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.