Estimation of the most suitable nitrogen concentration for sporocarp formation in Laccaria japonica colonizing Pinus densiflora seedlings through in vitro mycelial culture.

Many ectomycorrhizal (ECM) fungi produce commercially valuable edible sporocarps. However, the effects of nitrogen (N) application on ECM fungal sporocarp formation remain poorly understood. In this study, we investigated the effect of application of various N concentrations (0, 5, 25, 50, 100, and 200 mg/L) on the growth of Laccaria japonica mycelia in vitro for 1 month. The results showed that L. japonica mycelial biomass was highest in the 50 mg/L treatment and was significantly inhibited at N concentrations higher than 200 mg/L. Next, we investigated the effects of N application on mycorrhizal colonization and sporocarp formation in L. japonica colonizing Pinus densiflora seedlings in pots. The seedlings were watered with nutrient solutions containing 0, 5, 25, 50, or 100 mg N/L. The biomass, photosynthetic rate, and mycorrhizal colonization rates of the seedlings were measured at 45 days (first appearance of primordia), 65 days (sporocarp appearance on the substrate surface), and 4 months after seedlings were transplanted. The numbers of primordia and sporocarps were recorded during the experimental period. Total carbon (C) and N content were determined in seedlings at 4 months after transplantation, and in L. japonica sporocarps. Both mycelial growth and sporocarp production reached their maximum at an N application concentration of 50 mg/L, suggesting that the most suitable N concentration for ECM fungal sporocarp formation can easily be estimated in vitro during mycelial growth. This finding may help determine the most suitable N conditions for increasing edible ECM fungus sporocarp production in natural forests.

New findings on the fungal species Tricholoma matsutake from Ukraine, and revision of its taxonomy and biogeography based on multilocus phylogenetic analyses.

Matsutake mushrooms are among the best-known edible wild mushroom taxa worldwide. The representative Tricholoma matsutake is from East Asia and the northern and central regions of Europe. Here, we report the existence of T. matsutake under fir trees in Eastern Europe (i.e., Ukraine), as confirmed by phylogenetic analysis of nine loci on the nuclear and mitochondrial genomes. All specimens from Japan, Bhutan, China, North Korea, South Korea, Sweden, Finland, and Ukraine formed a T. matsutake clade according to the phylogeny of the internal transcribed spacer region. The European population of T. matsutake was clustered based on the ß2 tubulin gene, with a moderate bootstrap value. In contrast, based on analyses of three loci, i.e., rpb2, tef1, and the ß2 tubulin gene, T. matsutake specimens sampled from Bhutan and China belonged to a clade independent of the other specimens of this species, implying a genetically isolated population. As biologically available type specimens of T. matsutake have not been designated since its description as a new species from Japan in 1925, we established an epitype of this fungus, sampled in a Pinus densiflora forest in Nagano, Japan.

Dynamic evolution of eukaryotic mitochondrial and nuclear genomes: a case study in the gourmet pine mushroom Tricholoma matsutake.

Fungi, as eukaryotic organisms, contain two genomes, the mitochondrial genome and the nuclear genome, in their cells. How the two genomes evolve and correlate to each other is debated. Herein, taking the gourmet pine mushroom Tricholoma matsutake as an example, we performed comparative mitogenomic analysis using samples collected from diverse locations and compared the evolution of the two genomes. The T. matsutake mitogenome encodes 49 genes and is rich of repetitive and non-coding DNAs. Six genes were invaded by up to 11 group I introns, with one cox1 intron cox1P372 showing presence/absence dynamics among different samples. Bioinformatic analyses suggested limited or no evidence of mitochondrial heteroplasmy. Interestingly, hundreds of mitochondrial DNA fragments were found in the nuclear genome, with several larger than 500 nt confirmed by PCR assays and read count comparisons, indicating clear evidence of transfer of mitochondrial DNA into the nuclear genome. Nuclear DNA of T. matsutake showed a higher mutation rate than mitochondrial DNA. Furthermore, we found evidence of incongruence between phylogenetic trees derived from mitogenome and nuclear DNA sequences. Together, our results reveal the dynamic genome evolution of the gourmet pine mushroom.

Large-scale genome sequencing of mycorrhizal fungi provides insights into the early evolution of symbiotic traits.

Mycorrhizal fungi are mutualists that play crucial roles in nutrient acquisition in terrestrial ecosystems. Mycorrhizal symbioses arose repeatedly across multiple lineages of Mucoromycotina, Ascomycota, and Basidiomycota. Considerable variation exists in the capacity of mycorrhizal fungi to acquire carbon from soil organic matter. Here, we present a combined analysis of 135 fungal genomes from 73 saprotrophic, endophytic and pathogenic species, and 62 mycorrhizal species, including 29 new mycorrhizal genomes. This study samples ecologically dominant fungal guilds for which there were previously no symbiotic genomes available, including ectomycorrhizal Russulales, Thelephorales and Cantharellales. Our analyses show that transitions from saprotrophy to symbiosis involve (1) widespread losses of degrading enzymes acting on lignin and cellulose, (2) co-option of genes present in saprotrophic ancestors to fulfill new symbiotic functions, (3) diversification of novel, lineage-specific symbiosis-induced genes, (4) proliferation of transposable elements and (5) divergent genetic innovations underlying the convergent origins of the ectomycorrhizal guild.

The effects of co-colonising ectomycorrhizal fungi on mycorrhizal colonisation and sporocarp formation in Laccaria japonica colonising seedlings of Pinus densiflora.

Forest trees are colonised by different species of ectomycorrhizal (ECM) fungi that interact competitively or mutualistically with one another. Most ECM fungi can produce sporocarps. To date, the effects of co-colonising fungal species on sporocarp formation in ECM fungi remain unknown. In this study, we examined host plant growth, mycorrhizal colonisation, and sporocarp formation when roots of Pinus densiflora are colonised by Laccaria japonica and three other ECM fungal species (Cenococcum geophilum, Pisolithus sp., and Suillus luteus). Sporocarp numbers were recorded throughout the experimental period. The biomass, photosynthetic rate, and mycorrhizal colonisation rate of the seedlings were also measured at 45 days, 62 days, and 1 year after seedlings were transplanted. Results indicated that C. geophilum and S. luteus may negatively impact mycorrhizal colonisation and sporocarp formation in L. japonica. Sporocarp formation in L. japonica was positively correlated with conspecific mycorrhizal colonisation but negatively correlated with the biomass of seedlings of P. densiflora. The co-occurring ECM fungi largely competed with L. japonica, resulting in various effects on mycorrhizal colonisation and sporocarp formation in L. japonica. A variety of mechanisms may be involved in the competitive interactions among the different ECM fungal species, including abilities to more rapidly colonise root tips, acquire soil nutrients, or produce antibiotics. These mechanisms need to be confirmed in further studies.

Tricholoma matsutake may take more nitrogen in the organic form than other ectomycorrhizal fungi for its sporocarp development: the isotopic evidence.

Tricholoma matsutake is an ectomycorrhizal (ECM) fungus capable of in vitro saprotrophic growth, but the sources of C and N used to generate sporocarps in vivo are not well understood. We examined natural abundance isotope data to investigate this phenomenon. For this purpose, C, N and their stable isotopes (13C, 15N) content of fungal sporocarps and their potential nutrient sources (i.e., foliage, litter, fine roots, wood, and soil) were investigated from two well-studied sites in Finland and Japan. Our results show that δ13C values of T. matsutake and other fungal groups are consistent with those of most studies, but a very high δ15N value (16.8‰ ± 2.3) is observed in T. matsutake. Such isotopic pattern of fungal δ15N suggests that matsutake has a greater proteolytic potential to digest chemically complex 15N-enriched organic matter and hydrophobic hyphae. This assumption is further supported by a significant and positive correlation between δ13Ccap-stipe and δ15Ncap-stipe exclusively in T. matsutake, which suggests common C and N sources (protein) possible for isotopically enriched cap. The 13C increase of caps relative to stipe presumably reflects greater contents of 13C-enriched protein than 13C-depleted chitin. We conclude that T. matsutake is a typical ECM fungus which obtains for its sporocarp development for both C and N from a common protein source (vs. photosynthetic carbon) present in soil organic matter.

Microsatellite markers for the prized matsutake mushroom (Tricholoma matsutake, Tricholomataceae).

PREMISE OF THE STUDY: Novel and cost-effective microsatellite markers were developed to explore the population genetics, biogeographic structure, and evolutionary history of the prized Euro-Asian wild edible ectomycorrhizal fungus Tricholoma matsutake (Tricholomataceae). METHODS AND RESULTS: Eighteen new polymorphic simple sequence repeat loci, detected from a microsatellite-enriched genomic library, were used to characterize 131 individuals from eight T. matsutake populations. The number of alleles ranged from two to 10, with averages of 1.42 to 3.22. Levels of observed and expected heterozygosity ranged from 0.00-1.00 and from 0.00-0.83, with mean values of 0.21 and 0.26, respectively. In total, 50% of the loci showed interspecific transferability and polymorphism in the related species T. equestre. CONCLUSIONS: These newly developed markers will aid research into the genetic diversity and population structure of T. matsutake. They can also be used in other species of Tricholoma.

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.

Tricholoma matsutake can absorb and accumulate trace elements directly from rock fragments in the shiro.

Tricholoma matsutake, a highly valued delicacy in Japan and East Asia, is an ectomycorrhizal fungus typically found in a complex soil community of mycorrhizae, soil microbes, and host-tree roots referred to as the shiro in Japan. A curious characteristic of the shiro is an assortment of small rock fragments that have been implicated as a direct source of minerals and trace elements for the fungus. In this study, we measured the mineral content of 14 samples of shiro soil containing live matsutake mycelium and the extent to which the fungus can absorb minerals directly from the rock fragments. X-ray powder diffraction identified major phases of quartz, microcline, orthoclase, and albite in all shiro samples. PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting and direct sequencing confirmed the presence of T. matsutake on 32 of 33 rock fragments. Piloderma sp. co-occurred on 40% of fragments and was positively correlated with locations known to produce good mushroom crops. The ability of T. matsutake to absorb trace elements directly from rock fragments was examined in vitro on nutrient-agar plates supplemented with rock fragments from the shiro. In comparison to the mineral content of tissues grown on control media, the concentration of Al, Cu, Fe, Mn, P, and Zn increased from 1.1 to 106.4 times for both T. matsutake and Piloderma sp. Mineral content of dried sporocarps sampled from the study site partially reflected the results of the in vitro study. We discuss the implications of our results with respect to the natural development and artificial culture of this important fungus.

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.

Tricholoma matsutake dominates diverse microbial communities in different forest soils.

Fungal and actinobacterial communities were analyzed together with soil chemistry and enzyme activities in order to profile the microbial diversity associated with the economically important mushroom Tricholoma matsutake. Samples of mycelium-soil aggregation (shiro) were collected from three experimental sites where sporocarps naturally formed. PCR was used to confirm the presence and absence of matsutake in soil samples. PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting and direct sequencing were used to identify fungi and actinobacteria in the mineral and organic soil layers separately. Soil enzyme activities and hemicellulotic carbohydrates were analyzed in a productive experimental site. Soil chemistry was investigated in both organic and mineral soil layers at all three experimental sites. Matsutake dominated in the shiro but also coexisted with a high diversity of fungi and actinobacteria. Tomentollopsis sp. in the organic layer above the shiro and Piloderma sp. in the shiro correlated positively with the presence of T. matsutake in all experimental sites. A Thermomonosporaceae bacterium and Nocardia sp. correlated positively with the presence of T. matsutake, and Streptomyces sp. was a common cohabitant in the shiro, although these operational taxonomic units (OTUs) did not occur at all sites. Significantly higher enzyme activity levels were detected in shiro soil. These enzymes are involved in the mobilization of carbon from organic matter decomposition. Matsutake was not associated with a particular soil chemistry compared to that of nearby sites where the fungus does not occur. The presence of a significant hemicellulose pool and the enzymes to degrade it indicates the potential for obtaining carbon from the soil rather than tree roots.

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 situ and in vitro colonization of Cathaya argyrophylla (Pinaceae) by ectomycorrhizal fungi.

Cathaya argyrophylla, a critically endangered conifer, is found to grow at four isolated areas located in subtropical mountains of China. To examine the involvement and usefulness of mycorrhizas for sustaining the population of this tree, we compared the root system, morphology, and structure of mycorrhizal roots of C. argyrophylla, which were collected from a natural stand and an artificial stand, each grown at a different location. More mycorrhizal roots were found for trees from an artificial stand. The presence of extramatrical mycelium, mantle, and Hartig net revealed that C. argyrophylla formed an ectomycorrhizal association in both sampling sites. Starch granules were found in mycorrhizal roots collected only from a natural stand. The aseptic synthesis of C. argyrophylla and Cenococcum geophilum was established for the first time in vitro. Typical ectomycorrhizas formed on seedlings on RM medium containing 0.1 g/l glucose, 5 weeks after inoculation. By light microscopy, the synthesized mycorrhizas showed a thin mantle from which emanated extramatrical hyphae and highly branched Hartig net. A simple, rapid, and convenient mycorrhiza synthesis system was developed, which facilitates further studies on ectomycorrhizal development of C. argyrophylla.

Saprobic potential of Tricholoma matsutake: growth over pine bark treated with surfactants.

Saprotrophic growth of Tricholoma matsutake isolates was investigated over Pinus densiflora bark fragments either on soil or on agar media. Preferential colonization of pine bark fragments by hyphae, in glucose-deprived environments suggested that Matsutake was able to extract some nutrients to sustain its growth. This was confirmed in glucose-free liquid nutrient medium, where bark as sole carbon source significantly stimulated (up to two-fold) growth of T. matsutake isolates. The addition of surfactants (Tween 80 and Tween 40) in liquid medium further stimulated mycelium growth over pine bark by up to 55%. Such growth stimulation was associated with a sharp increase in protein and beta-glucosidase excretion by hyphae in culture filtrates. As T. matsutake has some saprotrophic ability, the initiation and extension of Matsutake Shiro in forest soil might require simultaneously nutrients derived from the host plant and from soil organic compounds. Data reported here may contribute to the formulation of new culture substrates adapted to the co-culture of T. matsutake and its host plant under controlled conditions.