a cluster of Elaeagnaceae-infective strains and exhibited a high degree of diversity. The three closely related local Frankia populations in Myrica nodules could be distinguished from one another using our approach. In addition to geographic separation and host selectivity for Frankia microsymbionts, edaphic factors such as soil moisture and organic matter content, which varied among locales, may account for differences in Frankia populations found in Myrica nodules.     

Molecular typing of native Bacillus thuringiensis isolates from diverse habitats in India using REP-PCR and ERIC-PCR analysis

Bacillus thuringiensis is a bacterium of great agronomic and scientific interest. The subspecies of this bacterium colonize and kill a large variety of host insects and even nematodes, but each strain does so with a high degree of specificity. Therefore molecular typing and diversity analysis of B. thuringiensis has enormous importance for discrimination of strains isolated from different sources. In this study, 113 native B. thuringiensis isolates collected from diverse habitats and locations in India and 27 B. thuringiensis type strains obtained from the Bacillus Genetic Stock Centre (BGSC), Ohio State University, USA and used as reference, were analyzed for molecular typing. Genotypic data of 140 B. thuringiensis isolates and type strains was generated by using REP-PCR and ERIC-PCR primers and unweighted pair group method with arithmetic mean (UPGMA) analysis using NTSYSpc2.2 and grouped into 4 main clusters. All the groups have isolates from diverse origins. No group was found to represent any specific origin or location. The observed patterns of REP-PCR and ERIC-PCR pattern were discriminatory enough to reveal differences in the B. thuringiensis isolates and reference strains. The resolution power and marker index of the ERIC-PCR (RP 9.39, MI 6.34) was found to be higher than that of the REP-PCR (RP 6.20, MI 4.48). The REP-PCR and ERIC-PCR markers have been found to be useful for discrimination of B. thuringiensis isolates and reference strains. ERIC-PCR was the more informative of the two techniques. This study showed that the B. thuringiensis isolates collected from diverse habitats in India had a high degree of genetic diversity.