Detection and Identification of Bacterial Endosymbionts in Arbuscular Mycorrhizal Fungi Belonging to the Family Gigasporaceae

Intracellular bacteria have been found previously in one isolate of the arbuscular mycorrhizal (AM) fungus Gigaspora margarita BEG 34. In this study, we extended our investigation to 11 fungal isolates obtained from different geographic areas and belonging to six different species of the family Gigasporaceae. With the exception of Gigaspora rosea, isolates of all of the AM species harbored bacteria, and their DNA could be PCR amplified with universal bacterial primers. Primers specific for the endosymbiotic bacteria of BEG 34 could also amplify spore DNA from four species. These specific primers were successfully used as probes for in situ hybridization of endobacteria in G. margarita spores. Neighbor-joining analysis of the 16S ribosomal DNA sequences obtained from isolates of Scutellospora persica, Scutellospora castanea, and G. margarita revealed a single, strongly supported branch nested in the genus Burkholderia.     

Restriction Analysis of PCR-Amplified Internal Transcribed Spacers of Ribosomal DNA as a Tool for Species Identification in Different Genera of the Order Glomales

A technique combining PCR and restriction fragment length polymorphism analysis was used to generate specific DNA fragment patterns from spore extracts of arbuscular mycorrhizal fungi. With the universal primers ITS1 and ITS4, DNA fragments were amplified from species of Scutellospora and Gigaspora that were approximately 500 bp long. The apparent lengths of the corresponding fragments from Glomus spp. varied between 580 and 600 bp. Within the genus Glomus, the restriction enzymes MboI, HinfI, and TaqI were useful for distinguishing species. Depending on the restriction enzyme used, groups of species with common fragment patterns could be found. Five tropical and subtropical isolates identified as Glomus manihotis and G. clarum could not be distinguished by their restriction patterns, corresponding to the morphological similarity of the spores. The variation of internal transcribed spacer sequences among the Gigaspora species under study was low. Fragment patterns of Scutellospora spp. showed their phylogenetic relationship with Gigaspora and revealed only a slightly higher degree of variation.     

Molecular analysis of Gigaspora (Glomales, Gigasporaceae)

This work presents a cooperative effort to integrate new molecular (isozyme and SSU analyses) characters into the morphological taxonomy of the genus Gigaspora (Glomales). Previous analyses of published Gigaspora SSU sequences indicated the presence of a few polymorphic nucleotides in the region delimited by primers NS71-SSU 1492'. In our study, the SSU of 24 isolates of arbuscular mycorrhizal (AM) fungi from the Gigasporaceae were amplified and the NS71-SSU 1492' region was directly sequenced. The corresponding sequences of four more isolates of AM fungi from Gigasporaceae, already published, were also included in our analyses. Three Gigaspora groups were identified on the basis of a 6 nucleotide-long 'molecular signature': Gigaspora rosea group ( G. rosea + G. albida ), Gigaspora margarita group ( G. margarita + G. decipiens ) and Gigaspora gigantea , which constituted a group by itself. The isozyme profiles (malate dehydrogenase, MDH) of 12 of these 28 isolates, and seven other isolates not sequenced, were compared. The results obtained further supported the grouping of isolates provided by the SSU analysis. Both SSU and MDH analysis indicated that two out of the 35 isolates had been misidentified, which was confirmed when their morphology was reassessed. The use of the Gigaspora intrageneric molecular signature as a quick, unambiguous and objective method to recognize Gigaspora isolates under any (field or laboratory) experimental conditions is suggested.     

PCR-denaturing gradient gel electrophoresis profiling of inter- and intraspecies 18S rRNA gene sequence heterogeneity is an accurate and sensitive method to assess species diversity of arbuscular mycorrhizal fungi of the genus Gigaspora

Despite the importance of arbuscular mycorrhizal fungi in the majority of terrestrial ecosystems, their ecology, genetics, and evolution are poorly understood, partly due to difficulties associated with detecting and identifying species. We explored the inter- and intraspecies variations of the 18S rRNA genes of the genus Gigaspora to assess the use of this marker for the discrimination of Gigaspora isolates and of Gigasporaceae populations from environmental samples. Screening of 48 Gigaspora isolates by PCR-denaturing gradient gel electrophoresis (DGGE) revealed that the V3-V4 region of the 18S rRNA gene contained insufficient variation to discriminate between different Gigaspora species. In contrast, the patterns of 18S ribosomal DNA (rDNA) heterogeneity within the V9 region of this marker could be used for reliable identification of all recognized species within this genus. PCR-DGGE patterns provided insight into some putative misidentifications and could be used to differentiate geographic isolates of G. albida, G. gigantea, and G. margarita but not G. rosea. Two major clusters were apparent based upon PCR-DGGE ribotype patterns, one containing G. albida, G. candida, G. ramisporophora, and G. rosea and the other containing G. decipiens and G. margarita. Dissection of the DGGE patterns by cloning, DGGE screening, and sequencing confirmed these groupings and revealed that some ribotypes were shared across species boundaries. Of the 48 isolates examined, only two displayed any spore-to-spore variation, and these exceptions may be indicative of coisolation of more than one species or subspecies within these cultures. Two Brazilian agricultural soils were also analyzed with a Gigasporaceae-specific nested PCR approach, revealing a dominance of G. margarita within this family.     

山东省不同植被区内野生植物根围AM菌的生态分布

ＡＭ菌是土壤习居菌 ,生态适应性强 ,可发生在各种生态环境。寄主范围也十分广泛 ,除少量植物如莎草科、苋科、灯心草科、藜科、石竹科等 2 0余科植物不能或不易形成ＡＭ外 ,大多数植物包括苔藓、蕨类、裸子植物、被子植物都能被菌根菌侵染。当前人们十分重视对野生植物上ＡＭ菌的调查[3 ,4 ,1 0 ,1 1 ,1 4 ]。研究发现 ,野生植物上可能有比栽培作物更多的ＡＭ菌种类[1 ]。我国野生植物资源丰富 ,开发和利用野生寄主植物上的ＡＭ菌潜力巨大。由于ＡＭ菌对寄主植物的选择性及对环境条件的适应性不同 ,或进化过程中的历史原因 ,造成了自然生态…     

A Burkholderia Strain Living Inside the Arbuscular Mycorrhizal Fungus Gigaspora margarita Possesses the vacB Gene,Which Is Involved in Host Cell Colonization by Bacteria

The arbuscular mycorrhizal (AM) fungus Gigaspora margarita harbors a resident population of endosymbiontic Burkholderia in its cytoplasm. Nothing is known about the acquisition of such bacteria and about the molecular bases which allow colonization of the fungus. We wondered whether the intracellular Burkholderia strain possesses genetic determinants involved in colonization of a eukaryotic cell. Using degenerated oligonucleotide primers for vacB, a gene involved in host cell colonization by pathogenic bacteria, an 842 bp DNA fragment was cloned, sequenced, and identified as a part of the vacB gene in Burkholderia sp. The insert was used as a probe to screen a fungal library that, because of the presence of intracellular Burkholderia cells, was also representative of the bacterial genome. The complete nucleotide sequence of vacB and flanking genes was determined. The bacterial origin of this genomic region was established by PCR, using specific vacB primers on DNA from Gigasporaceae that did or did not contain cytoplasmic Burkholderia, as well as on DNA from other bacteria, including free-living Burkholderia. We hypothesize that the vacB gene is part of a new genetic region acquired by a rhizospheric Burkholderia strain, which became able to establish a symbiotic interaction with the AM fungus G. margarita.     

Presymbiotic growth and sporal morphology are affected in the arbuscular mycorrhizal fungus Gigaspora margarita cured of its endobacteria

Some arbuscular mycorrhizal fungi contain endocellular bacteria. In Gigaspora margarita BEG 34, a homogenous population of beta-Proteobacteria is hosted inside the fungal spore. The bacteria, named Candidatus Glomeribacter gigasporarum, are vertically transmitted through fungal spore generations. Here we report how a protocol based on repeated passages through single-spore inocula caused dilution of the initial bacterial population eventually leading to cured spores. Spores of this line had a distinct phenotype regarding cytoplasm organization, vacuole morphology, cell wall organization, lipid bodies and pigment granules. The absence of bacteria severely affected presymbiotic fungal growth such as hyphal elongation and branching after root exudate treatment, suggesting that Ca. Glomeribacter gigasporarum is important for optimal development of its fungal host. Under laboratory conditions, the cured fungus could be propagated, i.e. could form mycorrhizae and sporulate, and can therefore be considered as a stable variant of the wild type. The results demonstrated that - at least for the G. margarita BEG 34 isolate - the absence of endobacteria affects the spore phenotype of the fungal host, and causes delays in the growth of germinating mycelium, possibly affecting its ecological fitness. This cured line is the first manipulated and stable isolate of an arbuscular mycorrhizal fungus.     

Characterization of root colonization profiles by a microcosm community of arbuscular mycorrhizal fungi using 25S rDNA-targeted nested PCR

The aim of the present work was to study colonization patterns in roots by different arbuscular mycorrhizal fungi developing from a mixed community in soil. As different fungi cannot be distinguished with certainty in planta on the basis of fungal structures, taxon-discriminating molecular probes were developed. The 5' end of the large ribosomal subunit containing the variable domains D1 and D2 was amplified by PCR from Glomus mosseae (BEG12), G. intraradices (LPA8), Gigaspora rosea (BEG9) and Scutellospora castanea (BEG1) using newly designed eukaryote-specific primers. Sequences of the amplification products showed high interspecies variability and PCR taxon-discriminating primers were designed to distinguish between each of these four fungi. A nested PCR, using universal eukaryotic primers for the first amplification and taxon-discriminating primers for the second, was performed on individual trypan blue-stained mycorrhizal root fragments of onion and leek, and root colonization by four fungi inoculated together in a microcosm experiment was estimated. More than one fungus was detected in the majority of root fragments and all four fungi frequently co-existed within the same root fragment. Root colonization by G. mosseae and G. intraradices was similar from individual and mixed inoculum, whilst the frequency of S. castanea and Gig. rosea increased in the presence of the two Glomus species, suggesting that synergistic interactions may exist between some arbuscular mycorrhizal fungi.     

长期定位施肥土壤中的丛枝菌根真菌

从连续26年长期定位施肥土壤中的玉米和小麦根围采集土样,从中鉴定出丛枝茵根(Arbuscular Myc- orrguza,AM)真菌5属19种,包括Glomus属11种:G．caledonium,G．constrictum,G．eburneum,G．ebunicatum, G．hyderabadensis,G．mossese,G．sinuosum,G．tortuosum,G．versiforme,G．sp1,G．sp2;Acaulospora属2种: A．excacota,A．sp;Gigaspora属2种:Gi．margarita,Gi．sp;Scutellospora属3种:S．cerradensis,S．coralloidea, S．pellucida;Archaespora属1种:Ar．leptoticha。     

AM真菌种间差异对枳壳生长及耐热性效应的研究※

用地表球囊霉、莫西球囊霉、珠状巨孢球囊霉及其混合菌剂接种无菌根枳壳幼苗进行盆栽试验,25℃培养4个月,观察对枳壳菌根形成和营养生长的影响,在40℃高温胁迫30d,调查分析菌根枳壳的耐热性。试验结果表明:接种AM真菌的根系形成了20%～80%的菌根侵染率;菌根枳壳的苗高、苗质量、节间长、茎基粗、须根数量和须根长度等营养生长显著增加;叶片中的SOD,POD活性和根系活力显著增强,可溶性蛋白、可溶性糖含量显著升高,叶片中的MDA含量降低,膜透性显著变小,枳壳苗的耐热性显著提高;但是,AM真菌在促进枳壳苗菌根化、营养生长和提高耐热性方面存在着种间差异,地表球囊霉、莫西球囊霉、珠状巨孢球囊霉、混合菌剂与枳壳根系形成丛枝菌根的侵染率依次为20.4%±1.2%、61.8%±3.4%、85.7%±2.7%、83.3%±2.2%,促进枳壳苗营养生长提高枳壳苗耐热能力的AM真菌依次为:地表球囊霉<莫西球囊霉<珠状巨孢球囊霉<混合菌剂,认为珠状巨孢球囊霉和莫西球囊霉是枳壳耐高温胁迫菌根化育苗的重要优良菌种。     

Isolation, free-living capacities, and genome structure of "Candidatus Glomeribacter gigasporarum," the endocellular bacterium of the mycorrhizal fungus Gigaspora margarita

"Candidatus Glomeribacter gigasporarum" is an endocellular beta-proteobacterium present in the arbuscular mycorrhizal (AM) fungus Gigaspora margarita. We established a protocol to isolate "Ca. Glomeribacter gigasporarum" from its host which allowed us to carry out morphological, physiological, and genomic investigations on purified bacteria. They are rod shaped, with a cell wall typical of gram-negative bacteria and a cytoplasm rich in ribosomes, and they present no flagella or pili. Isolated bacteria could not be grown in any of the 19 culture media tested, but they could be kept alive for up to 4 weeks. PCR-based investigations of purified DNA from isolated bacteria did not confirm the presence of all genes previously assigned to "Ca. Glomeribacter gigasporarum." In particular, the presence of nif genes could not be detected. Pulsed-field gel electrophoresis analyses allowed us to estimate the genome size of "Ca. Glomeribacter gigasporarum" to approximately 1.4 Mb with a ca. 750-kb chromosome and a 600- to 650-kb plasmid. This is the smallest genome known for a beta-proteobacterium. Such small genome sizes are typically found in endocellular bacteria living permanently in their host. Altogether, our data suggest that "Ca. Glomeribacter gigasporarum" is an ancient obligate endocellular bacterium of the AM fungus G. margarita.     

贵州烟草根围AM真菌多样性的初步研究

从贵州省内烟区不同土壤生态环境下采集烟草根际土样,湿筛离心法分离丛枝菌根（AM）真菌孢子,鉴定出烟草AM真菌4属20种,其中球囊霉属9种,无梗囊霉属7种,巨孢囊霉属3种,盾巨孢囊霉属1种。从土壤样品DNA中扩增AM真菌特异性片段并采用DGGE技术对AM真菌多样性进行分析。测序结果显示烟草根际土壤中菌根真菌主要菌群为球囊霉属,与湿筛离心法的鉴定结果一致。为进一步研究贵州地区AM真菌多样性以及开发应用提供了依据。     

红壤中VA菌根真菌(球囊霉目)的种类和生态分布

本文报道了发育于第四纪红色粘土母质上的红壤中的4属13种VA菌根真菌(球囊霉目):1.细齿无梗囊霉 Acaulospora denticulata Sieverding & Toro;2.丽孢无梗囊霉Acaulospora elegans Trappe & Gerdemann:3.光壁无梗囊霉Acaulospora laevis Gerdeman D & Trappe:4.巨大巨孢囊霉Gigaspora gigantea(Nicol.& Gerd.)Gerdemann & Trappe;5.珍珠巨孢囊霉Gigaspora margarita Becket & Hall;6.聚球囊霉Glomus aggregatum Schenck & Smith;7.明球囊霉Glomus clarum Nicolson & Schenck;8.集球囊霉Glomus fasiculatum(Thaxter)Gerdemann & Trappe;9.地球囊霉Glomus geosporum(Nicol.& Gerd.)Walker;10.木薯球囊霉Glomus manihot Howeler,Sieverding & Schenck;11.变形球囊霉Glomus versiforme(Karsten)Berch;12.美丽盾孢囊霉Scutellospora calospora(Nicol.& Gerd.)Walker & Sanders;13.异配盾孢囊霉Scutellospora heterogama(Nicol.& Gerd.)Walker & Sanders。其中细齿无梗囊霉(Acaulospora denticulata)、巨大巨孢囊霉(Gigaspora gigantea)、木薯球囊霉(Glomus manihot)和异配盾孢囊霉(Scutellospora heterogama)4种为国内新记录种。对这13种VA菌根真菌的形态进行了描述讨论,并对这些种群的出现频度、不同利用方式     

野生植物根围的丛枝菌根真菌I

１９９５－１９９７年作者比较系统地调查研究了山东省、北京和沈阳３个区域内野生植物根围丛枝菌根（Ａｒｂｕｓｃｕｌａｒｍｙｃｏｒｒｈｉｚａｅ,ＡＭ）菌的资源状况,对采自５０种野生植物根围的１８２个土样中ＡＭ菌进行了分离、培养和鉴定,共分离出Ａｃａｕｌｏｓｐｏｒａ属６种,分别为附柄无柄囊霉Ａｃａｕｌｏｓｐｏｒａａｐｐｅｎｄｉｃｕｌａ　Ｓｐａｉｎ,Ｓｉｅｖｅｒｄｉｎｇ　＆Ｓｃｈｅｎｃｋ、膨胀无柄囊霉ＡｃａｕｌｏｏｐｏｒａｄｉｌａｔａｔａＭｏｒｔｏｎ、浅窝无柄囊霉Ａ．ｌａｃｕｎｏｓａＭｏｒｔｏｎ、蜜色无柄囊霉Ａ．ｍｅｌｌｅａＳｐａｉｎ　＆Ｓｃｈｅｎｃｋ、细凹无柄囊霉Ａ．ｓｃｒｏｂｉｃｕｌａｔａＴｒａｐｐｅ、刺无柄囊霉Ａ．ｓｐｉｎｏｓａＷａｌｋｅｒ　＆Ｔｍｐｐｅ;Ｇｉｇａｓｐｏｒａ属的一个种,珍珠巨抱囊霉ＧｉｇａｓｐｏｒａｍａｔｙａｒｉｔａＢｅｃｋｅｒ＆Ｈａｌｌ;Ｓｃｕｔｅｌｌｏｓｐｏｒａ属的１个种,美丽盾巨孢囊霉Ｓｃｕｔｅｌｌｏｓｐｏｒａ　ｃａｌｏｓｐｏｒａ（Ｎｉｃｏｌ,＆Ｄｅｒｄ．）Ｇｅｕｌｅｍａｎｎ　＆Ｔｒａｐｐｅ,其中浅窝无柄囊霉为我国的一个新记录种。     

野生植物根围的丛枝菌根真菌I*

１９９５－１９９７年作者比较系统地调查研究了山东省、北京和沈阳３个区域内野生植物根围丛枝菌根（Ａｒｂｕｓｃｕｌａｒｍｙｃｏｒｒｈｉｚａｅ,ＡＭ）菌的资源状况,对采自５０种野生植物根围的１８２个土样中ＡＭ菌进行了分离、培养和鉴定,共分离出Ａｃａｕｌｏｓｐｏｒａ属６种,分别为附柄无柄囊霉Ａｃａｕｌｏｓｐｏｒａａｐｐｅｎｄｉｃｕｌａ　Ｓｐａｉｎ,Ｓｉｅｖｅｒｄｉｎｇ　＆Ｓｃｈｅｎｃｋ、膨胀无柄囊霉ＡｃａｕｌｏｏｐｏｒａｄｉｌａｔａｔａＭｏｒｔｏｎ、浅窝无柄囊霉Ａ．ｌａｃｕｎｏｓａＭｏｒｔｏｎ、蜜色无柄囊霉Ａ．ｍｅｌｌｅａＳｐａｉｎ　＆Ｓｃｈｅｎｃｋ、细凹无柄囊霉Ａ．ｓｃｒｏｂｉｃｕｌａｔａＴｒａｐｐｅ、刺无柄囊霉Ａ．ｓｐｉｎｏｓａＷａｌｋｅｒ　＆Ｔｍｐｐｅ;Ｇｉｇａｓｐｏｒａ属的一个种,珍珠巨抱囊霉ＧｉｇａｓｐｏｒａｍａｔｙａｒｉｔａＢｅｃｋｅｒ＆Ｈａｌｌ;Ｓｃｕｔｅｌｌｏｓｐｏｒａ属的１个种,美丽盾巨孢囊霉Ｓｃｕｔｅｌｌｏｓｐｏｒａ　ｃａｌｏｓｐｏｒａ（Ｎｉｃｏｌ,＆Ｄｅｒｄ．）Ｇｅｕｌｅｍａｎｎ　＆Ｔｒａｐｐｅ,其中浅窝无柄囊霉为我国的一个新记录种。     

An obligately endosymbiotic mycorrhizal fungus itself harbors obligately intracellular bacteria.

Arbuscular-mycorrhizal fungi are obligate endosymbionts that colonize the roots of almost 80% of land plants. This paper describes the employment of a combined morphological and molecular approach to demonstrate that the cytoplasm of the arbuscular-mycorrhizal fungus Gigaspora margarita harbors a further bacterial endosymbiont. Intracytoplasmic bacterium-like organisms (BLOs) were detected ultrastructurally in its spores and germinating and symbiotic mycelia. Morphological observations with a fluorescent stain revealed about 250,000 live bacteria inside each spore. The sequence for the small-subunit rRNA gene obtained for the BLOs from the spores was compared with those for representatives of the eubacterial lineages. Molecular phylogenetic analysis unambiguously showed that the endosymbiont of G. margarita was an rRNA group II pseudomanad (genus Burkholderia). PCR assays with specifically designed oligonucleotides were used to check that the sequence came from the BLOs. Successful amplification was obtained when templates from both the spores and the symbiotic mycelia were used. A band of the expected length was also obtained from spores of a Scutellospora sp. No bands were given by the negative controls. These findings indicate that mycorrhizal systems can include plant, fungal, and bacterial cells.     

西双版纳四种壳斗科植物丛枝菌根状况的初步研究

研究了西双版纳热带次生林中杯丝锥(Castanopsis calathiformis)、红锥(C.hys-trix)、印度锥(C.indica)和截果柯(Lithocarpus truncatus)4种壳斗科植物的丛枝菌根真菌(arbuscular mycorrhizal fungi,AMF)的侵染状况,并从这些植物的根际土壤中分离鉴定了隶属于球囊霉属(Glomus)、巨孢囊霉属(Gigaspora)、盾巨孢囊霉属(Scutellospora)和无梗囊霉属(Acaulospora)的10种丛枝菌根真菌。这4种壳斗科植物根际AMF的孢子密度为14~22个.100g-1土壤,种的丰富度在4~7,平均频度为60.00%,相对多度为4.41%~22.06%,丛枝菌根真菌的定居水平达46.26%~51.40%。     

不同pH水平下两种菌根真菌对紫云英生长的影响及其相互作用

设计在不同pH水平（4.3、5.1、5.8、6.8）下两种VA菌根真菌Glomus mosseae和Gigaspora margarita对紫云英Astragalus sinicus进行单接种、混合接种及无接种对照的盆栽实验。对紫云英地上和地下部分生物量、根部侵染率、SDH和ALP酶活进行了检测。实验结果表明：紫云英的生长效应与VA菌根真菌的侵染率及两种酶活成明显相关性。土壤pH升高,单接种Glomus mosseae和混合接种的侵染率也随之升高,而单接种Gigaspora margarita的侵染率呈现     

A molecular marker diagnostic of a specific isolate of an arbuscular mycorrhizal fungus,Gigaspora margarita

To investigate the auto-ecology of a strain of Gigaspora margarita in a commercial inoculum, we found a pair of PCR primers amplifying a sequence of 235 bp diagnostic of the isolate. We designed an oligonucleotide probe based on the DNA sequence. The combination of PCR and the probing successfully detected the diagnostic sequence from both DNA preparations of single spores and colonized roots. This protocol enabled us to distinguish the isolate among several isolates from Japan, Nepal and the USA.     

A comparison of nine species of arbuscular mycorrhizal fungi on the development and nutrition of micropropagated Rubus idaeus L. cv. Glen Prosen (Red Raspberry)

Growth, development and nutrient status of micropropagated Rubus idaeus cv. Glen Prosen in response to inoculation with nine species of arbuscular mycorrhizal (AM) fungi from three different genera was investigated. The nine species of AM fungi included, Glomus clarum, G. etunicatum, G. intraradices, Gigaspora rosea, Gi. gigantea, Gi. margarita, Scutellospora calospora, S. heterogama and S. persica. Plant responses to AM fungi varied from growth enhancement to growth depression. Depressive growth effects were specific to Gigaspora species. Furthermore, particular species of AM fungi had unique effects on the mineral status of the raspberry plants. Importance of isolate selection for inoculation of micropropagated raspberry plants is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.