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.     

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.     

丛枝菌根真菌对植物根尖分生区和根冠细胞的侵染*

一般说来,从枝菌根（ＡＭ）真菌大多数是从植物根系根毛区（成熟区）侵入和扩展的,在显微镜下往往看不到根尖分生区和根冠表皮细胞被ＡＭ真菌侵染的特征。这就很容易给人们造成一种假象,似乎ＡＭ真菌不能侵染根尖分生区和根冠表皮细胞,即它们对ＡＭ真菌是免疫的。然而笔者多次于显微镜下看到ＡＭ真菌侵染根尖分生区和根冠表皮细胞,并形成典型的泡囊、丛枝、菌丝等结构。这一现象导致作者在温室盆栽和大田条件下研究了玫瑰红巨孢囊霉（ Ｇｉｇａｓｐｏｒａ　ｒｏｓｅａ Ｎｉｃｏｌ　＆ Ｓｃｈｅｎｃｋ）、珠状巨孢囊霉（Ｇｉｇａｓｐｏｒａ　ｍａｒｇａｒｉｔａ　Ｂｅｃｋｅｒ　＆　Ｈａｌｌ）、根内球囊霉（Ｇｌｏｍｕｓ　ｏｍｔｒａｒａｄｉｃｅｓ　ｓｃｈｅｎｃｋ　＆　Ｓｍｉｔｈ、摩西球囊霉（Ｇｌｏｍｕｓ ｍｏｓｓｅａｅ （Ｎｉｃｏｌ　＆ Ｇｅｒｄ．） Ｇｅｒｄｅｍａｎｎ　＆ Ｔｒａｐｐｅ）、地表球囊霉（ Ｇｌｏｍｕｓ ｖｅｒｓｉｆｏｒｍｅ（ Ｋａｒｓｔｅｎ）Ｂｅｒｃｈ）和弯丝硬囊霉（ Ｓｃｌｅｒｏｃｙｓｔｉｓ　ｓｉｎｕｏｓａ　Ｇｅｒｄｅｍａｎｎ　＆ Ｂａｋｈｉ）对棉花（Ｇｏｓｓｙｐｉｕｍ　ｈｉｒｓｕｔｕｍ　Ｌ．）、烟草（Ｎｉｃｏｔｉａｎａ　 ｔａｂａｃｕｍ Ｌ．）和白     

Arbuscular mycorrhizal fungal community structures differ between co-occurring tree species of dry Afromontane tropical forest, and their seedlings exhibit potential to trap isolates suited for reforestation

The diversity of arbuscular mycorrhizal (AM) fungi and their broad or narrow association with distinct plant species in natural environments are crucial information in the understanding of the ecological role of AM fungi on plant co-existence. This knowledge is also needed for appropriate mycorrhization of nursery-grown seedlings for forestation efforts. Here, we report results from comparative studies on three co-occurring indigenous tree species of the dry Afromontane forests of Ethiopia and their seedlings grown under controlled conditions in soil collected from the sites. AM fungal SSU rDNA fragment was amplified and sequenced from mycorrhizas of adult plants and seedlings of Olea europaea subsp. cuspidata and Prunus africana, and from Podocarpus falcatus seedlings. AM fungal identity, diversity and community structure were analyzed based on sequence types defined by the NS31-AM1 SSU rDNA fragment similarity in order to compare with data from other habitats. A total of 409 sequences, grouped in 32 sequence types, belonging to Glomeraceae, Diversisporaceae and Gigasporaceae were found. Some sequence types are close to the widespread Glomus intraradices, G. hoi, G. etunicatum, G. cf. etunicatum and Gigaspora margarita. However, the majority (59%) of sequence types are so far specific for the sites including 11 new types when compared with previous data from the same area. The AM fungal community associated with adult plants, including data previously obtained from adult Podocarpus falcatus seedlings, and seedlings of a host species differed significantly, where seedlings trapping a surprising large number of native fungi. AM fungal community structure also differed significantly between host species and sites, respectively. The results confirm previous results from the same area indicating distinct fungal communities associated with the diverse tree species and suggests the potential of these indigenous tree seedlings to trap a wide range of AM fungi appropriate for successful afforestation.     

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。     

Arbuscular mycorrhizal colonization of tomato by Gigaspora and Glomus species in the presence of root flavonoids

The effect of flavonoids isolated from arbuscular mycorrhizal (AM) colonized and noncolonized clover roots on the number of entry points and percentage of root colonization of tomato (Lycopersicum esculentum L.) by Gigaspora rosea, Gi margarita, Glomus mosseae and G. intrarradices symbionts was determined. With fungi of both genera, a correlation between the number of entry points and the percentage of root colonization was found in the presence of some of the tested flavonoids. The flavonoids acacetin and rhamnetin, present in AM clover roots, inhibited the formation of AM penetration structures and the AM colonization of tomato roots, whereas the flavonoid 5,6,7,8,9-hydroxy chalcone, which could not be detected in AM clover root, inhibited both parameters. The flavonoid quercetin, which was present in AM clover roots, stimulated the penetration and root colonization of tomato by Gigaspora. However, the flavonoids 5,6,7,8-hydroxy-4'-methoxy flavone and 3,5,6,7,4'-hydroxy flavone, which was not found in AM clover root, increased the number of entry points and the AM colonization of tomato roots by Gigaspora. These results indicated that flavonoids could be imnplicated in the process of regulation of AM colonization in plant root, but its role is highly complex and depend not only on flavonoids, but also on AM fungal genus or even species.     

The pre-symbiotic growth of arbuscular mycorrhizal fungi is induced by a branching factor partially purified from plant root exudates

Arbuscular mycorrhizal (AM) symbiosis is an association between obligate biotrophic fungi and more than 80% of land plants. During the pre-symbiotic phase, the host plant releases critical metabolites necessary to trigger fungal growth and root colonization. We describe the isolation of a semipurified fraction from exudates of carrot hairy roots, highly active on germinating spores of Gigaspora gigantea, G. rosea, and G. margarita. This fraction, isolated on the basis of its activity on hyphal branching, contains a root factor (one or several molecules) that stimulates, directly or indirectly, G. gigantea nuclear division. We demonstrate the presence of this active factor in root exudates of all mycotrophic plant species tested (eight species) but not in those of nonhost plant species (four species). We negatively tested the hypothesis that it was a flavonoid or a compound synthesized via the flavonoid pathway. We propose that this root factor, yet to be chemically characterized, is a key plant signal for the development of AM fungi.     

A diffusible factor from arbuscular mycorrhizal fungi induces symbiosis-specific MtENOD11 expression in roots of Medicago truncatula

Using dual cultures of arbuscular mycorrhizal (AM) fungi and Medicago truncatula separated by a physical barrier, we demonstrate that hyphae from germinating spores produce a diffusible factor that is perceived by roots in the absence of direct physical contact. This AM factor elicits expression of the Nod factor-inducible gene MtENOD11, visualized using a pMtENOD11-gusA reporter. Transgene induction occurs primarily in the root cortex, with expression stretching from the zone of root hair emergence to the region of mature root hairs. All AM fungi tested (Gigaspora rosea, Gigaspora gigantea, Gigaspora margarita, and Glomus intraradices) elicit a similar response, whereas pathogenic fungi such as Phythophthora medicaginis, Phoma medicaginis var pinodella and Fusarium solani f.sp. phaseoli do not, suggesting that the observed root response is specific to AM fungi. Finally, pMtENOD11-gusA induction in response to the diffusible AM fungal factor is also observed with all three M. truncatula Nod(-)/Myc(-) mutants (dmi1, dmi2, and dmi3), whereas the same mutants are blocked in their response to Nod factor. This positive response of the Nod(-)/Myc(-) mutants to the diffusible AM fungal factor and the different cellular localization of pMtENOD11-gusA expression in response to Nod factor versus AM factor suggest that signal transduction occurs via different pathways and that expression of MtENOD11 is differently regulated by the two diffusible factors.     

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.     

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     

Role of the glutamic and aspartic residues in Na+-ATPase function in the ZrENA1 gene of Zygosaccharomyces rouxii

An in vitro system using Ri T-DNA transformed carrot roots as the host bearing simultaneously different arbuscular mycorrhizal (AM) fungi belonging to two genera Glomus intraradices (Smith and Schenck) and Gigaspora margarita (Becker and Hall) was developed. Co-existence appeared healthy and harmonious, as both the generic species showed extensive hyphal proliferation and sporulation. The co-culture model under the in vitro system appears especially appropriate for further investigations on the competition and on the interaction mechanism involved in such types of associations occurring in nature and also as a model approach towards mass production of multiple mycorrhizal fungal isolates. This is a first report of successful co-culture of two genera of AM fungi under in vitro conditions.     

Group I Intron-Mediated Trans-splicing in Mitochondria of Gigaspora rosea and a Robust Phylogenetic Affiliation of Arbuscular Mycorrhizal Fungi with Mortierellales

Gigaspora rosea is a member of the arbuscular mycorrhizal fungi (AMF; Glomeromycota) and a distant relative of Glomus species that are beneficial to plant growth. To allow for a better understanding of Glomeromycota, we have sequenced the mitochondrial DNA of G. rosea. A comparison with Glomus mitochondrial genomes reveals that Glomeromycota undergo insertion and loss of mitochondrial plasmid-related sequences and exhibit considerable variation in introns. The gene order between the two species is almost completely reshuffled. Furthermore, Gigaspora has fragmented cox1 and rns genes, and an unorthodox initiator tRNA that is tailored to decoding frequent UUG initiation codons. For the fragmented cox1 gene, we provide evidence that its RNA is joined via group I-mediated trans-splicing, whereas rns RNA remains in pieces. According to our model, the two cox1 precursor RNA pieces are brought together by flanking cox1 exon sequences that form a group I intron structure, potentially in conjunction with the nad5 intron 3 sequence. Finally, we present analyses that address the controversial phylogenetic association of Glomeromycota within fungi. According to our results, Glomeromycota are not a separate group of paraphyletic zygomycetes but branch together with Mortierellales, potentially also Harpellales.     

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的侵染率呈现出先上升后下降的趋势.本实验设计了特异性扩增Glomus mosseae和Gigaspora margarita的引物gml和gigl,在混合接种实验中,nested PCR扩增结果显示:在低pH水平下(4.3-5.1)大多数根段为Gigaspora margarita所侵染,在高pH水平下(5.8-6.8)Glomusmosseae表现出较强的竞争力,但并没有检测到两种VA真菌存在于同一条侵染根段;对比单接种实验,在低pH水平下,Glomus mosseae显著抑制了Gigaspora margarita的侵染,而在高pH水平下Gigasporamargarita明显促进Glomus mosseae的侵染.     

丛枝菌根真菌种间差异对柚苗营养生长及矿质含量的影响

在温室盆栽条件下研究了3种AM真菌Gigaspora margarita、Glomus mosseae和Glomus versiforme对长寿沙田柚(Citrus grandis cv. Changshou Shatian You)无菌苗营养生长及矿质含量的影响.结果表明,接种AM真菌的植株均有效地被感染;与对照相比,接种AM真菌能显著促进植株地上和地下部分生长,尤其促进了须根的生长,接种Glomus mosseae处理的主根长度比对照增加了22.7％,侧根数量增加了35.7％,须根数量和总长分别增加了160.8％和103.2％;接种AM真菌显著地提高了叶片的N、P、K、Ca、Mg、Zn、Cu和Mn含量,与对照相比,3种真菌处理的P含量分别增加了46.8％、88.7％和32.3％.3种AM真菌处理中,以接种Glomus mosseae处理营养生长最好,菌根依赖性最大,矿质元素N、P、K、Ca、Zn和Cu的含量最高,其效应顺序为Glomus mosseae＞Gigaspora margarita＞Glomus versiforme,可见,3种AM真菌对长寿沙田柚生长均有正效应,以Glomus mosseae最为显著,为长寿沙田柚适宜的优良菌种（株）.     

大豆毛状根-VA菌根真菌双重培养体系的建立

以大豆毛状根为宿主,接种VA菌根真菌珠状巨孢囊霉（Gigaspora margarita）,经过3.5个月的双重培养,观察到VA菌根真菌珠状巨孢囊霉对大豆毛状根的侵染,辅助细胞形成,并获得VA菌根真菌成熟孢子,在无菌条件下建立了大豆毛状根-VA菌根真菌双重培养体系,为研究菌根真菌侵染大豆根部形成共生体系及相关分子机制提供了一种有效的研究方法。     

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.     

GR24, a synthetic analog of strigolactones, stimulates the mitosis and growth of the arbuscular mycorrhizal fungus Gigaspora rosea by boosting its energy metabolism

Arbuscular mycorrhizal (AM) fungi are obligate biotrophs that participate in a highly beneficial root symbiosis with 80% of land plants. Strigolactones are trace molecules in plant root exudates that are perceived by AM fungi at subnanomolar concentrations. Within just a few hours, they were shown to stimulate fungal mitochondria, spore germination, and branching of germinating hyphae. In this study we show that treatment of Gigaspora rosea with a strigolactone analog (GR24) causes a rapid increase in the NADH concentration, the NADH dehydrogenase activity, and the ATP content of the fungal cell. This fully and rapidly (within minutes) activated oxidative metabolism does not require new gene expression. Up-regulation of the genes involved in mitochondrial metabolism and hyphal growth, and stimulation of the fungal mitotic activity, take place several days after this initial boost to the cellular energy of the fungus. Such a rapid and powerful action of GR24 on G. rosea cells suggests that strigolactones are important plant signals involved in switching AM fungi toward full germination and a presymbiotic state.     

Differential RNA accumulation of two β-tubulin genes in arbuscular mycorrhizal fungi

RNA was isolated from spores of different arbuscular mycorrhizal (AM) fungi and used for RT-PCR with degenerate primers for beta-tubulin genes. PCR products were cloned and the sequence of several clones was analysed for each fragment. Comparison of sequences identified two loci for beta-tubulin genes with different GC content and codon usage. Btub1 sequences were most similar to beta-tubulin genes from the Oomycota, while Btub2 sequences showed highest similarity to sequences from the Zygomycota. RT-PCR experiments were carried out to monitor RNA accumulation patterns of Btub1 and Btub2 in asymbiotic germinating spores and in symbiotic extraradical hyphae of three different AM fungi. This indicated that Btub1 is constitutively expressed in Gigaspora rosea, but down-regulated during symbiosis in Glomus mosseae and Glomus intraradices. In contrast, Btub2 showed constitutive expression in the two Glomus species, but down-regulation in G. rosea. Further analysis of different fungi indicated that Btub2 primers could be used to specifically monitor RNA accumulation of AM fungi in environmental samples.     

Glomus, the largest genus of the arbuscular mycorrhizal fungi (Glomales), is nonmonophyletic.

Arbuscular mycorrhizal (AM) fungi form a widespread and ecologically important symbiosis with plants in the land ecosystem. The phylogeny of the largest presently accepted genus, Glomus, of the monogeneric family Glomaceae (Glomales; AM fungi) was analyzed. Phylogenetic trees were computed from nearly full-length SSU rRNA gene sequences of 30 isolates, and show that "Glomus" is not monophyletic. Even after the very recent separation of Archaeospora and Paraglomus from "Glomus," the genus further separates into two suprageneric clades. One of them diverges further into two subclades, differing by phylogenetic distances equivalent to family level. The other, comprising Glomus versiforme, G. spurcum, and a species morphologically similar to G. etunicatum, is not closely related to the Glomaceae, but clusters together with the Acaulosporaceae and Gigasporaceae in a monophyletic clade. Based on the molecular evidence, a new family, separate from the Glomaceae, is required to accommodate this group of organisms, initially named Diversisporaceae fam. ined. The current taxonomic concept of the recently erected family Archaeosporaceae also requires future emendation, because Geosiphon pyriformis (Geosiphonaceae) renders Archaeospora, the sole genus formally included in this family, paraphyletic. The suborders Gigasporineae and Glominaeae are not congruent with the natural phylogeny of the AM fungi. Our data necessitate a general reexamination of the generic concepts within the Glomales. In addition to the new family structure hypothesized herein, establishment of at least three new genera will be necessary in the future.