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Cryptococcus neoformans is a basidiomycetous yeast which is an opportunistic pathogen responsible for life-threatening infections among immunocompromised persons. Our group has reported the unique cell cycle pattern of C. neoformans, different from that of the model budding yeast Saccharomyces cerevisiae. In C. neoformans, during exponential growth, budding and DNA synthesis occur simultaneously, but as the growth phase progresses during late log to early stationary phase, budding is delayed towards the end of the cell cycle at G2 phase, allowing cells to arrest at either G1 or G2 unbudded state. This phenomenon was also observed in response to stress conditions, and is therefore believed to be an important stress response mechanism which may play a role in pathogenesis. To clarify the cell cycle mechanisms, homologues of cell cycle control genes in C. neoformans were cloned. We have previously reported the cloning of the CDC28/Cdc2 homologue, CnCdk1. Here we report on the cloning and characterization of Cdk1 cyclin homologues. Using available genome sequence information we have identified at least three cell-cycle related cyclin homologues. The cDNAs were cloned using RT-PCR and RACE, sequenced and coding regions identified. Analysis of putative amino acid sequences showed that CnCln1 is a G1 cyclin homologue and CnClb1 and CnClb2 are B-type or G2/M cyclin homologues. Complementation tests in an S. cerevisiae G1 cyclin triple mutant confirmed that CnCln1 is able to complement S. cerevisiae G1 cyclin function while CnClb1 and CnClb2 cannot. Deletion of these genes in C. neoformans was carried out by biolistic transformation and analysis of deletion mutants is currently being done to confirm their role and function in C. neoformans cell cycle.

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Chestnut blight is one of the most famous and disastrous disease in chestnut tree plantations in Korea. The pathogenic fungus, Cryphonectria parasitica, sometimes contain dsRNA virus in the cytoplasm, and express hypovirulent traits. DsRNA virus is known to be transmitted by hyphal anastomosis between two vegetatively compatible hyphae, and subsequently the normal virulent strain converted into the hypovirulent one. In order to prove the role of dsRNA virus in the fungal cytoplasm indirectly, hypovirulent strains were cured by eliminating dsRNA virus artificially. Agar discs containing mycelium of hypovirulent strains were grown on 2% water agar supplemented with asparagine, glucose, and cAMP alone or in combinations. Sectored parts, which indicates the occurrence of genetic variation, were trans-cultured on fresh PDA plate, and then curing was confirmed by comparing with the original hypovirulent strains in cultural characteristics, mycelial growth, virulence, and the absence of dsRNA virus. Among three hypovirulent strains, one strain was only cured with the frequency of 23.5%. Virulence of cured and original hypovirulent strains was evaluated and compared on the bark and wood tissues of resistant, moderate and susceptible cultivars. Mycelial growth and virulence of the cured strains were completely restored as the normal virulent strains. DsRNA banding patterns showed that the genomic dsRNA virus was disappeared in the cured strain. Cured strains were converted into the hypovirulent strains again by pairing with the original hypovirulent strain.

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A novel ssRNA virus (we named FvSV)was isolated from cultivated Flammulina velutipes. This new virus was a 13nm spherical virus encapsidating a bipartite single-stranded RNA(ssRNA) of approximately 4 and 1.5kb with a coat protein of about 60 kDa. We made monoclonal antibodies and polyclonal antibodies against FvSV to makes TAS-ELISA Kit to screen the viruses in comercial farms.

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Root disease suppression by ectomycorrhizal(ECM) fungi, Pisolithus tinctorius, Rhizopogon rubescens, Hebeloma cylindrosporum, and Suillus bovinus, was investigated by in vitro examination and in vivo co-inoculation of both ECM fungi and root pathogenic fungus, Fusarium oxysporum, to the ectomycorrhizal-free Pinus densiflora seedlings. Mycelial growth and sporulation of F. oxysporum were inhibited on PDA containing culture filtrates of ECM fungi from 9.3 to 18.6% and from 20.1 to 58.9%, respectively, as compared with mycelial growth and sporulation on the control medium. Spore germination was also strongly inhibited by the culture filtrates of R. rubescens for 90 days upto 81.8%. Pre- or simultaneous inoculation of P. densiflora seedlings with ECM fungi resulted in resistance of seedling roots to the infection by root pathogenic fungus, F. oxysporum, as compared with the control seedlings. The survival rate of the seedlings inoculated with F. oxysporum, and subsequently with ECM fungi was averaged by 52%, but pre- or simultaneous inoculation of ECM fungi completely protected P. densiflora seedlings against root infection by F. oxysporum. Disease suppression by ECM fungi in P. densiflora is, therefore, associated with the increase of fungitoxic or fungistatic metabolites excreted by symbiotic ECM fungi to the rhizosphere of seedlings as well as mechanical barriers, i.e., fungal mantle and Hartig net.

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 Odaigahara is a mountainous area with the elevation of around 1,500 m in Nara Prefecture, Japan. Various kinds of explorations and investigations have been conducted by the direction of Ministry of the Environment to aim the regeneration and conservation of declined forest ecosystem. In order to estimate the difference in the forest regeneration potential among different types of vegetations, we conducted the survey of mycobiota of macrofungi and the quantification of mycorrhiza formation to the planted seedlings of Japanese spruce (Picea yezoensis var. hondoensis).
 There established seven vegetation types, I to VII, where the fences were put to protect the seedlings, buds and barks from deer's grazing. For the exploration of mycobiota of macrofungi, fruiting bodies found in the quadrates (30 m x 30 m) were recorded and identified monthly from June to October in 2004. For the investigation of mycorrhiza formation, 300 seeds each were sowed and 16 seedlings each grown for 5 month were planted in type I, II and III in June, 2005. Four month later, all of the seedlings were collected, root tips was counted and mycorrhizal frequency was determined.
 In type I, dominated by a bamboo grass (Sasa nipponica), no fruiting body was found. There found more fruiting bodies inside the fence than outside. Number of fruiting bodies of ectomycorrhizal fungi was greatest in type III, where Japanese spruce dominated, followed by type V, VI, VII, IV and II.
 Though the seedling growth was best in type I, mycorrhizal frequency was highest but less than 10% in type III, suggesting that Japanese spruce seedlings are not subject to ectomycorrhiza formation. The results indicate that the vegetation as well as the environmental condition affects the potential of mycorrhiza formation to Japanese spruce seedlings.
 This study was implemented as part of Odaigahara nature restoration promotion plan of Ministry of the Environment.

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Plant roots and their symbiotic and free-living microbial populations alter the physico-chemical characteristics of the rhizosphere making it different from bulk soil and affecting toxic metal speciation. The mechanisms by which fungi and plants obtain phosphate are of particular interest since solubilization of inorganic phosphates can result in release of associated metals. Zinc phosphate was of least toxicity and the most easily solubilized by axenic ericoid and ectomycorrhizal fungi among tested insoluble cadmium-, copper-, lead- and zinc-containing minerals. Solubilization of toxic metal minerals was related to metal tolerance of the fungal species. Zinc phosphate solubilization by Paxillus involutus/Pinus sylvestris ectomycorrhizal associations and protection of host plants against toxic metal mobilized from mineral depended on fungal tolerance and the phosphorus status in the growth matrix. Zinc, copper and lead mobilized from minerals were oxygen-coordinated within the fungal/ectomycorrhizal biomass. “Heterotrophic leaching” of toxic metal(s) from insoluble minerals combined acidification and ligand-promoted dissolution mediated by different organic acid anions: if oxalic acid was produced, precipitation of metal oxalates resulted.

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Recently, functional foods are more interesting among peoples for their health. Screening of physiological active substances against for allergy was studied by using mushrooms in this experiment. Degranuation is the discharge of histamine from mast cell stimulated by allergic factors. So, substances inhibited this degranuation were screened from the extracts of mushrooms.
(Material and methods)
The rat basophilic leukemia cell lines (RBL-2H3) were used and induced degranuation by using two chemicals; one is calcium ion and the other is antigen. Degranuation was estimated by the activity of β-hexosaminidase which discharged at the stage of degranuation. The cells were inoculated into a 96-well plate (1 x 10⁴ /well; Falcon, Nj, U.S.A.) and cultured overnight. After washing two times with the modified Tyrode HEPES buffer, each mushroom extract (80μl) diluted with Tyrode HEPES was added. And then, the cells were incubated in a growth medium containing 100ng/ml of mouse monoclonal anti-DNP (or 1μM calcium ionophore A23187) for 1 hour. The β-hexosaminidase activities of the supernatant were measured. Briefly, 25μl of the supernatant was mixed with 25μl of a β-hexosaminid 2.04μg./ml, pH4.5, and the mixture incubated in a 96-well plate at 37°C for 90 min. The reaction was terminated by adding a stop buffer (pH11, 150μl) and absorbance at 405nm was measured with a microplate reader (model 550; Bio-Rad, CA, U.S.A.). The histamine concentration was measured.
(Results)
In the experiment of both methods;calcium ionophore and antigen, Ganoderma lucidum, Pycnoporus coccineus and Russula sororia indicated the activity of inhibition of degranuation. About the other mushrooms, the experiments are going on.

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Medicinal mycology has its deep and firm roots in mushrooms used in the traditional medicine of the Far East. Both superoxide anion radical and hydroxyl radical belonging to the category of active oxygen cause various severe diseases such as arteriosclerosis, cardiac infarction, diabetes, and cancer, because they are strongly cytotoxic. The hitherto studies on searching for mushrooms which produce antioxidants possessing radical-scavenging activity have been done mostly on edible mushrooms, and a few on wild mushrooms. This time, we screened many wild mushrooms as potential sources for antioxidants. The methanolic extract of each sample of mushroom was prepared by extraction of each 1 g of dried mushroom with 10 ml of methanol at room temperature for 6 hours twice followed by evaporation of the solvent in vacuum. The methanolic extracts of over 100 samples of wild mushrooms belonging to 64 genera, 90 species collected in Japan were examined on the ordinary and reversed phase TLC plates using L-ascorbic acid as the positive control displaying the antioxidizing (radical-scavenging) activity against the radical reagent, 1, 1-diphenyl-2-picrylhydrazyl (DPPH). As the result, the extracts of about 50 samples of mushrooms including Boletus fraternus, Calvatia craniiformis, Ganoderma applanatum, Geastrum triplex, Lactarius flavidulus, Leucopaxillus septentrionalis, Microporus affinis, Pholiota flammans, and Xeromphalina campanella gave the spots exhibiting strong or moderate antioxidizing activity on the TLC plates. Among these extracts positively reacted, that of M. affinis (295 g) was partitioned with n-hexane and ethyl acetate under the guidance of the radical-scavenging activity. The ethyl acetate layer was then fractionated on the chromatographic silica gel columns and on the preparative TLC plates to afford a small amount of the active components.

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Aureobasidium pullulans is a yeast-like fungus commonly found in a variety of cosmopolitan environments such as plant leaf, soil, and damp indoor surface. In this investigation, Aureobasidium spp. were isolated from bathroom surfaces in several locations in Bangkok, Thailand and they were subsequently examined for antifungal agent production. Ten isolates obtained included KT1, BM1, PH1, TB1, JP1, VM1, HKW1, HKW2, HKW3 and HKW4. All isolates were identified as A. pullulans based on morphological observations, physiological properties and exopolysaccharide characteristics (IR-spectra). The antifungal agents, extracted from their fungal cells, were tested for their antifungal activities against some Aspergillus spp. by using paper disc method. The extracts from A. pullulans KT1 and PH1 showed activities against A. fumigatus and A. flavus, respectively, while the extract from A. pullulans BM1 had activities against both A. fumigatus and A. flavus. For A. pullulans TB1, VM1 and JP1, their extracts showed no inhibition activity against any of the Aspergilli tested. The extracts of A. pullulans isolates, containing antifungal activities, were further purified by using Thin Layer Chromatography (TLC) and High Performance Chromatography (HPLC) in comparison to the standard aureobasidin antibiotic.

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