Lu-Min Vaario

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

Abstract. 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 twofold) 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 β-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.

Rapid in vitro ectomycorrhizal infection on Pinus densiflora roots by Tricholoma matsutake.

The root systems of 11-wk-oldPinus densiflora seedlings were inoculated with a hyphal suspension ofTricholoma matsutake and aseptically incubated for 4 wk in a forest soil without supplying exogenous carbohydrates. One week following inoculation, fungal hyphae had colonized the root surface and bound soil particles together establishing a root-substrate continuum. Fungal hyphae were visible within the main root cortex following clearing bleaching and staining. In the ensuing days, fungal colonization was observed within elongating lateral roots in which Hartig net formation was confirmed 4 wk after inoculation. This is the first report of rapid ectomycorrhizal infection ofP. densiflora seedings byT. matsutake.

Genetic relationship of Tricholoma matsutake and T. nauseosum from the northern Hemisphere based on analyses of ribosomal DNA spacer regions

The genetic relationship among Tricholoma matsutake and T. nauseosum strains collected from various parts of the Northern Hemisphere was investigated using sequence analysis of the rDNA ITS region and PCR-RFLP analysis of the rDNA IGS-1 region. ITS sequence similarity between T. matsutake and T. nauseosum ranged between 98.1% and 100%. The strains of T. matsutake from coniferous forests and those from broad-leaved forests showed more than 99.8% similarity in their ITS sequences. Three distinct RFLP types were detected when IGS-1 regions were digested with Cfr13I. RFLP patterns showed no variability among the strains of T. nauseosum and those of T. matsutake from broad-leaved forests. This pattern corresponded to the dominant RFLP type in the Japanese population of T. matsutake. Thus, strains belonging to this RFLP type are widely distributed throughout East Asia and Europe and associated with many tree species of Pinaceae and Fagaceae. The result suggests that T. matsutake in coniferous and broad-leaved forests and T. nauseosum should be treated as the same species genetically.

Growth stimulation of a Shiro-like, mycorrhiza forming, mycelium of Tricholoma matsutake on solid substrates by non-ionic surfactants or vegetable oil

The incorporation of Tweens (1 %, 2 %, 5 %) or olive oil (1 %, 2 %) in soil or in soil-containing substrate strongly stimulated mycelial growth of the edible ectomycorrhizal mushroom Tricholoma matsutake (Matsutake) after 1 or 3 months, respectively. The growth responses to Tween 40 and Tween 80 were dose-dependent. Fungal biomass increased up to 15-fold as a result of olive oil incorporation. After 4 months of Matsutake/pine co-culture in the presence of olive oil (2 %), compact aggregates of substrate, hyphae, and surface-colonized roots were observed, recalling in some ways the mycelial mat structure of Matsutake in the field, i.e. Shiro. Olive oil did not prevent formation of well-developed Hartig net palmettis although those seemed rather less abundant than without oil addition. The incorporation of Tween 80 or olive oil (2 %) into nutrient agar induced the proliferation of peripheral hydrophilic-like hyphae penetrating the medium. Tricholoma matsutake growth stimulation, possibly related to the presence of fatty acids in surfactants and oil, could be a consequence of the higher hydrophilicity of treated hyphae, or of enhanced lytic enzyme excretion and activity. Parameters such as adjuvant type, concentration, and growth conditions will be further optimised to formulate culture substrates adapted to the co-culture of T. matsutake and its host plants.

Tricholoma matsutake Dominates Diverse Microbial Communities in Different Forest Soils

ABSTRACT 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.

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.

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.

Only Two Weeks Are Required for Tricholoma matsutake to Differentiate Ectomycorrhizal Hartig Net Structures in Roots of Pinus densiflora Seedlings Cultivated on Artificial Substrate

There has been conflicting debate over many years regarding the trophic status ofTricholoma matsutake (Ito et Imai) Sing., and further investigations are necessary to better understandT. matsutake physiology, particularly carbon nutrition, during ectomycorrhizal symbiosis. For this purpose, we developed a technique to rapidly synthesizein vitro ectomycorrhizas betweenPinus densiflora Sieb. et Zucc. andT. matsutake on artificial substrate (vermiculite: perlite: peat: beech sawdust; 5:5:1:1.), without added sugar in the nutrient solution. Only 1 week was required before the first rudimentary Hartig net ‘palmetti’ could be observed in roots. Well-developed Hartig net structures appeared in taproots after 2 weeks and in lateral roots after 3 weeks. Such rapid root infection may be attributed to the quality of the substrate and the inoculum used.

Aseptic ectomycorrhizal synthesis between Abies firma and Cenococcum geophilum in artificial culture

A simple in vitro system is described for the synthesis ofAbies firma-Cenococcum geophilum ectomycorrhizas. SterilizedA. firma seedlings on both MMN and FH media were inoculated with hyphal discs from actively growing margins ofC. geophilum colonies. Typical ectomycorrhizas formed on seedlings on FH medium after 3 mo of incubation. By light microscopy, the synthesized mycorrhizas were seen to possess a thin mantle from which emanated extraradicle hyphae and highly branched, rarely septate intracortical Hartig net mycelium, characteristic ectomycorrhizal features. This is the first report of aseptic ectomycorrhization ofA. firma seedlings byC. geophilum. This model system will facilitate detailed studies on ectomycorrhizal development ofAbies species.

In vitro ectomycorrhiza formation between Abies firma and Pisolithus tinctorius

Abstract The first in vitro aseptic synthesis of Abies firma Sieb. et Zucc. with Pisolithus tinctorius (Pers.) Coker & Couch is reported. Techniques were improved for the aseptic synthesis of ectomycorrhizas of A. firma, a slow-growing species in vitro, and Pisolithus tinctorius using a novel culture medium and both sterilized and re-rooted seedlings. After 2–3 months incubation, ectomycorrhizas were formed by both methods. The mycorrhizas possessed a mantle and a highly branched nonseptate Hartig net mycelium colonizing the intercellular spaces within the host cortex, features characteristic of ectomycorrhizas. These techniques will prove useful for addressing physiological and biochemical questions on the interactions of microbes with roots of whole plants.