Detections of reactive oxygen species (ROS) during ectomycorrhiza establishment between Rhizopogon roseolus (shoro) and Pinus thunbergii were made microscopically using a nitro blue tetrazolium (NBT) staining. Roots of P. thunbergii were aseptically infected with R. roseolus mycelium by using a Petri dish technique. From 2- to 4-week period after inoculation, initial mycorrhizal formation could be observed. Lateral root tips were treated with NBT and then observed under a light microscope. Depositions of blue formazan indicating O2- accumulation were detected mainly hyphal cells contacting with the roots surface. Observations of transverse section of the root revealed that depositions of blue formazan were also detected at the plasma membranes of the epidermal cells where the fungal hyphae were adhesively contacted. In the non-inoculated P. thunbergii roots, depositions of formazan were observed in root hair cells but not in epidermal cells. From 4- to 8-week period after inoculation, dichotomous mycorrhizas and extraradical mycelia were clearly observed. A section from the mycorrhiza treated with NBT showed that root tissue was surrounded by fungal mantle sheath, in which highly intensive reaction with NBT was demonstrated. The reactive formazan complexes were apparent in Hartig net hyphae between epidermal and cortical cells of the root. After 16 weeks following inoculation, morphology of mycorrhizas became variable, viz., initial, dichotomous and browned mycorrhizas. The browned mycorrhizas were characterized by wrinkled surfaces and sparse extraradical mycelia. The browned mycorrhizas were collected and treated with NBT. A section from the specimen showed that depositions were slightly observed only in the part of extraradical mycelia. These results suggest that O2- generations from both fungus and plant are involved with the early establishment of ectomycorrhizas between R. roseolus and P. thunbergii.

Rhizopogon roseolus (Corda) Th. M. Fr. (=R. rubescens Tul. & Tul.), known as “shoro” in Japan, is a hypogeous basidiomycete that is an important ectomycorrhizal symbiont of Pinaceae. In order to cultivate this edible ectomycorrhizal mushroom, several researches have tried to promote mycorrhization of this mushroom on host Pinus thunbergii roots: Pine seedlings were inoculated with mycelium in vitro, or with crushed fruiting bodies in nature. However, successful cultivation of this mushroom has not been fully refined. We have developed the useful mycelial inoculum that enable to produced abundant ectomycorrhizas and then to form fruiting body under greenhouse nursery conditions. We selected the superior strain that rapidly colonized and produced a lot of ectomycorrhizas in root of P. thunbergii． The mycelial inoculum was composed of mineral solution and homogenate of mycelium that had been cultured in liquid medium. Addition of surfactant in the mycelial inoculum resulted in stimulation of mycorrhzal formations in host roots. When the mycelial inoculum containng surfactant were introduced to the mother plant system in which the colonized seedling had been planted into in the nursery, stimulatally effects were observed on not only mycorrhzation of the seedlings but also fruiting body formation. Genotype analysis using microsatellite markers for R. roseolus showed that fruiting bodies produced in the nursery were originated from the inoculated strain. These results suggest that the mycelial inoculum containg surfactant could be the model of mycelial spawn for “shoro”.