Below-ground ectomycorrhizal communities: the effect of small scale spatial and short term temporal variation

Ectomycorrhizal (ECM) associations are an important form of symbiosis for the majority of tree species. In forest ecosystems where nutrients are often limited, ECM associations are vital for seedling establishment and tree survival. ECM communities are often very species rich and frequently follow the log normal distribution??with a few species being abundant and many species being rare. Much of the current knowledge of ECM communities has been revealed through a combination of above-ground sporocarp and below-ground root tip analyses. However, above-ground surveys of ECM communities reveal largely different findings regarding ECM diversity and community structure to below-ground surveys. Therefore below-ground surveys are vital to our understanding of ECM biology, ecology and community dynamics. In this article I review the recent findings regarding how ECM communities vary both spatially and temporally in forests. Spatial variation occurs at the centimetre scale both horizontally and vertically in forests, and can be explained by the separation of the ECM community into distinct niches. Temporal variation occurs over relatively short time scales, with ECM communities showing large changes even on a monthly basis. I then apply the niche concept to ECM fungi, and review a recent theory, ECM functional morphology, and examine how this may be used to explain a significant amount of spatial and temporal variation in forest ECM communities. The functional morphology theory is particularly useful in explaining patterns of ECM community variation across the distinct successional stages of the forest cycle. However, the effect of other abiotic and biological variables on ECM communities should not be ignored. Finally, as ECM communities are non-randomly distributed and vary widely in species richness over time, I lay out a sampling strategy to provide representative samples of the actual ECM community in the study area. Using (i) an extensive sampling methodology, (ii) separation of samples at distances greater than levels of spatial autocorrelation, and (iii) samples collected throughout the year, over a number of years, an ample picture of the ECM community in temperate forests can be collected.     

Intra-species genetic variability drives carbon metabolism and symbiotic host interactions in the ectomycorrhizal fungus Pisolithus microcarpus

Ectomycorrhizal (ECM) fungi are integral to boreal and temperate forest ecosystem functioning and nutrient cycling. ECM fungi, however, originate from diverse saprotrophic lineages and the impacts of genetic variation across species, and especially within a given ECM species, on function and interactions with the environment is not well understood. Here, we explore the extent of intra-species variation between four isolates of the ECM fungus Pisolithus microcarpus, in terms of gene regulation, carbon metabolism and growth, and interactions with a host, Eucalyptus grandis. We demonstrate that, while a core response to the host is maintained by all of the isolates tested, they have distinct patterns of gene expression and carbon metabolism, resulting in the differential expression of isolate-specific response pathways in the host plant. Together, these results highlight the importance of using a wider range of individuals within a species to understand the broader ecological roles of ECM fungi and their host interactions.     

Cryptic genetic structure and copy-number variation in the ubiquitous forest symbiotic fungus Cenococcum geophilum

Ectomycorrhizal (ECM) fungi associated with plants constitute one of the most successful symbiotic interactions in forest ecosystems. ECM support trophic exchanges with host plants and are important factors for the survival and stress resilience of trees. However, ECM clades often harbour morpho-species and cryptic lineages, with weak morphological differentiation. How this relates to intraspecific genome variability and ecological functioning is poorly known. Here, we analysed 16 European isolates of the ascomycete Cenococcum geophilum, an extremely ubiquitous forest symbiotic fungus with no known sexual or asexual spore-forming structures but with a massively enlarged genome. We carried out whole-genome sequencing to identify single-nucleotide polymorphisms. We found no geographic structure at the European scale but divergent lineages within sampling sites. Evidence for recombination was restricted to specific cryptic lineages. Lineage differentiation was supported by extensive copy-number variation. Finally, we confirmed heterothallism with a single MAT1 idiomorph per genome. Synteny analyses of the MAT1 locus revealed substantial rearrangements and a pseudogene of the opposite MAT1 idiomorph. Our study provides the first evidence for substantial genome-wide structural variation, lineage-specific recombination and low continent-wide genetic differentiation in C. geophilum. Our study provides a foundation for targeted analyses of intra-specific functional variation in this major symbiosis.     

Growth response of Afzelia africana Sm. seedlings to ectomycorrhizal inoculation in a nutrient-deficient soil

 The growth and mineral nutrition responses of seedlings of two provenances of Afzelia africana Sm. from Senegal and Burkina Faso, inoculated with four ectomycorrhizal (ECM) fungi (Scleroderma spp. and an unidentified isolate) from the same regions were assessed in a pot experiment in a savanna ECM-propagule-free soil deficient in NPK. There was little variation in the ability of the different fungal species to colonize roots of either provenance of A. africana or to produce external hyphal in soil. Root colonization by ECM fungi and their hyphal development were not related to mineral nutrition or ECM dependency. Differences in P, N, Mg and Ca concentrations in the leaves of inoculated and non-inoculated Afzelia seedlings were not always associated with production of biomass. Only leaf K concentration increased in both provenances after ECM inoculation. However, the Burkina Faso provenance responded better to inoculation with the two fungal isolates than the Senegal provenance in terms of biomass production. This was due to stimulation of root dry weight of the Burkina Faso provenance. Therefore, the hypothesis arises that non-nutritional rather than nutritional effects explain the contribution of ECM inoculation to the growth of A. africana seedlings. Accepted: 27 April 1999     

Diversity of ectomycorrhizal fungi in Britain: a test of the species–area relationship, and the role of host specificity

The host range of ectomycorrhizal (ECM) fungi in Britain was examined by compilation of a data matrix from published literature sources, based primarily on accounts of sporocarp associations with particular host genera. Information was gathered for 577 species of ECM fungi belonging to 51 genera, and 25 genera of host trees, representing the majority of ECM fungal species and host genera recorded in Britain.
Pronounced variation was recorded in the number of ECM fungal species associated with different host genera, with over 200 species recorded with Betula , Fagus , Pinus and Quercus . There was a positive linear relationship ( r ²=0·47, P =0·007) between the number of species of ECM fungi associated with different host genera and the total area occupied by each tree genus in Britain (both values log-transformed). There was also variation in the number of species of ECM fungi which were apparently specific to particular host genera, values ranging from zero (in 15 genera) to >40 in the case of Betula and Fagus . In total, 233 fungal species appeared to be specific to a single host genus (i.e. 40% of those surveyed). Comparison of the ECM mycota associated with different host genera by PCA accounted for 17% of the total variation, with genera belonging to the Fagaceae ( Quercus , Fagus and Castanea ) tending to cluster together, indicating a degree of overlap in their ECM associates. Exotic conifer species, which displayed a lower ECM diversity than would be expected from their distributional areas, were characterized by a high degree of overlap with the ECM associates of Pinus and Betula .
These results indicate that the abundance of different genera of host trees and variation in host specificity could provide a basis for understanding patterns of diversity in ECM fungi within Britain.     

Diversity and specificity of ectomycorrhizal fungi retrieved from an old-growth Mediterranean forest dominated by Quercus ilex

We analysed the ectomycorrhizal (ECM) fungal diversity in a Mediterranean old-growth Quercus ilex forest stand from Corsica (France), where Arbutus unedo was the only other ECM host. On a 6400 m2 stand, we investigated whether oak age and host species shaped below-ground ECM diversity. Ectomycorrhizas were collected under Q. ilex individuals of various ages (1 yr seedlings; 3-10 yr saplings; old trees) and A. unedo. They were typed by ITS-RFLP analysis and identified by match to RFLP patterns of fruitbodies, or by sequencing. A diversity of 140 taxa was found among 558 ectomycorrhizas, with many rare taxa. Cenococcum geophilum dominated (35% of ECMs), as well as Russulaceae, Cortinariaceae and Thelephoraceae. Fungal species richness was comparable above and below ground, but the two levels exhibited < 20% overlap in fungal species composition. Quercus ilex age did not strongly shape ECM diversity. The two ECM hosts, A. unedo and Q. ilex, tended to share few ECM species (< 15% of the ECM diversity). Implications for oak forest dynamics are discussed.     

华北山地6个外生菌根树种的根属性及其与菌根侵染率的关系

该研究以共存于同一暖温带森林的6个外生菌根(ECM)树种为研究对象,测定分析不同根序(1～5级)和功能根系(吸收细根和运输细根)的主要形态和构型属性及ECM侵染率,探究不同外生菌根树种的根属性变异模式及其与菌根真菌侵染程度的关系。结果表明:(1)随着根序的增加,不同树种根直径和单根长度均增加,而比根长和根分支强度均降低;根属性在同一根序下均存在显著的种间差异,尤其是2个裸子植物(落叶松和油松)的根直径较其他4个被子植物大。(2)同一树种的所有根属性在吸收细根和运输细根之间均有显著差异;吸收细根和运输细根的根直径、比根长和根组织密度在树种间均存在显著差异,而其单根长度和根分支强度在树种间无显著差异。(3)ECM侵染率以落叶松最高,千金榆和白桦最低,且与根尖直径呈显著正相关关系,与根尖比根长呈显著负相关关系。研究发现,基于根序或者功能根系,根属性在种间的变异模式不完全一致,单根长度和根分支强度在两个功能根系中均没有表现出显著的种间差异;吸收细根的比根长和根分支强度的变异系数较大,对环境变化有较敏感的响应;古老树种的根直径相对较粗,对菌根真菌的依赖性更高。     

Fungal diversity in ectomycorrhizal communities: sampling effort and species detection

A number of recent review articles on ectomycorrhizal (ECM) fungal community diversity have highlighted the unprecedented increase in the number of publications on this ecologically important but neglected area. The general features of these species-rich, highly dynamic and complex communities have been comprehensively covered but one aspect crucial to our assessment of diversity, namely the sampling of ECM communities has received less attention. This is a complex issue with two principal components, the physical sampling strategy employed and the life cycle traits of the ECM fungi being examined. Combined, these two components provide the image that we perceive as ECM diversity. This contribution will focus primarily on the former of these components using a recent study from a pine forest in central Sweden to highlight some sampling problems and also to discuss some features common to ECM communities. The two commonly used elements of diversity, species richness and community evenness, present rather different problems in the assessment of ECM diversity. The applicability of using current measures of abundance (number or percentage of root tips colonised) to determine community evenness is discussed in relation to our lack of knowledge on the size of individual genets of ECM fungi. The inherent structure of most ECM communities, with a few common species and a large number of rare species, severely limits our ability to accurately assess species richness. A discussion of theoretical detection limits is included that demonstrates the importance of the sampling effort (no. of samples or tips) involved in assessing species richness. Species area abundance plots are also discussed in this context. It is suggested that sampling strategy (bulk samples versus multiple collections of single tips) may have important consequences when sampling from communities where root tip densities differ. Finally, the need for studies of the spatial distribution of ECM on roots in relation to small-scale soil heterogeneity and of the temporal aspects of ECM community dynamics is raised.     

Growth on nitrate and occurrence of nitrate reductase-encoding genes in a phylogenetically diverse range of ectomycorrhizal fungi

Ectomycorrhizal (ECM) fungi are often considered to be most prevalent under conditions where organic sources of N predominate. However, ECM fungi are increasingly exposed to nitrate from anthropogenic sources. Currently, the ability of ECM fungi to metabolize this nitrate is poorly understood. Here, growth was examined among 106 isolates, representing 68 species, of ECM fungi on nitrate as the sole N source. In addition, the occurrence of genes coding for the nitrate reductase enzyme (nar gene) in a broad range of ectomycorrhizal fungi was investigated. All isolates grew on nitrate, but there was a strong taxonomic signature in the biomass production, with the Russulaceae and Amanita showing the lowest growth. Thirty-five partial nar sequences were obtained from 43 tested strains comprising 31 species and 10 genera. These taxa represent three out of the four clades of the Agaricales within which ECM fungi occur. No nar sequences were recovered from the Russulaceae and Amanita, but Southern hybridization showed that the genes were present. The results demonstrate that the ability to utilize nitrate as an N source is widespread in ECM fungi, even in those fungi from boreal forests where the supply of nitrate may be very low.     

A molecular survey of ectomycorrhizal hyphae in a California Quercus–Pinus woodland

Ectomycorrhizal (ECM) hyphal communities have not been well characterized. Furthermore, there have been few studies where the ECM hyphal community is compared to fungi detected as sporocarps or ECM-colonized root tips. We investigated fungi present as hyphae in a well-studied California Quercus–Pinus woodland. Hyphal species present were compared to those found as sporocarps and ECM root tips at the same site. Hyphae were extracted from root-restrictive nylon mesh in-growth bags buried in the soil near mature Quercus douglasii, Quercus wislizeni, and Pinus sabiniana. Taxa were identified using PCR, cloning, and DNA sequencing of internal transcribed spacer and 28s rDNA. Among the 33 species detected, rhizomorph-forming ECM fungi dominated the hyphal community, especially species of Thelephoraceae and Boletales. Most fungi in soils near Quercus spp. and P. sabiniana were ECM basidiomycetes, but we detected two ECM ascomycetes and three non-mycorrhizal fungi. Many ECM species present as hyphae were also previously detected at this site as sporocarps (18%) or on ECM root tips (58%). However, the hyphal community was mostly dominated by different taxa than either the sporocarp or ECM root communities.     

Fine-scale distribution of ectomycorrhizal fungi colonizing Tsuga diversifolia seedlings growing on rocks in a subalpine Abies veitchii forest

Numerous species of ectomycorrhizal (ECM) fungi coexist under the forest floor. To explore the mechanisms of coexistence, we investigated the fine-scale distribution of ECM fungal species colonizing root tips in the root system of Tsuga diversifolia seedlings in a subalpine forest. ECM root tips of three seedlings growing on the flat top surface of rocks were sampled after recording their positions in the root system. After the root tips were grouped by terminal-restriction fragment length polymorphism (T-RFLP) analysis of ITS rDNA, the fungal species representing each T-RFLP group were identified using DNA sequencing. Based on the fungal species identification, the distribution of root tips colonized by each ECM fungus was mapped. Significant clustering of root tips was estimated for each fungal species by comparing actual and randomly simulated distributions. In total, the three seedlings were colonized by 40 ECM fungal species. The composition of colonizing fungal species was quite different among the seedlings. Twelve of the 15 major ECM fungal species clustered significantly within a few centimeters. Some clusters overlapped or intermingled, while others were unique. Areas with high fungal species diversity were also identified in the root system. In this report, the mechanisms underlying generation of these ECM root tip clusters in the root system are discussed.     

Production of edible mushrooms in forests: trends in development of a mycosilviculture

Developing the production of ectomycorrhizal (ECM) mushrooms in forest has become a challenge. Only a few ECM species are currently cultivable. Controlled mycorrhization practices offer promising advance to produce currently uncultivable ECM mushrooms. The persistence of the production of edible species, either cultivated or wild, depends on both the tree and the ecological environment (fungal communities, climate, soil, tree development). Developing adapted forest management practices appears to be means to improve production of edible ECM mushrooms. This review summarises current knowledge on the development of a science-based mycosilviculture for the production of edible ECM mushrooms.     

Ectomycorrhizal networks and seedling establishment during early primary succession

Ectomycorrhizal (ECM) fungal mycelia are the main organs for nutrient uptake in many woody plants, and often connect seedlings to mature trees. While it is known that resources are shared among connected plants via common mycorrhizal networks (CMNs), the net effects of CMNs on seedling performance in the field are almost unknown. CMNs of individual ECM fungal species were produced in an early succession volcanic desert by transplanting current-year seedlings of Salix reinii with ECM mother trees that had been inoculated with one of 11 dominant ECM fungal species. Most seedlings were connected to individual CMNs without being infected by other ECM fungi. Although control seedlings showed poor growth under severe nutrient competition with larger nonmycorrhizal mother trees, nutrient acquisition and growth of seedlings connected to CMNs were improved with most fungal species. The positive effects of CMNs on seedling performance were significantly different among ECM fungal species; for example, the maximum difference in seedling nitrogen acquisition was 1 : 5.9. The net effects of individual CMNs in the field and interspecific variation among ECM fungal species are shown.     

Acclimation to temperature and temperature sensitivity of metabolism by ectomycorrhizal fungi

Ectomycorrhizal (ECM) fungi contribute significantly to ecosystem respiration, but little research has addressed the effect of temperature on ECM fungal respiration. Some plants have the ability to acclimate to temperature such that long‐term exposure to warmer conditions slows respiration at a given measurement temperature and long‐term exposure to cooler conditions increases respiration at a given measurement temperature. We examined acclimation to temperature and temperature sensitivity (Q₁₀) of respiration by ECM fungi by incubating them for a week at one of three temperatures and measuring respiration over a range of temperatures. Among the 12 ECM fungal isolates that were tested, Suillus intermedius, Cenococcum geophilum, and Lactarius cf. pubescens exhibited significant acclimation to temperature, exhibiting an average reduction in respiration of 20–45% when incubated at 23 °C compared with when incubated at 11 or 17 °C. The isolates differed significantly in their Q₁₀ values, which ranged from 1.67 to 2.56. We also found that half of the isolates significantly increased Q₁₀ with an increase in incubator temperature by an average of 15%. We conclude that substantial variation exists among ECM fungal isolates in their ability to acclimate to temperature and in their sensitivity to temperature. As soil temperatures increase, ECM fungi that acclimate may require less carbon from their host plants than fungi that do not acclimate. The ability of some ECM fungi to acclimate may partially ameliorate the anticipated positive feedback between soil respiration and temperature.     

Root symbioses of trees in African dry tropical forests

Abstract. The few surveys made and taxonomic considerations suggest that African dry forests are commonly diverse and dominated by vesicular-arbuscular mycorrhizal, VAM tree species. Ectomycorrhizal, ECM tree species are usually absent or occur in small numbers, but occasionally dominate. Nitrogen-fixing species (which also mostly form VAM) are, in general, few. Theoretical considerations and the limited data available suggest that phosphorus is the limiting nutrient. This probably explains the paucity of N₂-fixing species. It is more difficult to explain why VAM-species and not ECM-species dominate, or vice versa, in situations where P is limiting. A dominance of either type will be reinforced by processes mediated by hyphal networks. Because few taxa form ECM, such processes will promote the development of low diversity stands, whereas very many species can share the benefits of a VAM-network. Fertilizer trials are needed to identify precisely the limiting nutrient(s), and the reactions of species with different root symbioses to additions of N and P. In connection with such studies the fractional contribution of N₂ to N₂-fixing spp. can be estimated by ¹⁵N-methodology. Analogous possibilities do not exist for work on mycorrhizas, but comparative studies of A-values, or phosphatase and protease activities would be worthwhile. A promising approach would be studies of ECM-and VAM-seedlings transplanted into a variety of sites, including each other's habitats.     

Temporal variation of Bistorta vivipara‐associated ectomycorrhizal fungal communities in the High Arctic

Ectomycorrhizal (ECM) fungi are important for efficient nutrient uptake of several widespread arctic plant species. Knowledge of temporal variation of ECM fungi, and the relationship of these patterns to environmental variables, is essential to understand energy and nutrient cycling in Arctic ecosystems. We sampled roots of Bistorta vivipara ten times over two years; three times during the growing‐season (June, July and September) and twice during winter (November and April) of both years. We found 668 ECM OTUs belonging to 25 different ECM lineages, whereof 157 OTUs persisted throughout all sampling time‐points. Overall, ECM fungal richness peaked in winter and species belonging to Cortinarius, Serendipita and Sebacina were more frequent in winter than during summer. Structure of ECM fungal communities was primarily affected by spatial factors. However, after accounting for spatial effects, significant seasonal variation was evident revealing correspondence with seasonal changes in environmental conditions. We demonstrate that arctic ECM richness and community structure differ between summer (growing‐season) and winter, possibly due to reduced activity of the core community, and addition of fungi adapted for winter conditions forming a winter‐active fungal community. Significant month × year interactions were observed both for fungal richness and community composition, indicating unpredictable between‐year variation. Our study indicates that addressing seasonal changes requires replication over several years.     

Tree‐mycorrhizal associations detected remotely from canopy spectral properties

A central challenge in global ecology is the identification of key functional processes in ecosystems that scale, but do not require, data for individual species across landscapes. Given that nearly all tree species form symbiotic relationships with one of two types of mycorrhizal fungi – arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) fungi – and that AM‐ and ECM‐dominated forests often have distinct nutrient economies, the detection and mapping of mycorrhizae over large areas could provide valuable insights about fundamental ecosystem processes such as nutrient cycling, species interactions, and overall forest productivity. We explored remotely sensed tree canopy spectral properties to detect underlying mycorrhizal association across a gradient of AM‐ and ECM‐dominated forest plots. Statistical mining of reflectance and reflectance derivatives across moderate/high‐resolution Landsat data revealed distinctly unique phenological signals that differentiated AM and ECM associations. This approach was trained and validated against measurements of tree species and mycorrhizal association across ~130 000 trees throughout the temperate United States. We were able to predict 77% of the variation in mycorrhizal association distribution within the forest plots (P < 0.001). The implications for this work move us toward mapping mycorrhizal association globally and advancing our understanding of biogeochemical cycling and other ecosystem processes.     

Suprastructures of extracellular matrices: paradigms of functions controlled by aggregates rather than molecules

Extracellular matrices (ECM) not only serve as structural scaffolds in organs and tissues, but also determine critical cellular functions through cell-matrix interactions. These are mediated by cell surface receptors that recognise specific structural features of ECMs and, hence, overall physical properties of the acellular environment. ECM structures are subject to hierarchic organisations, which are tightly adapted to the functions of tissues and organs. Only a few specialised tasks are reserved for isolated ECM macromolecules. Instead, molecular ECM components attain their prominent functions only after polymerising into insoluble suprastructural elements, i.e. fibrils, microfibrils, or networks that, in turn, are assembled into regional tissue structures, such as fibres or basement membranes. As an outstanding feature, most, if not all, ECM suprastructures are co-polymers of more than one molecular species that differ in their identity and relative abundance. Thus, ECM suprastructures are composite biological amalgamates. The analogy to metal alloys refers to structural and functional characteristics of ECM composites, which differ from those of each homo-polymeric aggregate. At the tissue level, biological alloys can themselves be assembled into conglomerates that again assume properties distinct from those of each individual alloy. Nevertheless, most studies in matrix biology solely focus on molecular features and mechanisms. Progress has however been made in identifying principles of interactions within suprastructural elements and their functional consequences. We are now only beginning to understand the impact of suprastructural organisation on the assembly and the functions of whole tissues and many fundamental issues in this almost pristine field await discovery.     

Diversity of extracellular matrix morphology in vertebrate skeletal muscle

Existing data suggest the extracellular matrix (ECM) of vertebrate skeletal muscle consists of several morphologically distinct layers: an endomysium, perimysium, and epimysium surrounding muscle fibers, fascicles, and whole muscles, respectively. These ECM layers are hypothesized to serve important functional roles within muscle, influencing passive mechanics, providing avenues for force transmission, and influencing dynamic shape changes during contraction. The morphology of the skeletal muscle ECM is well described in mammals and birds; however, ECM morphology in other vertebrate groups including amphibians, fish, and reptiles remains largely unexamined. It remains unclear whether a multilayered ECM is a common feature of vertebrate skeletal muscle, and whether functional roles attributed to the ECM should be considered in mechanical analyses of non-mammalian and non-avian muscle. To explore the prevalence of a multilayered ECM, we used a cell maceration and scanning electron microscopy technique to visualize the organization of ECM collagen in muscle from six vertebrates: bullfrogs (Lithobates catesbeianus), turkeys (Meleagris gallopavo), alligators (Alligator mississippiensis), cane toads (Rhinella marina), laboratory mice (Mus musculus), and carp (Cyprinus carpio). All muscles studied contained a collagen-reinforced ECM with multiple morphologically distinct layers. An endomysium surrounding muscle fibers was apparent in all samples. A perimysium surrounding groups of muscle fibers was apparent in all but carp epaxial muscle; a muscle anatomically, functionally, and phylogenetically distinct from the others studied. An epimysium was apparent in all samples taken at the muscle periphery. These findings show that a multilayered ECM is a common feature of vertebrate muscle and suggest that a functionally relevant ECM should be considered in mechanical models of vertebrate muscle generally. It remains unclear whether cross-species variations in ECM architecture are the result of phylogenetic, anatomical, or functional differences, but understanding the influence of such variation on muscle mechanics may prove a fruitful area for future research.     

Viability of ectomycorrhizal fungi following cryopreservation

The use of ectomycorrhizal (ECM) fungi in biotechnological processes requires their maintenance over long periods under conditions that maintain their genetic, phenotypic, and physiological stability. Cryopreservation is considered as the most reliable method for long-term storage of most filamentous fungi. However, this technique is not widespread for ECM fungi since many do not survive or exhibit poor recovery after freezing. The aim of this study was to develop an efficient cryopreservation protocol for the long-term storage of ECM fungi. Two cryopreservation protocols were compared. The first protocol was the conventional straw protocol (SP). The mycelium of the ECM isolates was grown in Petri dishes on agar and subsequently collected by punching the mycelium into a sterile straw before cryopreservation. In the second protocol, the cryovial protocol (CP), the mycelium of the ECM isolates was grown directly in cryovials filled with agar and subsequently cryopreserved. The same cryoprotectant solution, freezing, and thawing process, and re-growth conditions were used in both protocols. The survival (positive when at least 60 % of the replicates showed re-growth) was evaluated before and immediately after freezing as well as after 1 week, 1 m, and 6 m of storage at −130 °C. Greater survival rate (80 % for the CP as compared to 25 % for the SP) and faster re-growth (within 10 d for the CP compared to the 4 weeks for the SP) were observed for most isolates with the CP suggesting that the preparation of the cultures prior to freezing had a significant impact on the isolates survival. The suitability of the CP for cryopreservation of ECM fungi was further confirmed on a set of 98 ECM isolates and displayed a survival rate of 88 % of the isolates. Only some isolates belonging to Suillus luteus, Hebeloma crustuliniforme, Paxillus involutus and Thelephora terrestris failed to survive. This suggested that the CP is an adequate method for the ultra-low cryopreservation of a large set of ECM fungi and that further studies are necessary for the more recalcitrant ones.