Diversity and host preference of fungi co-inhabiting Cenococcum mycorrhizae

Diverse fungal assemblages colonize the fine feeder roots of woody plants, including mycorrhizal fungi, fungal root endophytes and soil saprotrophs. The fungi co-inhabiting Cenococcum geophilum ectomycorrhizae (ECM) of Abies balsamea, Betula papyrifera and Picea glauca were studied at two boreal forest sites in Eastern Canada by direct PCR of ITS rDNA. 50 non-Cenococcum fungal sequence types were detected, including several potentially mycorrhizal species as well as fungal root endophytes. Non-melanized ascomycetes dominated, in contrast to the dark septate endophytes (DSE) reported in most culture dependent studies. The results demonstrate significant differences in root associated fungal assemblages among the host species studied. Fungal diversity was also host dependent, with P. glauca roots supporting a more diverse community than A. balsamea. Differences in root associated fungal communities may well influence ecological interactions among host plant species.     

Correspondence of ectomycorrhizal diversity and colonisation of willows (Salix spp.) grown in short rotation coppice on arable sites and adjacent natural stands

Willows (Salix spp.) are mycorrhizal tree species sometimes cultivated as short rotation coppice (SRC) on arable sites for energy purposes; they are also among the earliest plants colonising primary successional sites in natural stands. The objective of this study was to analyse the degree of colonisation and diversity of ectomycorrhizal (EM) communities on willows grown as SRC in arable soils and their adjacent natural or naturalized stands. Arable sites usually lack ectomycorrhizal host plants before the establishment of SRC, and adjacent natural or naturalized willow stands were hypothesized to be a leading source of ectomycorrhizal inoculum for the SRC. Three test sites including SRC stands (Salix viminalis, Salix dasyclados, and Salix schwerinii) and adjacent natural or naturalized (Salix caprea, Salix fragilis, and Salix?×?mollissima) stands in central Sweden were investigated on EM colonisation and morphotypes, and the fungal partners of 36 of the total 49 EM fungi morphotypes were identified using molecular tools. The frequency of mycorrhizas in the natural/naturalized stands was higher (two sites) or lower (one site) than in the corresponding cultivated stands. Correspondence analysis revealed that some EM taxa (e.g. Agaricales) were mostly associated with cultivated willows, while others (e.g. Thelephorales) were mostly found in natural/naturalized stands. In conclusion, we found strong effects of sites and willow genotype on EM fungi formation, but poor correspondence between the EM fungi abundance and diversity in SRC and their adjacent natural/naturalized stands. The underlying mechanism might be selective promotion of some EM fungi species by more effective spore dispersal.     

Arbuscular Mycorrhizae in association with aquatic and marshy plant species in Goa,India

Of the 20 plant species of hydrophytes screened for Arbuscular Mycorrhizal (AM) fungal root colonization, 10 aquatic plants (out of 14 species) and five marshy plants (out of six species) were found to be mycorrhizal, while the remaining species were non mycorrhizal. Vesicular colonization occurred in 12 plant species while arbuscular colonization was restricted to only three plant species. A rooted submerged pteridophyte viz., Isoetes coromandelina L. was found to be mycorrhizal exhibiting vesicular colonization. In all, two genera viz., Glomus and Scutellospora, the former being dominant, were recorded. The most common AM fungal species Glomus claroideum was recovered from 14 plant species.     

Nurse-plant and mulching effects on three conifer species in a Mexican temperate forest

Nurse-plant effects have been used as an effective reforestation and restoration strategy, and mulching has also effectively ameliorated soil-adverse conditions. However, use of nurse plants is limited by the presence of suitable nurse species before trees are planted, and use of mulching depends on availability of appropriate materials. The effects of Lupinus elegans as a nurse plant and pine-bark mulch were tested on three conifer species: Pinus montezumae, Pinus pseudostrobus, and Abies religiosa. We tested if nurse-plant type effects occurred if the lupines and the conifers were planted simultaneously. Overall survival was 53%, and significant differences among species were found. Survival regardless of cover type was significantly higher (P < 0.01) for P. pseudostrobus 17-month-old seedlings at planting (81%), followed by P. montezumae 7-month-old seedlings at planting (71%), P. pseudostrobus 7-month-old seedlings at planting (42%) and finally A. religiosa 7-month-old seedlings at planting (17%) the differences among the treatments were significant (P < 0.01). As a soil shading agent, lupines had a significant effect (P = 0.02), because they increased survival of P. pseudostrobus and A. religiosa, the effect of mulching with pine bark was not significant. The effects of a shading agent on growth differed among species. In particular lupines reduced height and stem diameter for P. montezumae and P. pseudostrobus, between 22% and 33%, and had no effect on growth of A. religiosa. The positive effects of the simultaneous planting of L. elegans on the survival of the trees suggests that nurse-plant facilitation can be used in areas where no suitable nurse plants are already present.     

Specificity of assemblage,not fungal partner species,explains mycorrhizal partnerships of mycoheterotrophic Burmannia plants

Mycoheterotrophic plants (MHPs) growing on arbuscular mycorrhizal fungi (AMF) usually maintain specialized mycorrhizal associations. The level of specificity varies between MHPs, although it remains largely unknown whether interactions with mycorrhizal fungi differ by plant lineage, species, and/or by population. Here, we investigate the mycorrhizal interactions among Burmannia species (Burmanniaceae) with different trophic modes using high-throughput DNA sequencing. We characterized the inter- and intraspecific dynamics of the fungal communities by assessing the composition and diversity of fungi among sites. We found that fully mycoheterotrophic species are more specialized in their fungal associations than chlorophyllous species, and that this specialization possibly results from the gradual loss of some fungal groups. In particular, although many fungal species were shared by different Burmannia species, fully MHP species typically host species-specific fungal assemblages, suggesting that they have a preference for the selected fungi. Although no apparent cophylogenetic relationship was detected between fungi and plants, we observe that evolutionarily closely related plants tend to have a greater proportion of shared or closely related fungal partners. Our findings suggest a host preference and specialization toward fungal assemblages in Burmannia, improving understanding of interactions between MHPs and fungi.Subject terms: Fungi, Plant sciences, Evolution     

Specificity and preference of mycorrhizal associations in two species of the genus Dendrobium (Orchidaceae)

Dendrobium is a large genus of tropical epiphytic orchids. Some members of this genus are in danger of extinction across China. To investigate orchid mycorrhizal associations of the genus Dendrobium, plants from two Dendrobium species (Dendrobium officinale and Dendrobium fimbriatum) were collected from two habitats in Guangxi Province, China, and clone libraries were constructed to identify the mycorrhizal fungi of individual plants. A low and high degree of specificity was observed in D. officinale and D. fimbriatum, respectively. Phylogenetic analysis revealed that the majority of Dendrobium mycorrhizal fungi are members of the Tulasnellaceae, but, in some plants, members of the Ceratobasidiaceae and Pluteaceae were also found. In D. officinale, individual plants associated with more than three fungi simultaneously, and, in some cases, associations with five fungi at the same time. One fungus was shared by individual plants of D. officinale collected from the two habitats. In D. fimbriatum, only one fungal partner was found in each population, and this fungus differed between populations. The two species of Dendrobium sampled from the same habitat did not share any fungal taxa. These results provide valuable information for conservation of these orchid species.     

Host specificity and biology ofMegacyllene mellyi [Col.: Cerambycidae] introduced into australia for the biological control ofBaccharis halimifolia [Compositae]

Biology and host plant specificity of the stem boring cerambycidMegacyllene mellyi (Chevrolat) were studied in Brazil to determine its suitability for introduction into Australia for control of the shrubBaccharis halimifolia L. Multiple choice host preference testing of plants related toBaccharis, of desirable plants from a range of plant families, and of the host plants of otherMegacyllene species, showed thatM. mellyi was restricted toBaccharis spp. It was introduced into Australia in 1975 and released in 1978. Recoveries were made 3 years after release and some stems were killed, although damage was slight relative to the number ofB. halimifolia plants in the release area.     

Untangling above‐ and belowground mycorrhizal fungal networks in tropical orchids

Orchids typically depend on fungi for establishment from seeds, forming mycorrhizal associations with basidiomycete fungal partners in the polyphyletic group rhizoctonia from early stages of germination, sometimes with very high specificity. This has raised important questions about the roles of plant and fungal phylogenetics, and their habitat preferences, in controlling which fungi associate with which plants. In this issue of Molecular Ecology, Martos et al. (2012) report the largest network analysis to date for orchids and their mycorrhizal fungi, sampling a total of over 450 plants from nearly half the 150 tropical orchid species on Reunion Island, encompassing its main terrestrial and epiphytic orchid genera. The authors found a total of 95 operational taxonomic units of mycorrhizal fungi and investigated the architecture and nestedness of their bipartite networks with 73 orchid species. The most striking finding was a major ecological barrier between above‐ and belowground mycorrhizal fungal networks, despite both epiphytic and terrestrial orchids often associating with closely related taxa across all three major lineages of rhizoctonia fungi. The fungal partnerships of the epiphytes and terrestrial species involved a diversity of fungal taxa in a modular network architecture, with only about one in ten mycorrhizal fungi partnering orchids in both groups. In contrast, plant and fungal phylogenetics had weak or no effects on the network. This highlights the power of recently developed ecological network analyses to give new insights into controls on plant–fungal symbioses and raises exciting new hypotheses about the differences in properties and functioning of mycorrhiza in epiphytic and terrestrial orchids.     

Mycorrhizal colonization impacts on phenolic content and antioxidant properties of artichoke leaves and flower heads two years after field transplant

Greenhouse and field experiments were carried out in order to investigate the influence of mycorrhizal inoculation on total phenolic content (TPC) and antioxidant activity, expressed as antiradical power (ARP), of artichoke (Cynara cardunculus L. var. scolymus F.) leaves and flower heads extracts. The establishment of mycorrhizal symbiosis was monitored in pot and field grown plants, and the persistence of the inoculated AMF in roots after 2 years’ growth in the field was assessed by fungal ITS sequencing. Both in the greenhouse and in the field, marked increases in TPC and ARP were detected in leaves and flower heads of artichoke plants inoculated with the AM fungal species Glomus intraradices, either alone or in mixture with Glomus mosseae. In the field, plants inoculated with Glomus mix showed flower heads ARP content increases of 52.7 and 30.0% in the first and second year, respectively, compared with uninoculated plants. After 2 years’ growth in the field ITS rDNA sequences clustering with those of G. mosseae and G. intraradices were retrieved only from inoculated plant roots. Our data show that mycorrhizal inoculation may represent an efficient and sustainable strategy to improve productivity and enhance plant biosynthesis of secondary metabolites with health promoting activities.     

Mycorrhizal features and fungal partners of four mycoheterotrophic Monotropoideae (Ericaceae) species from Yunnan, China

We provide a preliminary report of the mycobionts found within four Monotropoideae (Ericaceae) species from China: Monotropa uniflora, Hypopitys monotropa, Monotropastrum humile and Monotropastrum sciaphilum (a rare endemic species never previously studied for mycorrhizae). Such achlorophyllous Monotropoideae plants obtain their carbohydrates from mycorrhizal fungi linking them to surrounding trees, on which these fungi form ectomycorrhizae. Since Monotropoideae were rarely studied in continental Asia, the root systems of the four species sampled in Yunnan were examined using morphological and molecular methods. All the roots of these four species exhibit a typical monotropoid mycorrhizal morphology, including a fungal mantle, a Hartig net and hyphal pegs. In M. uniflora and M. humile mycorrhizae, cystidia typical of Russula symbionts covered the fungal mantle. ITS barcoding revealed that Russulales were the most frequent colonizers in all species, but Hypopitys monotropa displayed various additional mycorrhizal taxa. Moreover, a few additional ectomycorrhizal and saprotrophic Basidiomycota taxa were identified in the three other species, challenging that these four Monotropoideae species are as strictly fungal specific as the other Monotropoideae species hitherto studied. Moreover, a comparison with accompanying fungus sporocarps revealed that the fruiting fungal community significantly differed from that associated with the Monotropoideae roots, so that a clear fungal preference was evident. Finally, four fungal species were found on more than one Monotropoideae species: this contrasted with previous reports of sympatrically growing mycoheterotrophic plants, which did not reveal any overlap. This again challenges the idea of strict fungal specificity.     

Mycorrhizal colonization status of plant species established in an exposed area following the 2000 eruption of Mt. Usu, Hokkaido, Japan

We investigated the ectomycorrhizal (ECM) and arbuscular mycorrhizal (AM) colonization status of plant seedlings that established in areas devastated by the eruption in March 2000 of Mt. Usu, Hokkaido, Japan. In 2005, we estimated the mycorrhizal colonization ratios and frequencies in seedlings of twelve herbaceous and seven woody plant species. Although arbuscular mycorrhizas were found to colonize Equisetum arvense and Polygonum sachalinense, they were presented at very low frequencies and colonization ratios. Other herbaceous plants exhibited higher frequencies of AM associations and either AM and/or ECM fungal associations were observed in all of the woody plant seedlings investigated. The dominant woody plant species (Populus maximowiczii, Salix sachalinensis and Salix hultenii var. angustifolia) associated mainly with ECM fungi and exhibited variable associations with AM fungi. Mycorrhizal associations were common and significant events for plant establishment, even in the early stages of the revegetation process.     

Ectomycorrhizal pre-inoculation of <Emphasis Type="Italic">Pinus hartwegii</Emphasis> and <Emphasis Type="Italic">Abies religiosa</Emphasis> is replaced by native fungi in a temperate forest of central Mexico

Few studies have focused on analyzing the effect of native inoculated ectomycorrhizal (ECM) fungal strains on seedlings under field conditions in temperate forests. However, it is crucial to verify that the positive effects of ECM under nursery conditions also occur in the field, favoring their performance. We evaluated the short-term effect of ECM on three-year-old seedlings of Pinus hartwegii and Abies religiosa in central Mexico by subjecting them to four treatments: inoculation with Inocybe splendens, inoculation with Suillus brevipes (both native strains), inoculation with forest soil, and non-inoculated plants. Percentage of ECM colonization, plant growth (shoot height and stem diameter), and physiological (osmotic potential, stomatal conductance, CO₂ assimilation and water use efficiency) responses were evaluated. We found that these two ECM species were partial (P. hartwegii) or totally (A. religiosa) replaced after one and a half years in the field. P. hartwegii seedlings increased their water use efficiency during the dry season, but in A. religiosa seedlings, a clear strategy for avoiding water stress was not detected. This ECM replacement had a negative effect on the physiological performance Of A. religiosa. Our results emphasize the importance of selecting compatible fungal-host species combinations for nursery inoculation and of using sources of inoculum adapted to the environmental conditions of the transplant site, ensuring root colonization prior to field transplanting to minimize seedling mortality due to water stress.     

Culturable fungal endophytes in roots of Enkianthus campanulatus (Ericaceae)

Roots of plants in the genus Enkianthus, which belongs to the earliest diverging lineage in the Ericaceae, are commonly colonized by arbuscular mycorrhizal (AM) fungi. We documented the community of fungal root endophytes associated with Enkianthus species using a culture-based method for better understanding the members of root-colonizing fungi, except for AM fungi. Fungal isolates were successfully obtained from 610 out of 3,599 (16.9 %) root segments. Molecular analysis of fungal cultures based on ribosomal internal transcribed spacer (ITS) sequences yielded 63 operational taxonomical units (OTUs: 97 % sequence similarity cutoff) from 315 representative isolates. Further phylogenetic analysis showed that most (296 isolates) belonged to Ascomycota and were either members of Helotiales (Dermataceae, Hyaloscyphaceae, Phialocephala and Rhizoscyphus ericae aggregate), Oidiodendron, or other Pezizomycotina. Twenty-three out of 63 OTUs, which mainly consisted of Leotiomycetes, showed high similarities with reference sequences derived from roots of other ericaceous plants such as Rhododendron. The results indicated that Enkianthus houses variable root mycobionts including putative endophytic and mycorrhizal fungi in addition to AM fungi.     

Seasonal and altitude effects on the structure of arthropod communities associated with <Emphasis Type="Italic">Tillandsia violacea</Emphasis> Baker (Bromeliaceae) in a temperate forest of Mexico

The canopy of forests has been considered “the last biotic frontier,” and study of its elements is very important in explaining the global functionality in ecosystems. Epiphytic plants and arthropods are essential elements in canopy habitats, and their relationships have been studied in order to understand the high diversity in tropical forests. Nevertheless, there are few studies on this development in temperate forests. The arthropod community was studied during the rainy and dry seasons at two altitudes, and a total of 240 T. violacea plants of three sizes were collected from Abies religiosa and Quercus spp. host trees. A total of 163,043 arthropods were collected and about 200 morphospecies identified. The highest abundance was obtained during the dry season, while high diversity was found during the rainy season. There was a significant effect of plant size, host trees and collecting season on abundance and diversity, and there were seasonal variations in community composition. The community hosted on A. religiosa epiphytes showed higher abundance and density than that of Quercus.     

Geographical range and host breadth of Sebacina orchid mycorrhizal fungi associating with Caladenia in south‐western Australia

Specialized mycorrhizal interactions have the potential to limit the geographical range of plant species and contribute to reproductive isolation. We investigated these predictions in Caladenia (Orchidaceae) from south‐western Australia, a group known to have specialized mycorrhizal associations with the genus Sebacina s.l. Sequencing of fungal isolates from 47 of the 136 species of Western Australian Caladenia was undertaken to resolve the geographical range and habitat preferences of mycorrhizal fungal operational taxonomic units (OTUs) and their host breadth in Caladenia. Eight different fungal OTUs were used by Caladenia, with the more frequently detected OTUs occurring in a wide range of habitats and geographical regions. Given the comparatively narrow geographical ranges of most Western Australian Caladenia taxa, this suggests that the geographical ranges of fungal OTUs are unlikely to limit the geographical range of Caladenia spp. Extensive sharing of fungal OTUs between closely related orchid species was detected, suggesting that in the main there is little potential for mycorrhizal fungi to contribute to reproductive isolation between Caladenia spp. Our data mostly support previous work suggesting high mycorrhizal specificity in Caladenia, but this may not be the case in all subgenera, highlighting that Caladenia may offer powerful opportunities for investigating the evolution of specialized mycorrhizal associations.     

Diversity of arbuscular mycorrhizal fungi in the rhizosphere of three host plants in the farming–pastoral zone, north China

The spatial diversity and distribution of AM fungi were investigated in three plots located in farming–pastoral zone, north China. The rhizospheres of Caragana korshinskii, Artemisia sphaerocephala and Salix psammophila were sampled and thirty AM fungal species belonging to five genera were isolated. The study demonstrated that AM fungal diversity and distribution differed significantly among the three host plants and the three studied plots. Spore density of AM fungi ranged between 2 and 22 spores per g^?1 of soil and species richness between 8 and 23. Correlation coefficient analysis demonstrated that spore density was significantly and positively correlated with soil organic matter and available N (P?<?0.01). Species richness was significantly and positively correlated with soil organic matter and available P (P?<?0.01), but significantly and negatively correlated with soil pH (P?<?0.01). Finally, the Shannon–Weiner index was significantly and positively correlated with soil organic matter (P?<?0.05). In this farming–pastoral zone, Glomus reticulatum and G.melanosporum may be more adaptable to the arid conditions than other AM fungal species. This research into AM fungal diversity may lead to exploitation of AM fungi for the mitigation of soil erosion and desertification using mycorrhizal plants, such as C.korshinskii, A.sphaerocephala and S. psammophila. The results of this study support the conclusion that diversity and distribution of AM fungi might be useful to monitor desertification and soil degradation.     

Plant Kin Recognition Enhances Abundance of Symbiotic Microbial Partner

Background

The stability of cooperative interactions among different species can be compromised by cheating. In the plant-mycorrhizal fungi symbiosis, a single mycorrhizal network may interact with many plants, providing the opportunity for individual plants to cheat by obtaining nutrients from the fungi without donating carbon. Here we determine whether kin selection may favour plant investment in the mycorrhizal network, reducing the incentive to cheat when relatives interact with a single network.

Methodology/Principal Findings

We show that mycorrhizal network size and root colonization were greater when Ambrosia artemisiifolia L. was grown with siblings compared to strangers. Soil fungal abundance was positively correlated with group leaf nitrogen, and increased root colonization was associated with a reduced number of pathogen-induced root lesions, indicating greater benefit to plants grown with siblings.

Conclusions/Significance

Plants can benefit their relatives through investment in mycorrhizal fungi, and kin selection in plants could promote the persistence of the mycorrhizal symbiosis.     

Subcellular Nutrient Element Localization and Enrichment in Ecto- and Arbuscular Mycorrhizas of Field-Grown Beech and Ash Trees Indicate Functional Differences

Mycorrhizas are the chief organ for plant mineral nutrient acquisition. In temperate, mixed forests, ash roots (Fraxinus excelsior) are colonized by arbuscular mycorrhizal fungi (AM) and beech roots (Fagus sylvatica) by ectomycorrhizal fungi (EcM). Knowledge on the functions of different mycorrhizal species that coexist in the same environment is scarce. The concentrations of nutrient elements in plant and fungal cells can inform on nutrient accessibility and interspecific differences of mycorrhizal life forms. Here, we hypothesized that mycorrhizal fungal species exhibit interspecific differences in mineral nutrient concentrations and that the differences correlate with the mineral nutrient concentrations of their associated root cells. Abundant mycorrhizal fungal species of mature beech and ash trees in a long-term undisturbed forest ecosystem were the EcM Lactarius subdulcis, Clavulina cristata and Cenococcum geophilum and the AM Glomus sp. Mineral nutrient subcellular localization and quantities of the mycorrhizas were analysed after non-aqueous sample preparation by electron dispersive X-ray transmission electron microscopy. Cenococcum geophilum contained the highest sulphur, Clavulina cristata the highest calcium levels, and Glomus, in which cations and P were generally high, exhibited the highest potassium levels. Lactarius subdulcis-associated root cells contained the highest phosphorus levels. The root cell concentrations of K, Mg and P were unrelated to those of the associated fungal structures, whereas S and Ca showed significant correlations between fungal and plant concentrations of those elements. Our results support profound interspecific differences for mineral nutrient acquisition among mycorrhizas formed by different fungal taxa. The lack of correlation between some plant and fungal nutrient element concentrations may reflect different retention of mineral nutrients in the fungal part of the symbiosis. High mineral concentrations, especially of potassium, in Glomus sp. suggest that the well-known influence of tree species on chemical soil properties may be related to their mycorrhizal associates.     

Effects of Mycorrhizae and Nontarget Organisms on Restoration of a Seasonal Tropical Forest in Quintana Roo, Mexico: Factors Limiting Tree Establishment

We initiated a study of the effects of mycorrhizal fungal community composition on the restoration of tropical dry seasonal forest trees. Tree seedlings were planted in a severely burned experimental site (1995 fire) during the growing season of 1998 at the El Edén Ecological Reserve, in north Quintana Roo, Mexico. Seedlings of Leucaena leucocephala, Guazuma ulmifolia, Caesalpinia violacea, Piscidia piscipula, Gliricidia sepium, and Cochlospermum vitifolium were germinated in steam‐sterilized soil and either remained uninoculated (nonmycorrhizal at transplanting) or were inoculated with mycorrhizal fungi in soils from early‐seral (recently burned) or late‐seral (mature forest) inoculum. Inoculum from the early‐seral soil was largely Glomus spp., whereas a diverse community of arbuscular mycorrhizal fungi were reintroduced from the mature forest including species of Scutellospora, Gigaspora, Glomus, Sclerocystis, and Acaulospora. Plants grew better when associated with the mature forest inoculum, unlike a previous experiment in which plants grew taller with the early‐seral inoculum. Reasons for the different responses include a less‐intense burn resulting in more residual organic matter. In addition to mycorrhizal responses, plants were severely affected by deer browsing. One tree species, C. vitifolium found in the region but not in the reserve, was eliminated by a resident fungal facultative pathogen. Several practical conclusions for restoration can be made. The common nursery practice of soil sterilization may be detrimental because it eliminates beneficial mycorrhizal fungi; species not native to the site may not survive because they may not be adapted to the local pathogens; and herbivory can be severe depending on the landscape context of the restoration.     

Differences in nitrogen redistribution between early and late plant colonizers through ectomycorrhizal fungi on the volcano Mount Koma

Relationships involving the transfer of nitrogen (N) among Salix reinii (willow), Larix kaempferi (larch), and mycorrhizal fungi were investigated in a ridge and hillslope on the volcano Mount Koma in northern Japan using a two-pool fungal model. This model estimated N transfer among the examined taxa by measuring changes in the stable isotope ratio of N (δ¹⁵N). Although N content in tephra was low at both sites, it was higher on the ridge than on the hillslope, and higher in the willow patch than on bare ground or in the larch understory. The non-mycorrhizal sedge (Carex oxyandra) exhibited non-significant differences between the two sites regarding δ¹⁵N for N obtained from tephra. Larches developed a relationship with larch-specific Suillus mycorrhizal fungal species in the roots, and had a lower foliar δ¹⁵N on the hillslope than on the ridge. The larch δ¹⁵N increased during the growing season, while the willow δ¹⁵N remained stable. The dependence of larch on mycorrhizal fungi for N uptake was 3–5 % on the ridge and 56–76 % on the hillslope in autumn. Therefore, larches exhibited a flexible symbiotic relationship with mycorrhizal fungi for obtaining N. Over 45 % of the N taken up by willow plants was obtained from mycorrhizal fungi at both sites. In conclusion, willow plants promoted N deposition in tephra through the litter supply, and formed a stable relationship with mycorrhizal fungi. This enabled successful revegetation with larch plants, which exhibited flexibility in terms of N uptake (i.e., dependent on mycorrhizae or from tephra).