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。     

西藏沙棘5种不同组织内生细菌多样性

西藏沙棘(Hippophae tibetana)是分布于高寒高海拔地区的一类特殊的放线菌结瘤植物, 弗兰克氏菌能够侵染其根部形成根瘤, 因共生固氮等作用而增强其生态适应性。在西藏沙棘的根瘤中, 除了弗兰克氏菌之外还有其他内生菌, 而弗兰克氏菌又不仅仅在根瘤中有分布。为了探究弗兰克氏菌在西藏沙棘不同组织中的定殖及可能的迁移规律, 分析不同组织中内生细菌的群落结构及多样性, 本研究以生长在甘肃省天祝藏族自治县抓喜秀龙金强河河滩地的西藏沙棘为材料, 应用16S rRNA扩增子高通量测序技术, 对西藏沙棘根瘤、茎、枝、叶和种子等不同组织的内生细菌多样性进行了分析。研究结果表明, 西藏沙棘根瘤内生细菌群落丰富度及多样性最高, 种子内生细菌群落丰富度最低, 茎内生细菌群落多样性最低。西藏沙棘5种不同组织中的弗兰克氏菌和其他内生细菌多样性都具有一定差异, 变形菌门均为优势门, 弗兰克氏菌属(Frankia)为根瘤内生细菌群落的优势属, 弗莱德门菌属(Friedmanniella)为茎内生细菌群落的优势属, 寡养单胞菌属(Stenotrophomonas)为枝、叶和种子内生细菌群落的优势属。研究结果还表明, 弗兰克氏菌属不仅仅存在于西藏沙棘的根瘤, 还能够分布于其他组织, 且在同一种组织中存在弗兰克氏菌属的不同“种”; 而在西藏沙棘不同组织中, 也分布有弗兰克氏菌属的相同“种”。此外, 对西藏沙棘5种不同组织内生细菌中的功能菌株的分析表明, 不同组织中均存在着具有固氮、促生和抑菌功能的内生细菌, 但具有固氮作用的内生细菌主要分布于根瘤, 具有促生作用以及抑菌功能的内生细菌主要分布于枝和叶。综上, 西藏沙棘5种不同组织内生细菌具有丰富的多样性, 但各组织内生细菌的群落结构和优势种群有所不同, 且不同组织也能够定殖具有多种功能的内生细菌。     

三种胡颓子科植物根瘤内生菌培养条件研究

弗兰克氏菌与一些非豆科植物所建立的共生固氮作用,对于增加土壤肥力,提高林业生产力起着重要作用。近年来,国内外已从不同的非豆科植物根瘤中分离出一批弗兰克氏菌菌株。而非豆科植物的共生固氮,已成为生物     

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。     

一氧化氮对豆科植物结瘤及固氮的影响机制

豆科植物-根瘤菌共生过程受双方基因复杂且精细的调控, 能够产生特异的根瘤结构并可将大气中的惰性氮气(N₂)转化为可被植物直接利用的氨态氮。结瘤与固氮受多种因素影响, 其中, 一氧化氮(NO)作为一种自由基反应性气体信号分子, 可参与调节植物的许多生长发育过程, 如植物的呼吸、光形态建成、种子萌发、组织和器官发育、衰老以及响应各种生物及非生物胁迫。在豆科植物中, NO不仅影响寄主与菌共生关系的建立, 还参与调控根瘤菌对氮气的固定并提高植株氮素营养利用效率。该文主要从豆科植物及共生菌内NO的产生、降解及其对结瘤、共生固氮的影响和对环境胁迫的响应, 阐述了NO调控豆科植物共生体系中根瘤形成和共生固氮过程的作用机制, 展望了NO信号分子在豆科植物共生固氮体系中的研究前景。     

与某些非豆科植物共生的弗兰克氏菌(Frankia sp.)的泡囊及其固氮作用

非豆科植物的共生固氮作用大多数是由弗兰克氏菌的侵染形成根瘤引起。其中泡囊是弗氏菌在根瘤内的固氮场所(Akkermans等1977,Tjepkema等1981)。我们采用光学及电子显微镜对泡囊进行了观察,并对根瘤不同部位的泡囊数量及其固氮活性与其它生理活性的关系进行了研究。     

弗兰克氏菌的分类研究进展

弗兰克氏菌是非豆科植物共生固氮菌。到目前为止,已发现它们可以分别与8个科、25个属的200多种非豆科植物共生[1],其中包括赤杨、杨梅、沙棘、胡颓子、马桑、木麻黄、悬钩子、仙女木等。这些植物对于荒地的开垦和防止水土流失具有重要意义。而弗兰克氏菌与这些植物共生形成可     

弗兰克氏菌在构建新的共生固氮物种研究中的优越性

弗兰克氏菌在构建新的共生固氮物种研究中的优越性解放军石家庄医学高等专科学院０５００８１曲东明范浩南四川师范大学细胞研究室６１００６８韩善华弗兰克氏菌（Ｆｒａｎｋｉａ）能与多种木本被子植物建立共生固氮关系，它形成的根瘤具有较强的固氮能力，有些超过大豆根...     

沙棘通过自主选择塑造根瘤内生微生物组

【背景】解析植物微生物群落结构是利用微生物组工程强化植物抗生物和非生物胁迫水平、提高农林产品质量和品质的基础。固氮根瘤是沙棘具有抗旱、抗寒和抗贫瘠等多种优良生物性状的关键。【目的】比较分析沙棘根际土和根瘤内细菌群落结构的组成及影响因素,为揭示沙棘-弗兰克氏菌共生和植物-微生物互作协同抗逆机制提供理论基础。【方法】从辽宁、陕西和山西采集样品,通过16SrRNA基因V3–V4可变区的高通量测序技术,采用生物信息学方法比较分析沙棘根际土和根瘤内细菌群落组成和丰度差异,并探索土壤土理化性质对根际土细菌群落结构的影响。【结果】沙棘根际土和根瘤内的细菌群落均以放线菌门(Actinobacteria)和变形菌门(Proteobacteria)为主要优势菌门,且根瘤内弗兰克氏菌属(Frankia)为绝对优势菌属;根际土前10个优势菌门的丰度在三地样品间均存在显著差异,仅存在唯一共有的优势菌属(鞘氨醇单胞菌属,Sphingomonas),且前35个优势属中有27个属在三地间存在明显丰度差异;土壤pH和速效钾是沙棘根际土细菌群落多样性的主要影响因子;根瘤内优势门和属在三省份间存在高度的保守性,仅异根瘤菌属...     

新疆干旱地区根瘤菌资源研究I．根瘤菌种类及其共生固氮作用

从新疆不同生态条件下生长的31属109种豆科植物根瘤分离获得373株根瘤菌,其中88株是从未报道过结瘤情况的39种豆科植物根瘤分离获得。它们与豆科寄主植物共生结瘤,95％以上为有效根瘤。不同种的根瘤固氮活性差异较大,黄芪属根瘤固氮活性较高,最高者为大豆根瘤固氮活性的42倍。 20属37种豆科植物根瘤中97％有吸氢活性。根瘤固氮活性、吸氢活性均与寄主植物生长发育期有相关性。     

Chemical defense production in Lotus corniculatus L. I. The effects of nitrogen source on growth,reproduction and defense

Summary The carbon to nitrogen balance theory was examined for a legume, Lotus corniculatus L., which allocates carbon to nitrogen fixation. N-fixation can influence the ratio of carbon to nitrogen in legumes by providing nitrogen in nutrient-poor habitats, and by consuming carbon for support of symbiotic N-fixation. L. corniculatus clones (genotypes) were grown under two levels of nitrogen fertilization: a treatment which suppressed nodulation with fertilization and a treatment which received no additional fertilization. These plants relied solely on symbiotic N-fixation. Plants which supported symbionts had lower biomass and lower tannin concentrations than fertilized plants; this appears to be a result of the large carbon demand on N-fixation. Plants supporting symbionts often had relatively lower protein concentrations than fertilized plants. Cyanide concentration was influenced by plant genotype but not by nitrogen source. Although symbiotic N-fixing plants were smaller, they had three times the reproductive output of fertilized plants.     

非共生生物固氮微生物分子生态学研究进展

氮是限制生态系统生产力的主要元素,生物固氮是自然生态系统中氮的主要来源.生物固氮包括共生、联合和自生固氮3种类型,其中联合固氮和自生固氮统称为非共生固氮.相对于共生固氮而言,非共生固氮速率虽然较低,但其不需要与其他生物形成共生体系就可以生存并进行固氮,在时空分布上更加广泛,因此对生态系统氮循环特别是素输入具有重要贡献.本文对近年有关非共生固氮微生物的多样性、土壤和叶际固氮微生物的分布特征及影响因素等研究进展进行了综述,并在此基础上阐述了现有研究中存在的问题和发展前景.     

Soil moisture and potassium affect the performance of symbiotic nitrogen fixation in faba bean and common bean

Potassium (K) is reported to improve plant's resistance against environmental stress. A frequently experienced stress for plants in the tropics is water shortage. It is not known if sufficient K supply would help plants to partially overcome the effects of water stress, especially that of symbiotic nitrogen fixation which is often rather low in the tropics when compared to that of temperate regions. Thus, the impact of three levels of fertilizer potassium (0.1, 0.8 and 3.0 mM K) on symbiotic nitrogen fixation was evaluated with two legumes under high (field capacity to 25% depletion) and low (less than 50% of field capacity) water regimes. Plants were grown in single pots in silica sand under controlled conditions with 1.5 mM N (¹⁵N enriched NH₄NO₃). The species were faba bean (Vicia faba L.), a temperate, amide producing legume and common bean (Phaseolus vulgaris L.), a tropical, ureide producing species. In both species, 0.1 mM K was insufficient for nodulation at both moisture regimes, although plant growth was observed. The supply of 0.8 or 3.0 mM K allowed nodulation and subsequent nitrogen fixation which appeared to be adequate for respective plant growth. High potassium supply had a positive effect on nitrogen fixation, on shoot and root growth and on water potential in both water regimes. Where nodulation occurred, variations caused by either K or water supply had no consequences on the percentage of nitrogen derived from the symbiosis. The present data indicate that K can apparently alleviate water shortage to a certain extent. Moreover it is shown that the symbiotic system in both faba bean and common bean is less tolerant to limiting K supply than plants themselves. However, as long as nodulation occurs, N assimilation from the symbiotic source is not selectively affected by K as opposed to N assimilation from fertilizer.     

Nitrate levels affect the development of the black locust-Rhizobium symbiosis

Summary Experiments with black locust (Robinia pseudoacacia L.) seedlings grown under strictly controlled laboratory conditions indicated that the availability of nitrate has a marked impact on nitrogen fixation. When nitrate concentrations were very low, both nodulation and seedling growth were impaired, whereas nitrate concentrations high enough to promote plant growth strongly inhibited symbiotic nitrogen fixation. When nitrate was added to the growth medium after infection, nodulation and nitrogen fixation of the seedlings decreased. This effect was even more marked when nitrate was applied before infection with rhizobia. Higher nitrogen concentrations also reduced nodule number and nodule mass when applied simultaneously with the infecting bacteria. The contribution of symbiotic nitrogen fixation to black locust shoot mass by far exceeded its effects on shoot length and root mass. When nitrate availability was very low, specific nitrogen fixation (i. e. nitrogenase activity per nodule wet weight) was improved with increasing nitrogen supply, but rapidly decreased with higher nitrogen concentrations.     

Genetic diversity of Datisca cannabina-compatible Frankia strains as determined by sequence analysis of the PCR-amplified 16S rRNA gene.

The presence of Frankia strains in soil samples collected from northern areas of Pakistan was detected by inoculating Coriaria nepalensis and Datisca cannabina plants. The abundance of compatible Frankia strains in some areas was indicated by profuse nodulation of the host plants, whereas soil samples from other localities failed to result in nodulation. An oligonucleotide probe (COR/DAT) directed against the 16S rRNA gene of the endophytes of Coriaria and Datisca spp. that did not cross-react with the RNA gene of Frankia strains isolated from other hosts was developed. Genetic diversity among Frankia strains nodulating D. cannabina was determined by sequence analysis of the partial 16S rRNA gene amplified from nodules induced by soil samples from different localities by PCR. Four types of Frankia sequences and one non-Frankia sequence were detected by hybridization with a Frankia genus probe and the COR/DAT probe as well as by sequence analysis of the cloned PCR products.     

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.     

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 ,…     

The Sesbania Root Symbionts Sinorhizobium saheli and S. teranga bv. sesbaniae Can Form Stem Nodules on Sesbania rostrata, although They Are Less Adapted to Stem Nodulation than Azorhizobium caulinodans

Sesbania species can establish symbiotic interactions with rhizobia from two taxonomically distant genera, including the Sesbania rostrata stem-nodulating Azorhizobium sp. and Azorhizobium caulinodans and the newly described Sinorhizobium saheli and Sinorhizobium teranga bv. sesbaniae, isolated from the roots of various Sesbania species. A collection of strains from both groups were analyzed for their symbiotic properties with different Sesbania species. S. saheli and S. teranga bv. sesbaniae strains were found to effectively stem nodulate Sesbania rostrata, showing that stem nodulation is not restricted to Azorhizobium. Sinorhizobia and azorhizobia, however, exhibited clear differences in other aspects of symbiosis. Unlike Azorhizobium, S. teranga bv. sesbaniae and S. saheli did not induce effective stem nodules on plants previously inoculated on the roots, although stem nodulation was arrested at different stages. For Sesbania rostrata root nodulation, Sinorhizobium appeared more sensitive than Azorhizobium to the presence of combined nitrogen. S. saheli and S. teranga bv. sesbaniae were effective symbionts with all Sesbania species tested, while Azorhizobium strains fixed nitrogen only in symbiosis with Sesbania rostrata. In a simple screening test, S. saheli and S. teranga bv. sesbaniae were incapable of asymbiotic nitrogenase activity. Thus, Azorhizobium can easily be distinguished from Sinorhizobium among Sesbania symbionts on the basis of symbiotic and free-living nitrogen fixation. The ability of Azorhizobium to overcome the systemic plant control appears to be a stem adaptation function. This last property, together with its host-specific symbiotic nitrogen fixation, makes Azorhizobium highly specialized for stem nodulation of the aquatic legume Sesbania rostrata.