Alexis Guerin-Laguette

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.

Development of microsatellite markers from an ectomycorrhizal fungus, Tricholoma matsutake, by an ISSR-suppression-PCR method

Abstract. An inter-simple sequence repeat (ISSR)-suppression-PCR technique established to develop microsatellite markers of plant species was applied to an ectomycorrhizal fungus, Tricholoma matsutake. Six polymorphic SSR markers were developed. All six polymorphic SSR markers were single-locused and co-dominant. Alleles produced by these six single-locused markers ranged from two to nine per locus and the expected heterozygosities were calculated as values from 0.098 to 0.803. The results indicated that the ISSR-suppression-PCR technique was effective and applicable to the development of microsatellite markers from ectomycorrhizal fungi. Furthermore, the six microsatellite loci did not amplify DNA from any other ectomycorrhizal species investigated, except for Tricholoma nauseosum (Swedish matsutake) and Tricholoma fulvocastaneum, suggesting that population genetics and reproduction of T. matsutake could be investigated by the SSR markers developed in the present study.

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.

The mycorrhizal fungus Tricholoma matsutake stimulates Pinus densiflora seedling growth in vitro.

While it has been suggested that Matsutake mycorrhizae might not be functional and that Matsutake may behave as a saprobic fungus in soil or even have some pathogenic activity on seedlings, we investigated the consequences of Matsutake inoculation on Pinus densiflora growth. Seventy-five days after inoculation, hyphae were anchored on short roots and well-developed Hartig net palmettis were observed. Compared to both control treatments—seedlings treated with distilled water and seedlings treated with autoclaved mycelium—inoculation significantly stimulated seedling total dry weight by 70.9% and 98.0%, respectively. These findings attest that some type of symbiotic relationship must be functional and favour host growth, ruling out claims of pathogenicity under the sterile conditions used here.

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.

Detection of Tricholoma matsutake by specific ITS primers

Sequences of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA of ectomycorrhizal fungi were analysed and a specific PCR primer pair for Tricholoma matsutake was designed. Using the primer pair, part of the ITS region of T. matsutake (400 bp) was amplified, but no amplified fragment was detected for other ectomycorrhizal fungi. PCR was performed on DNA extracted from mycorrhizas of T. matsutake and Shiro soil (extramatrical mycelium of T. matsutake and soil complexes) and the 400 bp fragments were also specifically amplified. This indicates that presence of T. matsutake at any of its life stages can be easily confirmed by PCR typing.

Identification of a prevalent Tricholoma matsutake ribotype in Japan by rDNA IGS1 spacer characterization

The diversity of Tricholoma matsutake basidiomata and their distribution across Japan was investigated, for the first time, by using PCR-RFLP and sequencing. Eight distinct IGS1 rDNA types were identified by using the restriction endonuclease Cfr13I. Four (A, B, G, H) were characterized by one IGS1 dominant sequence, which could then be recorded by direct sequencing of the PCR products. In contrast, the four other types (C, D, E, F) were characterized by up to more than four codominant IGS1 copies that needed to be cloned before sequencing. Among the copies, one was always exhibiting the RFLP pattern of type A basidiomata. Pairwise nucleotide variation among IGS1 spacer copies, recorded in RFLP types A, B, C, D, F, and H, was very low and never exceeded 1.9%. In contrast, type G emerged and its IGS1 spacer sequence exhibited at least 10.6% nucleotide variation compared with all other types. While the data recorded suggests a higher genetic diversity in the southern than in the northern part of Japan, no host/matsutake ribotype specificity could be evidenced. In Japan, the T. matsutake basidiomata belonging to RFLP type A were by far the most frequent. They were found at most sites and were usually the most abundant at each site. Furthermore, this predominance persisted over time during ca 50 years. The dominant RFLP type A may represent the original Japanese population while the other genotypes may have been more recently introduced.

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.