Morphological and Molecular Evidence of Arbuscular Mycorrhizal Fungal Associations in Costa Rican Epiphytic Bromeliads1

Arbuscular mycorrhizal fungi influence the growth, morphology, and fitness of a variety of plant species, but little is known of the arbuscular mycorrhizal (AM) fungal associations of plant species in forest canopies. Plant species' associations with AM fungi are most often elucidated by examining the roots for fungal structures; however, morphological data may provide a limited resolution on a plant's mycorrhizal status. We combined a traditional staining technique with a molecular marker (the 18S ribosomal gene) to determine whether or not a variety of epiphytic bromeliads form arbuscular mycorrhizal fungal associations. Using these methods we show that the epiphytic bromeliad Vriesea werkleana forms arbuscular mycorrhizal fungal associations with members of the genus Glomus. AM fungal sequences of this plant species formed three distinct clades nested within a larger Glomus clade; two of the clades did not group with any previously sequenced lineage of Glomus. Novel clades may represent novel species. Although Vriesea werkleana is associated with multiple AM fungal species, each individual plant is colonized by a single lineage. The combination of morphological and molecular methods provides a practical approach to the characterization of the mycorrhizal status of epiphytic bromeliads, and perhaps other tropical epiphytes.     

Accumulation of reactive oxygen species in arbuscular mycorrhizal roots

We investigated the accumulation of reactive oxygen species (ROS) in arbuscular mycorrhizal (AM) roots from Medicago truncatula, Zea mays and Nicotiana tabacum using three independent staining techniques. Colonized root cortical cells and the symbiotic fungal partner were observed to be involved in the production of ROS. Extraradical hyphae and spores from Glomus intraradices accumulated small levels of ROS within their cell wall and produced ROS within the cytoplasm in response to stress. Within AM roots, we observed a certain correlation of arbuscular senescence and H₂O₂ accumulation after staining by diaminobenzidine (DAB) and a more general accumulation of ROS close to fungal structures when using dihydrorhodamine 123 (DHR 123) for staining. According to electron microscopical analysis of AM roots from Z. mays after staining by CeCl₃, intracellular accumulation of H₂O₂ was observed in the plant cytoplasm close to intact and collapsing fungal structures, whereas intercellular H₂O₂ was located on the surface of fungal hyphae. These characteristics of ROS accumulation in AM roots suggest similarities to ROS accumulation during the senescence of legume root nodules.     

The diversity of arbuscular mycorrhizal fungi in the subtropical forest of Huangshan (Yellow Mountain), East-Central China

The world heritage of Huangshan is located in the east-central China. In order to obtain a better overview of biodiversity in Huangshan, we investigated the diversity of arbuscular mycorrhizal fungi in the soil of Huangshan. Forty-two rhizosphere soil samples were collected and 989 arbuscular mycorrhizal fungal spore samples were obtained using the wet-sieving method. Twenty-five species of arbuscular mycorrhizal fungi were identified from the collections. The species were of the genera Acaulospora (6 species), Entrophospora (1 species), Glomus (16 species) and Scutellospora (2 species). Acaulospora and Glomus were dominant at the study site. The arbuscular mycorrhizal fungi spore density ranged from 45 to 3,250 per 100 g soil (average 839), and the species richness of arbuscular mycorrhizal fungi ranged from 1 to 9 (average 4.2) per soil sample. Shannon–Wiener index and Simpson’s index were calculated to evaluate the arbuscular mycorrhizal fungal diversity. The diversity of arbuscular mycorrhizal fungal community in the subtropical forest of Huangshan may be the result of mutual selection between arbuscular mycorrhizal fungi and the ecological environment.     

On the perils of mycorrhizal status lists: the case of <Emphasis Type="Italic">Buddleja davidii</Emphasis>

One observation in a mycorrhizal check-list that Buddleja davidii is nonmycorrhizal has been perpetuated in subsequent citations and used in a number of analyses of mycorrhizal ecology and evolution. Direct observation of B. davidii from New Zealand and the UK shows extensive arbuscular mycorrhizal fungal structures inside B. davidii roots. The suggestion that B. davidii is nonmycorrhizal is therefore not supported. The use of mycorrhizal checklists for analysis of plant traits and evolution needs to be undertaken with care to ensure the validity of underlying data.     

Trypan blue as a fluorochrome for confocal laser scanning microscopy of arbuscular mycorrhizae in three mangroves

Roots of three mangroves, Acanthus ilicifolius, Ceriops tagal and Excoecaria agallocha, collected from forests of the Sundarbans of India were stained with trypan blue to observe arbuscular mycorrhizal colonization. Spores of arbuscular mycorrhizal fungi isolated from rhizospheric soil, collected together with the root samples, also were stained for testing the suitability of the dye as a fluorochrome. Confocal laser scanning microscopy images were constructed. A. ilicifolius and E. agallocha exhibited “Arum” type colonization with highly branched arbuscules, whereas C. tagal showed “Paris” type association with clumped and collapsed arbuscules. We demonstrated that trypan blue is a suitable fluorochrome for staining arbuscular mycorrhizal fungal spores, fungal hyphae, arbuscules and vesicles, which presumably have a considerable amount of surface chitin. It appears that as the integration of chitin into the fungal cell wall changes, its accessibility to trypan blue dye also changes.     

Nested multiplex PCR—A feasible technique to study partial community of arbuscular mycorrhizal fungi in field-growing plant root

Plant can be infected by different arbuscular mycorrhizal fungi, but little is known about the interaction between them within root tissues mainly because different species cannot be distinguished on the basis of fungal structure. Accurate species identification of Arbuscular mycorrhizal fungi (AMF) colonized in plant roots is the comerstone of mycorrhizal study, yet this fundamental step is impossible through its morphological character alone. For accurate, rapid and inexpensive detection of partial mycorrhizal fungal community in plant roots, a nested multiplex polymerase chain reaction (PCR) was developed in this study. Five discriminating primers designed based on the variable region of the 5′ end of the large ribosomal subunit were used in the experiment for testing their specificity and the sensitivity in nested PCR by using spores from Glomus mosseae (BEG12), Glomus intraradices (BEG141), Scutellospora castaneae (BEG1) and two unidentified Glomus sp. HAUO3 and HAUO4. The feasibility assay of nested multiplex PCR was conducted by use of spore mixture, Astragalus sinicum roots co-inoculated with 4 species of arbuscular mycorrhizal fungi from pot cultures and 15 different field-growing plant roots respectively after analyses of the compatibility of primers. The result indicated that the sensitivity was in the same range as that of the corresponding single PCR reaction. Overall accuracy was 95%. The efficiency and sensitivity of this multiplex PCR procedure provided a rapid and easy way to simultaneously detect several of arbuscular mycorrhizal fungal species in a same plant root system.     

Nested multiplex PCR——A feasible technique to study partial community of arbuscular mycorrhizal fungi in field-growing plant root

Plant can be infected by different arbuscular mycorrhizal fungi, but little is known about the interaction between them within root tissues mainly because different species cannot be distinguished on the basis of fungal structure. Accurate species identification of Arbuscular mycorrhizal fungi (AMF) colonized in plant roots is the comerstone of mycorrhizal study, yet this fundamental step is impossible through its morphological character alone. For accurate, rapid and inexpensive detection of partial mycorrhizal fungal community in plant roots, a nested multiplex polymerase chain reaction (PCR) was developed in this study. Five discriminating primers designed based on the variable region of the 5′ end of the large ribosomal subunit were used in the experiment for testing their specificity and the sensitivity in nested PCR by using spores from Glomus mosseae (BEG12), Glomus intraradices (BEG141), Scutellospora castaneae (BEG1) and two unidentified Glomus sp. HAUO3 and HAUO4. The feasibility assay of nested multiplex PCR was conducted by use of spore mixture, Astragalus sinicum roots co-inoculated with 4 species of arbuscular mycorrhizal fungi from pot cultures and 15 different field-growing plant roots respectively after analyses of the compatibility of primers. The result indicated that the sensitivity was in the same range as that of the corresponding single PCR reaction. Overall accuracy was 95%. The efficiency and sensitivity of this multiplex PCR procedure provided a rapid and easy way to simultaneously detect several of arbuscular mycorrhizal fungal species in a same plant root system.     

Analysis of the cell cycle in an arbuscular mycorrhizal fungus by flow cytometry and bromodeoxyuridine labelling

Summary The cell cycle of an arbuscular mycorrhizal fungus,Glomus versiforme, was determined by flow cytometric analysis of nuclei isolated from spores and mycorrhizal roots of leek, and by immunogold staining after bromodeoxyuridine (BrdU) uptake by DNA. The aims of our work were to establish: (i) whether there are changes in ploidy during fungal growth and morphogenesis, (ii) when and where the cell cycle is activated. Our results demonstrate that nuclei isolated from quiescent spores ofG. versiforme are arrested in the GO/G1 phase (99.2%), whereas fungal nuclei from mycorrhizal roots are in the synthetic (S) (10.1%) and G2/M phase (3.9%). Nuclei undergoing DNA synthesis were detected in situ after BrdU uptake. Labelled nuclei were observed in intercellular hyphae and in large arbuscular trunks. This paper demonstrates that colonization of an arbuscular mycorrhizal fungus is linked to activation of its cell cycle.Abbreviations AM fungi arbuscular mycorrhizal fungi - BrdU 5-bromo-2-deoxyuridine - PI propidium iodide - DAPI 4,6-diamidino-2-phenylindole     

Fungal root endophytes of the carnivorous plant Drosera rotundifolia

As carnivorous plants acquire substantial amounts of nutrients from the digestion of their prey, mycorrhizal associations are considered to be redundant; however, fungal root endophytes have rarely been examined. As endophytic fungi can have profound impacts on plant communities, we aim to determine the extent of fungal root colonisation of the carnivorous plant Drosera rotundifolia at two points in the growing season (spring and summer). We have used a culture-dependent method to isolate fungal endophytes and diagnostic polymerase chain reaction methods to determine arbuscular mycorrhizal fungi colonisation. All of the roots sampled contained culturable fungal root endophytes; additionally, we have provided molecular evidence that they also host arbuscular mycorrhizal fungi. Colonisation showed seasonal differences: Roots in the spring were colonised by Articulospora tetracladia, two isolates of uncultured ectomycorrhizal fungi, an unidentified species of fungal endophyte and Trichoderma viride, which was present in every plant sampled. In contrast, roots in the summer were colonised by Alatospora acuminata, an uncultured ectomycorrhizal fungus, Penicillium pinophilum and an uncultured fungal clone. Although the functional roles of fungal endophytes of D. rotundifolia are unknown, colonisation may (a) confer abiotic stress tolerance, (b) facilitate the acquisition of scarce nutrients particularly at the beginning of the growing season or (c) play a role in nutrient signalling between root and shoot.     

Polygalacturonase activity and location in arbuscular mycorrhizal roots of Allium porrum L.

Polygalacturonase activity and location were analysed in leek roots (Allium porrum L.) colonized by Glomus versiforme (Karst.) Berch, an arbuscular mycorrhizal (AM) fungus. Polygalacturonase activity in mycorrhizal roots did not differ quantitatively from that found in nonmycorrhizal roots on all of the four harvesting dates. Fractionation of mycorrhizal root extracts by ion-exchange chromatography showed that expression of polygalacturonase was specific to the mutualistic association. Immunofluorescence and immunogold experiments were carried out to locate the polygalacturonase in mycorrhizal roots using a polyclonal antibody raised against a Fusarium moniliforme endopolygalacturonase. Immunolabelling was observed all over the arbuscules (intracellular fungal structures) but particularly at the interface between the arbuscule and the plant membrane. Since pectins are located in this area, we suggest that polygalacturonase produced during the symbiosis could play a role in plant pectin degradation.     

Diversity and Spatial Structure of Belowground Plant–Fungal Symbiosis in a Mixed Subtropical Forest of Ectomycorrhizal and Arbuscular Mycorrhizal Plants

Plant–mycorrhizal fungal interactions are ubiquitous in forest ecosystems. While ectomycorrhizal plants and their fungi generally dominate temperate forests, arbuscular mycorrhizal symbiosis is common in the tropics. In subtropical regions, however, ectomycorrhizal and arbuscular mycorrhizal plants co-occur at comparable abundances in single forests, presumably generating complex community structures of root-associated fungi. To reveal root-associated fungal community structure in a mixed forest of ectomycorrhizal and arbuscular mycorrhizal plants, we conducted a massively-parallel pyrosequencing analysis, targeting fungi in the roots of 36 plant species that co-occur in a subtropical forest. In total, 580 fungal operational taxonomic units were detected, of which 132 and 58 were probably ectomycorrhizal and arbuscular mycorrhizal, respectively. As expected, the composition of fungal symbionts differed between fagaceous (ectomycorrhizal) and non-fagaceous (possibly arbuscular mycorrhizal) plants. However, non-fagaceous plants were associated with not only arbuscular mycorrhizal fungi but also several clades of ectomycorrhizal (e.g., Russula) and root-endophytic ascomycete fungi. Many of the ectomycorrhizal and root-endophytic fungi were detected from both fagaceous and non-fagaceous plants in the community. Interestingly, ectomycorrhizal and arbuscular mycorrhizal fungi were concurrently detected from tiny root fragments of non-fagaceous plants. The plant–fungal associations in the forest were spatially structured, and non-fagaceous plant roots hosted ectomycorrhizal fungi more often in the proximity of ectomycorrhizal plant roots. Overall, this study suggests that belowground plant–fungal symbiosis in subtropical forests is complex in that it includes “non-typical” plant–fungal combinations (e.g., ectomycorrhizal fungi on possibly arbuscular mycorrhizal plants) that do not fall within the conventional classification of mycorrhizal symbioses, and in that associations with multiple functional (or phylogenetic) groups of fungi are ubiquitous among plants. Moreover, ectomycorrhizal fungal symbionts of fagaceous plants may “invade” the roots of neighboring non-fagaceous plants, potentially influencing the interactions between non-fagaceous plants and their arbuscular-mycorrhizal fungal symbionts at a fine spatial scale.     

Short-term response of arbuscular mycorrhizal association to spider mite herbivory

We examined effects of aboveground herbivory by spider mites (Tetranychus urticae) on colonization and activity of arbuscular mycorrhizal fungi (AMF; Gigaspora margarita) using potted plants (Lotus japonicus). We evaluated changes in arbuscular mycorrhizal (AM) association two ways: (1) conventional trypan blue staining of mycorrhizal hyphae to examine AMF biomass in roots (mycorrhizal colonization) and (2) vital staining for a mycorrhizal enzyme (succinate dehydrogenase, SDH) to examine mycorrhizal activity (SDH activity). Mycorrhizal colonization and SDH activity started to increase 4 days after aboveground herbivory, and returned to the initial levels in the absence of mite herbivory in 7 and 12 days, respectively. These results suggest that the change in AM association in response to mite herbivory is a short-term response.     

Arbuscular mycorrhizal structure and fungi associated with mosses

We investigated the colonization and diversity of arbuscular mycorrhizal (AM) fungi associated with 24 moss species belonging to 16 families in China. AM fungal structures, i.e. spores, vesicles, hyphal coils (including intracellular hyphae), or intercellular nonseptate hyphae, were found in 21 moss species. AM fungal structures (vesicles, hyphal coils, and intercellular nonseptate hyphae) were present in tissues of 14 moss species, and spores and nonseptate hyphae on the surface of gametophytes occurred in 15 species. AM fungal structures were present in 11 of the 12 saxicolous moss species and in six of the ten terricolous moss species, but absent in two epixylous moss species. AM fungal structures were only observed in moss stem and leaf tissues, but not in rhizoids. A total of 15 AM fungal taxa were isolated based on trap culture with clover, using 13 moss species as inocula. Of these AM fungi, 11 belonged to Glomus, two to Acaulospora, one to Gigaspora, and one to Paraglomus. Our results suggest that AM fungal structures commonly occur in most mosses and that diverse AM fungi, particularly Glomus species, are associated with mosses.     

Phialophora graminicola, a dark septate fungus, is a beneficial associate of the grass Vulpia ciliata ssp. ambigua

The influence of three organic compounds and bakers' dry yeast on growth of external mycelium and phosphorus uptake of the arbuscular mycorrhizal fungus Glomus intraradices Schenck & Smith (BEG 87) was examined. Two experiments were carried out in compartmentalized growth systems with root-free sand or soil compartments. The sand and soil in the root-free compartments were left untreated or uniformly mixed with one of the following substrates (0.5 mg g⁻¹ soil): bakers' dry yeast, bovine serum albumin, starch or cellulose. Effects of the organic substrates on biomass and hyphal length density of the arbuscular mycorrhizal fungus were examined by using specific fatty acid signatures in combination with direct microscopy. Micro-organisms other than the arbuscular mycorrhizal fungus were measured by fatty acid signatures, and radioactive ³³P labelling of the root-free soil was used to determine arbuscular mycorrhizal hyphal phosphorus uptake. In general, hyphal growth of G. intraradices was enhanced by yeast and bovine serum albumin, whereas the carbon sources, starch and cellulose, depressed fungal growth. By analysing the fatty acid 16:1ω5 from phospholipids (indicating mycelium) and neutral lipids (indicating storage structures) it was shown that increased fungal growth due to yeast was mainly in vegetative hyphae and less in storage structures. Arbuscular mycorrhizal hyphal phosphorus uptake was decreased by cellulose, but unaffected by the other substrates compared with the control. This means that both growth and phosphorus transport by the arbuscular mycorrhizal fungus were decreased under cellulose treatment. However, the composition of the microbial community varied under different substrate conditions indicating a possible interactive component with arbuscular mycorrhizal hyphal growth and phosphorus uptake.     

Tracking carbon from the atmosphere to the rhizosphere

Turnover rates of arbuscular mycorrhizal (AM) fungi may influence storage of soil organic carbon (SOC). We examined the longevity of AM hyphae in monoxenic cultures; and we also used ¹³C incorporation into signature fatty acids to study C dynamics in a mycorrhizal symbiosis involving Glomus intraradices and Plantago lanceolata. ¹³C enrichment of signature fatty acids showed rapid transfer of plant assimilates to AM fungi and a gradual release of C from roots to rhizosphere bacteria, but at a much slower rate. Furthermore, most C assimilated by AM fungi remained 32 days after labelling. These findings indicate that ¹³C labelled fatty acids can be used to track C flux from the atmosphere to the rhizosphere and that retention of C in AM fungal mycelium may contribute significantly to SOC.     

Arbuscular mycorrhiza of Arnica montana under field conditions—conventional and molecular studies

Two distinct populations of Arnica montana, an endangered medicinal plant, were studied under field conditions. The material was investigated using microscopic and molecular methods. The analyzed plants were always found to be mycorrhizal. Nineteen arbuscular mycorrhizal fungal DNA sequences were obtained from the roots. They were related to Glomus Group A, but most did not match any known species. Some showed a degree of similarity to fungi colonizing liverworts. Conventional analysis of spores isolated from soil samples allowed to identify different fungal taxa: Glomus macrocarpum, Glomus mosseae, Acaulospora lacunosa, and Scutellospora dipurpurescens. Their spores were also isolated from trap cultures.     

Symbiont identity matters: carbon and phosphorus fluxes between Medicago truncatula and different arbuscular mycorrhizal fungi

Many studies have scrutinized the nutritional benefits of arbuscular mycorrhizal associations to their host plants, while the carbon (C) balance of the symbiosis has often been neglected. Here, we present quantification of both the C costs and the phosphorus (P) uptake benefits of mycorrhizal association between barrel medic (Medicago truncatula) and three arbuscular mycorrhizal fungal species, namely Glomus intraradices, Glomus claroideum, and Gigaspora margarita. Plant growth, P uptake and C allocation were assessed 7 weeks after sowing by comparing inoculated plants with their non-mycorrhizal counterparts, supplemented with different amounts of P. Isotope tracing (³³P and ¹³C) was used to quantify both the mycorrhizal benefits and the costs, respectively. G. intraradices supported greatest plant P acquisition and incurred high C costs, which lead to similar plant growth benefits as inoculation with G. claroideum, which was less efficient in supporting plant P acquisition, but also required less C. G. margarita imposed large C requirement on the host plant and provided negligible P uptake benefits. However, it did not significantly reduce plant growth due to sink strength stimulation of plant photosynthesis. A simple experimental system such as the one established here should allow quantification of mycorrhizal costs and benefits routinely on a large number of experimental units. This is necessary for rapid progress in assessment of C fluxes between the plants and different mycorrhizal fungi or fungal communities, and for understanding the dynamics between mutualism and parasitism in mycorrhizal symbioses.     

The mycorrhizal status and colonization of 26 tree species growing in urban and rural environments

Urban environments are highly disturbed and fragmented ecosystems that commonly have lower mycorrhizal fungal species richness and diversity compared to rural or natural ecosystems. In this study, we assessed whether the mycorrhizal status and colonization of trees are influenced by the overall environment (rural vs. urban) they are growing in. Soil cores were collected from the rhizosphere of trees growing in urban and rural environments around southern Ontario. Roots were extracted from the soil cores to determine whether the trees were colonized by arbuscular mycorrhizal fungi, ectomycorrhizal fungi, or both, and to quantify the percent colonization of each type of mycorrhizal fungi. All 26 tree species were colonized by arbuscular mycorrhizal fungi, and seven tree species were dually colonized by arbuscular mycorrhizal and ectomycorrhizal fungi. Overall, arbuscular mycorrhizal and ectomycorrhizal fungal colonization was significantly (p < 0.001) lower in trees growing in urban compared to rural environments. It is not clear what ‘urban’ factors are responsible for the reduction in mycorrhizal fungal colonization; more research is needed to determine whether inoculating urban trees with mycorrhizal fungi would increase colonization levels and growth of the trees.     

Phylogenetic Analysis of a Dataset of Fungal 5.8S rDNA Sequences Shows That Highly Divergent Copies of Internal Transcribed Spacers Reported from Scutellospora castanea Are of Ascomycete Origin

Using a dataset comprising 5.8S rDNA sequences from a wide range of fungi, we show that some sequences reported recently from the arbuscular mycorrhizal (AM) fungus Scutellospora castanea most likely originate from Ascomycetes. Other ITS and 5.8S sequences which were previously reported are confirmed as being clearly of mycorrhizal origin and are variable within one isolate of S. castanea. However, these results mean that previous conclusions which were drawn regarding the heterokaryotic status of AM fungal spores remain unproven. We provide an enlarged 5.8S rDNA dataset that can be used to check ITS sequences for conflicts with well-established phylogenies of the organisms that they were obtained from.