沙棘属植物弗兰克氏菌研究进展

弗兰克氏菌(Frankia spp.)能够与沙棘等非豆科植物形成根瘤进行共生固氮,其固氮效率远远高于豆科植物根瘤菌,与沙棘共生的弗兰克氏菌还能够促进沙棘对旱寒等各种不同生境的适应性,是自然界一类具有开发潜力的放线菌资源。为了更好地开发利用弗兰克氏菌资源,推进弗兰克氏菌分类鉴定工作,加强弗兰克氏菌与寄主植物共生结瘤固氮的机制研究,促使弗兰克氏菌在农业生产中得到尽快应用,本文简要介绍沙棘属(Hippophae L.)物种多样性、结瘤状况与分布特点、沙棘根瘤形态结构与功能、弗兰克氏菌物种多样性与分布特征,讨论弗兰克氏菌的结瘤机制、生理生态效应与作用机制以及影响沙棘属植物与弗兰克氏菌共生的主要因子,以期为进一步开展沙棘属植物弗兰克氏菌的系统研究提供有价值的参考。     

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。     

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。     

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     

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。     

弗兰克氏菌分类学研究进展和现状

中国农科院油料作物研究所,武汉,430070．弗兰克氏菌（Frankia）是能与8科,24属,200多种非豆科树木共生结瘤的固氮放线菌,直到1978年才首次被分离或体外培养,进入实验室至今还不到20年。作为一种模式微生物,弗兰克氏菌的形态及培养特征、细胞化学组分、生理生化、浸染特性以及遗传学等方面已经进行了广泛的研究,以此积累的资料,已经并正在作为Fran切a分类的主要依据。本文介绍近几年Frankia分类学研究的进展和现状。IFrankia的系统发百（Phy－logeny）——弗兰克氏菌科的重新修订相对于放线菌结瘤植物在系统发育上的多起源,Fra…     

一株弗兰克氏菌分类鉴定的研究

从中国沙棘(Nippophae rhamnoidel LI s叩．tinensis Roun)根瘤中分离出一株生孢囊的放线菌Fran&㈦P．HRl04。经回接试验证实,它可以侵染寄主植物,形成能够固氮的有效根瘤。通过形态和培养特征、生理生化特性、细胞壁组份及DNA中G+c含量等的研究,表明这株菌与已经研究和描述过的弗兰克氏菌有差别,特别是胞壁型不同,可能属于一个尚未研究过的新属。本文还对弗兰克氏菌的分类标准和鉴定方法进行了初步探讨。     

几种非豆科植物根瘤内生菌的形态和培养特征

弗兰克氏菌可以侵染许多非豆科植物的根系形成根瘤。这种共生固氮作用是陆地生态系统中氮素的重要来源,对于改良土壤、提高土壤肥力有着重要意义,不少植物还具有较高的经济价值。多年来,由于这种根瘤内生菌的分离十分困难,因此人们一直认为这种固氮放线菌不能人工培养。1978年callaham等用酶     

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...     

几种非豆科植物根瘤内生菌侵染特征的研究

自不同科、属、种的非豆科植物根瘤分离内生菌,对其寄主植物进行了交叉侵染,结果表明,这些Frankia菌对不同寄主的侵染没有明显的专一性,供试菌可以进行跨越科、属、种的侵染,但有的菌株对于某些植物的侵染,可能存在一些特殊情况,相同菌株对不同植物的侵染能力,以及不同菌株对同一寄主的侵染能力是有差异的。从同一种植物根瘤中分离的不同菌株,侵染能力也有高低之分,供试菌随寄主植物的改变,侵染能力及所建立的共生系统固氮活性有所降低,侵染原寄主植物所形成的根瘤固氮活性较高的菌株,在改变寄主后所形成的根瘤固氮活性也比较高,在一定条件下,寄主植物的结瘤量与根瘤固氮活性呈正相关,而侵染不同寄主后,根瘤中菌体孢子的表面结构也发生了一定变化。     

Molecular analysis of actinorhizal symbiotic systems: Progress to date

The application of molecular tools to questions related to the genetics, ecology and evolution of actinorhizal symbiotic systems has been especially fruitful during the past two years. Host plant phylogenies based on molecular data have revealed markedly different relationships among host plants than have previously been suspected and have contributed to the development of new hypotheses on the origin and evolution of actinorhizal symbiotic systems. Molecular analyses of host plant gene expression in developing nodules have confirmed the occurrence of nodulin proteins and in situ hybridization techniques have been successfully adapted to permit the study of the spatial and temporal patterns of gene expression within actinorhizal nodules. The use of heterologous probes in combination with nucleotide sequence analysis have allowed a number of nif genes to be mapped on the Frankia chromosome which will ultimately contribute to the development of hypotheses related to nif gene regulation in Frankia. The use of both 16S and 23S rDNA nucleotide sequences has allowed the construction of phylogenetic trees that can be tested for congruence with symbiotic characters. In addition the development of Frankia-specific gene probes and amplification primers have contributed to studies on the genetic diversity and distribution of Frankia in the soil.     

Presence of sporangia of Frankia in the rhizosphere of Discaria (Rhamnaceae)

Discaria trinervis and Discaria americana are native actinorhizal plants in Argentina. Discaria seedlings growing in N-free liquid culture, inoculated with dry soils, developed Frankia colonies in the rhizosphere. The occurrence of hyphae, vesicles and sporangia characteristic of Frankia are described in these colonies. The presence of sporangia of Frankia has previously neither been reported in the genus Discaria, nor in the other genera of the tribe Colletieae inside root nodules or outside roots. The infective capacity of the colonies has been demonstrated.     

Preinfection cell wall formation in roots and developing nodules ofAlnus rubra Bong

Summary The establishment of actinorhizal root nodules involves penetration of host cell walls and intracellular colonization by the nitrogen-fixing endosymbiont,Frankia (Actinomycetales). In the early stages of the infection process inAlnus, unusual cell walls with undulate profiles were observed in root tip meristematic derivatives, and in early (preinfection) derivatives of the nodule lobe meristem, inFrankia-inoculated plants. The irregular cell walls attached obliquely to preexisting walls, but were not discontinuous. Serial sections revealed that the unusual walls divided two daughter cells. Microtubules in bundled arrays were abundant near the undulate walls, and radiated in several planes. In the root tips, the anomalous cell walls were observed within one day of inoculation withFrankia.     

Infectivity and effectivity of Frankia strains from the Rhamnaceae family on different actinorhizal plants

Ten strains of Frankia isolated from root nodules of plant species from five genera of the host family Rhamnaceae were assayed in cross inoculation assays. They were tested on host plants belonging to four actinorhizal families: Trevoa trinervis (Rhamnaceae), Elaeagnus angustifolia (Elaeagnaceae), Alnus glutinosa (Betulaceae) and Casuarina cunninghamiana (Casuarinaceae). All Frankia strains from the Rhamnaceae were able to infect and nodulate both T. trinervis and E. angustifolia. Strain ChI4 isolated from Colletia hystrix was also infective on Alnus glutinosa. All nodules showed a positive acetylene reduction indicating that the microsymbionts used as inoculants were effective in nitrogen fixation. The results suggest that Frankia strains from Rhamnaceae belong to the Elaeagnus-infective subdivision of the genus Frankia.     

Nodulation patterns of actinorhizal plants in the family rosaceae

Patterns of nodulation, growth, andFrankia — host specificity have not been well characterized for the actinorhizal genera in the family Rosaceae because of the scarcity ofFrankia isolates from these taxa. Furthermore, the few isolates available from actinorhizal Rosaceae have consistently failed to nodulate plants from the host genus. In a series of experiments, species of rosaceousDryas, Cowania, Cercocarpus, Fallugia, andPurshia were inoculated withFrankia isolates, crushedDryas actinorhizae, and neoglacial soils to ascertain whether any of these inocula would effectively induce nodulation. Neoglacial soils from Alaska and Canada nodulated not only the localDryas drummondii, but alsoCercocarpus betuloides, Cowania mexicana andPurshia tridentata from distant and ecologically diverse locales as well as nonrosaceous, actinorhizal species ofAlnus, Elaeagnus, Myrica, andShepherdia. But of eightFrankia isolates, including two fromPurshia tridentata and one fromCowania mexicana, none were able to induce nodulation onPurshia orCowania species. Globular, actinorhizae-like nodules incapable of acetylene reduction were produced onC. betuloides inoculated withFrankia isolates. Crushed nodule suspensions fromDryas drummondii nodulated rosaceousCowania, Dryas andPurshia, as well as non-rosaceousElaeagnus, Myrica, andShepherdia species. Nodules produced by inoculation ofCowania mexicana andPurshia tridentata with crushed, dried nodule suspensions fromDryas drummondii reduced acetylene to ethylene, indicating nitrogenase activity for these nodulated plants. These data suggest that a similar microsymbiont infects the actinorhizal genera in the family Rosaceae.     

Nodulation of actinorhizal plants byFrankia strains capable of both root hair infection and intercellular penetration

Summary A morphological analysis of the initiation and development of root nodules ofElaeagnus angustifolia andMyrica cerifera inoculated with pure-culturedFrankia strains DDB 011610 or DDB 020110 was undertaken. From ultrastructural observations it was determined that both of theseFrankia strains can infectElaeagnus by an intercellular penetration mechanism andMyrica by the root hair infection mechanism. This indicates that both of these strains have the ability to infect host plant roots by either of two mechanisms. The reverse, thatElaeagnus orMyrica could be infected by both mechanisms, was not observed. The infection and nodule development processes of these two plants in combination with these strains were similar to observations made in previous studies (Miller andBaker 1985,Torrey andCallaham 1979). However, one exception was identified in the development of the prenodule ofMyrica when infected with strain 011610, in that endophytic hyphae developed vesicles within the cells of the prenodule. This event has not been described before for any of the actinorhizal genera and may be an indication of less than optimal compatibility between the host plant and the symbiont.Contribution no. 876 of the Battelle-Kettering Laboratory.     

The initiation,development and structure of root nodules inElaeagnus angustifolia L. (Elaeagnaceae)

Summary A correlated light and electron microscopic study was undertaken of the initiation and development of root nodules of the actinorhizal tree species,Elaeagnus angustifolia L. (Elaeagnaceae).Two pure culturedFrankia strains were used for inoculation of plants in either standing water culture or axenic tube cultures. Unlike the well known root hair infection of other actinorhizal genera such asAlnus orMyrica the mode of infection ofElaeagnus in all cases was by direct intercellular penetration of the epidermis and apoplastic colonization of the root cortex. Root hairs were not involved in this process and were not observed to be deformed or curled in the presence of the actinomyceteFrankia. In response to the invasion of the root, host cells secreted a darkly staining material into the intercellular spaces. The colonizingFrankia grew through this material probably by enzymatic digestion as suggested by clear dissolution zones around the hyphal strands. A nodule primordium was initiated from the root pericycle, well in advance of the colonizingFrankia. No random division of root cortical cells, indicative of prenodule formation was observed inElaeagnus. As the nodule primordium grew in size it was surrounded by tanninised cells of a protoperiderm. The endophyte easily traversed this protoperiderm, and once inside the nodule primordium cortex ramified within the intercellular spaces at multiple cell junctions. Invasion of the nodule cortical cells occurred when a hyphal branch of the endophyte was initiated and grew through the plant cell wall, again by apparent enzymatic digestion. The plant cell plasmalemma of invaded cells always remained intact and numerous secretory vesicles fused with it to encapsulate the advancingFrankia within a fibrous cell wall-like material. Once within the host cell some endophyte cells began to differentiate into characteristic vesicles which are the presumed site of nitrogen fixation. This study clearly demonstrates that alternative developmental pathways exist for the development of actinorhizal nitrogen-fixing root symbioses.     

Studies of an effective strain ofFrankia fromAllocasuarina lehmanniana of the Casuarinaceae

Summary Seedlings ofCasuarina spp. andAllocasuarina spp. were grown from seed in the greenhouse and inoculated with a nodule suspension fromC. equisetifolia. Plants ofCasuarina spp. nodulated regularly and were effective in nitrogen-fixation. Only one species ofAllocasuariona, A. lehmanniana formed root nodules. Using these plants as source of inoculum, the isolation of a newFrankia sp. HFPA11I1 (HFP022 801) was made and the strain was grown in pure culture.Frankia sp. HFPA11I1 grows well in a defined medium and shows typical morphological characteristics. In media lacking combined nitrogen, the filamentours bacterium forms terminal vesicles in abundance and differentiaties large intrahyphal or terminal sporangia containing numerous spores. This strain, used as inoculum, nodulates effectively seedlings ofC. equisietifolia andC. cunninghamiana, forming nodules with verically-growing nodule roots. Although effective in acetylene reduction, the endophyte within the nodules is filamentous and lacks veiscles. When used to inoculated seedlings ofA llocasuarina lehmanniana, Frankia sp. HFPA11I1 induces root nodules which are coralloid and lacking nodule roots. The nodules are effective in acetylene reduction and the filamentous hyphae ofFrankia within the nodule lobes lack vesicles. Effective nodulation inA. Lehmanniana depends upon environmental conditions of the seedlings and proceeds much more slowly than in Casuariana.     

Devising an <Emphasis Type="Italic">in vitro</Emphasis> nodulation system for two actinorhizal plants: <Emphasis Type="Italic">Allocasuarina verticillata</Emphasis> (Lam.) L. Johnson and <Emphasis Type="Italic">Casuarina glauca</Emphasis> sieb. ex spreng (Casuarinaceae)

Summary The purpose of this study was to establish an efficient in vitro nodulation device for producing actinorhizal root nodules on Allocasuarina verticillata and Casuarina glauca. Seeds from the two species were germinated aseptically and seedlings with at least two photosynthetic branchlets and a 3–5 cm long root system were transferred into Petri dishes containing a biphasic (solid/liquid) medium. To assess the nodulation capacity, four different culture media were tested. As soon as the root system developed and spread adequately on the surface of the medium, plants were deprived of nitrogen for at least 1 wk and inoculated with the Frankia strain. The time course nodulation for A. verticillata showed that the basal Hoagland medium supplemented with CaCO₃ and KNO₃ was most efficient, with 83% of plantlets forming nodules, while the medium supplemented with CaCO₃ reached 100% nodulation for C. glauca. This procedure can provide a valuable tool for the study of early events of actinorhizal nodulation and spatio-temporal expression of symbiotic genes in transgenic Casuarinaceae.     

不同水稻组织内生细菌的群落多样性

[目的]为详细了解水稻不同组织内生细菌群落多样性。[方法]对宁粳43号内生细菌的总DNA提取后,采用高通量测序技术对水稻内生细菌的16S rRNA基因进行了序列测定,分析了水稻不同组织部位内生细菌群落结构特征。[结果]叶部共获得内生细菌OTUs 610个,茎部411个,根部174个。物种分类显示,叶部内生细菌种类隶属于22门40纲103目198科399属,其中优势类群是红球菌属（Rhodococcus）和乳酸杆菌属（Lactobacillus）,它们的相对丰度分别为21.00%和9.19%;茎部内生细菌种类隶属于19门31纲85目169科306属,其中优势类群是红球菌属和罗尔斯通菌属（Ralstonia）,它们的相对丰度分别为19.25%和13.52%;根部内生细菌种类隶属于9门19纲44目82科140属,其中优势类群是肠杆菌属（Enterobacter）和埃希氏杆菌属（Escherichia）,它们的相对丰度分别为81.13%和10.89%。根茎叶中相同的OTU有78个,放线菌门（Actinobacteria）与大多数细菌具有相关性。根系内生细菌中具有调控各种代谢网络功能的物种丰度高于茎部和叶部。[结论]不同水稻组织内生细菌具有丰富的群落多样性,其中叶部的内生细菌物种最丰富,根系参与各种代谢调控的细菌丰度最高,各个组织部位的优势菌属各不相同,变形菌门是最重要的水稻内生细菌。