As a member of ectomycorrhizal fungi, Tricholoma matsutake has a symbiotic relationship with its host, Pinus densiflora. To cultivate T. matsutake artificially, the co-cultivation of T. matsutake mycelia and bacteria from shiro was introduced. In this study, bacteria were isolated from soil samples in Bonghwa-gun, and seven bacterial isolates (B22_7_B05, B22_7_B06, B22_7_B07, B22_7_B08, B22_7_B10, B22_7_B13, and B22_7_B14) promoted the growth of T. matsutake mycelia (147.48, 232.11, 266.72, 211.43, 175.17, 154.62, and 177.92%, respectively). Sequencing of the 16S rRNA region of the isolated bacteria was performed. B22_7_B05 and B22_7_B10 were identified as Bacillus toyonensis, B22_7_B06 and B22_7_B08 as Paenibacillus taichungensis, B22_7_B07 and B22_7_B14 as P. gorilla, and B22_7_B13 as P. odorifer. These bacterial isolates were associated with the shiro community and are expected to contribute to the cultivation of T. matsutake.

Tricholoma matsutake is a traditional favorite food in East Asia, cultivated in fairy rings called “shiro,” which are found near Pinus densiflora. For effective artificial cultivation of Tri. matsutake, microorganisms from symbiotic fairy rings are co-cultivated. In this study, one bacterial isolate (Y22_B35) and two fungal isolates (Y22_F64 and Y22_F68) displayed growth-promoting effects on Tri. matsutake mycelium (158.47, 125.00, and 122.26% enhanced growth, respectively). For identification, 16S rRNA or ITS regions from the microorganisms¡¯ genomes were sequenced. Other sequences, including BenA, CaM, and RPB2 were sequenced in the fungal isolates. The bacterial isolate Y22_B35 was identified as Bacillus cereus. Y22_F64 and Y22_F68 were identified as Umbelopsis nana and Aspergillus parvulus, respectively. To identify the effects of the dominant microorganisms on Tri. Matsutake cultivation, metagenomic analyses were performed. Discovery of these Tri. matsutake mycelium growth-promoting microorganisms and metagenomics analyses are expected to contribute to our understanding of Tri. matsutake fruiting body growth and construction of biomimicry.

To cultivate pine mushroom (Tricholoma matsutake) artificially, co-cultivation with microorganisms has been introduced. Here, experiments were performed to assess the growth-promoting effect of bacteria on T. matsutake mycelia. Bacteria were isolated from soil samples collected in Yangyang County, Korea. Four of the bacterial isolates (Y22_B06, Y22_B11, Y22_B18, and Y22_B22) exhibited a growth-promoting effect on T. matsutake mycelia (154.67%, 125.91%, 134.06%, and 158.28%, respectively). To analyze the characteristics of the bacteria, especially the antifungal activity, -amylase and cellulase activity assays were performed. In comparison with the controls, the isolated bacteria exhibited low -amylase and cellulase activity. 16S rRNA gene sequencing was performed to identify the four bacterial isolates. The isolates belonged to the Terrabacteria group and were identified as Microbacterium paraoxydans, Paenibacillus castaneae, Peribacillus frigoritolerans, and P. butanolivorans. These bacterial isolates are expected to have contributed to the growth promotion of T. matsutake mycelia and the artificial cultivation of T. matsutake.

Tricholoma matsutake is a representative mushroom species with a characteristic pleasant aroma. The characteristic aroma component is methyl cinnamate, which is also produced in many plants. In basil, cinnamic acid is produced from l-phenylalanine (l-Phe) by phenylalanine ammonia-lyase (PAL) and converted to methyl cinnamate by a cinnamate/p-coumarate carboxyl methyltransferase. Two PAL genes, Tmpal1 and Tmpal2, have been isolated from T. matsutake. In this study, we aimed to clarify the relationships between l-Phe, methyl cinnamate production, and PAL expression in the mycelium of T. matsutake strain NBRC 30605. For this purpose, methyl cinnamate content, PAL activity, and transcript levels of Tmpal1 and Tmpal2 were examined in the mycelia of T. matsutake supplemented with l-Phe. The mycelia were cultured in 20 mL of a liquid medium (2% glucose, 0.15% yeast extract, and 0.15% Bacto Soytone) at 20 °C for 45 d, supplemented with 0.5-6 mM l-Phe, and then grown for a further 15 d. Mycelia cultured without l-Phe supplementation for 60 d in the medium were used as a control. Crude extracts were prepared from the mycelia harvested for enzymatic, protein, and methyl cinnamate assays. Methyl cinnamate was measured using gas chromatography. PAL activity was assayed by measuring the rate of trans-cinnamic acid formation as the absorbance at 290 nm (ɛ290 = 10,000 M−1 cm−1). The transcript levels of Tmpal1 and Tmpal2 were examined by performing real-time reverse transcriptase-quantitative PCR on the total RNA. Methyl cinnamate was detected in very low levels in cultures without l-Phe supplementation, but its content per mg of protein increased markedly with increasing concentrations of l-Phe, especially at 4-6 mM. When 6 mM l-Phe was added to the culture medium, the methyl cinnamate content was approximately 55-fold higher than that of the control sample. The specific activity of PAL also increased in cultures supplemented with l-Phe, especially at 4-6 mM. When l-Phe was added to the culture medium, the methyl cinnamate content in the mycelia was relatively well correlated with PAL activity. These results indicated that supplementation with l-Phe, a precursor of methyl cinnamate, increases the specific activity of PAL, leading to an increase in methyl cinnamate production in the mycelia of T. matsutake. The transcript level of Tmpal1 did not change markedly with l-Phe supplementation. In contrast, the transcript level of Tmpal2 increased greatly in cultures supplemented with 4-6 mM l-Phe. These results suggested that the expression of Tmpal1 and Tmpal2 was controlled by different regulatory mechanisms and that they may have different biological functions in T. matsutake. In addition, the pattern of PAL activity in the presence of l-Phe was similar to that of the transcript level of Tmpal2, but not Tmpal1, suggesting that the increase in PAL activity was dependent on the increased transcription of Tmpal2.

Effect of trehalose for the mycelial growth of Tricholoma matsutake were examined. When T. matsutake Z-1 strain was cultured in the partially modified matsutake liquid (PMML) medium and the Hamada matsutake liquid (HML) medium supplemented with trehalose at 24 ℃ for 80days, the vegetative mycelial dry weights showed higher value compared with those of PMML medium and HML medium supplemented with glucose (control). The range of the effect of 1.0-8.0% carbohydrate substrate on vegetative mycelial growth was investigated. The optimal concentration for mycelial growth was 2.0% for the glucose medium but 8.0% for the trehalose medium. To evaluate the potential of the production of trehalase from T. matsutake, the extracellular trehalase activity during the vegetative mycelial growth was measured. The activity of the extracellular trehalase increased during vegetative mycelial growth of T. matsutake and was maximal 70 days after inoculation. This extracellular enzyme was investigated for the purification and the characterization. The partially purified trehalase was obtained from about 1.53l static culture filtrate, with 19.1% recovery, and about 2,940 fold purification. The molecular mass was about 62.6kDa (SDS-PAGE) and 70kDa (Gel-filtration). The enzyme was most active around 40℃ and pH 5.0 and stable over a pH of 4.5~ 6.5 for 30min at 37℃. The enzyme readily hydrolyzed trehalose having α -1,1 glucosidic bond. However, it did not hydrolyze disaccharides such as maltose, iso-maltose, cellobiose, saccharose and lactose.