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.     

Sebacinales form ectendomycorrhizas with Cavendishia nobilis, a member of the Andean clade of Ericaceae, in the mountain rain forest of southern Ecuador

Cavendishia nobilis var. capitata is an endemic member of the Ericaceae growing as a hemiepiphyte in the tropical mountain rain forest of southern Ecuador. Mycorrhizas were collected from 20 individuals along an altitudinal gradient between 1850 and 2300 m. Transmission electron microscopy was used to study the symbiotic association in detail, and phylogenetic analyses based on nuclear rDNA coding for the ribosomal large subunit (nucLSU) were carried out to identify the associated mycorrhizal fungi. Microscopic and ultrastructural investigations showed the formation of a hyphal sheath, intercellular penetration of fine hyphae and colonization of the cortical cells by swollen hyphae of the same fungus. These structures were formed by hymenomycetes and ascomycetes. Molecular phylogenetic analysis detected seven groups of mycorrhizal fungi belonging to the Sebacinales. This is the first study to obtain evidence of ectendomycorrhizas in the Vaccinioideae. The ascomycetous nucLSU sequences belonged to members of the Leotiomycetes. The ectendomycorrhiza of C. nobilis with Sebacinales is discussed as a specific, hitherto undescribed mycorrhizal subcategory of ectomycorrhizas. We propose the term 'cavendishioid mycorrhiza'. This subcategory is most likely specific for the Andean clade of Ericaceae.     

The role of the motile tubular vacuole system in mycorrhizal fungi

Mycorrhizal fungi, to be effective for the plant, must be able to transfer mineral nutrient elements from sites of uptake at hyphal tips across various distances to the exchange region in the mycorrhiza. Vacuoles are likely to be important in this transport, since they contain elements of nutritional significance in abundance. In tip cells of hyphae of most fungi –- known to include three ectomycorrhizal basidiomycetes, an ericoid mycobiont, and two arbuscular mycorrhizal fungi –- the vacuoles form a motile tubular reticulum. The vacuoles are most active in hyphal tips, but non-motile vacuoles at a distance from the tip can be induced to become motile by environmental changes. Neither the tubular vacuolar reticulum nor its contents are properly preserved by conventional fixation and embedding. Vacuolar tubules are readily shown in vivo with fluorescent tracers, throughout the extramatrical mycelium and in outer hyphae of the sheath in eucalypt mycorrhizas synthesised with Pisolithus sp., but they have proved harder to label in field-collected ectomycorrhizas and ericoid mycorrhizas. Freeze-substitution does preserve the structure of vacuoles and vacuolar tubules, and careful anhydrous techniques allow them to be microanalysed, indicating high content of K and P in vacuoles of hyphal tips, and also in sheath and Hartig net of ectomycorrhizas. Vacuoles contain polyphosphate in diffuse, non-granular form. Polyphosphate is present right up to the tip region of hyphae as well as in sheath and Hartig net: thus important mineral nutrient elements are present at both ends of the long hyphal transport pathway. Exactly what happens in between, however, remains to be elucidated.     

Co-occurrence of three fungal root symbionts in Gaultheria poeppiggi DC in central Argentina

The roots of Gaultheria poeppiggi (Ericaceae) were examined for fungal symbiont colonization. Typical structures of ericoid mycorrhizas (hyphae and intracellular coil hyphae complexes), dark septate fungal endophytes (hyphae and sclerotia), and arbuscular mycorrhizas (hyphae, coils, vesicles and arbuscules) were found in the roots of all the individuals examined. The evolutionarily derived position of Gaultheria within the Ericales may suggest that G. poeppiggi recently acquired the ability to form arbuscular mycorrhizas rather than having retained it from ancestral lines.     

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.     

Ultrastructural investigations of Arbutus unedo-Laccaria amethystea mycorrhiza synthesized in vitro

Summary Anatomy and ultrastructure of the arbutoid mycorrhiza of Arbutus unedo-Laccaria amethystea from axenic culture are described. In comparison to non-inoculated roots, the rhizodermal cells of mycorrhizas are of greater volume, their nuclei are enlarged and show an irregular shape, plasmalemma and cytoplasm with mitochondria, plastids, endoplasmic reticulum and dictyosomes are increased. Several ontogenetical states are documented. The arbutoid mycorrhiza as a connecting link between ectomycorrhiza and ericoid mycorrhiza is discussed.     

Formation of structures resembling ericoid mycorrhizas by the root endophytic fungus Heteroconium chaetospira within roots of Rhododendron obtusum var. kaempferi

A resynthesis study was conducted to clarify the relationship between the root endophyte, Heteroconium chaetospira and the ericaceous plant, Rhododendron obtusum var. kaempferi. The host plant roots were recovered 2 months after inoculation, and the infection process and colonization pattern of the fungus were observed under a microscope. The hyphae of H. chaetospira developed structures resembling ericoid mycorrhizas, such as hyphal coils within the host epidermal cells. These structures were morphologically the same as previously reported ericoid mycorrhizal structures. The frequencies of hyphal coils within the epidermal cells of host roots ranged from 13 to 20%. H. chaetospira did not promote or reduce host plant growth. This is the first reported study that H. chaetospira is able to form structures resembling mycorrhizas within the roots of ericaceous plants.     

Morpho‐anatomical and molecular characterization of the mycorrhizas of European Polygala species

The mycorrhizas of 12 species of Polygala (Polygalaceae), including herbs, subshrubs and one shrub, collected from Germany, Mallorca (Spain) and Malta, were investigated by morpho‐anatomical and molecular methods. Aseptate hyphae, arbuscules and vesicles indicate an arbuscular mycorrhiza in all species examined. Hyphal spread in Polygala is predominantly, but not exclusively, intracellular and comprises three characteristic stages of colonization: (i) intracellular, linear hyphal growth in a cascading manner after penetration towards the penultimate parenchyma layer (layer 2), (ii) initially linear hyphal growth in the cells of layer 2 from where hyphal branches repeatedly penetrate the anatomically distinct innermost parenchyma layer (layer 1), forming arbuscule‐like structures therein which are subject to degeneration, (iii) more branches from the linear hyphae in layer 2 develop, but coil and make contact to the layer outside layer 2 (layer 3) in which arbuscule‐like structures similar to those in layer 1 form and degenerate. This general colonization pattern differs in details between the species, and critical comparisons, in particular between the woody P. myrtifolia, the herbaceous Polygala spp. and the mycoheterotrophic Epirixanthes spp. (Polygalaceae) suggest an evolutionary shift of mycorrhizal features within the family towards an optimization of plant benefit through the fungus. Based on the molecular marker 18S rDNA mycorrhizal fungi detected in roots of Polygala spp. are largely restricted to five clades of Glomeraceae 1 (Glomus Group A). This result rejects the hypothesis of a strict symbiotic specificity in Polygalaceae but may stimulate a discussion on functionally compatible groups of fungi.     

Morphological and molecular evidence supporting an arbutoid mycorrhizal relationship in the Costa Ricanpáramo

This study examines evidence for a particular arbutoid mycorrhizal interaction in páramo, a high-altitude neotropical ecosystem important in hydrological regulation but poorly known in terms of its fungal communities. Comarostaphylis arbutoides Lindley (Ericaceae) often forms dense thickets in Central American páramo habitats. Based on phylogenetic classification, it has been suggested that C. arbutoides forms arbutoid mycorrhizae with diverse Basidiomycetes and Ascomycetes; however, this assumption has not previously been confirmed. Based on field data, we hypothesized an arbutoid mycorrhizal association between C. arbutoides and the recently described bolete Leccinum monticola Halling & G.M. Mueller; in this study, we applied a rigorous approach using anatomical and molecular data to examine evidence for such an association. We examined root samples collected beneath L. monticola basidiomes for mycorrhizal structures, and we also compared rDNA internal transcribed spacer (ITS) sequences between mycorrhizal root tips and leaf or basidiome material of the suspected symbionts. Root cross sections showed a thin hyphal sheath and intracellular hyphal coils typical of arbutoid mycorrhizae. DNA sequence comparisons confirmed the identity of C. arbutoides and L. monticola as the mycorrhizal symbionts. In addition, this paper provides additional evidence for the widespread presence of minisatellite-like inserts in the ITS1 spacer in Leccinum species (including a characterization of the insert in L. monticola) and reports the use of an angiosperm-specific ITS primer pair useful for amplifying plant DNA from mycorrhizal roots without co-amplifying fungal DNA.     

Epiphytic and terrestrial mycorrhizas in a lower montane Costa Rican cloud forest

The epiphyte community is the most diverse plant community in neotropical cloud forests and its collective biomass can exceed that of the terrestrial shrubs and herbs. However, little is known about the role of mycorrhizas in this community. We assessed the mycorrhizal status of epiphytic (Araceae, Clusiaceae, Ericaceae, and Piperaceae) and terrestrial (Clusiaceae, Ericaceae) plants in a lower montane cloud forest in Costa Rica. Arbuscular mycorrhizas were observed in taxa from Araceae and Clusiaceae; ericoid mycorrhizas were observed in ericaceous plants. This is the first report of intracellular hyphal coils characteristic of ericoid mycorrhizas in roots of Cavendishia melastomoides, Disterigma humboldtii, and Gaultheria erecta. Ericaceous roots were also covered by an intermittent hyphal mantle that penetrated between epidermal cells. Mantles, observed uniquely on ericaceous roots, were more abundant on terrestrial than on epiphytic roots. Mantle abundance was negatively correlated with gravimetric soil water content for epiphytic samples. Dark septate endophytic (DSE) fungi colonized roots of all four families. For the common epiphyte D. humboldtii, DSE structures were most abundant on samples collected from exposed microsites in the canopy. The presence of mycorrhizas in all epiphytes except Peperomia sp. suggests that inoculum levels and environmental conditions in the canopy of tropical cloud forests are generally conducive to the formation of mycorrhizas. These may impact nutrient and water dynamics in arboreal ecosystems.     

Decomposition of organic matter by the ericoid mycorrhizal endophytes of Formosan rhododendron (Rhododendron formosanum Hemsl.)

Ericoid mycorrhizas are associated with a number of host plants in the Ericaceae in high-elevation regions of Taiwan. The ability of these microorganisms to thrive in harsh environmental conditions in the regions implies their capability of decomposing plant organic matter (raw humus). The objective of this study was to investigate the decomposition characteristics of three ericoid mycorrhizal endophytes isolated from the roots of Formosan rhododendron (Rhododendron formosanum Hemsl.). Molecular analysis indicated that strains Rf9 and Rf32 belong to the genus Cryptosporiopsis while strain Rf28 is a member of the genus Phialocephala. Mycorrhizal synthesis experiment showed that the roots of synthesized seedlings produced hyphal coils, a characteristic of ericoid mycorrhiza. Decomposition ability analysis revealed that strains Rf28 and Rf32 had the highest rates of decomposition of organic matter (up to 10.4% after 70 days) while the value for strain Rf9 was about 6.8%. Consistently, these strains secreted extracellular oxidases when cultured on tannic acid medium. Enzyme assay revealed that strains Rf28 and Rf32 secreted peroxidase, laccase, tyrosinase, and cellulase, but strain Rf9 secreted mainly peroxidase and tyrosinase. Apparently, the differences in secreted hydrolytic enzymes among the three endophytes are related to their ability to decompose organic matter. In the mycorrhizal synthesis experiment, all inoculated seedlings survived in the organic matter substrate for 70 days and exhibited a stronger vigor than the control. This study demonstrated that these three isolated endophytes, Rf9, Rf28, and Rf32, are ericoid mycorrhizal fungi, capable of forming ericoid mycorrhiza with Formosan rhododendron. Meanwhile, all three endophytes can secrete hydrolytic enzymes to decompose organic matter for growth, presumably a prerequisite for the adaptation of Formosan rhododendron to the harsh environments of high elevation.     

The mycorrhiza of <Emphasis Type="Italic">Schizocodon soldanelloides</Emphasis> var. <Emphasis Type="Italic">magnus</Emphasis> (Diapensiaceae) is regarded as ericoid mycorrhiza from its structure and fungal identities

Structure and fungal identities were examined in the mycorrhizal roots of Schizocodon soldanelloides var. magnus (Diapensiaceae) to determine the mycorrhizal category. Previous studies had suggested the mycorrhizae of Diapensiaceae could be categorized as ericoid, but the mycorrhizal fungi have never been identified. The diameter of the fine lateral roots, in which coiled hyphae were found in epidermal cells, was mostly less than 100 μm. Molecular analyses identified the fungal isolates to be Helotiales and Oidiodendron. From the structure and fungal identities, we confirmed that the mycorrhiza of S. soldanelloides is an ericoid mycorrhiza.     

The Co-occurrence and Morphological Continuum Between Ericoid Mycorrhiza and Dark Septate Endophytes in Roots of Six European Rhododendron Species

Ericaceae associate with a wide spectrum of root mycobionts, but the most common are ascomycetous ericoid mycorrhizal fungi and dark septate endophytes (DSE), followed by basidiomycetous fungi and glomeracean arbuscular mycorrhizal fungi. We investigated distribution and morphological diversity of ericoid mycorrhizae (ErM), DSE associations, ectomycorrhizae (EcM) and arbuscular mycorrhizae (AM) in hair roots of six European native Rhododendron species and found that i) while EcM and AM were absent, ErM and DSE associations were simultaneously present in all screened plants; ii) their levels were negatively correlated, suggesting Ericaceae preference for certain root-fungus association in certain habitats; iii) the highest ErM colonization occurred at sites in southern and central Europe, while the highest DSE colonization was found in a subarctic site in northern Finland and in a subalpine site in the Carpathians, suggesting a latitudinal/altitudinal shift in Ericaceae root-fungus associations; iv) some mycelia could simultaneously form structures corresponding to ErM and DSE association, which occasionally resulted in a unique ectendomycorrhizal colonization comprising an intercellular parenchymatous net and intracellular hyphal coils. These results indicate frequent interactions between ErM fungi and DSE in roots of European rhododendrons and a morphological continuum between ErM and DSE associations. The new ectendomycorrhizal type deserves further investigation.     

Mycorrhiza of the host-specific Lactarius deterrimus on the roots of Picea abies and Arctostaphylos uva-ursi

The ectomycorrhizal basidiomycete species Lactarius deterrimus Gröger is considered to be a strictly host-specific mycobiont of Picea abies (L.) Karst. However, we identified arbutoid mycorrhiza formed by this fungus on the roots of Arctostaphylos uva-ursi (L.) Spreng. in a mixed stand at the alpine timberline; typical ectomycorrhiza of P. abies were found in close relation. A. uva-ursi is known as an extremely unspecific phytobiont. The mycorrhizae of both associations are described and compared morphologically. The mycorrhiza formed by L. deterrimus on both A. uva-ursi and P. abies show typical ectomycorrhizal features such as a hyphal mantle and a Hartig net. The main difference between the mycorrhizal symbioses with the different phytobionts is the occurrence of intracellular hyphae in the epidermal cells of A. uva-ursi. This emphasizes the importance of the plant partner for mycorrhizal anatomy. This is the first report of a previously considered host-specific ectomycorrhizal fungus in association with A. uva-ursi under natural conditions. The advantages of this loose specificity between the fungus and plant species is discussed.     

Seasonal and environmental changes of mycorrhizal associations and heterotrophy levels in mixotrophic Pyrola japonica (Ericaceae) growing under different light environments

? Premise of the study: Mixotrophy is a strategy whereby plants acquire carbon both through photosynthesis and heterotrophic exploitation of mycorrhizal fungi. In Euro-American Pyroleae species studied hitherto, heterotrophy levels vary according to species, sites of study, and possibly light conditions. We investigated mycorrhizal association and mixotrophy in the Asiatic forest species Pyrola japonica, and their plasticity under different light conditions. ? Methods: Pyrola japonica was sampled bimonthly in sunny and shaded conditions from a deciduous broadleaf forest. We microscopically assessed the rate of fungal colonization and sequenced the ITS to identify the mycorrhizal fungi. We measured (13)C and (15)N isotopic abundances in P. japonica as compared with neighboring autotrophic and mycoheterotrophic plants, to evaluate P. japonica's heterotrophy level. ? Key results: Pyrola japonica formed arbutoid mycorrhizas devoid of fungal mantles, with intracellular hyphal coils and a Hartig net. It tended to be more colonized by mycorrhizal fungi in spring and summer. Most associated fungi belonged to ectomycorrhizal taxa, and 84% of identified fungi were Russula spp. Rate of mycorrhizal colonization and Russula frequency tended to be higher in shaded conditions. Both δ(13)C and δ(15)N values of P. japonica were significantly higher in autotrophic plants, showing that about half of the carbon on average was received from mycorrhizal fungi. Both isotopic values negatively correlated with light availability, suggesting higher heterotrophy levels in shaded conditions. ? Conclusions: The mixotrophic P. japonica undergoes changes in mycorrhizal symbionts and carbon nutrition according to light availability. Our results suggest that during Pyroleae evolution, a tendency to increased heterotrophy emerged in the Pyrola/Orthilia clade.     

Intracellular colonization of Rhododendron and Vaccinium roots by Cenococcum geophilum, Geomyces pannorum and Meliniomyces variabilis

Four in vitro experiments were set up to verify the colonization potential of ectomycorrhizal (EcM) Cenococcum geophilum FR. (strain CGE-4), saprotrophic Geomyces pannorum (LINK) SIGLER & CARMICHAEL (GPA-1) and a frequent root-associated, potentially ericoid mycorrhiza (ErM)-forming Meliniomyces variabilis Hambleton & Sigler (MVA-1) in roots of Rhododendron and Vaccinium. A typical ErM fungus, Rhizoscyphus ericae (Read) Zhuang & Korf (RER-1), was included for comparison. All fungal strains intracellularly colonized rooted Vaccinium microcuttings: GPA-1 occasionally produced hyphal loops similar to ErM, MVA-1 and RER-1 exhibited a typical ErM colonization pattern. CGE-4 hyphae grew vigorously on and around newly formed roots and rarely penetrated turgescent rhizodermal cells forming intracellular loose loops. Rooting of Rhododendron sp. microcuttings was not promoted by any fungal strain except CGE-4, which also promoted the most vigorous growth of Rhododendron ponticum L. seedlings. The widespread EcM fungus C. geophilum has a potential to colonize non-EcM roots and support their development which may influence overall growth of ericaceous plants. As shown for G. pannorum, structures resembling ErM may be formed by fungi that are to date not regarded as ericoid mycorrhizal.     

Ericoid fungal diversity: Challenges and opportunities for mycorrhizal research

Ericoid mycorrhiza occur only within the plant family Ericaceae, yet are globally widespread and contribute to carbon and nutrient cycling in many habitats where harsh conditions limit decomposition and plant nutrient uptake. An increasingly diverse range of fungi are recognized as ericoid symbionts and patterns in the distribution of ericoid taxa are beginning to emerge across scales. However, the true diversity of ericoid mycorrhizal fungi remains unresolved due to limited sampling from some regions and challenges associated with delineating mycorrhizal taxa from the broader fungal community associated with ericoid plants. Interpreting patterns in the diversity and distributions of ericoid mycorrhizal fungi will ultimately require improved understanding of their functional ecology and functional diversity, which is currently limited to a few well studied species. Fortunately, many ericoid taxa are amenable to experimental manipulation and continued ericoid mycorrhizal research promises to improve general understanding of the ecology and evolution of mycorrhizal symbioses.     

Distribution of different mycorrhizal classes on Mount Koma, northern Japan

To investigate the role of mycorrhizae in nutrient-poor primary successional volcanic ecosystems, we surveyed mycorrhizal frequencies on the volcano Mount Koma (42°04N, 140°42E, 1,140 m elevation) in northern Japan. After the 1929 eruptions, plant community development started at the base of the volcano. Ammonia and nitrate levels, along with plant cover, decreased with increasing elevation, whereas phosphorus did not. In total, 305 individuals of 56 seed plant species were investigated in three elevational zones (550–600 m, 650–700 m, and 750–800 m). Five mycorrhizal classes were classified based on morphological traits: ecto- (ECM), arbuscular (AM), arbutoid, ericoid, and orchid mycorrhiza. All plant species were mycorrhizal to at least some extent, with most widespread tree species being heavily ectomycorrhizal. In addition, of 16 tree species collected in all three zones, 6 differed in the frequencies of ECM on roots between elevational zones, and 3 of these 6 species increased in frequency with increasing elevation. These results suggest that ECM colonization in some tree species is related to establishment in nutrient-poor habitats. All species of Ericaceae and Pyrolaceae had ericoid mycorrhizae, and an Orchidaceae species had orchid mycorrhizae. Herbaceous species, except for the low mycorrhizal frequency of Carex oxyandra and two Polygonaceae species, and ericoid and orchid mycorrhizal species, were generally AM. Of herbaceous species, Anaphalis margaritacea var. angustior increased AM frequency and decreased ECM frequency with increasing elevation, and Hieracium umbellatum increased ECM frequency. In total, the establishment of herbaceous species was not sufficiently explained by AM colonization on roots. Tree individuals developed 2–3 classes of mycorrhizae more than herbs at each elevational zone. We conclude that the symbiosis between seed plants and mycorrhizae, ECM in particular, greatly influences plant community structures on Mount Koma. Not only a single mycorrhizal class, but combinations of mycorrhizal classes should be studied to clarify effects on plant community dynamics.     

Fungal associates of <Emphasis Type="Italic">Pyrola rotundifolia</Emphasis>, a mixotrophic Ericaceae,from two Estonian boreal forests

Pyrola rotundifolia (Ericaceae, Pyroleae tribe) is an understorey subshrub that was recently demonstrated to receive considerable amount of carbon from its fungal mycorrhizal associates. So far, little is known of the identity of these fungi and the mycorrhizal anatomy in the Pyroleae. Using 140 mycorrhizal root fragments collected from two Estonian boreal forests already studied in the context of mixotrophic Ericaceae in sequence analysis of the ribosomal DNA internal transcribed spacer region, we recovered 71 sequences that corresponded to 45 putative species in 19 fungal genera. The identified fungi were mainly ectomycorrhizal basidiomycetes, including Tomentella, Cortinarius, Russula, Hebeloma, as well as some ectomycorrhizal and/or endophytic ascomycetes. The P. rotundifolia fungal communities of the two forests did not differ significantly in terms of species richness, diversity and nutritional mode. The relatively high diversity retrieved suggests that P. rotundifolia does not have a strict preference for any fungal taxa. Anatomical analyses showed typical arbutoid mycorrhizae, with variable mantle structures, uniseriate Hartig nets and intracellular hyphal coils in the large epidermal cells. Whenever compared, fungal ultrastructure was congruent with the molecular identification. Similarly to other mixotrophic and autotrophic pyroloids in the same forests, P. rotundifolia shares its mycorrhizal fungal associates with surrounding trees that are likely a carbon source for pyroloids.