Observation and Distribution of Ectomycorrhizal Fungi in Pinus Roots

Detailed structures of ectomycorrhizae formed in Pinus roots were observed with various microscopes: light, fluorescence, and scanning electron microscopes. The mantles and Hartig nets commonly found in the structure of ectomycorrhiza were newly observed according to developmental stage by various staining. The mycelia were observed to be composed of coiled types on the surface of epidermal root during early stage and fused to form mantles of smooth fungal layers, loosing mycelia with some viscous liquid secreted. The ectomycorrhizal hyphae in anatomical roots penetrated the cortical layer and formed obviously mantle and Hartig net. The round spots of ectomycorrhizal mycelia were observed morphological distribution from the cortical layer to vascular bundle of stele in the ectomycorrhizal roots of Pinus species and especially scattered at the area of meristem at the root tip as longitudinal sections. Those mycelia penetrated seemed to move into other roots by means of vascular bundle of ectomycorrhizal roots and newly form ectomycorrhizal roots of dichotomous branches.

ITS Primers with Enhanced Specificity to Detect the Ectomycorrhizal Fungi in the Roots of Wood Plants

With universal primer ITS1-F, the specific DHJ2 primer was developed to detect the Ectomycorrhizal (ECM) root tips in soil and to identify the species of ECM fungi, as based on DNA sequences of rDNA stored in GeneBank of NCBI. This primer was designed with the common sites of rDNA of Amanita and Boletus, and was also designed with several DNA programs provided by NCBI. The DNA fragments synthesized by PCR were calculated to be 1,000 to 1,200 bps of DNA located to 18s to 28s rDNA to contain two variable sites of ITS, indicating much diversities for specific species or ecotypes of ECM fungi. The primer DHJ2 reacted with the genomic DNA's extracted from the tissues of basidiocarp at the rate of 73 of 80 fungi collected produced single bands with a 1,100 bps length. The DNA fragment synthesized with the genomic DNA that extracted from eight ECM tips of Pinus densiflora was confirmed and analysized to the rDNAs of ECM in full sequences, and informed to be a ECM fungal species in the forest.

Mycorrhizal Formations and Seedling Growth of Pinus desiflora by in vitro Synthesis with the Inoculation of Ectomycorrhizal Fungi

The mycelia were directly isolated from eight species of fungal basidiocarps, confirmed to the ectomycorrhiza in the roots from the fields (forestry); Suillus bovinus, Paxillus involutus, Lactarius hysginus, Russula fragilis, Lepista nuda, Lyophyllum shimeji, Tricholoma matsutake, and Russula integra. The mycelia were pure-cultured with several transferring in various agars, and inoculated to the roots of pine (Pinus densiflora) seedling by in vitro method. After ten months growth under artificially aseptic conditions, all pine seedlings inoculated were stimulated at the growth-height, whereas those not inoculated were nearly dead. Also, the ramifications of ectomycorrhizal pine roots formed in the synthetic in vitro systems and were various according to the different mycelia. Synthesis of ectomycorrhiza were clearly confirmed in ten months growth, but not distinguished at this moment. It was clearly proved that the mycelia isolated caused the ectomycorrhizae in the roots of pine seedlings.

Ectomycorrhizal Roots Collected from the Bases of the four Edible Basidiocarps Around Mt. Wol Ak

The ectomycorrhizal roots were collected from the soils around the bases of basidiocarps of the four edible mushrooms in the stands of Pinus densiflora or Querus acutissima communities (Mt. Wol-Ak in Eastern Chung-Puk): The basidiocarps of Tricholoma matsutake (TM), Sarcodon asparatus (SA), S. imbricatum (SI), and Polyozellus multiplex (PM) are usually collected. The ectomycorrhizal roots of TM, PM and SI were related to the roots of P. densiflora, but the other to the roots of Q. acutissima in Korea. Particularly, the basidiocarps of PM were collected in the mixed stand of both P. densiflora and Q. acutissima. The morphologies of the ectomycorrhizal roots were observed to be the yellowish brown coral (dichromatous) or pyramid types in the roots of the pine, but dark brown un-branched sticks (roots) in the ends of ectomycorrhizal roots of Querus plants. The un-branched roots were covered with the dark mycelia (rhizomorph) around them. Therefore, the ectomyorrhizal roots of PM were observed to have two kinds of types; The single blackish un-branched roots were observed to be attached to the yellowish coral type roots. The bundles of TM mycelia were filled with cortical cells (in the roots of P. densiflora), but the mycelia of the other fungi (Aphylloporales) were massed between the cortical cells of P. densiflora or Q. acutissima. Their anatomical and gross features were considered to be simailar but very important in the ectomycorrhizal roots for these edible mushrooms.

Observations on the Ectomycorrhizal Roots Collected from the Bases of the Basiodiocarps in Chungbuk

Various types of ectomycorrihzal root were observed from the bases of the basidiocarps collected from the four different soils of WalAk Mt., JoRyeong Mt, MinJuJi Mt. and hills near Korea National University of Education; The dichotomous ectomycorrihzal roots were mainly found to be from the interactions of the Pinus roots with the basidiocarps of Boletus and Amanita species. The black single bat-like ectomycorrihzal roots with the darken rhizomorphic mycelia were mainly found to be from the interactions of the Quercus roots with the basidiocarps of Russula species. The pyramid and the monopodial-pinnate ectomycorrihzal roots were also found to be from the interactions of the Pinus and deciduous tree roots with the basidiocarps of Russula and Boletus species. The distinguished structures of the mantle and Hartig net in the cortex layers were observed to be from the roots related to the genus of Pinus in the anatomical sections. However, the aggregated (premeated) structures of mantle and Hartig net in the cortex layer were found to be in the black and bat-like ectomycorrihzal roots. The structures of cross section in the ectomycorrihzal roots, especially the roots related to the host-plants of Pinus, were observed to be different from those according to the fungal species of the basidiocarps collected. The ectomycorrihzal root not reported or confirmed by the anatomical sections were newly found to be from the bases of the basidocarps of Amanita castanopsidis, Boletus laetissimus, B. violaceofuscus, B. speciosus, Clitopilus prunulus, Leccinum rugosiceps, Russula. alboareolata, R. bella, Strobilomyces confusus, Tylropilus neofelleus, and T. plumbeoviolaceus. From the observations mentioned above, the various types of ectomycomhzal roots were speculated to be formed between the different host plants and the different species of basidiocarps.