Visualization of Arbuscular Mycorrhizal Fungal Extraradical Hyphae and Spores Vitality and Activity.

The hyphae and spores of arbuscular mycorrhizal (AM) fungi represent an essential component in the extraradical zone due to their role in nutrients and water uptake and as propagules that allow the perpetuation of the AM symbiosis over time, respectively. However, the attention of scientific literature is usually more focused on root colonization than on the study of the extraradical components of AM fungi, especially their vital, active, or functional fractions. This chapter presents some easy-to-use alternatives for staining vital, active, or functional structures of AM fungi for their subsequent microscopic visualization, such as the application of enzyme-based stains, NADPH formation, and also nucleus staining. Some modified methods for the extraction of mycelium from the soil are also presented.

Tuber brennemanii and Tuber floridanum: Two new Tuber species are among the most commonly detected ectomycorrhizal taxa within commercial pecan (Carya illinoinensis) orchards.

Truffles are sequestrate hypogeous fungi, and most form ectomycorrhizal (ECM) associations with trees. Truffles belonging to the genus Tuber (Pezizales, Ascomycota), "true truffles," associate with diverse plant hosts, including economically important species such as pecan (Carya illinoinensis). Morphological and phylogenetic studies delimited several major lineages of Tuber, which include many cryptic and undescribed species. One of these, the Maculatum clade, is a speciose group characterized by relatively small, light-colored ascomata that have alveolate-reticulate spores. Here, we describe two new species in the Maculatum clade, Tuber brennemanii and T. floridanum (previously identified as Tuber sp. 36 and Tuber sp. 47). We delineate these two species by phylogenetic analyses of nuc ITS1-5.8S-ITS2 (= ITS) and partial 28S rDNA (= LSU), and through morphological analysis. A recent collection of T. floridanum from a pecan orchard in Brazil indicates that this species was introduced there on the roots of pecan seedlings. Systematic studies of ascomata and ECM fungal communities indicate that these species are geographically widespread and common ECM symbionts of pecans and other members of the Fagales, particularly in sites with disturbed soils and nutrient enrichment.

Arbuscular mycorrhizas and dark septate endophytes associated with grasses from the Argentine Puna.

The Andean Puna is an arid, high-elevation plateau in which plants such as grasses experience high abiotic stress and distinctive environmental conditions. We assessed colonization by arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE) in the roots of 20 native grass species and examined the relationship between root-associated fungi (AMF and DSE) as a function of the elevation of study sites, the photosynthetic pathways of the grass hosts, and the hosts' life cycles. In general, grasses were co-colonized by AMF and DSE and the colonization by AMF and DSE was not extensive. The extension of colonization of AMF and that of DSE were positively correlated, as were number of arbuscules and DSE colonization extension. The extension of AMF colonization differed among sites with different elevations, but DSE colonization was similar across sites. Overall, AMF and DSE patterns shifted as a function of elevation in most grass species, with no general trends observed with respect to host photosynthetic pathway or life cycle. In general, our observations differ from previous studies in the Northern Hemisphere. Variation among sites in AMF and DSE colonization was greater than variation that could be explained by the other factors considered here, suggesting a strong influence of environmental factors. We predict that both AMF and DSE may have established synergistic and beneficial associations with grasses in these distinctive and harsh ecosystems.

Cadmium induced changes in Solidago chilensis Meyen (Asteraceae) grown on organically fertilized soil with reference to mycorrhizae, metabolism, anatomy and ultrastructure.

Solidago chilensis Meyen (Asteraceae) is a medicinal important plant with few studies on nutrition and metabolism and none information on cadmium phytotoxicity. The objective of this study was to investigate Cd induced responses on the growth and metabolism in S. chilensis and on arbuscular mycorrhiza (AM). The experiment was carried out in a greenhouse, consisting of a 5 × 4 factorial with five doses of manure (0, 3.5, 7, 14 and 21gdm-3) and four doses of cadmium (0, 25, 50 and 75mgdm-3) applied to a Dystrophic Ultisol. After 250 days of plant cultivation, biomass, nutrient content, photosynthetic rate, guaiacol peroxidase activity, mycorrhizal colonization, glomalin content, anatomical and ultrastucture were evaluated. Plants were significantly affected by interaction of manure and Cd doses with anatomical, ultrastructural, physiological and nutritional modifications. Manure applied into Cd contaminated soil significantly improved mycorrhizal colonization and glomalin production. The highest organic manure dose (21gdm-3) alleviated toxicity symptoms of Cd on S. chilensis.

Two new species of Lactarius associated with Alnus acuminata subsp. arguta in Mexico.

In pure stands of Alnus acuminata subsp. arguta trees from Sierra Norte de Puebla (central Mexico) two undescribed ectomycorrhizal species of Lactarius were discovered. Distinction of the two new species is based on morphological characters and supported with phylogenetic analyses of the nuclear ribosomal DNA ITS region and part of the gene that encodes for the second largest subunit of RNA polymerase II (rpb2). The phylogenies inferred recovered the two species in different clades strongly supported by posterior probabilities and bootstrap values. The new Lactarius species are recognized as part of the assemblage of ectomycorrhizal fungi associated with Alnus acuminata. Information about these taxa includes the morphological variation achieved along 16 monitories 2010-2013. Descriptions are provided. They are accompanied by photos including SEM photomicrographs of basidiospores and information on differences between them and other related taxa from Europe and the United States.

Anastomosis behavior differs between asymbiotic and symbiotic hyphae of Rhizophagus clarus.

The life history of arbuscular mycorrhizal fungi (AMF, Glomeromycota) consists of a short asymbiotic phase when spores germinate and a longer symbiotic phase where hyphae form a network within roots and subsequently in the rhizosphere. Hyphal anastomosis contributes to colony formation, yet this process has been studied mostly in the asymbiotic phase rather than in mycorrhizal plants because of methodological limitations. We sought to compare patterns of anastomosis during each phase of fungal growth by measuring hyphal fusions in genetically identical and different single spore isolates of Rhizophagus clarus from different environments and geographic locations. These isolates were genotyped with two anonymous markers of microsatellite-flanking regions. Anastomosis of hyphae from germinating spores was examined in axenic Petri dishes. A rhizohyphatron consisting of agar-coated glass slides bridging single or paired mycorrhizal sorghum plants allowed evaluation of anastomosis of symbiotic hyphae. Anastomosis of hyphae within a colony, defined here as a mycelium from an individual germinating spore or from mycorrhizal roots of one plant, occurred with similar frequencies (8-38%). However, anastomosis between paired colonies was observed in germinating spores from either genetically identical or different isolates, but it was never detected in symbiotic hyphae. The frequency of anastomosis in asymbiotic hyphae from paired interactions was low, occurring in fewer than 6% of hyphal contacts. These data suggest that anastomosis is relatively unconstrained when interactions occur within a colony but is confined to asymbiotic hyphae when interactions occur between paired colonies. This pattern of behavior suggests that asymbiotic and symbiotic phases of mycelium development by R. clarus may differ in function. Anastomosis in the asymbiotic phase may provide brief opportunities for gene flow between populations of this and possibly other AMF species.

Ectomycorrhizal association of three Lactarius species with Carpinus and Quercus trees in a Mexican montane cloud forest.

Ectomycorrhizal (EM) fungi are being monitored in the Santuario del Bosque de Niebla in the central region of Veracruz (eastern Mexico). Based on the comparison of DNA sequences (ITS rDNA) of spatiotemporally co-occurring basidiomes and EM root tips, we discovered the EM symbiosis of Lactarius indigo, L. areolatus and L. strigosipes with Carpinus caroliniana, Quercus xalapensis and Quercus spp. The host of the EM tips was identified by comparison of the large subunit of the ribulose-bisphosphate carboxylase gene (rbcL). Descriptions coupled with photographs of ectomycorrhizas and basidiomes are presented.

New Elaphomyces species (Elaphomycetaceae, Eurotiales, Ascomycota) from Guyana.

Elaphomyces compleximurus sp. nov. and E. digitatus sp. nov. are described from the Pakaraima Mountains of Guyana. Macromorphological, micromorphological, habitat and DNA sequence data are provided for each new species. This is the first report of Elaphomyces ascomata associated with ectomycorrhizal members of the Fabaceae and also for the genus from the lowland South American tropics.

The species of Scleroderma from Argentina, including a new species from the Nothofagus forest.

Five ectomycorrhizal species of Scleroderma were identified from herbarium and field-collected specimens from Argentina. A new hypogeous species, Scleroderma patagonicum, was recorded in association with native Nothofagus spp. in Patagonia. The epigeous species S. albidum, S. areolatum, S. bovista and S. citrinum were associated with various exotic tree species. A phylogenetic analysis based on the ITS region of Scleroderma species, including S. patagonicum, illustrates its distinct status within Scleroderma, including its placement among species with reticulate spores. Descriptions with SEM images of the spores and a key to the species are provided.

Blastospora in the Americas: a new combination and anamorphic genus and species.

This paper presents the new teleomorphic combination Blastospora colombiana as well as a description of a new anamorph genus Pelastoma with two species, all on Apocynaceae from Central and South America.

The arbuscular mycorrhizal Paraglomus majewskii sp. nov. represents a distinct basal lineage in Glomeromycota.

Paraglomus majewskii sp. nov. (Glomeromycota) is described and illustrated. It forms single spores, which are hyaline through their life cycle, globose to subglobose, (35-)63(-78) µm diam, sometimes egg-shaped, 50-70 × 65-90 µm, and have an unusually narrow, (3.2-)4.6(-5.9) µm, cylindrical to slightly flared subtending hypha. The spore wall of P. majewskii consists of an evanescent, short-lived outermost layer, a laminate middle layer, and a flexible innermost layer, which adheres tightly to the middle layer. None of the spore wall layers stain in Melzer's reagent. In single-species cultures with Plantago lanceolata as the host plant P. majewskii formed arbuscular mycorrhizae staining violet in trypan blue. P. majewskii has been isolated from several, distant geographic regions and from different habitats. In phylogenetic analyses of partial nrDNA SSU and LSU sequences the fungus formed mono-phyletic group with Paraglomus species; however it represents a well separated distinct lineage. Its nrDNA sequences are highly similar to in planta arbuscular mycorrhizal fungal sequences from different habitats in Spain and Ecuador.

New species of Clavulina (Cantharellales, Basidiomycota) with resupinate and effused basidiomata from the Guiana Shield.

Three new species of Clavulina (Cantharellales, Basidiomycota) are described from rainforests dominated by ectomycorrhizal trees of the leguminous genus Dicymbe (Fabaceae subfam. Caesalpinioideae) from the central Guiana Shield. Species of Clavulina typically form branched, coralloid basidiomata with amphigenous hymenia. However, the three species described here form resupinate or effuso-coralloid basidiomata, macromorphological forms previously unknown in Clavulina. Macromorphological, micromorphological, habitat and DNA sequence data are provided for each new species. Micromorphological features and DNA sequence data from the second largest subunit of DNA-dependant RNA polymerase II (rpb2) and internal transcribed spacer (ITS) and large subunit (28S) of the ribosomal repeat justify placement of these new species in Clavulina. Comparisons with described Clavulina species and other resupinate taxa within the Cantharellales are provided.

New species and distribution records of Clavulina (Cantharellales, Basidiomycota) from the Guiana Shield.

Two new species of Clavulina Schroet. (Clavulinaceae, Cantharellales, Basidiomycota) and new distribution records for Clavulina amazonensis Corner and Clavulina sprucei (Berk.) Corner are described from the Pakaraima Mountains of Guyana, in the central Guiana Shield region. These fungi occur in rainforests dominated by ectomycorrhizal (ECM) trees of the leguminous genus Dicymbe (Fabaceae subfam. Caesalpinioideae). Macromorphological, micromorphological and habitat data are provided for each species. Nuclear ribosomal DNA sequences of the internal transcribed spacer region and 28S subunit were obtained for new species and from representative collections of C. amazonensis and C. sprucei. The two new species, Clavulina kunmudlutsa sp. nov. and Clavulina tepurumenga sp. nov., constitute important edible fungi for the Patamona Amerindians. Our specimens of C. sprucei represent the first reports of the species since 1853 as well as a range extension of nearly 1500 km, while sequence data from basidiomata as well as ECM roots suggest that this taxon consists of a cryptic species complex.

Basidiosporogenesis, meiosis, and post-meiotic mitosis in the ectomycorrhizal fungus Pisolithusmicrocarpus.

Pisolithus microcarpus (Cooke and Massee) G. Cunn. is a model organism for the studies on the ecology, physiology, and genetics of the ectomycorrhizal associations. However, little is known about the basidiosporogenesis in this species and, in particular, the nuclear behavior after karyogamy. In this work, the events involved in basidiosporogenesis and meiosis in P. microcarpus were analyzed using fluorescence and scanning electron microscopy. The basidia are formed inside peridioles by the differentiation of the cells along the whole hyphae. Basidial cells measure 12-18 microm in length and 6-7 microm in diameter. P. microcarpus produces eight basidiospores per basidium imbibed in a gelatinous matrix in the basidiocarp. The basidiospores are globose, equinate, with blunt spines, and measure 6-8 microm. Karyogamy can take place inside basidia as well as in undifferentiated hyphal cells followed by nuclear migration to a newly developed basidium where meiosis takes place. After the formation of the meiotic tetrad, one round of post-meiotic mitosis occurs, resulting in the production of eight nuclei per basidium. The newly-formed nuclei migrate into the basidiospores asynchronously, resulting in the production of eight uninucleate spores. This corresponds to pattern A of post-meiotic mitosis. This work is the first report on meiosis and post-meiotic mitosis during basidiosporogenesis in P. microcarpus and contributes to clarify some aspects of the biology and genetics of this ectomycorrhizal species.

Atractiellomycetes belonging to the 'rust' lineage (Pucciniomycotina) form mycorrhizae with terrestrial and epiphytic neotropical orchids.

Distinctive groups of fungi are involved in the diverse mycorrhizal associations of land plants. All previously known mycorrhiza-forming Basidiomycota associated with trees, ericads, liverworts or orchids are hosted in Agaricomycetes, Agaricomycotina. Here we demonstrate for the first time that Atractiellomycetes, members of the 'rust' lineage (Pucciniomycotina), are mycobionts of orchids. The mycobionts of 103 terrestrial and epiphytic orchid individuals, sampled in the tropical mountain rainforest of Southern Ecuador, were identified by sequencing the whole ITS1-5.8S-ITS2 region and part of 28S rDNA. Mycorrhizae of 13 orchid individuals were investigated by transmission electron microscopy. Simple septal pores and symplechosomes in the hyphal coils of mycorrhizae from four orchid individuals indicated members of Atractiellomycetes. Molecular phylogeny of sequences from mycobionts of 32 orchid individuals out of 103 samples confirmed Atractiellomycetes and the placement in Pucciniomycotina, previously known to comprise only parasitic and saprophytic fungi. Thus, our finding reveals these fungi, frequently associated to neotropical orchids, as the most basal living basidiomycetes involved in mycorrhizal associations of land plants.

Sebacinales are common mycorrhizal associates of Ericaceae.

Previous reports of sequences of Sebacinales (basal Hymenomycetes) from ericoid mycorrhizas raised the question as to whether Sebacinales are common mycorrhizal associates of Ericaceae, which are usually considered to associate with ascomycetes. Here, we sampled 239 mycorrhizas from 36 ericoid mycorrhizal species across the world (Vaccinioideae and Ericoideae) and 361 mycorrhizas from four species of basal Ericaceae lineages (Arbutoideae and Monotropoideae) that do not form ericoid mycorrhizas, but ectendomycorrhizas. Sebacinales were detected using sebacinoid-specific primers for nuclear 28S ribosomal DNA, and some samples were investigated by transmission electron microscopy (TEM). Diverging Sebacinales sequences were recovered from 76 ericoid mycorrhizas, all belonging to Sebacinales clade B. Indeed, some intracellular hyphal coils had ultrastructural TEM features expected for Sebacinales, and occurred in living cells. Sebacinales belonging to clade A were found on 13 investigated roots of the basal Ericaceae, and TEM revealed typical ectendomycorrhizal structures. Basal Ericaceae lineages thus form ectendomycorrhizas with clade A Sebacinales, a clade that also harbours ectomycorrhizal fungi. This further supports the proposition that Ericaceae ectendomycorrhizas involve ectomycorrhizal fungal taxa. When ericoid mycorrhizas evolved secondarily in Ericaceae, a shift of mycobionts occurred to ascomycetes and clade B Sebacinales, hitherto not described as ericoid mycorrhizal fungi.

Hyphal healing mechanism in the arbuscular mycorrhizal fungi Scutellospora reticulata and Glomus clarum differs in response to severe physical stress.

The hyphal healing mechanism (HHM) has been shown to differ between Gigasporaceae and Glomeraceae. However, this process has not been considered under (severe) physical stress conditions. Scutellospora reticulata and Glomus clarum strains were cultured monoxenically. The impact of long distance separating cut extremities of hyphae and of multiple injuries within hyphae on the HHM was monitored. For long distances (>5000 microm) separating cut extremities, hyphae healing was observed in half the cases in S. reticulata and was absent in G. clarum. For multiple-injured hyphae, the HHM was always oriented towards the complete recovery of hyphae integrity in S. reticulata, while in G. clarum, the growing hyphal tips (GHTs) could indifferently reconnect cut sections, by-pass cut sections or develop into the environment. Hyphae behaviour under severe physical stress clearly differentiated S. reticulata from G. clarum, suggesting that both fungi have developed different strategies for colony growth to survive under adverse conditions.

Diverse tulasnelloid fungi form mycorrhizas with epiphytic orchids in an Andean cloud forest.

The mycorrhizal state of epiphytic orchids has been controversially discussed, and the state and mycobionts of the pleurothallid orchids, occurring abundantly and with a high number of species on stems of trees in the Andean cloud forest, were unknown. Root samples of 77 adult individuals of the epiphytic orchids Stelis hallii, S. superbiens, S. concinna and Pleurothallis lilijae were collected in a tropical mountain rainforest of southern Ecuador. Ultrastructural evidence of symbiotic interaction was combined with molecular sequencing of fungi directly from the mycorrhizas and isolation of mycobionts. Ultrastructural analyses displayed vital orchid mycorrhizas formed by fungi with an imperforate parenthesome and cell wall slime bodies typical for the genus Tulasnella. Three different Tulasnella isolates were obtained in pure culture. Phylogenetic analysis of nuclear rDNA sequences from coding regions of the ribosomal large subunit (nucLSU) and the 5.8S subunit, including parts of the internal transcribed spacers, obtained directly from the roots and from the fungal isolates, yielded seven distinct Tulasnella clades. Tulasnella mycobionts in Stelis concinna were restricted to two Tulasnella sequence types while the other orchids were associated with up to six Tulasnella sequence types. All Tulasnella sequences are new to science and distinct from known sequences of mycobionts of terrestrial orchids. The results indicate that tulasnelloid fungi, adapted to the conditions on tree stems, might be important for orchid growth and maintenance in the Andean cloud forest.