Widespread association between the ericoid mycorrhizal fungus Rhizoscyphus ericae and a leafy liverwort in the maritime and sub-Antarctic

A recent study identified a fungal isolate from the Antarctic leafy liverwort Cephaloziella varians as the ericoid mycorrhizal associate Rhizoscyphus ericae. However, nothing is known about the wider Antarctic distribution of R. ericae in C. varians, and inoculation experiments confirming the ability of the fungus to form coils in the liverwort are lacking. Using direct isolation and baiting with Vaccinium macrocarpon seedlings, fungi were isolated from C. varians sampled from eight sites across a 1875-km transect through sub- and maritime Antarctica. The ability of an isolate to form coils in aseptically grown C. varians was also tested. Fungi with 98-99% sequence identity to R. ericae internal transcribed spacer (ITS) region and partial large subunit ribosomal (r)DNA sequences were frequently isolated from C. varians at all sites sampled. The EF4/Fung5 primer set did not amplify small subunit rDNA from three of five R. ericae isolates, probably accounting for the reported absence of the fungus from C. varians in a previous study. Rhizoscyphus ericae was found to colonize aseptically-grown C. varians intracellularly, forming hyphal coils. This study shows that the association between R. ericae and C. varians is apparently widespread in Antarctica, and confirms that R. ericae is at least in part responsible for the formation of the coils observed in rhizoids of field-collected C. varians.     

A novel ascomycetous endophytic association in the rhizoids of the leafy liverwort family, Schistochilaceae (Jungermanniidae, Hepaticopsida)

Liverworts form diverse associations with endophytic fungi similar to mycorrhizas in vascular plants. Whereas the widespread occurrence of glomeromycotes in the basal liverwort lineages is well documented, knowledge of the distribution of ascomycetes and basidiomycetes in derived thalloid and leafy clades is more fragmented. Our discovery that the ramified and septate rhizoids of the Schistochilaceae, the sister group to all other ascomycete-containing liverworts, are packed with fungal hyphae prompted this study on the effects of the fungi on rhizoid morphology, host specificity, the cytology of the association, and a molecular analysis of the endophytes. Two species of Pachyschistochila and their fungi were grown axenically. Axenic rhizoids were unbranched and nonseptate. Reinfected with their own fungus and that from the other species, both Pachyschistochila species produced branched and septate rhizoids identical to those in nature. Woronin bodies and simple septa identified the fungus as an ascomycete referable, according to phylogenetic analyses of ITS sequences, to the Rhizoscyphus (Hymenoscyphus) ericae aggregate, also found in other liverwort-ascomycete associations and in mycorrhizas in the Ericales. Healthy hyphae and host cytoplasm suggest that the Schistochila-fungus association reflects a balanced mutualistic relationship. The recent dating of the divergence of the Jungermanniales from the fungus-free Porellales in the Permian and the origins of the Schistochilaceae in the Triassic indicate that these associations in liverworts predate the appearance of the Ericales.     

Structural changes to a mycothallus along a latitudinal transect through the maritime and sub-Antarctic

Previous studies have shown the leafy liverwort Cephaloziella varians to associate consistently with fungi, typically the ericoid mycorrhizal symbiont Rhizoscyphus ericae, across a wide latitudinal gradient in the maritime and sub-Antarctic. Hitherto, however, there are no quantitative data on the intensity of colonisation of C. varians by fungal structures in the natural environment and how colonisation might vary with changing environmental conditions. A study is hence reported showing that the frequency of colonisation by fungal structures of C. varians alters along a latitudinal transect from South Georgia (54° S, 38° W) to Moutonnée Valley on Alexander Island (71° S, 68° W). The percentage of stem length colonised by dark septate (DS) hyphae increased significantly along the transect, from 30% at South Georgia to 97% at Moutonnée Valley. In contrast, the percentage of stem length colonised by hyaline hyphae decreased significantly, from 85% at South Georgia to 13% at Moutonnée Valley, and that colonised by hyphal coils similarly decreased from 71% at the former location to 15% at the latter. The frequencies of DS hyphae were negatively associated with mean annual and seasonal air temperatures, whereas those of hyaline septate hyphae and hyphal coils were positively associated with air temperatures. Coils at northerly locations were more convoluted than those at southerly locations. The data indicate that hyphal coils, usually associated with nutrient exchange between partners in ericoid mycorrhizas, do form in C. varians tissues in the maritime and sub-Antarctic, but that the frequency of these structures diminishes in colder habitats.     

New Neotropical Sebacinales Species from a Pakaraimaea dipterocarpacea Forest in the Guayana Region,Southern Venezuela: Structural Diversity and Phylogeography

Pakaraimaea dipterocarpacea, a member of the Dipterocarpaceae endemic in the Guayana region, is associated with a diverse community of ectomycorrhizal (ECM) fungi. Amongst the 41 ECM fungal species detected in a 400 m² P. dipterocarpacea ssp. nitida plot in Southern Venezuela, three species belonged to the Sebacinales. We tested whether ECM anatomotype characterization can be used as a feasible element in an integrative taxonomy in this diverse fungal group, where the relevance of fruitbody morphology for species delimitation seems limited. Using a combination of ECM morpho-anatomical characterizations and phylogenetic analyses based on nuclear ITS and LSU sequences, we report three new species. The main distinguishing features of Sebacina guayanensis are the yellowish cell walls together with conspicuous undifferentiated, uniform compact (type B) rhizomorphs. Staghorn-like hyphae are characteristic of S. tomentosa. The combination of clusters of thick-walled emanating hyphae, including hyphae similar to awl-shaped cystidia with basal dichotomous or trichotomous ramifications, and the presence of type B rhizomorphs were characteristic of a third, yet unnamed species. The three species belong to three different, possibly specifically tropical clades in Sebacinales Group A. The geographic distribution of phylogenetically related strains was wide, including a Dicymbe forest in Guyana and an Ecuadorian rainforest with Coccoloba species. We show that ECM morpho-anatomy can be used, in combination with other analyses, to delineate species within Sebacinales Group A. In addition to phylogenetic information, type B rhizomorphs observed in different Sebacinales clades have important ecological implications for this fungal group. The phylogeography of Sebacinales suggests that dispersion and host jump are important radiation mechanisms that shaped P. dipterocarpacea ECM fungal community. This study emphasizes the need for more sequence data to evaluate the hypothesis that phylogeographic relationships between neo- and paleotropical ECM fungal species could be attributed to the vicariance of cross-continental hosts such as the Dipterocarpacae.     

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.     

Conservative ecological and evolutionary patterns in liverwort–fungal symbioses

Liverworts, the most ancient group of land plants, form a range of intimate associations with fungi that may be analogous to the mycorrhizas of vascular plants. Most thalloid liverworts contain arbuscular mycorrhizal glomeromycete fungi similar to most vascular plants. In contrast, a range of leafy liverwort genera and one simple thalloid liverwort family (the Aneuraceae) have switched to basidiomycete fungi. These liverwort switches away from glomeromycete fungi may be expected to parallel switches undergone by vascular plants that target diverse lineages of basidiomycete fungi to form ectomycorrhizas. To test this hypothesis, we used a cultivation-independent approach to examine the basidiomycete fungi associated with liverworts in varied worldwide locations by generating fungal DNA sequence data from over 200 field collections of over 30 species. Here we show that eight leafy liverwort genera predominantly and consistently associate with members of the Sebacina vermifera species complex and that Aneuraceae thalloid liverworts associate nearly exclusively with Tulasnella species. Furthermore, within sites where multiple liverwort species co-occur, they almost never share the same fungi. Our analyses reveal a strikingly conservative ecological and evolutionary pattern of liverwort symbioses with basidiomycete fungi that is unlike that of vascular plant mycorrhizas.     

Combining nested PCR and restriction digest of the internal transcribed spacer region to characterize arbuscular mycorrhizal fungi on roots from the field

Identification of arbuscular mycorrhizal fungi (AMF) on roots is almost impossible with morphological methods and, due to the presence of contaminating fungi, it is also difficult with molecular biological techniques. To allow broad investigation of the population structure of AMF in the field, we have established a new method to selectively amplify the internal transcribed spacer (ITS) region of most AMF with a unique primer set. Based on available sequences of the rDNA, one primer pair specific for AMF and a few other fungal groups was designed and combined in a nested PCR with the already established primer pair ITS5/ITS4. Amplification from contaminating organisms was reduced by an AluI restriction after the first reaction of the nested PCR. The method was assessed at five different field sites representing different types of habitats. Members of all major groups within the Glomeromycota (except Archaeosporaceae) were detected at the different sites. Gigasporaceae also proved detectable with the method based on cultivated strains.     

Two widespread green Neottia species (Orchidaceae) show mycorrhizal preference for Sebacinales in various habitats and ontogenetic stages

Plant dependence on fungal carbon (mycoheterotrophy) evolved repeatedly. In orchids, it is connected with a mycorrhizal shift from rhizoctonia to ectomycorrhizal fungi and a high natural ¹³C and ¹⁵N abundance. Some green relatives of mycoheterotrophic species show identical trends, but most of these remain unstudied, blurring our understanding of evolution to mycoheterotrophy. We analysed mycorrhizal associations and ¹³C and ¹⁵N biomass content in two green species, Neottia ovata and N. cordata (tribe Neottieae), from a genus comprising green and nongreen (mycoheterotrophic) species. Our study covered 41 European sites, including different meadow and forest habitats and orchid developmental stages. Fungal ITS barcoding and electron microscopy showed that both Neottia species associated mainly with nonectomycorrhizal Sebacinales Clade B, a group of rhizoctonia symbionts of green orchids, regardless of the habitat or growth stage. Few additional rhizoctonias from Ceratobasidiaceae and Tulasnellaceae, and ectomycorrhizal fungi were detected. Isotope abundances did not detect carbon gain from the ectomycorrhizal fungi, suggesting a usual nutrition of rhizoctonia‐associated green orchids. Considering associations of related partially or fully mycoheterotrophic species such as Neottia camtschatea or N. nidus‐avis with ectomycorrhizal Sebacinales Clade A, we propose that the genus Neottia displays a mycorrhizal preference for Sebacinales and that the association with nonectomycorrhizal Sebacinales Clade B is likely ancestral. Such a change in preference for mycorrhizal associates differing in ecology within the same fungal taxon is rare among orchids. Moreover, the existence of rhizoctonia‐associated Neottia spp. challenges the shift to ectomycorrhizal fungi as an ancestral pre‐adaptation to mycoheterotrophy in the whole Neottieae.     

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.     

Two sebacinoid ectomycorrhizae on Chinese pine

Sebacinoid fungi show a broad mycorrhizal capacity; therefore, they play a very important role in natural systems. Worldwide, fungi of Sebacinales are present under different environmental conditions and associate with diverse plant hosts, however, are hitherto poorly studied in China. Two sebacinoid ectomycorrhizae (ECM), Pinirhiza multifurcata and Pinirhiza nondextrinoidea, are described in detail morphologically and anatomically in the present study. They share a plectenchymatous outer mantle with multiply ramified hyphae in a gelatinous matrix, clampless, thin, thick-walled emanating hyphae with mostly Y-shaped ramifications and triangular inflations at the point of ramification. P. multifurcata and P. nondextrinoidea can be distinguished by thick cells in mantle layers, the ramification of emanating hyphae, the presence or absence of rhizomorphs, as well as the differing color reaction in Melzer's reagent. The putative molecular phylogenetic relationships of P. multifurcata and P. nondextrinoidea were inferred by analyses of the partial large subunit nuclear rDNA (nLSU); however, an affiliation to fungal species was not possible. This is the first report of sebacinoid ECM on Chinese pine.     

Endophytic fungi associated with the Antarctic grass <Emphasis Type="Italic">Deschampsia antarctica</Emphasis> Desv. (<Emphasis Type="Italic">Poaceae</Emphasis>)

Deschampsia antarctica Desv. (Poaceae) represents one of the two vascular plants that have colonized the Antarctic continent, which is usually exposed to extreme environmental conditions. In this work, we have characterized the endophytic fungi associated with the leaves of D. antarctica. Endophytic fungi were recovered from 91 individual plants from different points of Admiralty Bay at King George Island, Antarctica. A total of 26 fungal isolates were obtained from 273 leaf fragments. All isolates were identified by analysis of the sequences of the internal transcribed spacer region (ITS) of the rDNA. Alternaria and Phaeosphaeria were the most frequent genera associated with the plant. Other fungal isolates were identified as Entrophospora sp. and several undescribed Ascomycete species. An interesting result was obtained for the isolates UFMGCB 215 and UFMGCB 262, which were related to fungi associated with bryophytes present in boreal ecosystems. Some isolates showed low identity in the ITS sequences to sequences of fungal species deposited in GenBank, suggesting that these fungi could be new species. This work is the first report on fungal endophytes associated with leaves of the Antarctic grass D. antarctica.     

Metabarcoding of fungal communities associated with bark beetles

Many species of fungi are closely allied with bark beetles, including many tree pathogens, but their species richness and patterns of distribution remain largely unknown. We established a protocol for metabarcoding of fungal communities directly from total genomic DNA extracted from individual beetles, showing that the ITS3/4 primer pair selectively amplifies the fungal ITS. Using three specimens of bark beetle from different species, we assess the fungal diversity associated with these specimens and the repeatability of these estimates in PCRs conducted with different primer tags. The combined replicates produced 727 fungal Operational Taxonomic Units (OTUs) for the specimen of Hylastes ater, 435 OTUs for Tomicus piniperda, and 294 OTUs for Trypodendron lineatum, while individual PCR reactions produced on average only 229, 54, and 31 OTUs for the three specimens, respectively. Yet, communities from PCR replicates were very similar in pairwise comparisons, in particular when considering species abundance, but differed greatly among the three beetle specimens. Different primer tags or the inclusion of amplicons in separate libraries did not impact the species composition. The ITS2 sequences were identified with the Lowest Common Ancestor approach and correspond to diverse lineages of fungi, including Ophiostomaceae and Leotiomycetes widely found to be tree pathogens. We conclude that Illumina MiSeq metabarcoding reliably captures fungal diversity associated with bark beetles, although numerous PCR replicates are recommended for an exhaustive sample. Direct PCR from beetle DNA extractions provides a rapid method for future surveys of fungal species diversity and their associations with bark beetles and environmental variables.     

Distribution of dominant arbuscular mycorrhizal fungi among five plant species in undisturbed vegetation of a coastal grassland

Most plant species in mixed grassland vegetation are colonized by arbuscular mycorrhizal (AM) fungi. Previous studies have reported differences in host preferences among AM fungi, although the fungi are known to lack host specificity. In the present study, the distribution of phylogenetic groups of AM fungi belonging to a clade of Glomus species was studied in five plant species from a coastal grassland in Denmark. The occurrence of the fungi was determined by PCR analyses of fungal large subunit ribosomal DNA sequences amplified from root fragments using a specific primer set. The results showed that the dominant Glomus species were able to colonize all the studied plant species, supporting the view that the AM fungi represent a large underground interconnecting mycelial network.     

Molecular analysis of fungal diversity associated with three bryophyte species in the Fildes Region, King George Island, maritime Antarctica

The fungal communities associated with three bryophytes species (the liverwort Barbilophozia hatcheri, the mosses Chorisodontium aciphyllum and Sanionia uncinata) in the Fildes Region, King George Island, maritime Antarctica, were studied using clone library analysis. Fungal communities showed low diversity; the 680 clones belonged to 93 OTUs. Of these, 78 belonged to the phylum Ascomycota, 13 to the phylum Basidiomycota, 1 to the phylum Zygomycota, and 1 to an unknown phylum. Among the OTUs, the most common orders in the Ascomycota were Helotiales (42 OTUs) and Chaetothyriales (14 OTUs) and the most common orders in the Basidiomycota were Sebacinales (3 OTUs) and Platygloeales (3 OTUs). Most OTUs clustered within clades that contained phylotypes identified from samples in Antarctic or Arctic ecosystems or from bryophytes in other ecosystems. In addition, we found that host-related factor may shape the fungal communities associated with bryophytes in this region. This is the first systematic study of the fungal community in Antarctic bryophytes to be performed using culture-independent method and the results may improve understanding of the endophytic fungal evolution and ecology in the Antarctic ecosystem.     

银叶杜鹃和繁花杜鹃根部真菌的多样性

植物根系与真菌形成菌根, 在自然生态系统的物质能量循环中具有重要的生态功能。作者在四川省的中国杜鹃园选取银叶杜鹃(Rhododendron argyrophyllum)和繁花杜鹃(R. floribundum), 通过直接扩增杜鹃花根部真菌rDNA-ITS区片段, 来揭示该地区杜鹃花属植物根部真菌的多样性。ITS序列分析结果表明: 从两种杜鹃的根部共检测到41个真菌分类单元, 分别属于子囊菌纲的柔膜菌目(Helotiales)、散囊菌目(Eurotiales)、盘菌目(Pezizales)、假球壳目(Pleosporales)和担子菌纲的蜡壳耳目(Sebacinales)、伞菌目(Agaricales)、Erythrobasidiales、线黑粉菌目(Filobasidiales)。银叶杜鹃和繁花杜鹃根部真菌种类丰富, 包括了杜鹃花类菌根真菌、外生菌根真菌和其他类型真菌, 其中担子菌纲的蜡壳耳目和子囊菌纲的柔膜菌目占有较大比例。     

Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts

Background  

The Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in distinguishing fungal species by PCR analysis. Previously published PCR primers available for amplifying these sequences from environmental samples provide varying degrees of success at discriminating against plant DNA while maintaining a broad range of compatibility. Typically, it has been necessary to use multiple primer sets to accommodate the range of fungi under study, potentially creating artificial distinctions for fungal sequences that amplify with more than one primer set.     

Fungal community diversity and soil health in intensive potato cropping systems of the east Po valley, northern Italy

An ecological approach was used to investigate the relationship between diversity of soil fungal communities and soil‐borne pathogen inoculum in a potato growing area of northern Italy affected by yield decline. The study was performed in 14 sites with the same tillage management practices: 10 named ‘potato sites’, that for many years had been intensely cultivated with potatoes, and 4 named ‘rotation sites’, subject to a 4‐year rotation without potatoes or any recurrent crop for many years. Fungal communities were recorded using conventional (soil fungi by plate count and endophytic fungi as infection frequency on pot‐grown potato plant roots in soil samples) and molecular approaches [Basidiomycetes and Ascomycetes with specific and denaturing gradient gel electrophoresis (DGGE) analysis]. Diversity of fungal communities in potato sites was significantly lower than that in rotation sites. In addition, fungal communities in rotation sites showed lower Berger–Parker dominance than those in the potato sites, suggesting that rotation sites had a higher diversity as well as a better fungal community balance than potato sites. The ANalysis Of SIMilarity test of soil fungi and root endophytic fungi revealed that the two cropping systems differed significantly for species composition. Root endophytic fungal communities showed a greater ability to colonise potato roots in soil samples from potato sites than those from rotation sites. Moreover, the majority of endophytic root fungal community species in potato sites belonged to the potato root rot complex and storage disease (Colletotrichum coccodes, Fusarium solani and Fusarium oxysporum), while those in rotation sites were mainly ubiquitous or saprobic fungi. Soil rDNA analyses showed that Ascomycetes were much more frequent than Basidiomycetes in all the soils examined. DGGE analysis, with the Ascomycete‐specific primer (ITS1F/ITS4A), did not reveal distinctions between the communities found at the potato and rotation sites, although the same analysis showed differences between the communities of Basidiomycetes (specific primer ITS1F/ITS4B). These findings showed that recurrent potato cropping affected diversity and composition of soil fungal communities and induced a shift in specialisation of the endophytic fungi towards potato.     

Members of Sebacinales subgroup B form mycorrhizae with epiphytic orchids in a neotropical mountain rain forest

Previous investigations revealed that epiphytic orchids in a mountain rain forest in southern Ecuador formed mycorrhizae with diverse members of Tulasnellales. Using specific primers, we now show that the same orchids are also associated with Sebacinales. Ultrastructural observations confirmed the Sebacinales mycobionts in situ. Mycorrhizae of flowering individuals of Stelis hallii, S. superbiens, S. concinna and Pleurothallis lilijae were sampled in different forest types of the mountain rain forest of southern Ecuador along an altitudinal gradient between 1,850 and 2,100 m a.s.l. Phylogenetic analysis of fungal nuclear rDNA sequences coding for the ribosomal large subunit (nucLSU) showed the presence of eight sequence types based on proportional differences of <1% bp. All sequence types clustered in the Sebacinales subgroup B which also contained sequences of mycobionts from ericads and terrestrial orchids. Sequences of the nuclear rDNA 5.8S subunit, including parts of the internal transcribed spacers ITS1 and ITS2 (5.8-ITS) from the mycobionts of the epiphytic orchids, were distinct from published sequences of sebacinoid mycobionts of green terrestrial orchids and ericads. Sebacinales sequences from different epiphytic orchid species differed at least by 1% bp as was previously found for Tulasnella sequences. Sebacinales occurred less frequently and with a lower number of sequence types than Tulasnellales, but distribution along the altitudinal gradient was similar.