RESUMEN

A new glomeromycotan fungus, Archaeospora ecuadoriana sp. nov., was found in the south Ecuadorian mountain rainforest region, a global plant biodiversity hotspot. It was cultivated as single spore isolate originating from nursery-grown native tree seedlings inoculated with mixed soil from pristine forest and agricultural fields. The new species is known from the Loja area, southern Ecuador, at about 2100 m above mean sea level (mamsl) and has been detected in potato roots from an Andean region in Peru at 2658 mamsl by previous molecular data. The fungus forms small, colourless to frosted white, mainly globose spores, averaging 61 × 60 µm, formed singly or very rarely in clusters. There is no reaction to Melzer's reagent, other than a slight unspecific overall yellow iodine staining. The spores are very similar to those of Archaeospora trappei and A. schenckii. However, molecular phylogenetic analysis shows the species to be clearly separate from all other described Archaeospora species. The analysis of the available Archaeospora sequence data shows that sequences of Palaeospora spainiae, of the monospecific genus Palaeospora, cluster within the genus Archaeospora. Palaeospora therefore is synonymised with Archaeospora and P. spainiae is transferred to Archaeospora, as A. spainiae comb. nov.

Asunto(s)

Glomeromycota/clasificación , Clasificación , Ecuador , Glomeromycota/genética , Perú , ARN de Hongos/análisis , ARN Ribosómico/análisis , Análisis de Secuencia de ARN

RESUMEN

Nutrient acquisition and transfer are essential steps in the arbuscular mycorrhizal (AM) symbiosis, which is formed by the majority of land plants. Mineral nutrients are taken up by AM fungi from the soil and transferred to the plant partner. Within the cortical plant root cells the fungal hyphae form tree-like structures (arbuscules) where the nutrients are released to the plant-fungal interface, i.e., to the periarbuscular space, before being taken up by the plant. In exchange, the AM fungi receive carbohydrates from the plant host. Besides the well-studied uptake of phosphorus (P), the uptake and transfer of nitrogen (N) plays a crucial role in this mutualistic interaction. In the AM fungus Rhizophagus irregularis (formerly called Glomus intraradices), two ammonium transporters (AMT) were previously described, namely GintAMT1 and GintAMT2. Here, we report the identification and characterization of a newly identified R. irregularis AMT, GintAMT3. Phylogenetic analyses revealed high sequence similarity to previously identified AM fungal AMTs and a clear separation from other fungal AMTs. Topological analysis indicated GintAMT3 to be a membrane bound pore forming protein, and GFP tagging showed it to be highly expressed in the intraradical mycelium of a fully established AM symbiosis. Expression of GintAMT3 in yeast successfully complemented the yeast AMT triple deletion mutant (MATa ura3 mep1Δ mep2Δ::LEU2 mep3Δ::KanMX2). GintAMT3 is characterized as a low affinity transport system with an apparent Km of 1.8 mM and a V max of 240 nmol(-1) min(-1) 10(8) cells(-1), which is regulated by substrate concentration and carbon supply.

RESUMEN

Arbuscular mycorrhizal fungi (AMF) are obligate symbionts of most land plants. They have great ecological and economic importance as they can improve plant nutrition, plant water supply, soil structure, and plant resistance to pathogens. We describe two approaches for the DNA-based characterization and identification of AMF, which both can be used for single fungal spores, soil, or roots samples and resolve closely related AMF species: (a) Sanger sequencing of a 1.5 kb extended rDNA-barcode from clone libraries, e.g., to characterize AMF isolates, and (b) high throughput 454 GS-FLX+ pyrosequencing of a 0.8 kb rDNA fragment, e.g., for in-field monitoring.

Asunto(s)

Secuenciación de Nucleótidos de Alto Rendimiento , Micorrizas/genética , Raíces de Plantas/microbiología , Análisis de Secuencia de ADN/métodos , ADN de Hongos/genética , Micorrizas/clasificación , Micorrizas/aislamiento & purificación , Plantas/genética , Esporas Fúngicas/genética

RESUMEN

In many deforested regions of the tropics, afforestation with native tree species could valorize a growing reservoir of degraded, previously overused and abandoned land. The inoculation of tropical tree seedlings with arbuscular mycorrhizal fungi (AM fungi) can improve tree growth and viability, but efficiency may depend on plant and AM fungal genotype. To study such effects, seven phylogenetically diverse AM fungi, native to Ecuador, from seven genera and a non-native AM fungus (Rhizophagus irregularis DAOM197198) were used to inoculate the tropical potential crop tree (PCT) species Handroanthus chrysanthus (synonym Tabebuia chrysantha), Cedrela montana, and Heliocarpus americanus. Twenty-four plant-fungus combinations were studied in five different fertilization and AMF inoculation treatments. Numerous plant growth parameters and mycorrhizal root colonization were assessed. The inoculation with any of the tested AM fungi improved seedling growth significantly and in most cases reduced plant mortality. Plants produced up to threefold higher biomass, when compared to the standard nursery practice. AM fungal inoculation alone or in combination with low fertilization both outperformed full fertilization in terms of plant growth promotion. Interestingly, root colonization levels for individual fungi strongly depended on the host tree species, but surprisingly the colonization strength did not correlate with plant growth promotion. The combination of AM fungal inoculation with a low dosage of slow release fertilizer improved PCT seedling performance strongest, but also AM fungal treatments without any fertilization were highly efficient. The AM fungi tested are promising candidates to improve management practices in tropical tree seedling production.

Asunto(s)

Micorrizas/fisiología , Plantones/crecimiento & desarrollo , Plantones/microbiología , Árboles/crecimiento & desarrollo , Árboles/microbiología , Biodiversidad , Biomasa , Ecuador , Fertilizantes , Genotipo , Glomeromycota/crecimiento & desarrollo , Micorrizas/clasificación , Micorrizas/genética , Filogenia , Desarrollo de la Planta , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/microbiología , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/microbiología , Plantones/efectos de los fármacos , Semillas/crecimiento & desarrollo , Semillas/microbiología , Suelo , Árboles/efectos de los fármacos

RESUMEN

For more than 450 million years, arbuscular mycorrhizal fungi (AMF) have formed intimate, mutualistic symbioses with the vast majority of land plants and are major drivers in almost all terrestrial ecosystems. The obligate plant-symbiotic AMF host additional symbionts, so-called Mollicutes-related endobacteria (MRE). To uncover putative functional roles of these widespread but yet enigmatic MRE, we sequenced the genome of DhMRE living in the AMF Dentiscutata heterogama. Multilocus phylogenetic analyses showed that MRE form a previously unidentified lineage sister to the hominis group of Mycoplasma species. DhMRE possesses a strongly reduced metabolic capacity with 55% of the proteins having unknown function, which reflects unique adaptations to an intracellular lifestyle. We found evidence for transkingdom gene transfer between MRE and their AMF host. At least 27 annotated DhMRE proteins show similarities to nuclear-encoded proteins of the AMF Rhizophagus irregularis, which itself lacks MRE. Nuclear-encoded homologs could moreover be identified for another AMF, Gigaspora margarita, and surprisingly, also the non-AMF Mortierella verticillata. Our data indicate a possible origin of the MRE-fungus association in ancestors of the Glomeromycota and Mucoromycotina. The DhMRE genome encodes an arsenal of putative regulatory proteins with eukaryotic-like domains, some of them encoded in putative genomic islands. MRE are highly interesting candidates to study the evolution and interactions between an ancient, obligate endosymbiotic prokaryote with its obligate plant-symbiotic fungal host. Our data moreover may be used for further targeted searches for ancient effector-like proteins that may be key components in the regulation of the arbuscular mycorrhiza symbiosis.

Asunto(s)

Bacterias/genética , Técnicas de Transferencia de Gen , Mosaicismo , Micorrizas , Genoma Bacteriano , Datos de Secuencia Molecular

RESUMEN

The world's fourth largest food crop, potato, originates in the Andes. Here, the community composition of arbuscular mycorrhizal fungi (AMF) associated with potato in Andean ecosystems is described for the first time. AMF were studied in potato roots and rhizosphere soil at four different altitudes from 2,658 to 4,075 m above mean sea level (mamsl) and in three plant growth stages (emergence, flowering, and senescence). AMF species were distinguished by sequencing an approx. 1,500 bp nuclear rDNA region. Twenty species of AMF were identified, of which 12 came from potato roots and 15 from rhizosphere soil. Seven species were found in both roots and soil. Interestingly, altitude affected species composition with the highest altitude exhibiting the greatest species diversity. The three most common colonizers of potato roots detected were Funneliformis mosseae, an unknown Claroideoglomus sp., and Rhizophagus irregularis. Notably, the potato-associated AMF diversity observed in this Andean region is much higher than that reported for potato in other ecosystems. Potato plants were colonized by diverse species from 8 of the 11 Glomeromycota families. Identification of the AMF species is important for their potential use in sustainable management practices to improve potato production in the Andean region.

Asunto(s)

Biodiversidad , Micorrizas/clasificación , Micorrizas/aislamiento & purificación , Solanum tuberosum/microbiología , Altitud , Análisis por Conglomerados , ADN de Hongos/química , ADN de Hongos/genética , ADN Ribosómico/química , ADN Ribosómico/genética , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Genes de ARNr , Datos de Secuencia Molecular , Micorrizas/genética , Perú , Filogenia , ARN de Hongos/genética , ARN Ribosómico/genética , ARN Ribosómico 18S/genética , ARN Ribosómico 5.8S/genética , Rizosfera , Análisis de Secuencia de ADN , Microbiología del Suelo

RESUMEN

The nuclear ribosomal internal transcribed spacer (ITS) region is the formal fungal barcode and in most cases the marker of choice for the exploration of fungal diversity in environmental samples. Two problems are particularly acute in the pursuit of satisfactory taxonomic assignment of newly generated ITS sequences: (i) the lack of an inclusive, reliable public reference data set and (ii) the lack of means to refer to fungal species, for which no Latin name is available in a standardized stable way. Here, we report on progress in these regards through further development of the UNITE database (http://unite.ut.ee) for molecular identification of fungi. All fungal species represented by at least two ITS sequences in the international nucleotide sequence databases are now given a unique, stable name of the accession number type (e.g. Hymenoscyphus pseudoalbidus|GU586904|SH133781.05FU), and their taxonomic and ecological annotations were corrected as far as possible through a distributed, third-party annotation effort. We introduce the term 'species hypothesis' (SH) for the taxa discovered in clustering on different similarity thresholds (97-99%). An automatically or manually designated sequence is chosen to represent each such SH. These reference sequences are released (http://unite.ut.ee/repository.php) for use by the scientific community in, for example, local sequence similarity searches and in the QIIME pipeline. The system and the data will be updated automatically as the number of public fungal ITS sequences grows. We invite everybody in the position to improve the annotation or metadata associated with their particular fungal lineages of expertise to do so through the new Web-based sequence management system in UNITE.

Asunto(s)

Bases de Datos de Ácidos Nucleicos , Hongos/clasificación , Filogenia , Código de Barras del ADN Taxonómico , ADN de Hongos/genética , ADN Espaciador Ribosómico/genética , Hongos/genética , Internet

RESUMEN

Members of the Glomeromycota form the arbuscular mycorrhiza (AM) symbiosis. They supply plants with inorganic nutrients, including nitrogen, from the soil. To gain insight into transporters potentially facilitating nitrogen transport processes, ammonium transporters (AMTs) of Geosiphon pyriformis, a glomeromycotan fungus forming a symbiosis with cyanobacteria, were studied. Three AMT genes were identified, and all three were expressed in the symbiotic stage. The localization and functional characterization of the proteins in a heterologous yeast system revealed distinct characteristics for each of them. AMT1 of G. pyriformis (GpAMT1) and GpAMT2 were both plasma membrane localized, but only GpAMT1 transported ammonium. Neither protein transported the ammonium analogue methylammonium. Unexpectedly, GpAMT3 was localized in the vacuolar membrane, and it has as-yet-unknown transport characteristics. An unusual cysteine residue in the AMT signature of GpAMT2 and GpAMT3 was identified, and the corresponding residue was demonstrated to play an important role in ammonium transport. Surprisingly, each of the three AMTs of G. pyriformis had very distinct features. The localization of an AMT in the yeast vacuolar membrane is novel, as is the described amino acid residue that clearly influences ammonium transport. The AMT characteristics might reflect adaptations to the lifestyle of glomeromycotan fungi.

Asunto(s)

Compuestos de Amonio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Proteínas Fúngicas/metabolismo , Glomeromycota/metabolismo , Secuencia de Aminoácidos , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Glomeromycota/genética , Membranas Intracelulares/metabolismo , Datos de Secuencia Molecular , Vacuolas/metabolismo

RESUMEN

The publication of a large number of taxon names at all levels within the arbuscular mycorrhizal fungi (Glomeromycota) has resulted in conflicting systematic schemes and generated considerable confusion among biologists working with these important plant symbionts. A group of biologists with more than a century of collective experience in the systematics of Glomeromycota examined all available molecular-phylogenetic evidence within the framework of phylogenetic hypotheses, incorporating morphological characters when they were congruent. This study is the outcome, wherein the classification of Glomeromycota is revised by rejecting some new names on the grounds that they are founded in error and by synonymizing others that, while validly published, are not evidence-based. The proposed "consensus" will provide a framework for additional original research aimed at clarifying the evolutionary history of this important group of symbiotic fungi.

Asunto(s)

Glomeromycota/clasificación , Micorrizas/clasificación , Consenso , Evolución Molecular , Glomeromycota/genética , Glomeromycota/crecimiento & desarrollo , Glomeromycota/aislamiento & purificación , Micorrizas/genética , Micorrizas/crecimiento & desarrollo , Micorrizas/aislamiento & purificación , Filogenia , Esporas Fúngicas/clasificación , Esporas Fúngicas/genética , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/aislamiento & purificación

RESUMEN

In legumes rhizobial infection during root nodule symbiosis (RNS) is controlled by a conserved set of receptor proteins and downstream components. MtSYMREM1, a protein of the Remorin family in Medicago truncatula, was shown to interact with at least three receptor-like kinases (RLKs) that are essential for RNS. Remorins are comprised of a conserved C-terminal domain and a variable N-terminal region that defines the six different Remorin groups. While both N- and C-terminal regions of Remorins belonging to the same phylogenetic group are similar to each other throughout the plant kingdom, the N-terminal domains of legume-specific group 2 Remorins show exceptional high degrees of sequence divergence suggesting evolutionary specialization of this protein within this clade. We therefore identified and characterized the MtSYMREM1 ortholog from Lotus japonicus (LjSYMREM1), a model legume that forms determinate root nodules. Here, we resolved its spatio-temporal regulation and showed that over-expression of LjSYMREM1 increases nodulation on transgenic roots. Using a structure-function approach we show that protein interactions including Remorin oligomerization are mainly mediated and stabilized by the Remorin C-terminal region with its coiled-coil domain while the RLK kinase domains transiently interact in vivo and phosphorylate a residue in the N-terminal region of the LjSYMREM1 protein in vitro. These data provide novel insights into the mechanism of this putative molecular scaffold protein and underline its importance during rhizobial infection.

Asunto(s)

Proteínas Portadoras/química , Proteínas Portadoras/fisiología , Lotus , Fosfoproteínas/química , Fosfoproteínas/fisiología , Proteínas de Plantas/química , Proteínas de Plantas/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Evolución Molecular , Fabaceae/genética , Fabaceae/metabolismo , Regulación de la Expresión Génica de las Plantas , Especiación Genética , Lotus/genética , Lotus/metabolismo , Lotus/fisiología , Medicago truncatula/genética , Medicago truncatula/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Unión Proteica , Multimerización de Proteína/genética , Estructura Terciaria de Proteína/fisiología , Nódulos de las Raíces de las Plantas/genética , Nódulos de las Raíces de las Plantas/metabolismo , Relación Estructura-Actividad , Simbiosis/genética , Simbiosis/fisiología , Transfección

RESUMEN

Although the molecular phylogeny, evolution and biodiversity of arbuscular mycorrhizal fungi (AMF) are becoming clearer, phylotaxonomically reliable sequence data are still limited. To fill this gap, a data set allowing resolution and environmental tracing across all taxonomic levels is provided. Two overlapping nuclear DNA regions, totalling c. 3 kb, were analysed: the small subunit (SSU) rRNA gene (up to 1800 bp) and a fragment spanning c. 250 bp of the SSU rDNA, the internal transcribed spacer (ITS) region (c. 475-520 bp) and c. 800 bp of the large subunit (LSU) rRNA gene. Both DNA regions together could be analysed for 35 described species, the SSU rDNA for c. 76 named and 18 as yet undefined species, and the ITS region or LSU rDNA, or a combination of both, for c. 91 named and 16 as yet undefined species. Present phylogenetic analyses, based on the three rDNA markers, provide reliable and robust resolution from phylum to species level. Altogether, 109 named species and 27 cultures representing as yet undefined species were analysed. This study provides a reference data set for molecular systematics and environmental community analyses of AMF, including analyses based on deep sequencing.

Asunto(s)

Micorrizas/clasificación , Micorrizas/genética , Filogenia , Clasificación/métodos , ADN de Hongos , ADN Ribosómico , Hongos/clasificación , Hongos/genética , Glomeromycota/clasificación , Glomeromycota/genética , Datos de Secuencia Molecular , ARN Ribosómico , ARN Ribosómico 5.8S

RESUMEN

Sequence analysis of the ribosomal RNA operon, particularly the internal transcribed spacer (ITS) region, provides a powerful tool for identification of mycorrhizal fungi. The sequence data deposited in the International Nucleotide Sequence Databases (INSD) are, however, unfiltered for quality and are often poorly annotated with metadata. To detect chimeric and low-quality sequences and assign the ectomycorrhizal fungi to phylogenetic lineages, fungal ITS sequences were downloaded from INSD, aligned within family-level groups, and examined through phylogenetic analyses and BLAST searches. By combining the fungal sequence database UNITE and the annotation and search tool PlutoF, we also added metadata from the literature to these accessions. Altogether 35,632 sequences belonged to mycorrhizal fungi or originated from ericoid and orchid mycorrhizal roots. Of these sequences, 677 were considered chimeric and 2,174 of low read quality. Information detailing country of collection, geographical coordinates, interacting taxon and isolation source were supplemented to cover 78.0%, 33.0%, 41.7% and 96.4% of the sequences, respectively. These annotated sequences are publicly available via UNITE (http://unite.ut.ee/) for downstream biogeographic, ecological and taxonomic analyses. In European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena/), the annotated sequences have a special link-out to UNITE. We intend to expand the data annotation to additional genes and all taxonomic groups and functional guilds of fungi.

Asunto(s)

ADN Espaciador Ribosómico/genética , Bases de Datos de Ácidos Nucleicos , Geografía , Internet , Micorrizas/genética , Operón de ARNr/genética , Ecología , Cooperación Internacional , Raíces de Plantas/genética

RESUMEN

BACKGROUND: Understanding the mechanisms underlying biological phenomena, such as evolutionarily conservative trait inheritance, is predicated on knowledge of the natural relationships among organisms. However, despite their enormous ecological significance, many of the ubiquitous soil inhabiting and plant symbiotic arbuscular mycorrhizal fungi (AMF, phylum Glomeromycota) are incorrectly classified. METHODOLOGY/PRINCIPAL FINDINGS: Here, we focused on a frequently used model AMF registered as culture BEG47. This fungus is a descendent of the ex-type culture-lineage of Glomus epigaeum, which in 1983 was synonymised with Glomus versiforme. It has since then been used as 'G. versiforme BEG47'. We show by morphological comparisons, based on type material, collected 1860-61, of G. versiforme and on type material and living ex-type cultures of G. epigaeum, that these two AMF species cannot be conspecific, and by molecular phylogenetics that BEG47 is a member of the genus Diversispora. CONCLUSIONS: This study highlights that experimental works published during the last >25 years on an AMF named 'G. versiforme' or 'BEG47' refer to D. epigaea, a species that is actually evolutionarily separated by hundreds of millions of years from all members of the genera in the Glomerales and thus from most other commonly used AMF 'laboratory strains'. Detailed redescriptions substantiate the renaming of G. epigaeum (BEG47) as D. epigaea, positioning it systematically in the order Diversisporales, thus enabling an evolutionary understanding of genetical, physiological, and ecological traits, relative to those of other AMF. Diversispora epigaea is widely cultured as a laboratory strain of AMF, whereas G. versiforme appears not to have been cultured nor found in the field since its original description.

Asunto(s)

Glomeromycota/clasificación , Micorrizas/clasificación , Secuencia de Bases , ADN Ribosómico/genética , Glomeromycota/citología , Glomeromycota/genética , Datos de Secuencia Molecular , Micorrizas/citología , Micorrizas/genética , Filogenia , Esporas Fúngicas/citología , Esporas Fúngicas/fisiología

RESUMEN

Spores of two supposedly arbuscular mycorrhizal fungal species, new to the United Kingdom and recently described as Acaulospora alpina and Ambispora brasiliensis (Glomeromycota), were discovered in soil samples from moorland in upland Scotland. Soil and plant trap pot cultures were established, but attempts to establish these fungi in single-species pot cultures with Plantago lanceolata as host were unsuccessful. Nevertheless, based on a 1.5-kb DNA fragment spanning part of the small subunit rRNA gene, the internal transcribed spacer region and part of the large subunit rRNA gene, both these species could be detected directly in field-sampled roots, together with one uncultured species each of Scutellospora, Rhizophagus (former Glomus group Ab, or 'Glomus intraradices clade') and Acaulospora. Whereas A. alpina has characteristic morphological similarities to other species in its genus, A. brasiliensis morphologically has little in common with any other species in Ambispora. The molecular phylogeny, DNA barcoding and morphological evidence clearly place A. brasiliensis in the genus Acaulospora. We therefore rename the species, reported from Brazil and Scotland, as Acaulospora brasiliensis comb. nov., and discuss ecological aspects of the very different environments from which A. brasiliensis and A. alpina have been reported.

Asunto(s)

ADN de Hongos/genética , Glomeromycota/clasificación , Glomeromycota/aislamiento & purificación , Micorrizas/clasificación , Micorrizas/aislamiento & purificación , Raíces de Plantas/microbiología , Microbiología del Suelo , ADN Espaciador Ribosómico/genética , Glomeromycota/genética , Glomeromycota/crecimiento & desarrollo , Datos de Secuencia Molecular , Técnicas de Tipificación Micológica , Micorrizas/genética , Micorrizas/crecimiento & desarrollo , Filogenia , Plantas/microbiología , Escocia , Esporas Fúngicas/clasificación , Esporas Fúngicas/genética , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/aislamiento & purificación

RESUMEN

SUMMARY: *Currently, no official DNA barcode region is defined for the Fungi. The COX1 gene DNA barcode is difficult to apply. The internal transcribed spacer (ITS) region has been suggested as a primary barcode candidate, but for arbuscular mycorrhizal fungi (AMF; Glomeromycota) the region is exceptionably variable and does not resolve closely related species. *DNA barcoding analyses were performed with datasets from several phylogenetic lineages of the Glomeromycota. We tested a c. 1500 bp fragment spanning small subunit (SSU), ITS region, and large subunit (LSU) nuclear ribosomal DNA for species resolving power. Subfragments covering the complete ITS region, c. 800 bp of the LSU rDNA, and three c. 400 bp fragments spanning the ITS2, the LSU-D1 or LSU-D2 domains were also analysed. *Barcode gap analyses did not resolve all species, but neighbour joining analyses, using Kimura two-parameter (K2P) distances, resolved all species when based on the 1500 bp fragment. The shorter fragments failed to separate closely related species. *We recommend the complete 1500 bp fragment as a basis for AMF DNA barcoding. This will also allow future identification of AMF at species level based on 400 or 1000 bp amplicons in deep sequencing approaches.

Asunto(s)

ADN de Hongos/genética , Procesamiento Automatizado de Datos/métodos , Glomeromycota/genética , Micorrizas/genética , Análisis de Secuencia de ADN/métodos , Secuencia de Bases , Núcleo Celular/genética , ADN Espaciador Ribosómico/genética , Bases de Datos Genéticas , Variación Genética , Filogenia , Especificidad de la Especie

RESUMEN

Arbuscular mycorrhizal fungi (AMF) have been symbionts of land plants for at least 450 Myr. It is known that some AMF host in their cytoplasm Gram-positive endobacteria called bacterium-like organisms (BLOs), of unknown phylogenetic origin. In this study, an extensive inventory of 28 cultured AMF, from diverse evolutionary lineages and four continents, indicated that most of the AMF species investigated possess BLOs. Analyzing the 16S ribosomal DNA (rDNA) as a phylogenetic marker revealed that BLO sequences from divergent lineages all clustered in a well-supported monophyletic clade. Unexpectedly, the cell-walled BLOs were shown to likely represent a sister clade of the Mycoplasmatales and Entomoplasmatales, within the Mollicutes, whose members are lacking cell walls and show symbiotic or parasitic lifestyles. Perhaps BLOs maintained the Gram-positive trait whereas the sister groups lost it. The intracellular location of BLOs was revealed by fluorescent in situ hybridization (FISH), and confirmed by pyrosequencing. BLO DNA could only be amplified from AMF spores and not from spore washings. As highly divergent BLO sequences were found within individual fungal spores, amplicon libraries derived from Glomus etunicatum isolates from different geographic regions were pyrosequenced; they revealed distinct sequence compositions in different isolates. Our results show a vertically inherited, monophyletic and globally distributed lineage of endobacteria thriving in AMF cytoplasm. These bacteria split from their sister groups more than 400 Myr ago, colonizing their fungal hosts already before main AMF lineages separated. The BLO-AMF symbiosis can, therefore, be dated back at least to the time when AMF formed the ancestral symbiosis with emergent land plants.

Asunto(s)

Citoplasma/microbiología , Evolución Molecular , Glomeromycota/ultraestructura , Micorrizas , Tenericutes/clasificación , Glomeromycota/clasificación , Glomeromycota/genética , Glomeromycota/fisiología , Hibridación Fluorescente in Situ , Filogenia , Plantas/microbiología , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Esporas Fúngicas/ultraestructura , Simbiosis , Tenericutes/genética , Tenericutes/aislamiento & purificación

RESUMEN

Glomus intraradices-like fungi are the most intensely studied arbuscular mycorrhizal (AM) fungi. However, there are several AM fungi named as G. intraradices that may not be conspecific. Therefore, the hypothesis was tested that DAOM197198 and similar AM fungi, such as BEG195, correspond to the type of G. intraradices. The G. intraradices isotype material, a descendant (INVAM FL208) of the type culture, and a morphologically corresponding AM fungus (MUCL49410) isolated from the type locality were studied and compared with several cultures of DAOM197198 and BEG195. Phylogenetic analyses of the partial small subunit (SSU), complete internal transcribed spacer (ITS) and partial large subunit (LSU) nuclear rDNA regions revealed two clades, one including G. intraradices FL208 and MUCL49410, the other containing DAOM197198 and BEG195. The two clades were clearly separated by sequence analyses, despite the high intraspecific and intrasporal ITS region sequence divergence of up to > 23%. We conclude that the AM fungi with the identifiers DAOM197198 and BEG195 are not G. intraradices, but fall in a clade that contains the recently described species G. irregulare.

Asunto(s)

ADN de Hongos , Glomeromycota/clasificación , Micorrizas , Filogenia , Secuencia de Bases , ADN Ribosómico , ADN Espaciador Ribosómico , Glomeromycota/genética , Micorrizas/genética , Micorrizas/aislamiento & purificación , Subunidades Ribosómicas , Análisis de Secuencia de ADN , Especificidad de la Especie

RESUMEN

* At present, molecular ecological studies of arbuscular mycorrhizal fungi (AMF) are only possible above species level when targeting entire communities. To improve molecular species characterization and to allow species level community analyses in the field, a set of newly designed AMF specific PCR primers was successfully tested. * Nuclear rDNA fragments from diverse phylogenetic AMF lineages were sequenced and analysed to design four primer mixtures, each targeting one binding site in the small subunit (SSU) or large subunit (LSU) rDNA. To allow species resolution, they span a fragment covering the partial SSU, whole internal transcribed spacer (ITS) rDNA region and partial LSU. * The new primers are suitable for specifically amplifying AMF rDNA from material that may be contaminated by other organisms (e.g., samples from pot cultures or the field), characterizing the diversity of AMF species from field samples, and amplifying a SSU-ITS-LSU fragment that allows phylogenetic analyses with species level resolution. * The PCR primers can be used to monitor entire AMF field communities, based on a single rDNA marker region. Their application will improve the base for deep sequencing approaches; moreover, they can be efficiently used as DNA barcoding primers.

Asunto(s)

Cartilla de ADN , ADN Ribosómico , Genes Fúngicos , Glomeromycota/genética , Micorrizas/genética , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , ADN Intergénico , Filogenia , Subunidades Ribosómicas Grandes de Eucariotas , Subunidades Ribosómicas Pequeñas de Eucariotas , Especificidad de la Especie

RESUMEN

The increasing numbers of taxonomically unassigned phylotypes reported in molecular ecological studies contrast with the few formally described arbuscular mycorrhizal fungi (AMF; Glomeromycota). Here, a species new to science with Glomus-like spores is phylogenetically, morphologically and ecologically characterized. From single spore isolates of a previously recognized member of the Diversisporaceae from Swiss agricultural grassland, 17 new nuclear internal transcribed spacer (ITS), large subunit (LSU) and small subunit (SSU) ribosomal RNA (rRNA) gene sequences were determined and compared with 14 newly generated sequences of two close relatives and public database sequences, including environmental sequences, of known geographic origin. SSU ribosomal DNA (rDNA) sequence signatures and phylogenies based on ITS, LSU and SSU rDNA sequences show that the fungus belongs to the genus Diversispora. It is described as Diversispora celata sp. nov. Comparison with environmental sequences in the public domain confirmed its molecular genetic distinctiveness and revealed a cross-continental distribution of close relatives. The value of combining morphology and phylogeny to characterize AMF was reinforced by the morphological similarity to other species and the inconspicuous nature of D. celata spores and mycorrhizas. Inclusion of all three nuclear rDNA regions in species descriptions will facilitate species determination from environmental phylotypes.

Asunto(s)

Glomeromycota/clasificación , Micorrizas/clasificación , Filogenia , Geografía , Glomeromycota/genética , Glomeromycota/aislamiento & purificación , Micorrizas/genética , Micorrizas/aislamiento & purificación , ARN Ribosómico , Análisis de Secuencia de ARN , Esporas Fúngicas , Suiza

RESUMEN

The AM fungal family Archaeosporaceae and the genus Archaeospora are rendered paraphyletic by the relationship with the Geosiphonaceae. This problem led to a more detailed study of the Archaeosporales. Members of the Archaeosporaceae were described as forming both glomoid and acaulosporoid spores, or solely acaulosporoid spores. However, we found that Glomus callosum fell into the same phylogenetic clade as A. leptoticha and A. gerdemannii, but exclusively formed glomoid spores. To resolve these inconsistencies, a genus, Ambispora gen. nov., typified by Ambispora fennica sp. nov., is erected based on morphological evidence and SSU and ITS region rDNA data. Ambispora contains three species known to produce both acaulosporoid and glomoid spores: A. fennica, A. leptoticha comb. nov. (basionym G. leptotichum), and A. gerdemannii comb. nov. (basionym G. gerdemannii). Another species, A. callosa comb. nov. (basionym G. callosum), is known only from glomoid spores. Ambispora is placed in a new family, the Ambisporaceae fam. nov. The Archaeosporaceae is maintained with the type species, Archaeospora trappei (basionym Acaulospora trappei), along with Intraspora schenckii (basionym Entrophospora schenckii). Acaulospora nicolsonii, known only from acaulosporoid spores, is discussed and is considered likely to belong in the Ambisporaceae, but is retained within its present genus because of inadequate morphological information and a lack of molecular data.

Asunto(s)

Micorrizas/genética , Microbiología del Suelo , Secuencia de Bases , Clasificación , ADN de Hongos/química , ADN de Hongos/genética , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Datos de Secuencia Molecular , Micorrizas/clasificación , Micorrizas/ultraestructura , Filogenia , Reacción en Cadena de la Polimerasa , Alineación de Secuencia , Esporas Fúngicas/clasificación , Esporas Fúngicas/genética , Esporas Fúngicas/ultraestructura