In vitro ectomycorrhizal synthesis of Tricholoma matsutake with host plants has been widely conducted to elucidate fungal symbiotic properties for future cultivation practices. Here, we report on the importance of basidiospore inocula for this fungus to provide ectomycorrhizal seedlings in vitro. Ectomycorrhizal pine seedlings synthesized in vitro with cultured mycelium of T. matsutake (isolate #45 or #84) in a 250-mL culture vessel (soil volume) were transplanted to a large 1-L culture vessel. Fresh basidiospores of this fungus were aseptically inoculated on the ectomycorrhizal root system. The ectomycorrhizal seedlings in the 1-L vessel were grown for 9 months, and some plants were further grown for 6 more months under non-aseptic conditions in 4.1-L jars. The ectomycorrhizal seedlings previously inoculated with isolate #84 in the 1-L vessel showed significant ectomycorrhizal biomass (mycorrhizal root length) after spore inoculation. The ectomycorrhizal seedlings in the 4.1-L vessel showed large shiro structures (> 10 cm in diameter). PCR amplification of intergenic spacer 1 of the rRNA gene and long terminal repeat retroelement of T. matsutake in ectomycorrhizal root tips in both the 1-L vessels and 4.1-L jars revealed the presence of amplicons of the previously inoculated culture isolate of T. matsutake and the new genet(s) that established via germination of the inoculated basidiospores. This is the first report that inoculated basidiospores of T. matsutake germinated and colonized the host root to generate ectomycorrhizae in vitro.

松茸与宿主植物的体外外生菌根合成已被广泛进行，以阐明真菌的共生特性，以供将来的栽培实践。在这里，我们报告了担子孢子接种对于这种真菌在体外提供外生菌根幼苗的重要性。松茸菌丝体体外合成的外生菌根松树苗将250 mL培养皿（土壤体积）中的（45号或84号分离株）移植到1 L大的培养皿中。将这种真菌的新鲜担子孢子无菌接种在根外生根系统上。1-L容器中的外生菌根幼苗生长9个月，并且在非无菌条件下在4.1 L广口瓶中将一些植物进一步生长6个月。孢子接种后，先前在1-L容器中接种隔离株＃84的外生菌根幼苗显示出显着的外生菌根生物量（菌根根长）。4.1-L容器中的外生菌根幼苗显示出大的shiro结构（直径> 10 cm）。rRNA基因的基因间隔区1的PCR扩增和松茸的长末端重复序列在1-L容器和4.1-L广口瓶中的外生菌根根尖中，发现了先前接种的松茸培养分离物和通过接种的担子孢子萌发而建立的新种的扩增子。这是首次报道的接种担孢子T.松茸发芽并定殖宿主根生成菌根体外。