Studies On Inducing To Form Artificial Shiro Of Tricholoma Matsutake

This study was made to establish a culture system of Shiros of matsutake (Tricholoma matsutake) on artificial substrates. Firstly, the ecology of matsutake growing in the foothills of Mount Changbai-shan, which is one of major production areas of matsutake in China, was investigated and 10 fruit-bodies of matsutake were collected. Subsequently W2, W3, F1, F2 and F4 strains were isolated. The average isolation rate was 50% with the highest rate of 62% from lamella parts and the lowest rate of 38% from tissues between lamellae and pilei. Secondly, the capability to utilize carbon sources was investigated in culture media in vitro in three isolated matsutake strains of W3, F1, and F2 as well as three strains of Y1, A, and T945 obtained from different sources. It was found for the first time that cellobiose was as good a carbon source as glucose for hypha growth of matsutake in liquid medium. Xylan was utilized at a limited rate. No growth in tested strains except for T945 was found in cellulose-containing medium. Lastly, a new method of sterilizing culture containers was established by combination of ultraviolet-light radiation, 70% ethanol spray, and 0.1% NaClO solution spray, without using high pressure. Subsequently, a culture system of artificial Shiro of matsutake was sought to develop. The soil (pH 6.43) taken from the forest floor of Mount Maoer-shan in Heilongjiang Province was chosen after screening test to induce hyphae growth of matsutake. In strains of W2, F2, and A remarkable growth in hypha was found on the soil supplemented with olive oil at 0.5 - 2 % in volume. The growth of hyphae near the soil surface in these strains, which was estimated from measurements of matsutake ergosterol content by high performance liquid chromatography, increased as the concentrations of olive oil were higher. However, growth of hyphae was inhibited at 5 % olive oil in soil. Seedlings of Pinus densiflora grew normally in the soil containing 0.5 and 1% olive oil, but turned to yellow when the concentration was increased to 2%. In strains of W2, W3, F1, F2, F4, and A, the ergosterol contents of hyphae growing on the soil containing 1% olive oil were higher than those on the soil without olive oil. Three months after hyphae were inoculated in soil with 1 % olive oil, mycelia A extended to cover the whole soil surface and formed aggregation with soil. The mixture of hyphae and soil resembled a Shiro, which is the white or pale gray soil within or around host rhizosphere intermixed with compact mass of mycelia in situ. Pine seedlings grew well for two months after transplanted to this artificial shiro. Loose hyphae were observed microscopically to connect to some lateral roots of a seedling without root hairs, when lateral roots turned to dark brown. Therefore, it was shown that the artificial Shiro performed very well.