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.     

Agrobacterium-mediated transformation of the ectomycorrhizal basidiomycete Tricholoma matsutake that produces commercially valuable fruit bodies, matsutake

Using agroinfection with a T-DNA vector carrying a hygromycin resistance marker, the recombinants were generated for the first time from the ectomycorrhizal basidiomycete Tricholoma matsutake, which produces commercially valuable fruit bodies, matsutake, during association with Pinus sp. plants. The transformation system may be useful in the genetic analysis of T. matsutake.     

Ectomycorrhiza formation ofTricholoma matsutake onPinus densiflora

Mycorrhizal association ofTricholoma matsutake withPinus densiflora was studied. A naturally establishedP. densiflora stand (age: ca. 45 yr) where occurrences ofT. matsutake sporocarps had been confirmed was studied in lbaraki Prefecture, Japan. Pine root systems connected withT. matsutake sporocarps via the fungal white mycelia were sampled in October 1997. The sampled pine roots were covered overall with mycelia. Under a dissecting microscope, the mycelia were confirmed to form fungal sheaths on the lateral roots. Under a light microscope, transverse and longitudinal sections of these roots showed the presence of both fungal sheaths and Hartig nets, which are typical of ectomycorrhizas. The fungal sheath was ca. 1.5–20 μm. in thickness, and felt prosenchymatous in texture. Hartig nets developed continuously at the cortex and extended to the boundary between cortical cells and endodermal cells. The same ectomycorrhizal morphotype on the pine was also recovered from inside the same mycelial colony (i.e., “shiro”) ofT. matsutake from winter to summer. These results suggest thatT. matsutake has a perennial ectomycorrhizal association withP. densiflora.     

Productivity of hydrolytic enzymes by mycorrhizal mushrooms

To survey the potential for production of extracellular hydrolytic enzymes by mycorrhizal mushrooms, productivities of these exo-enzymes from mycelia on potato-dextrose liquid medium were determined.Tricholoma matsutake produced relatively high levels of CM-cellulase and avicelase activities in all test strains. It also produced higher activity of acid proteinase than neutral proteinase. Its xylanase activities seemed to be higher than those of the other carbohydrases. The productivities ofLyophyllum shimeji strains were at similar levels to those ofT. matsutake strains. CM-cellulase and avicelase activities ofL. shimeji were higher than those ofT. matsutake. Neutral proteinase inL. shimeji strains showed higher activity levels than acid proteinase. The relative productivities of hydrolytic enzymes between the groups of mycorrhizal mushrooms and wood-rotting mushrooms were also examined.T. matsutake andL. shimeji both produce many kinds of hydrolytic enzymes in their culture broth, and the potential for production of hydrolytic enzymes by mycorrhizal mushrooms was judged to be relatively high.     

Identification of ectomycorrhizae formed betweenTricholoma matsutake andPinus densiflora by polymerase chain reaction (PCR) targeting retroelement coding regions

We previously reported that conservation and diversification of repetitive sequences carrying motifs of retroposons have occurred inTricholoma matsutake and related ectomycorrhizal basidiomycetes through their evolution. Here we report that the polymerase chain reaction using primers designed to amplify retroelement coding regions specified ectomycorrhizae formed betweenT. matsutake andPinus densiflora.     

Detection of the ectomycorrhizal fungusTricholoma matsutake and some related species with specific ITS primers

Oligonucleotide primers (Tm1 and Tm4) were designed to amplify a 447–448 base pair fragment, comprising sections of the rDNA internal transcribed spacers (ITS) and the entire 5.8S rDNA, ofTricholoma matsutake. PCR products of predicted size were produced for six of eight isolates ofT. matsutake from across its natural range in Asia, and for isolates of some closely related fungi includingT. bakamatsutake, T. magnivelare, andT. caligatum. The closely relatedT. robustum could be discriminated fromT. matsutake by PCR fragment size. No PCR products were produced where the primers were tested against 16 species of ectomycorrhizal fungi associated withPinus spp. in the Southern Hemisphere. The specific primers were also used successfully to produce PCR products from matsutake infected roots collected in natural forests in China and Japan, and from pure culture synthesisedPinus radiata-T. matsutake material. These primers will be useful in research directed at establishing matsutake in the Southern Hemisphere, and also have the potential to be applied to the study of matsutake within its natural range.     

Successful inoculation of mature pine with Tricholoma matsutake

Our finding demonstrates, for the first time, that the roots of mature pine trees can be successfully inoculated with a symbiotic ectomycorrhizal fungus, the valuable matsutake mushroom. Long root segments (ca. 5–10 mm in diameter, ca. 50 cm in length) of 50-year-old Pinus densiflora trees were excavated, washed, auxin-treated (2–5 mg indole butyric acid, IBA, per root) and incubated in moist Spagnum moss. Twelve months later, short roots were regenerated, of which approximately 90% were free of mycorrhizae. Mycorrhiza-free short roots were inoculated with mycelial pieces of Tricholoma matsutake and incubated further in a sterilized substrate. Four-and-a-half months later, roots putatively colonized by Matsutake were sampled near the inoculation points. A T. matsutake-specific ITS-rDNA fragment was amplified by nested PCR from approximately 80% of the root samples analyzed, whereas approximately 66% of the root samples processed for staining with Chlorazol black E displayed characteristic T. matsutake Hartig net structures. These results confirm the symbiotic infection of mature P. densiflora roots by matsutake.     

Characterization of the carbohydrase productions of an ectomycorrhizal fungus, Tricholoma matsutake

To evaluate the potential of the production of the ectomycorrhizal fungus Tricholoma matsutake to produce carbohydrases, (1) the distribution of carbohydrase activities among the different strains (18 strains) was investigated and (2) the abilities of T. matsutake and saprophytic fungi to produce β-glucosidase were compared. The results showed that the carbohydrase productions patterns of T. matsutake still resemble one another. Moreover, this fungus exhibited markedly higher β-glucosidase than did the saprophytic mushrooms. Tricholoma matsutake showed weak production of α-amylase and α-glucosidase in a static cultur filtrate. On the other hand, glucoamylase activity was not observed. Surprisingly, we discovered that β-glucosidase demonstrated strong activity. This finding suggests that this fungus has saprotrophic abilities. The carbohydrase production systems in T. matsutake were characterized from our experimental results. Also, we point out some weak points in the carbohydrase production systems of T. matsutake.     

Intra- and inter-specific variations in the copy number of two types of retrotransposons from the ectomycorrhizal basidiomycete Tricholoma matsutake

To explore intra- and inter-specific variations of the ectomycorrhizal basidiomycete Tricholoma matsutake that produces the fruit body matsutake, we carried out real-time PCR analysis based on two types of retrotransposons, one designated marY1, which resembles a retrovirus carrying the long terminal repeat (LTR) and the other marY2N, which resembles mRNA carrying the polyadenylated tail. Calculation based on the average genome size of homobasidiomycetes (34 Mbp) shows that ca. 5.5% of the total genome of T. matsutake isolated from Asia is made up of these retrotransposons, whereas they occupy ca. 1.4% in the isolates from Morocco, ca. 0.8% in isolates from Mexico, and ca. 0.5% in Tricholoma magnivelare, the species which produces American matsutake. Other Tricholoma spp. that produce fruit bodies similar to those of T. matsutake, such as T. bakamatsutake, T. fulvocastaneum, and T. robustum, carry them in the region less than 0.05% of their total genome. Copy number of LTR of marY1 is consistently and markedly higher than that of the coding regions of marY1 and marY2N. Data suggest that retrotransposons are deeply involved in evolution of the ectomycorrhizal symbiont.     

Rapid in vitro ectomycorrhizal infection onPinus densiflora roots byTricholoma 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.     

Ectomycorrhizal symbiosis in vitro between Tricholoma matsutake and Pinus densiflora seedlings that resembles naturally occurring ‘shiro’

We established an in vitro ectomycorrhizal symbiosis between Tricholoma matsutake and Pinus densiflora. Mycorrhiza formed in a substrate of Modified Norkrans' C medium and granite-based soil had features similar to those observed previously only in naturally occurring mycorrhizal system called ‘shiro,’ and promoted the growth of plants with smaller root/shoot ratios. The in vitro formation of ‘shiro’ is essential for the development of T. matsutake system to produce mushrooms and is useful for the propagation and plantation of the mycorrhizal seedlings.     

Isolation ofTricholoma matsutake andT. bakamatsutake cultures from field-collected ectomycorrhizas

Tricholoma matsutake was isolated into pure cultures from field samples of ectomycorrhizas onPinus densiflora. The mycorrhizal tips were collected at different times of the year from a colony ofT. matsutake in aP. densiflora stand. The mycorrhizal tips were continuously washed with sterilized distilled water and diluted Tween 80 solution, surface-sterilized with calcium hypochlorite solution, and inoculated on several kinds of nutrient agar media. Most of the mycorrhizal tips collected in winter and spring produced colonies that were morphologically similar to cultures ofT. matsutake isolated from basidiocarps. The identity of isolates obtained from mycorrhizas was further confirmed to beT. matsutake based on fungal morphology and RFLP patterns of PCR amplified rDNA. The feasibility ofT. bakamatsutake isolation into pure culture from ectomycorrhizas onQuercus serrata was also confirmed. These results indicated that mycelium of matsutake mushrooms can be isolated into pure culture from ectomycorrhizas at different times of the year. Mycorrhizas of bothT. matsutake andT. bakamatsutake were not observed to have any specific association with soil fungi such asMortierella spp.     

Detection of β-glucosidase as saprotrophic ability from an ectomycorrhizal mushroom, Tricholoma matsutake

We investigated extracellular carbohydrase production in the medium of an ectomycorrhizal fungus, Tricholoma matsutake, to reveal its ability to utilize carbohydrates such as starch as a growth substrate and to survey the saprotrophic aspects. We found β-glucosidase activity in the static culture filtrate of this fungus. The β-glucosidase was purified and characterized. The purified enzyme was obtained from about 2.1 l static culture filtrate, with 9.0% recovery, and showed a single protein band on SDS-PAGE. Molecular mass was about 160 kDa. The enzyme was most active around 60°C and pH 5.0, and stable over a pH of 4.0–8.0 for 30 min at 37°C. The purified enzyme was activated by the presence of Ca²⁺ and Mn²⁺ ions (about 2–3 times that of the control). The enzyme readily hydrolyzed oligosaccharides having a β-1,4-glucosidic linkage such as cellobiose and cellotriose. However, it did not hydrolyze polysaccharides such as avicel and CM-cellulose or oligosaccharides having an α-glucosidic linkage. Moreover, cellotriose was hydrolyzed by the enzyme for various durations, and the resultant products were analyzed by TLC. We concluded that the enzyme from T. matsutake seems to be a β-glucosidase because cellotriose with a β-1,4-glucosidic linkage decomposed to glucose during the enzyme reaction.     

Effects of carbohydrate substrate on the vegetative mycelial growth of an ectomycorrhizal mushroom, <Emphasis Type="Italic">Tricholoma matsutake</Emphasis>, isolated from <Emphasis Type="Italic">Quercus</Emphasis>

We studied the characteristics of the utilization of carbohydrate substrates and the production of those hydrolyzing enzymes of the Tricholoma matsutake J-1 strain isolated from hardwood (Quercus sp.). In the culture medium, 5% glucose inhibited mycelial growth. The growth inhibition rate was remarkable in the Z-1 strain from softwood (Pinus densiflora) compared with that of the J-1 strain from hardwood. α-Amylase production varied with starches from different origins in contrast to mycelial growth. The range of the effect of 0.5%–15% soluble starch on vegetative mycelial growth was also investigated. The optimal concentration for mycelial growth was 15% for the J-1 strain but 10% for the Z-1 strain. Mycelial growth of the J-1 strain was strongly inhibited in PMML medium containing Sunpeal-CP prepared from sulfite pulp softwood waste, but that of the Z-1 strain was not inhibited by Sunpeal-CP. Moreover, mycelial growth of the J-1 strain from Quercus sp. dramatically decreased with the addition of CNF-HWSF (hot water-soluble fractions from corn fiber) to the PMML and PDL medium. However, inhibition by CNF-HWSF was not shown in the Z-1 strain from P. densiflora.     

The LTR retrotransposon micropia in the cardini group of Drosophila (Diptera: Drosophilidae): a possible case of horizontal transfer

The presence of the micropia retroelement from the Ty1-copia family of LTR retroelements was investigated in three species of the Drosophila cardini group. Southern blot analysis suggested the existence of at least four micropia copies in the genomes of D. cardinoides, D. neocardini and D. polymorpha populations. The high sequence similarity between dhMiF2 and Dm11 clones (micropia retroelements isolated from D. hydei and D. melanogaster, respectively) with micropia sequences amplified from D. cardini group genome supports the hypothesis that this retroelement plays an active role in horizontal transfer events between D. hydei and the D. cardini group. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Highly polymorphic DNA markers to specify strains of the ectomycorrhizal basidiomycete Tricholoma matsutake based on σ marY1 , the long terminal repeat of gypsy-type retroelement marY1

The ectomycorrhizal basidiomycete Tricholoma matsutake produces commercially valuable fruit bodies—matsutake—in Pinus sp. forest. Here we report that PCR with outward facing primers designed based on sequences comprising _marY1, the long terminal repeat of the gypsy-type retroelement marY1, specifies strains of T. matsutake. PCR with a primer based on the 22-bp sequence conserved at the 5-end of _marY1 conferred 73 reliable bands overall whose profiles depend upon strains of T. matsutake and T. magnivelare, the latter known as American matsutake. This PCR system gave no detectable band in any other species of Tricholoma tested, including T. bakamatsutake and T. fulvocastaneum, symbionts closely related to T. matsutake, as well as a host plant, Pinus densiflora. Similarly, PCR with a set of primers based on 26-bp and 28-bp sequences at bp 48–73 and bp 281–308 of _marY1, internal regions that are mutated in a variant of marY1, conferred 90 reliable bands only in strains of T. matsutake. Theoretically, PCR with the 22-bp primer would allow generation of 2⁷³, or 9.4×10²¹, types of polymorphism, and PCR with a combination of 26- and 28-bp primers, 2⁹⁰, or 1.2×10²⁷ types. The probability of falsely specifying two different isolates as the same strain is <1/10²¹. While polymorphisms conferred by the primer based on the 5 end of _marY1 rather exhibit genetic conservation of a group of T. matsutake, those resulting from primers based on the internal sequences more clearly demonstrate intra-specific diversification. Both systems revealed that T. matsutake is divergent within the species. Ectomycorrhizas formed between P. densiflora and T. matsutake were identified by the PCR systems developed in the present study. This method, using _marY1 as a genetic marker, is useful in analyzing the diversity of T. matsutake, monitoring the behavior of individual mycorrhizas, and specifying the ecological background of fruit bodies traded in markets.     

Tricholoma matsutake 1-Ocen-3-ol and methyl cinnamate repel mycophagous Proisotoma minuta (Collembola: Insecta)

Two major volatiles produced by the mycelia and fruiting bodies of Tricholoma matsutake (1-octen-3-ol and methyl cinnamate) repel a mycophagous collembolan, Proisotoma minuta. Aggregation of the collembolans on their diet was significantly inhibited by exposure to 1 ppm methyl cinnamate or 10 to 100 ppm 1-octen-3-ol. The aggregation activity decreased dose-dependently upon exposure to 1-octen-3-ol at concentrations higher than 0.01 ppm. Aggregation in the presence of methyl cinnamate exhibited three phases: no significant effect at concentrations ranging from 0.001 to 0.1 ppm, significant inhibition from 1 to 100 ppm, and strong inhibition at 1,000 ppm. These results may explain why certain collembolan species do not prefer T. matsutake fruiting bodies.     

Tricholoma matsutake– an assessment of in situ and in vitro infection by observing cleared and stained whole roots

 Structures present within field-collected Tricholoma matsutake/Pinus densiflora ectomycorrhizas and in vitro infections of P. densiflora roots by T. matsutake were observed by clearing, bleaching and staining whole lateral roots and mycorrhizas. Field mycorrhizas were characterized by a lack of root hairs, by the presence of a sparse discontinuous mantle composed of irregularly darkly staining hyphae over the root surface, primarily behind the root cap, and by the presence of Hartig net mycelium within the root cortex. Hartig net 'palmettis' were classified into three basic structures, each with distinctive morphologies. Aerial hyphae, bearing terminal swellings, were observed emanating from the mantle. Cleared, bleached and stained in vitro-infected roots possessed multibranched hyphal structures within the host root cortex and aerial hyphae bearing terminal swellings were observed arising from the mycelium colonizing the root surface. T. matsutake on P. densiflora conforms to the accepted morphology of an ectomycorrhiza. This staining protocol is particularly suited to the study of Matsutake mycorrhizal roots and gives rapid, clear, high-contrast images using standard light microscopy while conserving spatial relationships between hyphal elements and host tissues. Accepted: 26 August 1999     

Enzymatic characterization of an extracellular glucoamylase from Tricholoma matsutake and its cloning and secretory expression in Pichia pastoris

ABSTRACT

A glucoamylase from the ectomycorrhizal fungus Tricholoma matsutake (TmGLA) was purified 33.2-fold to homogeneity as a single monomeric glycoprotein with a molecular mass of 63.9 kDa. Maximum activity was observed at 60°C and pH 5.0. The enzyme is active down to 50°C and in the pH range of 4.0–6.0, and its activity is strongly inhibited by Ag⁺. It degrades α-1,4- and α-1,6-glycosidic linkages in various polysaccharides. Its gene (TmGlu1) was cloned using information from the enzyme’s internal amino acid sequences and the whole genome sequence of T. matsutake NBRC 30605. The deduced amino acid sequence showed clear homology with those of GH family 15 proteins. Pichia pastoris transformed with TmGlu1 secreted the active enzyme in a glycosylated form, and its characteristics were the same as the native enzyme.     

Sustainable fruit-body formation of edible mycorrhizal <Emphasis Type="Italic">Tricholoma</Emphasis> species for 3 years in open pot culture with pine seedling hosts

Three edible mycorrhizal mushrooms, Tricholoma portentosum, T. saponaceum, and T. terreum, that had formed ectomycorrhizas with Pinus densiflora seedlings in vitro, were maintained in open pot culture for 3 years under laboratory conditions. Tricholoma portentosum and T. saponaceum produced fruit bodies several times. For T. terreum, which produced a single fruit body in the third year, this is the first report of mushroom production under controlled conditions. Morphological observation of fruit bodies indicated that they were mature, i.e., well-organized cap, stem, and gills, and basidiospores. These results suggest that cultivation of these three edible Tricholoma mushrooms is feasible.