Edible ectomycorrhizal fungi and Cistaceae. A study on compatibility and fungal ecological strategies.

Wild edible mycorrhizal mushrooms are among the most appreciated and prized mushrooms in the world. Despite the cultivation of ectomycorrhizal (ECM) mushrooms has been a growing subject of study worldwide, it has been hampered by the mutualistic lifestyle of the fungi. Although not being obligate symbionts, most of the species of ECM mushrooms only produce fruit bodies in association with trees or shrubs. In the present study, we aimed at understanding certain aspects of the ecology of four different edible ECM fungi: Lactarius deliciosus, Tricholoma equestre, T. portentosum and Boletus fragrans. Despite having a broad distribution worldwide, these fungi inhabit also Mediterranean habitats with understories typically dominated by rockroses (Cistaceae). Studying the ecology of these mutualistic fungi as well as the interaction with these species of shrubs is not only scientifically relevant but also pivotal for the discovery of profitable cultivation protocols. We evaluated the compatibility of these ECM species with five species within Cistaceae family - Cistus ladanifer, C. psilosepalus, C. salviifolius, Halimium halimifolium and Tuberaria lignosa. Each species of fungi proved to be able to establish mycorrhizas with at least 2 different plants species but varied in their host range of the tested Cistaceae. The dissimilarity in terms of host specificity between some fungal species seemed to be connected with the phylogenetic distances of the fungi. A correlation between the colonization percentage of the root systems and the mycelial growth rates in pure culture was found. The connection of these traits might be an important key to understanding the ecological competitor-colonizer tradeoffs of these ECM fungal species. Altogether, our study reports unknown plant-fungi combinations with economical relevance and also adds new insights about the ecology of these species of ECM fungi.

Diversity and effect of Trichoderma isolated from the roots of Pinus densiflora within the fairy ring of pine mushroom (Tricholoma matsutake).

Pine mushroom (PM, Tricholoma matsutake) is an important ectomycorrhizal fungus in Asia primarily due to its value as a food delicacy. Recent studies have shown that fairy rings of PM have distinctive fungal communities, which suggests that other fungi influence the growth of PM. Trichoderma is a well-known saprotrophic fungus commonly found in pine roots within PM fairy rings; however, little is known about the diversity of Trichoderma associated with PM and how these species influence PM growth. This study focused on diversity of Trichoderma isolated from pine roots within PM fairy rings and how these species affect the growth of PM isolate. Based on tef1a phylogenetic analyses, nine Trichoderma species (261 isolates) were identified. Trichoderma songyi and T. spirale were the dominant species, and Trichoderma community varied geographically. Growth experiments indicated that metabolites from five Trichoderma species had a significant influence on the growth of PM isolates. Metabolites of two Trichoderma species increased PM growth, while those of three Trichoderma species suppressed the growth. Within the fairy rings, Trichoderma that had a positive or neutral effect comprised the majority of Trichoderma communities. The results of this study suggest that various Trichoderma species co-exist within PM fairy rings and that these species influence PM growth.

Root-associated bacteria influencing mycelial growth of Tricholoma matsutake (pine mushroom).

Tricholoma matsutake is an ectomycorrhizal fungus usually associated with Pinus densiflora in South Korea. Fruiting bodies (mushrooms) of T. matsutake are economically important due to their attractive aroma; yet, T. matsutake is uncultivatable and its habitat is rapidly being eradicated due to global climate change. Root-associated bacteria can influence the growth of ectomycorrhizal fungi that co-exist in the host rhizosphere and distinctive bacterial communities are associated with T. matsutake. In this study, we investigated how these bacterial communities affect T. matsutake growth by isolating bacteria from the roots of P. densiflora colonized by ectomycorrhizae of T. matsutake and co-culturing rootassociated bacteria with T. matsutake isolates. Thirteen species of bacteria (27 isolates) were found in pine roots, all belonging to the orders Bacillales or Burkholderiales. Two species in the genus Paenibacillus promoted the growth of T. matsutake in glucose poor conditions, likely using soluble metabolites. In contrast, other bacteria suppressed the growth of T. matsutake using both soluble and volatile metabolites. Antifungal activity was more frequent in glucose poor conditions. In general, pine rhizospheres harbored many bacteria that had a negative impact on T. matsutake growth and the few Paenibacillus species that promoted T. matsutake growth. Paenibacillus species, therefore, may represent a promising resource toward successful cultivation of T. matsutake.

Prediction of the potential geographic distribution of the ectomycorrhizal mushroom Tricholoma matsutake under multiple climate change scenarios.

Effective conservation and utilization strategies for natural biological resources require a clear understanding of the geographic distribution of the target species. Tricholoma matsutake is an ectomycorrhizal (ECM) mushroom with high ecological and economic value. In this study, the potential geographic distribution of T. matsutake under current conditions in China was simulated using MaxEnt software based on species presence data and 24 environmental variables. The future distributions of T. matsutake in the 2050s and 2070s were also projected under the RCP 8.5, RCP 6, RCP 4.5 and RCP 2.6 climate change emission scenarios described in the Special Report on Emissions Scenarios (SRES) by the Intergovernmental Panel on Climate Change (IPCC). The areas of marginally suitable, suitable and highly suitable habitats for T. matsutake in China were approximately 0.22 × 106 km2, 0.14 × 106 km2, and 0.11 × 106 km2, respectively. The model simulations indicated that the area of marginally suitable habitats would undergo a relatively small change under all four climate change scenarios; however, suitable habitats would significantly decrease, and highly suitable habitat would nearly disappear. Our results will be influential in the future ecological conservation and management of T. matsutake and can be used as a reference for studies on other ectomycorrhizal mushroom species.

Is the switch to an ectomycorrhizal state an evolutionary key innovation in mushroom-forming fungi? A case study in the Tricholomatineae (Agaricales).

Although fungi are one of the most diverse groups of organisms, little is known about the processes that shape their high taxonomic diversity. This study focuses on evolution of ectomycorrhizal (ECM) mushroom-forming fungi, symbiotic associates of many trees and shrubs, in the suborder Tricholomatineae of the Agaricales. We used the BiSSE model and BAMM to test the hypothesis that the ECM habit represents an evolutionary key innovation that allowed the colonization of new niches followed by an increase in diversification rate. Ancestral state reconstruction (ASR) supports the ancestor of the Tricholomatineae as non-ECM. We detected two diversification rate increases in the genus Tricholoma and the Rhodopolioid clade of the genus Entoloma. However, no increases in diversification were detected in the four other ECM clades of Tricholomatineae. We suggest that diversification of Tricholoma was not only due to the evolution of the ECM lifestyle, but also to the expansion and dominance of its main hosts and ability to associate with a variety of hosts. Diversification in the Rhodopolioid clade could be due to the unique combination of spore morphology and ECM habit. The spore morphology may represent an exaptation that aided spore dispersal and colonization. This is the first study to investigate rate shifts across a phylogeny that contains both non-ECM and ECM lineages.

Distinctive Feature of Microbial Communities and Bacterial Functional Profiles in Tricholoma matsutake Dominant Soil.

Tricholoma matsutake, the pine mushroom, is a valuable forest product with high economic value in Asia, and plays an important ecological role as an ectomycorrhizal fungus. Around the host tree, T. matsutake hyphae generate a distinctive soil aggregating environment called a fairy ring, where fruiting bodies form. Because T. matsutake hyphae dominate the soil near the fairy ring, this species has the potential to influence the microbial community. To explore the influence of T. matsutake on the microbial communities, we compared the microbial community and predicted bacterial function between two different soil types-T. matsutake dominant and T. matsutake minor. DNA sequence analyses showed that fungal and bacterial diversity were lower in the T. matsutake dominant soil compared to T. matsutake minor soil. Some microbial taxa were significantly more common in the T. matsutake dominant soil across geographic locations, many of which were previously identified as mycophillic or mycorrhiza helper bacteria. Between the two soil types, the predicted bacterial functional profiles (using PICRUSt) had significantly distinct KEGG modules. Modules for amino acid uptake, carbohydrate metabolism, and the type III secretion system were higher in the T. matsutake dominant soil than in the T. matsutake minor soil. Overall, similar microbial diversity, community structure, and bacterial functional profiles of the T. matsutake dominant soil across geographic locations suggest that T. matsutake may generate a dominance effect.

A qPCR assay that specifically quantifies Tricholoma matsutake biomass in natural soil.

Tricholoma matsutake is an ectomycorrhizal basidiomycete that produces prized, yet uncultivable, "matsutake" mushrooms along densely developed mycelia, called "shiro," in the rhizosphere of coniferous forests. Pinus densiflora is a major host of this fungus in Japan. Measuring T. matsutake biomass in soil allows us to determine the kinetics of fungal growth before and after fruiting, which is useful for analyzing the conditions of the shiro and its surrounding mycorrhizosphere, predicting fruiting timing, and managing forests to obtain better crop yields. Here, we document a novel method to quantify T. matsutake mycelia in soil by quantifying a single-copy DNA element that is uniquely conserved within T. matsutake but is absent from other fungal species, including close relatives and a wide range of ectomycorrhizal associates of P. densiflora. The targeted DNA region was amplified quantitatively in cultured mycelia that were mixed with other fungal species and soil, as well as in an in vitro co-culture system with P. densiflora seedlings. Using this method, we quantified T. matsutake mycelia not only from shiro in natural environments but also from the surrounding soil in which T. matsutake mycelia could not be observed by visual examination or distinguished by other means. It was demonstrated that the core of the shiro and its underlying area in the B horizon are predominantly composed of fungal mycelia. The fungal mass in the A or A0 horizon was much lower, although many white mycelia were observed at the A horizon. Additionally, the rhizospheric fungal biomass peaked during the fruiting season.

Dehydrogenase genes in the ectomycorrhizal fungus Tricholoma vaccinum: A role for Ald1 in mycorrhizal symbiosis.

Ectomycorrhizal symbiosis is important for forest ecosystem functioning with tree-fungal cooperation increasing performance and countering stress conditions. Aldehyde dehydrogenases (ALDHs) are key enzymes for detoxification and thus may play a role in stress response of the symbiotic association. With this focus, eight dehydrogenases, Ald1 through Ald7 and TyrA, of the ectomycorrhizal basidiomycete Tricholoma vaccinum were characterized and phylogenetically investigated. Functional analysis was performed through differential expression analysis by feeding different, environmentally important substances. A strong effect of indole-3-acetic acid (IAA) was identified, linking mycorrhiza formation and auxin signaling between the symbiosis partners. We investigated ald1 overexpressing strains for performance in mycorrhiza with the host tree spruce (Picea abies) and observed an increased width of the apoplast, accommodating the Hartig' net hyphae of the T. vaccinum over-expressing transformants. The results support a role for Ald1 in ectomycorrhiza formation and underline functional differentiation within fungal aldehyde dehydrogenases in the family 1 of ALDHs.

Tricholoma vaccinum host communication during ectomycorrhiza formation.

The genome sequence of Tricholoma vaccinum was obtained to predict its secretome in order to elucidate communication of T. vaccinum with its host tree spruce (Picea abies) in interkingdom signaling. The most prominent protein domains within the 206 predicted secreted proteins belong to energy and nutrition (52%), cell wall degradation (19%) and mycorrhiza establishment (9%). Additionally, we found small secreted proteins that show typical features of effectors potentially involved in host communication. From the secretome, 22 proteins could be identified, two of which showed higher protein abundances after spruce root exudate exposure, while five were downregulated in this treatment. The changes in T. vaccinum protein excretion with first recognition of the partner were used to identify small secreted proteins with the potential to act as effectors in the mutually beneficial symbiosis. Our observations support the hypothesis of a complex communication network including a cocktail of communication molecules induced long before physical contact of the partners.

Hartig' net formation of Tricholoma vaccinum-spruce ectomycorrhiza in hydroponic cultures.

For re-forestation of metal-contaminated land, ectomycorrhizal trees may provide a solution. Hence, the study of the interaction is necessary to allow for comprehensive understanding of the mutually symbiotic features. On a structural level, hyphal mantle and the Hartig' net formed in the root apoplast are essential for plant protection and mycorrhizal functioning. As a model, we used the basidiomycete Tricholoma vaccinum and its host spruce (Picea abies). Using an optimized hydroponic cultivation system, both features could be visualized and lower stress response of the tree was obtained in non-challenged cultivation. Larger spaces in the apoplasts could be shown with high statistical significance. The easy accessibility will allow to address metal stress or molecular responses in both partners. Additionally, the proposed cultivation system will enable for other experimental applications like addressing flooding, biological interactions with helper bacteria, chemical signaling, or other biotic or abiotic challenges relevant in the natural habitat.

Ectomycorrhizas in vitro between Tricholoma matsutake, a basidiomycete that associates with Pinaceae, and Betula platyphylla var. japonica, an early-successional birch species, in cool-temperate forests.

Tricholoma matsutake is an ectomycorrhizal basidiomycete that associates with Pinaceae in the Northern Hemisphere and produces prized "matsutake" mushrooms. We questioned whether the symbiont could associate with a birch that is an early-successional species in boreal, cool-temperate, or subalpine forests. In the present study, we demonstrated that T. matsutake can form typical ectomycorrhizas with Betula platyphylla var. japonica; the associations included a Hartig net and a thin but distinct fungal sheath, as well as the rhizospheric mycelial aggregate "shiro" that is required for fruiting in nature. The in vitro shiro also emitted a characteristic aroma. This is the first report of an ectomycorrhizal formation between T. matsutake and a deciduous broad-leaved tree in the boreal or cool-temperate zones that T. matsutake naturally inhabits.

Variation among matsutake ectomycorrhizae in four clones of Pinus sylvestris.

Tricholoma matsutake is an ectomycorrhizal fungus that forms commercially important mushrooms in coniferous forests. In this study, we explored the ability of T. matsutake to form mycorrhizae with Pinus sylvestris by inoculating emblings produced through somatic embryogenesis (SE) in an aseptic culture system. Two months after inoculation, clones with less phenolic compounds in the tissue culture phase formed mycorrhizae with T. matsutake, while clones containing more phenols did not. Effects of inoculation on embling growth varied among clones; two of the four tested showed a significant increase in biomass and two had a significant increase in root density. In addition, results suggest that clones forming well-developed mycorrhizae absorbed more Al, Fe, Na, P, and Zn after 8 weeks of inoculation. This study illustrates the value of SE materials in experimental work concerning T. matsutake as well as the role played by phenolic compounds in host plant response to infection by mycorrhizal fungi.

The host ranges of conifer-associated Tricholoma matsutake, Fagaceae-associated T. bakamatsutake and T. fulvocastaneum are wider in vitro than in nature.

Tricholoma matsutake is the most commercially important edible mushroom in pine forests in Japan. Tricholoma bakamatsutake and T. fulvocastaneum, species closely related to T. matsutake, occur in Fagaceae forests. We examined ectomycorrhizal (EM) formation by these Tricholoma species by in vitro synthesis among seven strains (two of T. matsutake, four of T. bakamatsutake, one of T. fulvocastaneum) and axenic plants of pine (Pinus densiflora) and oak (Quercus serrata, Q. phillyraeoides). All strains, except for one of T. matsutake, formed EM associations with both pine and oak. Plant growth and mycelial development were differently affected by EM formation depending on the plant-fungus combination.

Root endophyte symbiosis in vitro between the ectomycorrhizal basidiomycete Tricholoma matsutake and the arbuscular mycorrhizal plant Prunus speciosa.

We previously reported that Tricholoma matsutake and Tricholoma fulvocastaneum, ectomycorrhizal basidiomycetes that associate with Pinaceae and Fagaceae, respectively, in the Northern Hemisphere, could interact in vitro as a root endophyte of somatic plants of Cedrela odorata (Meliaceae), which naturally harbors arbuscular mycorrhizal fungi in South America, to form a characteristic rhizospheric colony or "shiro". We questioned whether this phenomenon could have occurred because of plant-microbe interactions between geographically separated species that never encounter one another in nature. In the present study, we document that these fungi formed root endophyte interactions and shiro within 140 days of inoculation with somatic plants of Prunus speciosa (=Cerasus speciosa, Rosaceae), a wild cherry tree that naturally harbors arbuscular mycorrhizal fungi in Japan. Compared with C. odorata, infected P. speciosa plants had less mycelial sheath surrounding the exodermis, and the older the roots, especially main roots, the more hyphae penetrated. In addition, a large number of juvenile roots were not associated with hyphae. We concluded that such root endophyte interactions were not events isolated to the interactions between exotic plants and microbes but could occur generally in vitro. Our pure culture system with a somatic plant allowed these fungi to express symbiosis-related phenotypes that varied with the plant host; these traits are innately programmed but suppressed in nature and could be useful in genetic analyses of plant-fungal symbiosis.

The ectomycorrhizal fungus Tricholoma matsutake is a facultative saprotroph in vitro.

Tricholoma matsutake is an economically important ectomycorrhizal fungus of coniferous woodlands. Mycologists suspect that this fungus is also capable of saprotrophic feeding. In order to evaluate this hypothesis, enzyme and chemical assays were performed in the field and laboratory. From a natural population of T. matsutake in southern Finland, samples of soil-mycelium aggregate (shiro) were taken from sites of sporocarp formation and nearby control (PCR-negative) spots. Soil organic carbon and activity rates of hemicellulolytic enzymes were measured. The productivity of T. matsutake was related to the amount of utilizable organic carbon in the shiro, where the activity of xylosidase was significantly higher than in the control sample. In the laboratory, sterile pieces of bark from the roots of Scots pine were inoculated with T. matsutake and the activity rates of two hemicellulolytic enzymes (xylosidase and glucuronidase) were assayed. Furthermore, a liquid culture system showed how T. matsutake can utilize hemicellulose as its sole carbon source. Results linked and quantified the general relationship between enzymes secreted by T. matsutake and the degradation of hemicellulose. Our findings suggest that T. matsutake lives mainly as an ectomycorrhizal symbiont but can also feed as a saprotroph. A flexible trophic ecology confers T. matsutake with a clear advantage in a heterogeneous environment and during sporocarp formation.

Modulation of ethanol stress tolerance by aldehyde dehydrogenase in the mycorrhizal fungus Tricholoma vaccinum.

We report the first mycorrhizal fungal aldehyde dehydrogenase gene, ald1, which was isolated from the basidiomycete Tricholoma vaccinum. The gene, encoding a protein Ald1 of 502 amino acids, is up-regulated in ectomycorrhiza. Phylogenetic analyses using 53 specific fungal aldehyde dehydrogenases from all major phyla in the kingdom of fungi including Ald1 and two partial sequences of T. vaccinum were performed to get an insight in the evolution of the aldehyde dehydrogenase family. By using competitive and real-time RT-PCR, ald1 is up-regulated in response to alcohol and aldehyde-related stress. Furthermore, heterologous expression of ald1 in Escherichia coli and subsequent in vitro enzyme activity assay demonstrated the oxidation of propionaldehyde and butyraldehyde with different kinetics using either NAD(+) or NADP(+) as cofactors. In addition, overexpression of ald1 in T. vaccinum after Agrobacterium tumefaciens-mediated transformation increased ethanol stress tolerance. These results demonstrate the ability of Ald1 to circumvent ethanol stress, a critical function in mycorrhizal habitats.

Ectomycorrhization of Tricholoma matsutake and two major conifers in Finland-an assessment of in vitro mycorrhiza formation.

This study aimed to test the ability of Tricholoma matsutake isolates to form mycorrhizas with aseptic seedlings of Pinus sylvestris L. and Picea abies (L.) Karst. Germinated seedlings of Scots pine and Norway spruce were separately inoculated with either isolates originating from Finland or Japan. Eight months after inoculation, the Finnish isolate had formed a sheath and Hartig net on both host species. Ectomycorrhizal Scots pine seedlings inoculated with the Finnish isolate showed the same shoot height and dry mass as the controls. Ectomycorrhizal Norway spruce seedlings inoculated with the Finnish isolate had similar shoot height but slightly less dry mass than the control seedlings. For both tree species, inoculation with the Finnish isolate resulted in reduced total nitrogen content per seedling, but carbon content was unaffected. Inoculation with the Japanese isolate resulted in an initial Hartig net-like structure in pine but not in spruce. No typical Hartig net was observed on either tree species. Furthermore, seedlings of both species inoculated with the Japanese isolate showed significantly reduced growth, dry mass, nitrogen, and carbon content per seedling and shoot height (in spruce) compared to the controls. This study documents and describes the in vitro ectomycorrhization between T. matsutake and Scots pine or Norway spruce and the variable mycorrhizal structures that matsutake isolates can form.

In vitro ectomycorrhizal specificity between the Asian red pine Pinus densiflora and Tricholoma matsutake and allied species from worldwide Pinaceae and Fagaceae forests.

Tricholoma matsutake produces commercially valuable, yet uncultivable, mushrooms (matsutake) in association with pines in the Far East and Scandinavia and with both pines and oaks in the foothills of Tibet. Other matsutake mushrooms, such as Tricholoma anatolicum from the Mediterranean regions and Tricholoma magnivelare and Tricholoma sp. from the North Pacific Coast area of Canada and North America as well as Mexico, respectively, are associated with pines or oaks in their natural habitats. Tricholoma bakamatsutake and Tricholoma fulvocastaneum from Asia produce moderately valuable matsutake mushrooms and are solely associated with Fagaceae in nature. In this study, we demonstrate for the first time that matsutake mushrooms from Scandinavia, Mediterranean regions, North America, and Tibet form ectomycorrhizae with Pinus densiflora similar to the Far East T. matsutake. In general, worldwide T. matsutake and the symbionts of Pinaceae colonize the rhizospheres of P. densiflora as well as T. matsutake isolated from the host plant. However, T. fulvocastaneum and T. bakamatsutake formed a discontinuous Hartig net and no Hartig net, respectively, and colonized to a lesser extent as compared to T. matsutake. The data suggest that conifer-associated matsutake mushrooms in their native habitat will associate symbiotically with the Asian red pine.