Geosiphon pyriforme,an Endosymbiotic Association of Fungus and Cyanobacteria: the Spore Structure Resembles that of Arbuscular Mycorrhizal (AM) Fungi

The zygomycete Geosiphon pyriforme is the only known endocyanosis of a fungus. The Nostoc spp. filaments are included in photosynthetically active and nitrogen fixing, multinucleated bladders, which grow on the soil surface. The spores of the fungus are white or slightly brownish. They are about 250 μm in diameter and develop singly on hyphal ends or, less frequently, intercalarly. The wall of the spores consists of a thin innermost layer, a laminated inner layer with a thickness of about 10–13 μm, and an evanescent outer layer. The laminated layer is composed of helicoidally arranged microfibrils, and is separated from the evanescent outer layer by a thin electron-dense sublayer. Polarisation microscopy indicates the occurrence of chitin. Shape and wall ultrastructure of the Geosiphon spores and their cytoplasm resemble that of Glomus spores, but are different from that of other genera of the Glomales and Endogonales. Germination occurs by a single thick hyphal outgrowth directly through the spore wall. Like various AM forming fungi, Geosiphon pyriforme contains endocytic bacteria-like organisms, which are not surrounded by a host membrane. Our observations indicate that Geosiphon is a potential AM fungus.     

The fungal bladders of the endocyanosisGeosiphon pyriforme,aGlomus-related fungus: cell wall permeability indicates a limiting pore radius of only 0.5 nm

Summary Geosiphon pyriforme, a consortium of aGlomiw-like fungus andNostoc spp., forms syncytial, up to 2 mm long bladders accommodating the endosymbiotic cyanobacteria. The bladders are bordered by an elastic cell wall and have a turgor of about 0.6 MPa, as measured by piercing them with oil filled microcapillaries within different osmolarities of sorbitol. In the presence of certain organic osmolytes in the surrounding medium, the bladders collapsed, i.e., showed cytorrhysis. We studied systematically the cytorrhytic effectivity of the diverse osmolytes in relation to their hydrodynamic molecule radii by a solute-exclusion method with living bladders and those which have been extracted by different methods. The results suggest that the cell wall of the bladders has an unusually small limiting pore size thus representing an effective diffusion barrier for glucose and is virtually impermeable for sucrose for at least 8 h. The pore radii of the cell wall are estimated to be about 0.5 nm. Na₂CO₃ extraction, frequently used to partially extract pectic substances from plant cell walls, strongly increases wall permeability. Electron microscopic observations show an electron-dense outer cell wall layer, perhaps responsible for the low permeability. The finding that the cell wall of theGeosiphon bladders represents an effective osmotic barrier provides not only new insights into the cell physiology ofGeosiphon but may also contribute more generally to a better understanding of the mechanisms of selectivity of transport across the cell walls of AM fungi.Abbreviations AM arbuscular mycorrhiza - DMSO dimethyl sulfoxyde - EDTA ethylenediaminetetraacetic acid - PEG polyethylene glycol - res Einstein-Stokes hydrodynamic radius     

Molecular phylogeny,taxonomy, and evolution of Geosiphon pyriformis and arbuscular mycorrhizal fungi

Geosiphon pyriformis(Kütz.) v. Wettstein is the only known example of a fungus living in endocytobiotic association with a cyanobacterium. The close phylogenetic relationship of Geosiphonwith some arbuscular mycorrhizal fungi (AMF) and the phylogenetic position of Geosiphonare shown in detail. Comprehensive small subunit (SSU) rRNA sequence analyses allow the erection of a new, molecular phylogeny-based taxonomic system for the AMF, including Geosiphon (Geosiphonaceae). Within the recently described phylum Glomeromycota (with one class, Glomeromycetes), a system including four orders was proposed. The erection of several new families will also be necessary. Evolutionary implications are discussed, referring to different possibilities of the influence of AMF on the colonization of the terrestrial habitat by plants.     

Studies on initiation and development of the partner association inGeosiphon pyriforme (Kütz.) v. Wettstein,a unique endocytobiotic system of a fungus (Glomales) and the cyanobacteriumNostoc punctiforme (Kütz.) Hariot

Summary Phagocytosis ofNostoc filaments byGeosiphon, a fungus closely related to AM forming Glomales, was observed under light microscopes. Incorporation can only be performed ifNostoc primordia come into contact with growing hyphal tips of the fungus. The fungal wall just below the apex softens, and fungal cytoplasm is bulged out repeatedly covering the vegetativeNostoc cells but not the heterocytes. New heterocytes are differentiated by the internalised filament whose cells can increase up to ten times in volume after recovering from incorporation strain. TheNostoc cells are coated stepwise by short finger-shaped protuberances of the fungal hypha. These hernia-like outgrowths first remain separated, after 1 to 2 days they merge. Adjacent hyphal walls inside the complex covering disintegrate. Periphal fungal wall portions are united to form a smooth strong outer envelope. Internalisation is categorised as phagocytosis. The partnership is partly specific,Nostoc strains capable of living endocytobiotically are often partners in other symbioses besidesGeosiphon.Abbreviations AM arbuscular mycorrhiza (formerly VAM vesicular arbuscular mycorrhiza) - DIC differential interference contrast - LD light/dark Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Seasonal variation of arbuscular mycorrhizal fungi in temperate grasslands along a wide hydrologic gradient

We studied seasonal variation in population attributes of arbuscular mycorrhizal (AM) fungi over 2 years in four sites of temperate grasslands of the Argentinean Flooding Pampas. The sites represent a wide range of soil conditions, hydrologic gradients, and floristic composition. Lotus glaber, a perennial herbaceous legume naturalised in the Flooding Pampas, was dominant at the four plant community sites. Its roots were highly colonised by AM fungi. Temporal variations in spore density, spore type, AM root colonisation, floristic composition and soil chemical characteristics occurred in each site and were different among sites. The duration of flooding had no effect on spore density but depressed AM root colonisation. Eleven different types of spores were recognized and four were identified. Two species dominated at the four sites: Glomus fasciculatum and Glomus intraradices. Spore density was highest in summer (dry season) and lowest in winter (wet season) with intermediate values in autumn and spring. Colonisation of L. glaber roots was highest in summer or spring and lowest in winter or autumn. The relative density of G. fasciculatum and G. intraradices versus Glomus sp. and Acaulospora sp. had distinctive seasonal peaks. These seasonal peaks occurred at all four sites, suggesting differences among AM fungus species with respect to the seasonality of sporulation. Spore density and AM root colonisation when measured at any one time were poorly related to each other. However, spore density was significantly correlated with root colonisation 3 months before, suggesting that high colonisation in one season precedes high sporulation in the next season.     

Arbuscular mycorrhizal fungi associated with common pteridophytes in Dujiangyan,southwest China

The colonization and diversity of arbuscular mycorrhizal (AM) fungi associated with common pteridophytes were investigated in Dujiangyan, southwest China. Of the 34 species of ferns from 16 families collected, 31 were colonized by AM fungi. The mean percentage root length colonized was 15%, ranging from 0 to 47%. Nineteen species formed Paris-type and 10 intermediate-type AM. In two ferns, only rare intercellular non-septate hyphae or vesicles were observed in the roots and AM type could not be determined. Of the 40 AM fungal taxa belonging to five genera isolated from rooting-zone soils, 32 belonged to Glomus, five to Acaulospora, one to Archaeospora, one to Entrophospora, and one to Gigaspora. Acaulospora and Glomus were the dominant genera and Glomus versiforme was the most common species. The average AM spore density was 213 per 100 g air-dried soil and the average species richness was 3.7 AM species per soil sample. There was no correlation between spore density and percentage root length colonized by AM fungi.     

Phylogenetic position of an arbuscular mycorrhizal fungus,Acaulospora gerdemannii, and its synanamorphGlomus leptotichum, based upon 18S rRNA gene sequence

We examined the phylogenetic position of an arbuscular mycorrhizal fungus which produces two types of spore,Acaulospora gerdemannii andGlomus leptotichum, based upon the DNA sequence of the 18S rRNA gene. DNA was extracted separately from bothGlomus-like orAcaulospora-like spores and partial 5′-terminus segments of 18S rRNA gene were amplified by the PCR method. Several clones derived from each spore type were sequenced and compared. The sequences from both spore types agreed well, confirming that these morphologically different spores were formed by the same fungus. Nucleotide substitutions were found among several clones, suggesting polymorphism of the rRNA gene in glomalean fungi. Further phylogenetic analysis based upon the whole sequence of the 18S rRNA gene showed thatA. gerdemannii may be within the order Glomales but is far from the fungi that have been analyzed and probably should be in a new family.     

Vesicular-arbuscular mycorrhizal fungi in coastal dune plant communities I. Spore formation ofGlomus spp. predominates under a patch ofElymus mollis

Vesicular-arbuscular (VA) mycorrhizal fungi in pure patches of coastal dune plantsElymus mollis, Wedelia prostrata andZoysia macrostachya were examined for frequency of occurrence and number of spores of VA mycorrhizal fungi over one year. Six species in three genera of VA mycorrhizal fungi were recovered. Under a patch ofE. mollis, spores ofAcaulospora sp. 1,Glomus tortuosum, Glomus sp. 1,Glomus sp. 2 andScutellospora gregaria were recovered. Spores ofGlomus spp. were most common. In patches ofW. prostrata andZ. macrostachya spores ofAcaulospora sp. 1,G. tortuosum, Glomus sp. 1,Glomus sp. 2,S. gregaria andScutellospora sp. 1 were found.Contribution No. 112, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Geosiphon pyriforme,an Endosymbiotic Consortium of a Fungus and a Cyanobacterium (Nostoc), Fixes Nitrogen

The bladders of Geosiphon pyriforme, an endosymbiotic consortium of a fungus and the cyanobacterium Nostoc punctiforme, show nitrogenase activity. This suggests that the organism is capable of nitrogen fixation.     

Identification of arbuscular mycorrhizal fungi in soils and roots of plants colonizing zinc wastes in southern Poland

 Analysis of the community of arbuscular mycorrhizal (AM) fungi in roots of Fragaria vesca growing in a heavy metal contaminated site was carried out on a Zn waste site near Chrzanow (southern Poland). The waste substratum was characterized by high contents of Pb, Zn, Cd, Cu and As, and by low levels of N, P and organic matter. Spores of Glomales were isolated by wet sieving and DNA was isolated from individual spores. Nested polymerase chain reaction (PCR) with taxon-specific primers was used to identify the species Glomus mosseae, Glomus intraradices and Glomus claroideum. Spores of other fungi were morphologically characterized and new taxon-discriminating molecular probes were developed for two of them (Glomus sp. HM-CL4 and HM-CL5) based on variations in the large ribosomal subunit (25S rDNA). High sequence similarities were found between Glomus sp. HM-CL4 and Glomus gerdemanii, and between Glomus sp. HM-CL5 and Glomus occultum. The designed primers were used to characterize the population of AM fungi colonizing the roots of F. vesca collected from the Zn waste site. The analysis, carried out on roots stained with trypan blue, showed that the most effective colonizer was closely related to G. gerdemannii. G. claroideum and the G. occultum-like fungus were slightly less common whilst frequencies of G. intraradices and G. mosseae in roots were much lower. The analysis of mycorrhiza stained with rhodizoniate to localize heavy metal accumulation showed that the stain does not influence the PCR reaction. Seventy percent of the root samples containing positively stained fungal hyphae were found to be colonized by G. mosseae. The data obtained demonstrate the usefulness of nested PCR for studies carried out in polluted areas. It will enable selection of AM fungi which are able to colonize plant roots under heavy metal stress conditions, as well as the identification of fungi showing high in situ accumulation of potentially toxic elements. Accepted: 7 July 2000     

Photosynthetic carbon assimilation in Geosiphon pyriforme (Kützing) F. v. Wettstein,an endosymbiotic association of fungus and cyanobacterium

Geosiphon pyriforme, an endosymbiotic association between a fungus and the cyanobacterium Nostoc, was shown by tracer studies to acquire carbon photosynthetically from CO₂ or bicarbonate. The organism also fixes inorganic carbon in darkness, at lower rates than in the light. The patterns of label distribution are indicative of the operation of the reductive pentose-phosphate pathway in the light and of the phosphoenolpyruvate-carboxylase reaction in the dark. The results are discussed in relation to the evolution of photoautotrophic endosymbiotic associations.This investigation has been supported by the Deutsche Forschungsgemeinschaft. We are indepted to Professor G.B. Feige (Institut für Pflanzenphysiologie, Universität Essen, FRG) for helpful discussion, Karin Faist for her excellent technical assitance and to Doris Schäfer for drawing the figures.     

Characterization of theGeosiphon pyriforme symbiosome by affinity techniques: confocal laser scanning microscopy (CLSM) and electron microscopy

Summary Geosiphon pyriforme represents a photoautotrophic endosymbiosis of aGlomus-like fungus with the cyanobacteriumNostoc punctiforme. The fungus forms unicellular bladders of up to 2 mm in length and 0.5 mm in diameter growing on the soil surface and harboring the endosymbioticNostoc filaments. The cyanobacteria are located in a compartment (the symbiosome) bordered by a host membrane. The space between this symbiosome membrane (SM) and theNostoc cell wall is filled with an about 30–40 nm thick layer of amorphous material, which is present also in the regions of the symbiosome where noNostoc filaments are located. At these sites the amorphous material consists of a 20–30 nm thick layer separating the SM. The region between the SM and the cyanobacterium is defined as symbiosome space (SS). Fungal bladders, hyphae and free livingNostoc were analyzed by affinity techniques as well as the material occurring in the SS. FITC-coupled lectins with sugar specificity to -D-mannosyl/-D-glucosyl (Con A), N-acetyl--D-glucosamine oligomers (WGA), -L-fucosyl (UEA-I), -D-galactosyl (RCA-120), -D-galactosyl (BS-I-B₄), N-acetyl--D-galactosamine (HPA), and sialic acid (EBL) residues were tested. WGA binding and calcofluor white staining demonstrated that the bladder wall as well as the SS contain fibrillar chitin. Of the other lectins only Con A clearly labeled the symbiosome. On the contrary, the lectin binding properties of the slime produced by free livingNostoc-colonies indicate the presence of mannose, fucose, GalNAc, sialic acid, and galactose, while chitin or GlucNAc-oligomers could not be detected. The symbiosome was also investigated electron microscopically. WGA-gold binding confirmed the presence of chitin, while a slight PATAg reaction indicated some polysaccharidic molecules within the SS. Our results show that the amorphous material within the SS contains molecules typical of the fungal cell wall and suggest that the SM is related to the fungal plasma membrane. The applied lectins all bind to the hyphal surface, indicating a high molecular complexity. Mannosyl, -galactosyl, and sialic acid residues are strongly exposed at the outer cell wall layer, whereas GlucNAc, GalNAc, and -galactosyl residues seem to be present in smaller amounts. The symbiotic interface established between the fungus andNostoc inGeosiphon shows many similarities to that occurring between fungi and root cells in arbuscular mycorrhizas.Abbreviations AM arbuscular mycorrhiza - BS-I-B₄ Bandeiraea simplicifolia lectin I isolectin B₄ - CLSM confocal laser scanning microscopy - Con A Concanavalin A - EBL elderberry bark lectin I - FITC fluorescein isothiocyanate - HPA Helix pomatia agglutinin - PATAg periodic acid-thiocarbohydrazide-Ag proteinate - SM symbiosome membrane - SS symbiosome space - RCA-120 Ricinus communis agglutinin 120 - UEA-I Ulex europaeus agglutinin I - WGA wheat germ agglutinin Dedicated to Professor Dr. Peter Sitte at the occasion of his 65th birthday     

Arbuscular mycorrhizal colonization of the dominant plant species in primary successional volcanic deserts on the Southeast slope of Mount Fuji

Arbuscular mycorrhizal (AM) colonization was observed on four plant species in primary successional volcanic deserts on the Southeast slope of Mount Fuji. The AM colonization of the dominant species, Polygonum cuspidatum, contradicts the conclusion that Polygonaceae are often regarded as being non-mycorrhizal species. The secondary dominant species, Polygonum weyrichii var. alpinum, formed no mycorrhizas. The roots of Cirsium purpuratum, Clematis stans and Campanula punctata ssp. hondoensis, showed a higher percentage of AM colonization than P. cuspidatum. AM colonization and spore density in the rhizosphere soil of P. cuspidatum significantly decreased as elevation increased. AM colonization in roots of Cirsium purpuratum and Clematis stans also tended to decrease with increased altitudes. Cirsium purpuratum and Campanula punctata ssp. hondoensis formed single structural types of Arum- and Paris-type, respectively, whereas P. cuspidatum and Clematis stans formed both Arum- and Paris-type morphologies.     

Nothobartsia,a new genus ofScrophulariaceae from southwest Europe

Recent systematic studies ofBartsia andOdontites showed the necessity to exclude the closely related perennial speciesBartsia aspera (Portugal and northern Morocco) andBartsia spicata (Central Pyrenees) from the genusBartsia as a segregate genus namedNothobartsia. Morphologically this new genus combines characteristic features ofBartsia and ofOdontites. Its autonomous systematic position is supported by the cladistic analysis, showing thatNothobartsia represents a relatively primitive genus standing close to the common ancestral root ofBartsia, Euphrasia, andOdontites.     

Improvement by soil yeasts of arbuscular mycorrhizal symbiosis of soybean (<Emphasis Type="Italic">Glycine max</Emphasis>) colonized by<Emphasis Type="Italic"> Glomus mosseae</Emphasis>

The effects of the soil yeasts Rhodotorula mucilaginosa, Cryptococcus laurentii and Saccharomyces kunashirensis on the arbuscular mycorrhizal (AM) fungus Glomus mosseae (BEG 12) was studied in vitro and in greenhouse trials. The presence of yeasts or their soluble and volatile exudates stimulated the percentage spore germination and hyphal growth of G. mosseae. Percentage root length colonized by G. mosseae and plant dry matter of soybean (Glycine max L. Merill) were increased only when the soil yeasts were inoculated prior to the AM fungus. Higher beneficial effects on AM colonization and plant dry matter were found when the soil yeasts were inoculated as an aqueous solution rather than as a thin agar slice. Although soluble and volatile exudates of yeasts benefited the AM symbiosis, their modes of action were different.This revised version was published online in May 2004 with corrections to the section of the article.     

Assessment of arbuscular mycorrhizal fungi diversity in the rhizosphere of <Emphasis Type="Italic">Viola calaminaria</Emphasis> and effect of these fungi on heavy metal uptake by clover

 The ability of arbuscular mycorrhizal (AM) fungi from a metal-tolerant plant (Viola calaminaria, violet) to colonise and reduce metal uptake by a non-tolerant plant (Trifolium subterraneum, subterranean clover) in comparison to a metal-tolerant AM fungus isolated from a non-tolerant plant was studied. AM spores from the violet rhizosphere and from violet roots were characterised by polymerase chain reaction (PCR) amplification of the SSU rDNA, and sequencing. Subterranean clover was grown in pots containing a soil supplemented with Cd and Zn salts and inoculated either with a mixture of spores extracted from the violet rhizosphere or with spores of a Cd-tolerant Glomus mosseae P2 (BEG 69), or non-inoculated. The diversity of fungi, including AM fungi, colonising clover roots was assessed and analysed using terminal-restriction fragment length polymorphism. At least four different Glomus species were found in the violet rhizosphere. After 8 weeks in a growth chamber, colonisation of clover roots with spores from the violet rhizosphere increased Cd and Zn concentrations in clover roots without significantly affecting the concentrations of metals in the shoot and plant growth. G. mosseae P2 reduced plant growth and slightly increased the Cd concentration. Only one AM fungus (Glomus b) from the violet rhizosphere colonised clover roots, but other fungi were present. AM fungi from heavy metal-contaminated soils and associated with metal-tolerant plants may be effective in accumulating heavy metals in roots in a non-toxic form. Accepted: 7 July 2000     

Presence of different arbuscular mycorrhizal infection patterns in roots of<Emphasis Type="Italic"> Lotus glaber</Emphasis> plants growing in the Salado River basin

Morphological types of arbuscular mycorrhizal (AM) fungi associated with Lotus glaber in sodic soils of the Salado River basin were studied. At least eight colonization patterns (IP) of AM fungi in roots of L. glaber were observed after 30 plants were analyzed. Arum- and Paris-type infection were found in the same plant species. This result supports the idea that AM morphology is not solely under plant control, but is also influenced by fungal identity. One infection pattern, presumably corresponding to Glomus intraradices, and a second, possibly assignable to Glomus tenue, were the most commonly found. Our results reinforce previous suggestions that G. intraradices is well adapted to sodic-saline conditions and may play a role in the resistance of L. glaber to these soils.     

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.     

Studies on the diversity of the distinct phylogenetic lineage encompassing Glomus claroideum and Glomus etunicatum

Morphological and molecular characters were analysed to investigate diversity within isolates of the Glomus claroideum/Glomus etunicatum species group in the genus Glomus. The inter- and intra-isolate sequence diversity of the large subunit (LSU) rRNA gene D2 region of eight isolates of G. claroideum and G. etunicatum was studied using PCR-single strand conformational polymorphism (SSCP)-sequencing. In addition, two isolates recently obtained from Southern China were included in the analysis to allow for a wider geographic screening. Single spore DNA isolation confirmed the magnitude of gene diversity found in multispore DNA extractions. An apparent overlap of spore morphological characters was found between G. claroideum and G. etunicatum in some isolates. Analysis of the sequence frequencies in all G. etunicatum and G. claroideum isolates (ten) showed that four LSU D2 sequences, representing 32.1% of the clones analysed for multispore extraction (564) were found to be common to both species, and those sequences were the most abundant in four of the ten isolates analysed. The frequency of these sequences ranged between 23.2% and 87.5% of the clones analysed in each isolate. The implications for the use of phenotypic characters to define species in arbuscular mycorrhizal fungi are discussed. The current position of G. claroideum/G.etunicatum in the taxonomy of the Glomeromycota is also discussed.     

Phylogeny of theAsterales sensu lato based onrbcL sequences with particular reference to theGoodeniaceae

TherbcL gene of 25 taxa was sequenced and analyzed cladistically in order to define more precisely the orderAsterales s.l. and to reconstruct the phylogeny ofGoodeniaceae. The cladistic analyses show that theAsterales comprise the familiesAbrophyllaceae, Alseuosmiaceae, Argophyllaceae, Asteraceae, Calyceraceae, Campanulaceae s.l.,Donatiaceae, Goodeniaceae (includingBrunoniaceae),Menyanthaceae, Pentaphragmataceae, andStylidiaceae. Abrophyllaceae, Alseuosmiaceae, Brunoniaceae, andDonatiaceae have previously not been studied in this respect. Within theGoodeniaceae, four groups supported by therbcL data can be distinguished: the genusLechenaultia, theAnthotium-Dampiera-group, the genusBrunonia, and a group formed by the remaining genera, theScaevola-Goodenia-group.