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.

외생균근은 접종 3일 후 뿌리방향으로 이동하여 접종 5일 후 균사가 뿌리 표면에 감염되어 균사 다발이 뭉쳐진 상태를 나타내었다. 균근 감염 6~9일 후에는 균사가 토양 전면에 퍼졌다. 균근 감염 9~14일 후 균사가 뿌리를 감싸기 시작하였다. 균근 감염 15~19일 후 균사는 뿌리의 외피를 감싸고 피층 세포 사이로 침입하여 균근을 형성하였다, 균근이 형성된 후에는 새로운 뿌리가 형성되었으며 균근이 뿌리에 활착되어 Y자형으로 뿌리 끝이 부풀면서 균근이 형성된 것을 확인할 수 있었고 해송의 생장이 촉진되었다. 해송(P. thunbergii)을 파종하여 외생균근 균인 모래밭버섯(P. tinctorius)과 황소비단그물버섯(S. bovinus)을 접종하여 4개월 생장한 결과, 대조구는 줄기 길이가 7.4cm인 반면 외생균근 균인 모래밭버섯(P. tinctorius)을 접종한 것은 11.4cm로 34.2% 생장이 촉진되었고, 외생균근 황소비단그물버섯(S. bovinus)을 접종한 것은 10.5cm로 28.6% 생장이 촉진되었다. 이외에도 줄기와 잎의 개체수가 많아졌다.