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.     

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.     

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。     

不同属宿主来源的Frankia菌株形态特征的研究

利用光学显微镜和扫描电镜系统研究了从赤扬、香蕨木、扬梅、木麻黄、异木麻黄、沙棘、胡颓子等七个属中13个树种根瘤中分离出来的15株Frankia菌株。发现不同属、种来源的Frankia菌株除具有相似的形态特征外,在菌丝的粗细、菌丝体发育的稀疏、微密程度、孢子囊的大小,顶囊的形状、大小和顶囊柄的长短等均有着明显的差别。不同属来源的Frankia菌株在培养特征上有明显的差异。赤扬属不同种来源的Frankia菌株在以丙酸钠为碳源的Bap液体无氮培养基中均生长良好,但在以丙酮酸钠为碳源时,则不能生长,而木麻黄属、异木麻黄属来源的Frankia菌株,在丙酸钠或丙酮酸钠为碳源时均生长良好。沙棘属、胡颓子属来源的Frankia菌株在丙酸钠或丙酮酸钠为碳源时均生长良好并产生棕褐色色素。根据Frankia菌的形态和培养特征,可将七个属来源的Frankia菌株划分为三个类群:即赤扬香蕨木,扬梅类群;木麻黄、异木麻黄类群;沙棘和胡颓子类群。     

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。     

沙棘——弗氏放线菌共生固氮体系研究——Ⅰ环境因子影响沙棘结瘤和生长

环境因子包括盐度、pH值、化合氮和磷素供给显著影响了Frankia菌株Hr32对水培沙棘小苗的侵染结瘤。沙棘能在0.8％盐度下生长但受抑,0.2％盐度下开始有根瘤发生,0.1％盐度下结瘤较好。Hr32纯培养下耐盐力不强,0.5％盐度下已不能生长,导致了高盐浓度下不能结瘤。pH=7.0—8.0为沙棘结瘤最佳范围,pH值高至9.0或低至4.2,沙棘不能结瘤。低磷供给限制了根瘤发生,当供给10ppm或更高的磷时,沙棘成功结瘤。低于6ppm化合氮供给促进结瘤,当供给16ppm化合氮时,结瘤完全抑制。结瘤使沙棘获得近二倍的增长效果,水培条件下人工根瘤活性低于自然生境根瘤。结瘤使沙棘的落叶延迟。     

一株木麻黄根瘤内生菌的培养条件研究

1982年,Diem等人从木麻黄根瘤中分离出Frankia菌株DI_1和G_2,虽然纯培养的形态与典型Frankia菌株相似,但回接原宿主未获成功,而在沙棘上回接成功结瘤。同年,他又获得了CjI-82菌株,回接木麻黄结瘤并固氮。1985年,蒋建德与朱宝琴报道从木麻黄根瘤中分离到IFS 020601菌株,并回接成功。作者用四氧化锇法处理木麻黄根瘤,接种于     

长白山三种赤杨根瘤内Frankia基因多样性的REP分析

利用REP PCR方法研究了长白山不同海拔处 3种赤杨根瘤内Frankia菌基因多样性。结果表明 ,赤杨根瘤内Frankia菌存在丰富的基因多样性。宿主和海拔因素共同影响长白山赤杨属Frankia菌的基因多样性 :东北赤杨Frankia菌的宿主专一性很强 ,西伯利亚赤杨和色赤杨的Frankia菌的有较近的亲缘关系。不同海拔的同种宿主共生的Frankia菌基因类型不同 ,相同海拔的西伯利亚赤杨和色赤杨的Frankia菌出现宿主混乱现象     

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。     

基于高通量测序分析西藏沙棘根瘤内生菌的多样性

根瘤是微生物侵染植物根部并与之形成的共生结构,这些微生物都可被称为植物内生菌。豆科植物根瘤中的内生菌常常又被称为根瘤菌,而侵染非豆科植物形成根瘤的主要是放线菌弗兰克氏菌,这些非豆科植物又被称为放线菌结瘤植物。西藏沙棘是一种典型的放线菌结瘤植物,由于其分布生境的特殊性,对其根瘤内生菌的研究具有重要的生态意义。对于西藏沙棘根瘤内生菌的研究,培养方法因难以模拟自然条件而不易获得纯培养,高通量测序技术对其多样性的研究提供了便利。因此,本研究以生长在甘肃省天祝县金强河河滩地的西藏沙棘根瘤为材料,采用16S rRNA基因扩增子高通量测序方法,结合OTU分析,对西藏沙棘根瘤内生菌的多样性进行探讨。实验结果表明,西藏沙棘根瘤内生菌具有丰富的多样性,根瘤内的优势属为共生固氮的弗兰克氏菌属（Frankia）,其相对丰度为47.63%,共检测到7个弗兰克氏菌属的OTUs;根瘤内除弗兰克氏菌外,还存在大量的非弗兰克氏菌,共检测到1523个OTUs,隶属于22个门、33个纲、69个目、113个科和202个属,相对丰度排名前9的属中有25个非弗兰克氏菌属的OTUs。该研究也表明,西藏沙棘根瘤内生菌具有丰富的多样性,西藏沙棘根瘤中不仅存在着可共生固氮的弗兰克氏菌,并且还分布着非弗兰克氏菌;在同一根瘤样品中,弗兰克氏菌属还具有不同的物种。本研究不仅拓展了西藏沙棘根瘤内生菌多样性的研究方法,还为同一寄主植物中弗兰克氏菌多样性的研究提供了分析思路。     

Divergence in symbiotic interactions between same genotypic PCR–RFLP Frankia strains and different Casuarinaceae species under natural conditions

The symbiotic interactions between Frankia strains and their associated plants from the Casuarinaceae under controlled conditions are well documented but little is known about these interactions under natural conditions. We explored the symbiotic interactions between eight genotypically characterized Frankia strains and five Casuarinaceae species in long-term field trials. Characterization of strains was performed using the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) for the nifD – nifK intergenic transcribed spacer (ITS) and 16S–23S ITS. Assessments of the symbiotic interactions were based on nodulation patterns using nodule dry weight and viability, and on actual N₂ fixation using the δ¹⁵N method. The PCR–RFLP patterns showed that the analyzed strains belonged to the same genotypic group (CeD group), regardless of the host species and environment of origin. The nodule viability index is introduced as a new tool to measure the viability of perennial nodules and to predict their effectiveness. The host Casuarinaceae species was a key factor influencing both the actual N₂-fixing activity of the associated Frankia strain and the viability of nodules within a location. This is the first study providing information on the symbiotic interactions between genotypically characterized Frankia strains and actinorhizal plants under natural conditions. The results revealed a way to improve a long-term management of the Casuarinaceae symbiosis.     

Distribution and N2-fixing activity of Frankia strains in relation to soil depth

The qualitative and quantitative distribution of Frankia strains infective on Elaeagnus angustifolia L. from different depths in the same soil (10–20 cm; 30–40 cm; 50–60 cm) were compared. The soil samples collected from a natural stand devoid of Elaeagnaceae were planted with Elaeagnus angustifolia L. seedlings. All plants became nodulated, demonstrating that Frankia strains were present at least down to 60 cm in the soil. The decreased density of Frankia strains was correlated to the decline of soil organic matter content with soil depth. DNA extracted from nodules produced on Elaeagnus angustifolia L. seedlings were polymerase chain reaction (PCR) characterized by amplifying the nif D-K intergenic spacer (IGS), using the polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis. This showed the presence of seven nif-Hae III profiles within this Frankia community. Diversity of Frankia strains was maintained throughout the soil column, but the relative distribution of strains varied. Some nif-Hae III profiles were only found in the deeper soil, suggesting different selective advantages to withstand the constraints of soil depth. ¹⁵N₂ experiments indicated that all the strains tested had N₂-fixing activity. However, efficiency was not significantly different among nodules of different nif-Hae III profiles. Therefore, N₂-fixing activity does not seem to be the main factor responsible for the different distribution of Frankia strains at different soil depths.     

Geographic distribution and genetic diversity of Ceanothus-infective Frankia strains

Little is known about Ceanothus-infective Frankia strains because no Frankia strains that can reinfect the host plants have been isolated from Ceonothus spp. Therefore, we studied the diversity of the Ceonothus-infective Frankia strains by using molecular techniques. Frankia strains inhabiting root nodules of nine Ceanothus species were characterized. The Ceanothus species used represent the taxonomic diversity and geographic range of the genus; therefore, the breadth of the diversity of Frankia strains that infect Ceanothus spp. was studied. DNA was amplified directly from nodular material by using the PCR. The amplified region included the 3' end of the 16S rRNA gene, the intergenic spacer, and a large portion of the 23S rRNA gene. A series of restriction enzyme digestions of the PCR product allowed us to identify PCR-restriction fragment length polymorphism (RFLP) groups among the Ceanothus-infective Frankia strains tested. Twelve different enzymes were used, which resulted in four different PCR-RFLP groups. The groups did not follow the taxonomic lines of the Ceanothus host species. Instead, the Frankia strains present were related to the sample collection locales.     

Genetic heterogeneity among Frankia isolates from root nodules of individual actinorhizal plants

Abstract Genetic variations among selected Frankia isolates from nitrogen-fixing root nodules harvested from an individual actinorhizal plant ( Elaeagnus angustifolia L. or Shepherdia argentea Nutt.) were estimated by restriction fragment analysis of their total genomic DNA. The presence of plasmids and their restriction enzyme patterns were used as additional criteria. Certain isolates from separate nodules on the same plant were found indistinguishable, being probably clones of the same strain. An endophytic passage of a strain isolated from S. argentea on another host plant, Hippophaë rhamnoides L., did not modify the structural characteristics of the genome in the reisolates obtained. However, in some cases, especially when restriction endonucleases cleaving Frankia DNA into relatively small fragments were used, multiple infection of the actinorhizal plants with different Frankia strains and the presence of more than one strain in a nodule were demonstrated. Some aspects of variability in natural populations of Frankia are discussed.     

Polymorphism in Alnus based Frankia of Darjeeling

DNA samples extracted from the root nodules of Alnus nepalensis, collected from 10 different locations of Darjeeling hills, were used to assess the genetic diversity of Frankia. The DNA samples from the nodules of naturally growing plants were used as templates in PCR, targeting different genomic regions of Frankia, namely distal, middle and proximal parts of 16S rRNA gene and nifH-D IGS region with locus specific primers. The PCR products were digested with a number of frequent (4-base) cutter restriction endonucleases. Bands were scored as present (1) or absent (0) and the clustering was done using NTSYSpc. Distinct polymorphism was found among the nodules collected from different parts of the region and those of same geographic area. These results demonstrate that genetic diversity is indeed present among the naturally occurring Frankia of Darjeeling, India.     

非豆科固氮树种-沙棘与微生物联合共生体的纯培养研究

Ｆｒａｎｋｉａ 非豆科树木共生固氮体系是自然界中重要的固氮生物资源 ,具有与Ｒｈｉｚｏｂｉｕｍ 豆科植物相似的固氮能力 ,在自然界能量循环和生态平衡中起着重要作用 ,而且与形成共生固氮的非豆科植物其抗逆性强 ,耐干旱、盐碱 ,抗高寒、瘠薄 ,能在一般豆科植物不能生长的环境下正常生长结瘤固氮。非豆科固氮树种 沙棘是三北地区重要垦荒先锋树种1) ,其果实富含多种维生素 ,具有重要的经济开发价值。同时 ,沙棘又是具有内生菌根真菌的固氮树种[1] 。本文通过对沙棘菌根、根瘤联合共生体人工构建技术以及沙棘联合共生的增效作用的研究 ,…     

Evidence that some Frankia sp. strains are able to cross boundaries between Alnus and Elaeagnus host specificity groups.

Phenotypic and genotypic methods were used to prove the existence of Frankia strains isolated from an Elaeagnus sp. that are able to cross the inoculation barriers and infect Alnus spp. also. Repeated cycles of inoculation, nodulation, and reisolation were performed under axenic conditions. Frankia wild-type strain UFI 13270257 and three of its coisolates did exhibit complete infectivity and effectiveness on Elaeagnus spp. and Hippophaë rhamnoides and variable infectivity on Alnus spp. Microscopical observation of host plant roots showed that these strains are able to infect Alnus spp. by penetrating deformed root hairs. Reisolates obtained from nodules induced on monoxenic Alnus glutinosa, Alnus incana, and Elaeagnus angustifolia resembled the parent strains in host infectivity range, in planta and in vitro morphophysiology, isoenzymes, and nif and rrn restriction fragment length polymorphisms, thus fulfilling Koch's postulates on both host plant genera. Alnus and Elaeagnus group-specific polymerase chain reaction DNA amplifications, DNA-DNA hybridizations, and partial gene sequences coding for 16S rRNA provided evidence for the genetic uniformity of wild-type strains and their inclusion into one and the same genomic species, clearly belonging to the Elaeagnus group of Frankia species.     

Recent advances in the biogeography and genecology of symbiotic Frankia and its host plants

Molecular phylogenetic approaches have begun to outline the origin, distribution and diversity of actinorhizal partners. Geographic isolation of Frankia and its host plants resulting from shifting continents and dispersal patterns have apparently led to the development of Frankia genotypes with differing affinities for host genera, even within the same plant family. Actinorhizal plant genera of widespread global distribution tend to nodulate readily even outside their native ranges. These taxa may maintain infective Frankia populations of considerable diversity on a broad scale. Arid environments seem to have distinctive actinorhizal partnerships, with smaller and more specific sets of Frankia symbionts. This has led to the hypothesis that some host families have taxa that are evolving towards narrow strain specificity, perhaps because of drier habitats where fewer Frankia strains would be able to survive. Harsh conditions such as water-saturated soils near lakes, swamps or bogs that are typically acidic and low in oxygen may similarly lessen the diversity of Frankia strains present in the soil, perhaps limiting the pool of frankiae available for infection locally and, at a larger scale, for natural selection of symbiotic partnerships with host plants. Recent molecular ecological studies have also provided examples of Frankia strain sorting by soil environment within higher order cluster groupings of Frankia host specificity. Future frontiers for ecological research on Frankia and actinorhizal plants include the soil ecosystem and the genome of Frankia and its hosts.     

Characterization of genetic diversity ofFrankia strains in nodules ofAlnus nepalensis (D. Don) from the Hengduan Mountains on the basis of PCR-RFLP analysis of thenifD-nifK IGS

Nodule samples from 90A. nepalensis individuals were collected at five sites in the Hengduan Mountains. PCR-RFLP analysis of IGS betweennifD andnifK genes was directly applied to unculturedFrankia strains in the nodules. Sizes of thenifD-nifK IGS amplicons and genetic distance between the RFLP patterns from these samples were noticeably different, indicating significant genetic variation in theFrankia population. There were some nodule samples, which produced more than one PCR fragment, and compound RFLP patterns, indicating thatFrankia strains with different PCR-RFLP patterns coexisted in the same host plant under natural conditions. Among the 29 restriction patterns obtained, 5 patterns were found in more than one population and occurred in the majority of samples, while each of the other 24 patterns were represented by only one or two samples and were endemic to a particular population. From the calculatedGst and UPGMA cluster analysis, genetic diversity ofFrankia strains was inferred to be related to climate and glaciation history in the Hengduan Mountains.