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The pine mushroom ( Tricholoma matsutake ; Agaricales, Tricholomataceae) is an ectomycorrhizal fungus that produces a commercially valuable, edible mushrooms. Attempts to artificially cultivate  T. matsutake  has so far been unsuccessful. One method used to induce  T. matsutake  to produce fruiting bodies of in the wild is shiro (mycelial aggregations of  T. matsutake ) transplantation.  In vitro  ectomycorrhization of  T. matsutake  with seedlings of  Pinus densiflora  has been successful, but field trials showed limited production of fruiting bodies. Few studies have been done to test what happens after transplantation in the wild, whether  T. matsutake  persists on the pine seedling roots or gets replaced by other fungi. Here, we investigated the composition and the interaction of the root fungal microbiome of  P. densiflora  seedlings inoculated with  T. matsutake  over a 3 year period after field transplantation, using high-throughput sequencing. We found a decline of  T. matsutake  colonization on pine roots and succession of mycorrhizal fungi as  P. densiflora  seedlings grew. Early on, roots were colonized by fast-growing, saprotrophic Ascomycota, then later replaced by early stage ectomycorrhiza such as  Wilcoxina . At the end, more competitive  Suillus  species dominated the host roots. Most of the major OTUs had negative or neutral correlation with  T. matsutake , but several saprotrophic/plant pathogenic/mycoparasitic species in genera  Fusarium ,  Oidiodendron , and  Trichoderma  had positive correlation with  T. matsutake . Four keystone species were identified during succession; two species ( Fusarium oxysporum , and  F. trincintum ) had a positive correlation with  T. matsutake , while the other two had a negative correlation ( Suillus granulatus ,  Cylindrocarpon pauciseptatum ). These findings have important implications for further studies on the artificial cultivation of  T. matsutake .

Successional Change of the Fungal Microbiome Pine Seedling Roots Inoculated With Tricholoma matsutake