Exploring structural and molecular diversity of Ericaceae hair root mycobionts: a comparison between Northern Bohemia and Argentine Patagonia.

Core Ericaceae produce delicate hair roots with inflated rhizodermal cells that host plethora of fungal symbionts. These poorly known mycobionts include various endophytes, parasites, saprobes, and the ericoid mycorrhizal (ErM) fungi (ErMF) that form the ErM symbiosis crucial for the fitness of their hosts. Using microscopy and high-throughput sequencing, we investigated their structural and molecular diversity in 14 different host × site combinations in Northern Bohemia (Central Europe) and Argentine Patagonia (South America). While we found typical ericoid mycorrhiza in all combinations, we did not detect ectomycorrhiza and arbuscular mycorrhiza. Superficial mantles of various thickness formed by non-clamped hyphae were observed in all combinations except Calluna vulgaris from N. Bohemia. Some samples contained frequent intercellular hyphae while others possessed previously unreported intracellular haustoria-like structures linked with intracellular hyphal coils. The 711 detected fungal OTU were dominated by Ascomycota (563) and Basidiomycota (119), followed by four other phyla. Ascomycetes comprised Helotiales (255), Pleosporales (53), Chaetothyriales (42), and other 19 orders, while basidiomycetes Sebacinales (42), Agaricales (28), Auriculariales (7), and other 14 orders. While many dominant OTU from both hemispheres lacked close relatives in reference databases, many were very similar to identical to unnamed sequences from around the world. On the other hand, several significant ericaceous mycobionts were absent in our dataset, incl. Cairneyella, Gamarada, Kurtia, Lachnum, and Leohumicola. Most of the detected OTU could not be reliably linked to a particular trophic mode, and only two could be reliably assigned to the archetypal ErMF Hyaloscypha hepaticicola. Probable ErMF comprised Hyaloscypha variabilis and Oidiodendron maius, both detected only in N. Bohemia. Possible ErMF comprised sebacinoid fungi and several unnamed members of Hyaloscypha s. str. While H. hepaticicola was dominant only in C. vulgaris, this model ErM host lacked O. maius and sebacinoid mycobionts. Hyaloscypha hepaticicola was absent in two and very rare in six combinations from Patagonia. Nine OTU represented dark septate endophytes from the Phialocephala fortinii s. lat.-Acephala applanata species complex, including the most abundant OTU (the only detected in all combinations). Statistical analyses revealed marked differences between N. Bohemia and Patagonia, but also within Patagonia, due to the unique community detected in a Valdivian temperate rainforest. Our results show that the ericaceous hair roots may host diverse mycobionts with mostly unknown functions and indicate that many novel ErMF lineages await discovery. Transhemispheric differences (thousands of km) in their communities may be evenly matched by local differences (scales of km, m, and less).

Fungi in hair roots of Vaccinium spp. (Ericaceae) growing on decomposing wood: colonization patterns, identity, and in vitro symbiotic potential.

Most of our knowledge on the ericoid mycorrhizal (ErM) symbiosis comes from temperate heathlands characterized by acidic peaty soils and many experiments with a few ascomycetous fungi. However, ericaceous plants thrive in many other ecosystems and in temperate coniferous forests, their seedlings often prosper on decomposing wood. While wood is typically exploited by basidiomycetous ectomycorrhizal (EcM) and saprobic fungi, the role of ErM fungi (ErMF) is much less clear. We explored the cultivable mycobiota of surface sterilized hair roots of Vaccinium spp. growing on decomposing wood in two coniferous forests in Mid-Norway (Scandinavia) and Northern Bohemia (Central Europe). Obtained isolates were identified using molecular tools and their symbiotic potential was tested in vitro. While the detected community lacked the archetypal ErMF Hyaloscypha hepaticicola and the incidence of dark septate endophytes and EcM fungi was negligible, it comprised other frequent asexual ascomycetous ErMF, namely H. variabilis and Oidiodendron maius, together with several isolates displaying affinities to sexual saprobic H. daedaleae and H. fuckelii. Ascomycete-suppressing media revealed representatives of the saprobic basidiomycetous genera Coprinellus, Gymnopilus, Mycena (Agaricales), and Hypochnicium (Polyporales). In the resyntheses, the tested basidiomycetes occasionally penetrated the rhizodermal cells of their hosts but never formed ericoid mycorrhizae and in many cases overgrew and killed the inoculated seedlings. In contrast, a representative of the H. daedaleae/H. fuckelii-related isolates repeatedly formed what morphologically appears as the ErM symbiosis and supported host's growth. In conclusion, while basidiomycetous saprobic fungi have a potential to colonize healthy-looking ericaceous hair roots, the mode(-s) of their functioning remain obscure. For the first time, a lineage in Hyaloscypha s. str. (corresponding to the former Hymenoscyphus ericae aggregate) where sexual saprobes are intermingled with root symbionts has been revealed, shedding new light on the ecology and evolution of these prominent ascomycetous ErMF.

Hyaloscypha gabretae and Hyaloscypha gryndleri spp. nov. (Hyaloscyphaceae, Helotiales), two new mycobionts colonizing conifer, ericaceous and orchid roots.

Historically, Hyaloscypha s. lat. (Hyaloscyphaceae, Helotiales) included various saprobes with small apothecia formed on decaying plant matter, usually wood, that were defined by chemical and (ultra)structural aspects. However, recent molecular phylogenetic and resynthesis studies have narrowed the concept of the genus and shown that it contains several widely distributed species with unknown sexual morphs that form ectomycorrhizae, ericoid mycorrhizae, and mycothalli and also grow endophytically in plant roots and hypogeous ectomycorrhizal (EcM) fruitbodies (i.e., the historical Hymenoscyphus ericae aggregate). Hence, some of the sexually reproducing saprobic Hyaloscypha s. lat. and the symbionts belong to the monophyletic Hyaloscypha s. str. Here, we introduce two new root-symbiotic Hyaloscypha s. str. species, i.e., H. gabretae and H. gryndleri spp. nov. While the former was isolated only from ericaceous hosts (Vaccinium myrtillus from Southern Bohemia, Czechia and Calluna vulgaris from England, UK), the latter was obtained from a basidiomycetous EcM root tip of Picea abies (Pinaceae), roots of Pseudorchis albida (Orchidaceae), and hair roots of V. myrtillus from Southern Bohemia and C. vulgaris from England. Hyaloscypha gryndleri comprises two closely related lineages, suggesting ongoing speciation, possibly connected with the root-symbiotic life-style. Fungal isolates from ericaceous roots with sequences similar to H. gabretae and H. gryndleri have been obtained in Japan and in Canada and Norway, respectively, suggesting a wide and scattered distribution across the Northern Hemisphere. In a series of in vitro experiments, both new species failed to form orchid mycorrhizal structures in roots of P. albida and H. gryndleri repeatedly formed what morphologically corresponds to the ericoid mycorrhizal (ErM) symbiosis in hair roots of V. myrtillus, whereas the ErM potential of H. gabretae remained unresolved. Our results highlight the symbiotic plasticity of root-associated hyaloscyphoid mycobionts as well as our limited knowledge of their diversity and distribution, warranting further ecophysiological and taxonomic research of these important and widespread fungi.

Fungal symbionts may modulate nitrate inhibitory effect on orchid seed germination.

Many orchid species are threatened, while some disappear from their natural habitats without obvious reasons. Eutrophication has been suggested as a possible factor and nitrate, which is able to suppress non-symbiotic orchid seed germination even at very low concentrations, and could pose a serious threat for natural orchid populations. Early ontogenesis of all orchids entirely depends on orchid mycorrhizal symbiosis, and at this initial mycoheterotrophic stage, many terrestrial green orchids associate with polyphyletic fungal symbionts (i.e., mycobionts), collectively called "rhizoctonias." We asked whether these fungi might also have some non-nutritional roles, i.e., whether they might confer resistance to eutrophication. To test this hypothesis, we co-cultivated seeds of the terrestrial orchid Dactylorhiza majalis with five rhizoctonias (two Tulasnella, two Ceratobasidium and one Serendipita isolate) at various ecologically meaningful nitrate concentrations (0 to 100 mg/L). With the exception of one Tulasnella isolate, all mycobionts supported the growth of protocorms and formed orchid mycorrhiza, i.e., intracellular hyphal pelotons, in the protocorms. Nitrate suppressed asymbiotic, as well as symbiotic, seed germination in all but one fungal treatment; the seeds co-cultivated with one of the Ceratobasidium isolates were indeed insensitive to nitrate. We conclude that nitrates also negatively affect symbiotic orchid germination, depending on the available compatible mycobionts. Thus, eutrophication with nitrate may decrease the number of orchid mycobionts capable of supporting seed germination.

Marinomyxa Gen. Nov. Accommodates Gall-Forming Parasites of the Tropical to Subtropical Seagrass Genus Halophila and Constitutes a Novel Deep-Branching Lineage Within Phytomyxea (Rhizaria: Endomyxa).

Marine representatives of Phytomyxea (SAR: Rhizaria: Endomyxa), a peculiar class of obligate endobiotic parasites, are a greatly understudied ecological group of protists infecting many algal, diatom, and seagrass species. Very little is known about the actual diversity, ecology, and pathogenic potential of these organisms and their taxonomic treatment in many cases follows outdated morphotaxonomic concepts. Here we focused on resolving the phylogenetic relations of the phytomyxean parasites of the widespread seagrass genus Halophila. We report the first finding of Plasmodiophora halophilae, the parasite of ovate-leaf Halophila species, after more than 100 years since its original description in 1913. We provide additional information on its anatomy, morphology, distribution, and host range, together with a phylogenetic evidence that it is congeneric with the recently rediscovered species infecting the invasive seagrass Halophila stipulacea in the Mediterranean Sea. Despite the previously hypothesized affiliation of the latter to Tetramyxa, our phylogenetic analyses of the 18S rRNA gene place Tetramyxa parasitica (a parasite of brackish water phanerogams and the type species of the genus) in the freshwater/terrestrial phytomyxean order Plasmodiophorida and reveal that phytomyxids associated with Halophila spp. form a separate deep-branching clade within the class proposed here as Marinomyxa gen. nov. We further argue that M. marina infecting H. stipulacea is most likely a species-specific parasite and implies their comigration through the Suez Canal.

Ericoid mycorrhizal symbiosis: theoretical background and methods for its comprehensive investigation.

Despite decades of intensive research (especially from 1970s to 1990s), the ericoid mycorrhizal (ErM) hair root is still largely terra incognita and this simplified guide is intended to revive and promote the study of its mycobiota. Basic theoretical knowledge on the ErM symbiosis is summarized, followed by practical advices on Ericaceae root sample collection and handling, microscopic observations and photo-documentation of root fungal colonization, mycobiont isolation, maintenance and identification and resynthesis experiments with ericoid plants. The necessity of a proper selection of the root material and its surface sterilization prior to mycobiont isolation is stressed, together with the need of including suitable control treatments in inoculation experiments. The culture-dependent approach employing plating of single short (~ 2 mm) hair root segments on nutrient media is substantiated as a useful tool for characterization of Ericaceae root-associated fungal communities; it targets living mycelium and provides metabolically active cultures that can be used in physiological experiments and taxonomic studies, thus providing essential reference material for culture-independent approaches. On the other hand, it is stressed that not every mycobiont isolated from an ericoid hair root necessarily represent an ErM fungus. Likewise, not every intracellular hyphal coil formed in the Ericaceae rhizodermis necessarily represents the ErM symbiosis. Taxonomy of the most important ericoid mycobionts is updated, mutualism in the ErM symbiosis is briefly discussed from the mycobiont perspective, and some interesting lines of possible future research are highlighted.

Enigmatic Phytomyxid Parasite of the Alien Seagrass Halophila stipulacea: New Insights into Its Ecology, Phylogeny, and Distribution in the Mediterranean Sea.

Marine phytomyxids represent often overlooked obligate biotrophic parasites colonizing diatoms, brown algae, and seagrasses. An illustrative example of their enigmatic nature is the phytomyxid infecting the seagrass Halophila stipulacea (a well-known Lessepsian migrant from the Indo-Pacific to the Mediterranean Sea). In the Mediterranean, the occurrence of this phytomyxid was first described in 1995 in the Strait of Messina (southern Italy) and the second time in 2017 in the Aegean coast of Turkey. Here we investigated, using scuba diving, stereomicroscopy, light and scanning electron microscopy, and molecular methods, whether the symbiosis is still present in southern Italy, its distribution in this region and its relation to the previous reports. From the total of 16 localities investigated, the symbiosis has only been found at one site. A seasonal pattern was observed with exceptionally high abundance (> 40% of the leaf petioles colonized) in September 2017, absence of the symbiosis in May/June 2018, and then again high infection rates (~ 30%) in September 2018. In terms of anatomy and morphology as well as resting spore dimensions and arrangement, the symbiosis seems to be identical to the preceding observations in the Mediterranean. According to the phylogenetic analyses of the 18S rRNA gene, the phytomyxid represents the first characterized member of the environmental clade "TAGIRI-5". Our results provide new clues about its on-site ecology (incl. possible dispersal mechanisms), hint that it is rare but established in the Mediterranean, and encourage further research into its distribution, ecophysiology, and taxonomy.

Extensive sampling and high-throughput sequencing reveal Posidoniomycesatricolor gen. et sp. nov. (Aigialaceae, Pleosporales) as the dominant root mycobiont of the dominant Mediterranean seagrass Posidoniaoceanica.

Seagrasses provide invaluable ecosystem services yet very little is known about their root mycobiont diversity and distribution. Here we focused on the dominant Mediterranean seagrass Posidoniaoceanica and assessed its root mycobiome at 32 localities covering most of the ecoregions in the NW Mediterranean Sea using light and scanning electron microscopy and tag-encoded 454-pyrosequencing. Microscopy revealed that the recently discovered dark septate endophytic association specific for P.oceanica is present at all localities and pyrosequencing confirmed that the P.oceanica root mycobiome is dominated by a single undescribed pleosporalean fungus, hitherto unknown from other hosts and ecosystems. Its numerous slow-growing isolates were obtained from surface-sterilised root segments at one locality and after prolonged cultivation, several of them produced viable sterile mycelium. To infer their phylogenetic relationships we sequenced and analysed the large (LSU) and small (SSU) subunit nrDNA, the ITS nrDNA and the DNA-directed RNA polymerase II (RPB2). The fungus represents an independent marine biotrophic lineage in the Aigialaceae (Pleosporales) and is introduced here as Posidoniomycesatricolor gen. et sp. nov. Its closest relatives are typically plant-associated saprobes from marine, terrestrial and freshwater habitats in Southeast Asia and Central America. This study expands our knowledge and diversity of the Aigialaceae, adds a new symbiotic lifestyle to this family and provides a formal name for the dominant root mycobiont of the dominant Mediterranean seagrass.

Ontogenetic transition from specialized root hairs to specific root-fungus symbiosis in the dominant Mediterranean seagrass Posidonia oceanica.

Terrestrial plants typically take up nutrients through roots or mycorrhizae while freshwater plants additionally utilize leaves. Their nutrient uptake may be enhanced by root hairs whose occurrence is often negatively correlated with mycorrhizal colonization. Seagrasses utilize both leaves and roots and often form root hairs, but seem to be devoid of mycorrhizae. The Mediterranean seagrass Posidonia oceanica is an exception: its adults commonly lack root hairs and regularly form a specific association with a single pleosporalean fungus. Here we show that at two sites in the southern Adriatic, all its seedlings possessed abundant root hairs with peculiar morphology (swollen terminal parts) and anatomy (spirally formed cell walls) as apparent adaptations for better attachment to the substrate and increase of breaking strain. Later on, their roots became colonized by dark septate mycelium while root hairs were reduced. In adults, most of terminal fine roots possessed the specific fungal association while root hairs were absent. These observations indicate for the first time that processes regulating transition from root hairs to root fungal colonization exist also in some seagrasses. This ontogenetic shift in root traits may suggests an involvement of the specific root symbiosis in the nutrient uptake by the dominant Mediterranean seagrass.

When the ribosomal DNA does not tell the truth: The case of the taxonomic position of Kurtia argillacea, an ericoid mycorrhizal fungus residing among Hymenochaetales.

The nuclear ribosomal DNA (nuc-rDNA) is widely used for the identification and phylogenetic reconstruction of Agaricomycetes. However, nuc-rDNA-based phylogenies may sometimes be in conflict with phylogenetic relationships derived from protein coding genes. In this study, the taxonomic position of the basidiomycetous mycobiont that forms the recently discovered sheathed ericoid mycorrhiza was investigated, because its nuc-rDNA is highly dissimilar to any other available fungal sequences in terms of nucleotide composition and length, and its nuc-rDNA-based phylogeny is inconclusive and significantly disagrees with protein coding sequences and morphological data. In the present work, this mycobiont was identified as Kurtia argillacea (= Hyphoderma argillaceum) residing in the order Hymenochaetales (Basidiomycota). Bioinformatic screening of the Kurtia ribosomal DNA sequence indicates that it represents a gene with a non-standard substitution rate or nucleotide composition heterogeneity rather than a deep paralogue or a pseudogene. Such a phenomenon probably also occurs in other lineages of the Fungi and should be taken into consideration when nuc-rDNA (especially that with unusual nucleotide composition) is used as a sole marker for phylogenetic reconstructions. Kurtia argillacea so far represents the only confirmed non-sebacinoid ericoid mycorrhizal fungus in the Basidiomycota and its intriguing placement among mostly saprobic and parasitic Hymenochaetales begs further investigation of its eco-physiology.

First record of Rhizoscyphus ericae in Southern Hemisphere's Ericaceae.

Ericoid mycorrhiza is arguably the least investigated mycorrhizal type, particularly when related to the number of potential hosts and the ecosystems they inhabit. Little is known about the global distribution of ericoid mycorrhizal (ErM) fungi, and this holds true even for the prominent ErM mycobiont Rhizoscyphus ericae. Earlier studies suggested R. ericae might be low in abundance or absent in the roots of Southern Hemisphere's Ericaceae, and our previous investigations in two Argentine Patagonian forests supported this view. Here, we revisited the formerly investigated area, albeit at a higher altitude, and screened fungi inhabiting hair roots of Gaultheria caespitosa and Gaultheria pumila at a treeless alpine site using the same methods as previously. We obtained 234 isolates, most of them belonging to Ascomycota. In contrast to previous findings, however, among 37 detected operational taxonomic units (OTUs), OTU 1 (=R. ericae s. str.) comprised the highest number of isolates (87, ∼37 %). Most of the OTUs and isolates belonged to the Helotiales, and 82.5 % of isolates belonged to OTUs shared between both Gaultheria species. At the alpine site, ericoid mycorrhizal fungi dominated, followed by dark septate endophytes and aquatic hyphomycetes probably acting as root endophytes. Our results suggest that the distribution of R. ericae is influenced, among others, by factors related to altitude such as soil type and presence/absence and type of the neighboring vegetation. Our study is the first report on R. ericae colonizing Ericaceae roots in the Southern Hemisphere and extends the known range of this prominent ErM species to NW Patagonia.

Experimental evidence of ericoid mycorrhizal potential within Serendipitaceae (Sebacinales).

The Sebacinales are a monophyletic group of ubiquitous hymenomycetous mycobionts which form ericoid and orchid mycorrhizae, ecto- and ectendomycorrhizae, and nonspecific root endophytic associations with a wide spectrum of plants. However, due to the complete lack of fungal isolates derived from Ericaceae roots, the Sebacinales ericoid mycorrhizal (ErM) potential has not yet been tested experimentally. Here, we report for the first time isolation of a serendipitoid (formerly Sebacinales Group B) mycobiont from Ericaceae which survived in pure culture for several years. This allowed us to test its ability to form ericoid mycorrhizae with an Ericaceae host in vitro, to describe its development and colonization pattern in host roots over time, and to compare its performance with typical ErM fungi and other serendipitoids derived from non-Ericaceae hosts. Out of ten serendipitoid isolates tested, eight intracellularly colonized Vaccinium hair roots, but only the Ericaceae-derived isolate repeatedly formed typical ericoid mycorrhiza morphologically identical to ericoid mycorrhiza commonly found in naturally colonized Ericaceae, but yet different from ericoid mycorrhiza formed in vitro by the prominent ascomycetous ErM fungus Rhizoscyphus ericae. One Orchidaceae-derived isolate repeatedly formed abundant hyaline intracellular microsclerotia morphologically identical to those occasionally found in naturally colonized Ericaceae, and an isolate of Serendipita (= Piriformospora) indica produced abundant intracellular chlamydospores typical of this species. Our results confirm for the first time experimentally that some Sebacinales can form ericoid mycorrhiza, point to their broad endophytic potential in Ericaceae hosts, and suggest possible ericoid mycorrhizal specificity in Serendipitaceae.

Communities of Cultivable Root Mycobionts of the Seagrass Posidonia oceanica in the Northwest Mediterranean Sea Are Dominated by a Hitherto Undescribed Pleosporalean Dark Septate Endophyte.

Seagrasses, a small group of submerged marine macrophytes, were reported to lack mycorrhizae, i.e., the root-fungus symbioses most terrestrial plants use for nutrient uptake. On the other hand, several authors detected fungal endophytes in seagrass leaves, shoots, rhizomes, and roots, and an anatomically and morphologically unique dark septate endophytic (DSE) association has been recently described in the roots of the Mediterranean seagrass Posidonia oceanica. Nevertheless, the global diversity of seagrass mycobionts is not well understood, and it remains unclear what fungus forms the DSE association in P. oceanica roots. We isolated and determined P. oceanica root mycobionts from 11 localities in the northwest Mediterranean Sea with documented presence of the DSE association and compared our results with recent literature. The mycobiont communities were low in diversity (only three species), were dominated by a single yet unreported marine fungal species (ca. 90 % of the total 177 isolates), and lacked common terrestrial and freshwater root mycobionts. Our phylogenetic analysis suggests that the dominating species represents a new monotypic lineage within the recently described Aigialaceae family (Pleosporales, Ascomycota), probably representing a new genus. Most of its examined colonies developed from intracellular microsclerotia occupying host hypodermis and resembling microsclerotia of terrestrial DSE fungi. Biological significance of this hitherto overlooked seagrass root mycobiont remains obscure, but its presence across the NW Mediterranean Sea and apparent root intracellular lifestyle indicate an intriguing symbiotic relationship with the dominant Mediterranean seagrass. Our microscopic observations suggest that it may form the DSE association recently described in P. oceanica roots.

Anatomically and morphologically unique dark septate endophytic association in the roots of the Mediterranean endemic seagrass Posidonia oceanica.

Roots of terrestrial plants host a wide spectrum of soil fungi that form various parasitic, neutral and mutualistic associations. A similar trend is evident in freshwater aquatic plants and plants inhabiting salt marshes or mangroves. Marine vascular plants (seagrasses), by contrast, seem to lack specific root-fungus symbioses. We examined roots of two Mediterranean seagrasses, Posidonia oceanica and Cymodocea nodosa, in the northwestern Mediterranean Sea for fungal colonization using light and scanning and transmission electron microscopy. We found that P. oceanica, but not C. nodosa, is regularly associated with melanized septate hyphae in a manner resembling colonization by the ubiquitous dark septate endophytes (DSE) in roots of most terrestrial plants. P. oceanica roots were found to be colonized by sparse dematiaceous running hyphae as well as dense parenchymatous nets/hyphal sheaths on the root surface, intracellular melanized microsclerotia and occasionally also intra- and intercellular hyphae. The colonization was most prominent in the thick-walled hypodermis of the thinnest healthy looking roots, and the mycobiont seemed to colonize both living and dead host cells. Dark septate hyphae infrequently occurred also inside rhizodermal cells, but never colonized vascular tissues. The biological significance of this overlooked marine symbiosis remains unknown, but its morphology, extent, distribution across the NW Mediterranean Sea and absence in C. nodosa indicate an intriguing relationship between the dominant Mediterranean seagrass and its dark septate root mycobionts.

The potential of Dark Septate Endophytes to form root symbioses with ectomycorrhizal and ericoid mycorrhizal middle European forest plants.

The unresolved ecophysiological significance of Dark Septate Endophytes (DSE) may be in part due to existence of morphologically indistinguishable cryptic species in the most common Phialocephala fortinii s. l.--Acephala applanata species complex (PAC). We inoculated three middle European forest plants (European blueberry, Norway spruce and silver birch) with 16 strains of eight PAC cryptic species and other DSE and ectomycorrhizal/ericoid mycorrhizal fungi and focused on intraradical structures possibly representing interfaces for plant-fungus nutrient transfer and on host growth response. The PAC species Acephala applanata simultaneously formed structures resembling ericoid mycorrhiza (ErM) and DSE microsclerotia in blueberry. A. macrosclerotiorum, a close relative to PAC, formed ectomycorrhizae with spruce but not with birch, and structures resembling ErM in blueberry. Phialocephala glacialis, another close relative to PAC, formed structures resembling ErM in blueberry. In blueberry, six PAC strains significantly decreased dry shoot biomass compared to ErM control. In birch, one A. macrosclerotiorum strain increased root biomass and the other shoot biomass in comparison with non-inoculated control. The dual mycorrhizal ability of A. macrosclerotiorum suggested that it may form mycorrhizal links between Ericaceae and Pinaceae. However, we were unable to detect this species in Ericaceae roots growing in a forest with presence of A. macrosclerotiorum ectomycorrhizae. Nevertheless, the diversity of Ericaceae mycobionts was high (380 OTUs) with individual sites often dominated by hitherto unreported helotialean and chaetothyrialean/verrucarialean species; in contrast, typical ErM fungi were either absent or low in abundance. Some DSE apparently have a potential to form mycorrhizae with typical middle European forest plants. However, except A. applanata, the tested representatives of all hitherto described PAC cryptic species formed typical DSE colonization without specific structures necessary for mycorrhizal nutrient transport. A. macrosclerotiorum forms ectomycorrhiza with conifers but not with broadleaves and probably does not form common mycorrhizal networks between conifers with Ericaceae.

Is the prominent ericoid mycorrhizal fungus Rhizoscyphus ericae absent in the Southern Hemisphere's Ericaceae? A case study on the diversity of root mycobionts in Gaultheria spp. from northwest Patagonia, Argentina.

Ericaceae diversity hotspots are in the mountains of the Neotropics and Papua New Guinea, South Africa's fynbos and Southeast Asia but majority of references to their root mycobionts come from the Northern Hemisphere. Here, typical cultivable ericoid mycorrhizal (ErM) fungi comprise Rhizoscyphus ericae, Meliniomyces variabilis, and Oidiodendron maius. It is however unclear whether this is true also for the Southern Hemisphere. Our study focused on cultivable mycobionts from hair roots of Gaultheria mucronata and Gaultheria poeppigii (Ericaceae) from two natural forests in NW Patagonia, Argentina, differing in mycorrhizal preferences of their tree dominants. We detected 62 well-defined OTUs mostly belonging to Helotiales and Hypocreales; the most frequent were Phialocephala fortinii s. l., Pochonia suchlasporia, and Ilyonectria radicicola. Only one out of 257 isolates showed ITS nrDNA similarity to members of the R. ericae aggregate (REA) but was not conspecific with R. ericae, and only five isolates were conspecific with O. maius. Microscopic observations showed that the screened roots were frequently colonized in a manner differing from the pattern typically produced by R. ericae and O. maius. A re-synthesis experiment with selected isolates showed that only O. maius formed colonization resembling ericoid mycorrhiza. Amplification of root fungal DNA with REA-specific and Sebacinaceae-specific primers showed that REA mycobionts were present in some of the screened samples while Sebacinaceae were present in all samples. These results suggest that Gaultheria spp. from NW Patagonia form ericoid mycorrhizae predominantly with the difficult-to-cultivate Sebacinaceae while the incidence of REA is relatively low and may be masked by other most likely non-mycorrhizal cultivable mycobionts.

Novel root-fungus symbiosis in Ericaceae: sheathed ericoid mycorrhiza formed by a hitherto undescribed basidiomycete with affinities to Trechisporales.

Ericaceae (the heath family) are widely distributed calcifuges inhabiting soils with inherently poor nutrient status. Ericaceae overcome nutrient limitation through symbiosis with ericoid mycorrhizal (ErM) fungi that mobilize nutrients complexed in recalcitrant organic matter. At present, recognized ErM fungi include a narrow taxonomic range within the Ascomycota, and the Sebacinales, basal Hymenomycetes with unclamped hyphae and imperforate parenthesomes. Here we describe a novel type of basidiomycetous ErM symbiosis, termed 'sheathed ericoid mycorrhiza', discovered in two habitats in mid-Norway as a co-dominant mycorrhizal symbiosis in Vaccinium spp. The basidiomycete forming sheathed ErM possesses clamped hyphae with perforate parenthesomes, produces 1- to 3-layer sheaths around terminal parts of hair roots and colonizes their rhizodermis intracellularly forming hyphal coils typical for ErM symbiosis. Two basidiomycetous isolates were obtained from sheathed ErM and molecular and phylogenetic tools were used to determine their identity; they were also examined for the ability to form sheathed ErM and lignocellulolytic potential. Surprisingly, ITS rDNA of both conspecific isolates failed to amplify with the most commonly used primer pairs, including ITS1 and ITS1F + ITS4. Phylogenetic analysis of nuclear LSU, SSU and 5.8S rDNA indicates that the basidiomycete occupies a long branch residing in the proximity of Trechisporales and Hymenochaetales, but lacks a clear sequence relationship (>90% similarity) to fungi currently placed in these orders. The basidiomycete formed the characteristic sheathed ErM symbiosis and enhanced growth of Vaccinium spp. in vitro, and degraded a recalcitrant aromatic substrate that was left unaltered by common ErM ascomycetes. Our findings provide coherent evidence that this hitherto undescribed basidiomycete forms a morphologically distinct ErM symbiosis that may occur at significant levels under natural conditions, yet remain undetected when subject to amplification by 'universal' primers. The lignocellulolytic assay suggests the basidiomycete may confer host adaptations distinct from those provisioned by the so far investigated ascomycetous ErM fungi.

Surprising spectra of root-associated fungi in submerged aquatic plants.

Similarly to plants from terrestrial ecosystems, aquatic species harbour wide spectra of root-associated fungi (RAF). However, comparably less is known about fungal diversity in submerged roots. We assessed the incidence and diversity of RAF in submerged aquatic plants using microscopy, culture-dependent and culture-independent techniques. We studied RAF of five submerged isoetid species collected in four oligotrophic freshwater lakes in Norway. Levels of dark septate endophytes (DSE) colonization differed among the lakes and were positively related to the organic matter content and negatively related to pH. In total, we identified 41 fungal OTUs using culture-dependent and culture-independent techniques, belonging to Mucoromycotina, Chytridiomycota, Glomeromycota, Ascomycota as well as Basidiomycota. Sequences corresponding to aquatic hyphomycetes (e.g. Nectria lugdunensis, Tetracladium furcatum and Varicosporium elodeae) were obtained. Eight arbuscular mycorrhizal taxa belonging to the orders Archaeosporales, Diversisporales and Glomerales were also detected. However, the vast majority of the fungal species detected (e.g. Ceratobasidium sp., Cryptosporiopsis rhizophila, Leptodontidium orchidicola, and Tuber sp.) have previously been known only from roots of terrestrial plants. The abundance and phylogenetic distribution of mycorrhizal as well as nonmycorrhizal fungi in the roots of submerged plants have reshaped our views on the fungal diversity in aquatic environment.

Ericaceous dwarf shrubs affect ectomycorrhizal fungal community of the invasive Pinus strobus and native Pinus sylvestris in a pot experiment.

This study aimed to elucidate the relationship between ericaceous understorey shrubs and the diversity and abundance of ectomycorrhizal fungi (EcMF) associated with the invasive Pinus strobus and native Pinus sylvestris. Seedlings of both pines were grown in mesocosms and subjected to three treatments simulating different forest microhabitats: (a) grown in isolation and grown with (b) Vaccinium myrtillus or (c) Vaccinium vitis-idaea. Ericaceous plants did not act as a species pool of pine mycobionts and inhibited the ability of the potentially shared species Meliniomyces bicolor to form ectomycorrhizae. Similarly, Ericaceae significantly reduced the formation of Thelephora terrestris ectomycorrhizae in P. sylvestris. EcMF species composition in the mesocosms was strongly affected by both the host species and the presence of an ericaceous neighbour. When grown in isolation, P. strobus root tips were predominantly colonised by Wilcoxina mikolae, whereas those of P. sylvestris were more commonly colonised by Suillus and Rhizopogon spp. Interestingly, these differences were less evident (Suillus + Rhizopogon spp.) or absent (W. mikolae) when the pines were grown with Ericaceae. P. strobus exclusively associated with Rhizopogon salebrosus s.l., suggesting the presence of host specificity at the intrageneric level. Ericaceous plants had a positive effect on colonisation of P. strobus root tips by R. salebrosus s.l. This study demonstrates that the interaction of selective factors such as host species and presence of ericaceous plants may affect the realised niche of the ectomycorrhizal fungi.

Interactions between testate amoebae and saprotrophic microfungi in a Scots pine litter microcosm.

In all terrestrial ecosystems, testate amoebae (TA) encounter fungi. There are strong indications that both groups engage in multiple interactions, including mycophagy and decomposition of TA shells, processes which might be fundamental in nutrient cycling in certain ecosystems. Here, we present the results of an experiment focusing on interactions between TA and saprotrophic microfungi colonizing Scots pine (Pinus sylvestris L.) litter needles. The needles were collected from a temperate pine forest and cultivated in damp chambers. Over a few weeks, melanized mycelium of Anavirga laxa Sutton started to grow out of some needles; simultaneously, the common forest-soil TA Phryganella acropodia (Hertwig and Lesser) Hopkinson reproduced and spread around the mycelium. We investigated whether a potential relationship between TA and saprotrophic microfungi exists by comparing the composition of TA communities on and around the needles and testing the spatial relationship between the A. laxa mycelium and P. acropodia shells in the experimental microcosm. Additionally, we asked whether P. acropodia utilized the A. laxa mycelium as a nutrient source and screened whether P. acropodia shells were colonized by the microfungi inhabiting the experimental microcosm. Our results indicate that saprotrophic microfungi may affect the composition of TA communities and their mycelium may affect distribution of TA individuals in pine litter. Our observations suggest that P. acropodia did not graze directly on A. laxa mycelium, but rather fed on its exudates or bacteria associated with the exudates. The fungus Pochonia bulbillosa (Gams & Malla) Zare & Gams was often found parasitising encysted shells or decomposing already dead individuals of P. acropodia. TA and pine litter microfungi engage in various direct and indirect interactions which are still poorly understood and deserve further investigation. Their elucidation will improve our knowledge on fundamental processes influencing coexistence of soil microflora and microfauna.