A basidiomycetous wood rotting fungus Pleurocybella porrigens (angel wings) has been well known as an excellent edible mushroom and named as Sugihiratake, in Japanese common name based on its morphological and ecological features. This mushroom has been favorably consuming by Japanese people. In 2004, an outbreak of serious acute encephalopathy exclusively occurred in patients with chronic kidney diseases after the intake of this mushroom. Thereafter, many researchers have conducted to examine the factor of onset of the encephalopathy, but the exact factors that induced the encephalopathy still remain unclear. Matsumoto et al. (2005) reported the mushroom specimens from different geographical areas in Japan were grouped into two distinct clusters based on ITS rDNA sequences. Vegetative growth of P. porrigens is extremely slow even cultivation on natural media surveyed and its physiological characteristic gives a disadvantage for the development of various fields of researches on this mushroom. Thus, we attempted to find a medium accelerating the vegetative growth of P. porrigens for further research to elucidate the possible association between its chemical constituent(s) and the onset of encephalopathy. Fifteen isolates of P. porrigens collected from different geographical areas in Japan were cultivated on PDA. The growth of the isolates was extremely slow as we know and most tested isolates formed mycelia look like those often observed in the cultivation of basidiomycetous ectomycorrhizal fungi. Among them, we chose five isolates based on the ranking of their higher growth rates. The top five isolates were cultured in five kinds of liquid media, PD medium, MY medium, Carrot medium, Ohta’s medium for ectomycorrhizal fungi (Ohta, 1990) and Amazake medium (Ishihara, 2007), and on a solid medium, PDA, at 20℃ in the dark. The dry biomasses of the isolates cultured in the liquid media were determined after 6 weeks of the stationary cultivation whereas the growth rates of the isolates cultured on PDA were compared by the colony diameter after 8 weeks of the cultivation. Among the tested liquid media, PD medium was the most suitable medium for their biomass growth (17 mg dry mycelium/flask - 127 mg dry mycelium/flask) and followed by Ohta’s medium (13 mg dry mycelia/flask - 79 mg dry mycelia/flask), MY medium (10 mg dry mycelium/flask - 72 mg dry mycelium/flask), Amazake medium (8 mg dry mycelium/flask - 47 mg dry mycelium/flask) and Carrot medium (4 mg dry mecelium/flask - 18 mg dry mycelium/flask). The average biomass growths of the isolates cultured in the synthetic medium, Ohta’s medium were 20-92% of those of the isolates cultured in PD medium. No correlation was observed between the cultivation in PD medium (liquid medium) and that on PDA (solid medium). Extremely large biomass variation was observed among the same isolates cultured on the same kind of the liquid medium. Large variation in colony features associated with their growth rates was also observed among the isolates cultured on PDA. These results suggest that PD medium would be a suitable one for most researches and Ohta’s medium for researches requiring clarity of its chemical constituents. The above results also indicate that the Japanese population of P. porrigens has large variation in vegetative growth although we have not yet examined whether the tested isolates comprise cryptic species or not.

Genomic DNA prepared from fruitbdies of 25 Grifola frondosa strains were amplified with the ITS primers and the PCR reaction products were enzymed. The ITS bands have same electrophoresis patterns but part of them have different restriction enzyme cutting site. These strains were divided into three broad categories. SRAP amplification employing 47 SRAP primer pairs were carried on and 138 polymorphic bands were detected. The phylogram tree showed that: ten strains were differentiated from the others effectively and fifteen strains have no obvious differences between each other. The results implied that the ITS-RFLP and SRAP markers were effective methods for strains identifica tion and germplasm evaluation but other markers were also needed to be used in conjunction.

Lipoxygenase (LOX) is considered to be a key enzyme in the biosynthetic pathways of the most important mushroom aroma, 1-octen-3-ol. In previous work, we purified and characterized a LOX from Pleurotus ostreatus (probably H1 strain) fruit bodies [1] and also determined its partial amino acid sequence. In this study, to clarify the biosynthetic mechanism of 1-octen-3-ol, we isolated cDNA and genomic DNA corresponding to a LOX (Polox1) gene of P. ostreatus H1, and analyzed the expression of the gene in the fruit bodies. A commercial P. ostreatus H1 strain (Onuki kinjin, Utsunomiya, Japan) was used in this study. To isolate the Polox1 cDNA, RT-PCR was done using degenerate primers designed from the partial amino acid sequence. This approach generated a single DNA band of approximately 1.1 kbp, which was cloned and sequenced. The deduced amino acid sequence showed high similarity to LOXs of some ascomycetes fungi. To obtain the full-length cDNA of Polox1, clones corresponding to the Polox1 gene were isolated by plaque hybridization from a cDNA library of the P. ostreatus H1 fruit body. DNA sequences of all clones were determined. The 5’ end of the Polox1 cDNA was amplified by the 5’ RACE method and cloned. The full-length cDNA of Polox1 is 2,031 bp long and contains 640 amino acid residues. The deduced amino acid sequence contains LOX iron-binding catalytic domain signature sequences. Next, to determine the genomic DNA sequence of the Polox1 gene, inverse PCR and PCR was done with P. ostreatus H1 genomic DNA. After inverse PCR and PCR, 3.3 and 1.9 kbp DNA fragments, respectively, were amplified and sequenced. Sequence comparison between cDNA and genomic DNA showed that Polox1 gene contained one intron. To investigate expression of the Polox1 gene, northern blot analysis and measurement of LOX activity were performed. P. ostreatus fruit bodies were produced in a sawdust medium containing beech sawdust and rice bran and separated into pileus and stipe. Two transcripts were detected by northern blot analysis in both pileus and stipe. The band intensities were relatively higher in the stipe than in the pileus. The level of LOX activity in the stipe was 3.8 times higher than that in the pileus. By Southern blot analysis, several major bands were detected after the digestion of 4 restriction enzymes. These blot analyses suggest that the Polox1 gene is probably a member of a small gene family. [1] T. Kuribayashi et al., J. Agric. Food Chem., 50, 1247 (2002).

Four Ganoderma lucidum strains, Chizhi 05, Jingda, Huizhou and Xinzhou, were screened out as hybrid parent in order to establish G. lucidum cross breeding system that based on protoplast monokaryogenesis method. Monokaryotic strains of each parental strains were obtained and mating type of each monokaryotic strains were determined. One to three monokaryotic strains that have different mating types were mated, and hybrids were identified by clamp connection observation and antagonist response. The results showed that the number of monokaryon came from Chizhi 05, Jingda, Huizhou and Xinzhou was 9, 14, 40 and 38, respectively. Only one mating type was obtained from Jingda, and two mating types were obtained from the other three strains, Chizhi 05, Huizhou and Xinzhou, respectively. Chi-square test showed that the ratio of two mating types of the three strains was 1:1. Fourteen monokaryotic strains of different mating types from 4 parental strains were select as a cross- breeding materia, and 17 hybrids were obtained, which were identified by clamp connection observation and antagonist response. This study proclaimed that the practicality of the hybridization breeding of G. lucidum by protoplast monokaryogenesis method.

Lentinula edodes is an important cultivated mushroom in China. The development of Lentinula edodes production promotes more studies on it. In our previous work, degenerate PCR and chromosome walking technologies were used to obtain one pheromone receptor gene and one pheromone precursor gene from Lentinula edodes. In this study, four pairs of specific primers were designed according to the whole genome sequencing of the protoplast monokaryon of Lentinula edodes strain 135, to amplify STE3-like pheromone receptor gene and its flanking conserved genes in the protoplast monokaryon strain SUP2 derived from Lentinula edodes strain Suxiang and 33655bp DNA sequence was obtained. By BlastX search, seven putative genes were identified, and three of them are pheromone receptor encoded genes. Furthermore, near to two pheromone receptor genes, four genes encoding proteins with conserved motifs of pheromone precursors were found. This study firstly reveals the molecular organization of the B mating type locus of Lentinula edodes.

The mating-type genes control formation of the dikaryon from two haploid strains. These genes are now used in mating-type-assisted breeding programs for economically important mushrooms, especially the oyster mushroom, Pleurotus ostreatus, aiming at high-yield and high-quality standard mushroom production. However, it improves the breeding program when the breeder is able to quickly identify compatible strains in a given set of progeny. The two mating factors with their mating-type loci are used as markers for breeding and have been incorporated in a chromosome mapping investigation. The linkage maps include not only genetic markers such as the mating types that can be cored, but also molecular markers such as PCR-assisted approaches, e.g. RAPD analyses, or RFLP markers. Once mating-type genes within progeny may be more easily identified by the use of PCR-directed cloning of partial mating-type genes. We analyzed homeodomain (HD1 and HD2) and pheromone receptor(rcb1, 2 and 3) genes as molecular markers for breeding using mating type A and B of Pleurotus eryngii and Pleurotus ferulae by direct PCR.

Microsatellite loci are increasingly used as markers in the human, animal and plant genomes. Being highly mutable, microsatellite regions are able to differentiate between related taxa, even at the level of individual isolates in a single species. Studies on mushroom population structure, gene flow and dispersal between natural and cultivated species have become central in breeding programmes and the knowledge of new polymorphic, codominant markers will be a promising avenue to exploit wild genetic resources. The molecular phylogeny in 50 different commercial cultivated strains of Pleurotus eryngii using PCR amplification with URP primers and mitochondrial microsatellite primer was studed. The sizes of the polymorphic fragments obtained were in the range of 200 to 2000 bp. RAPD analysis techniques were able to detect genetic variation among the tested strains. With these isolated PCR amplification with URP primers we intend to analyse the population structure of the P. eryngii species complex and investigate the structure of the basidiomycete genome which deserves. A few single-locus microsatellite markers have been isolated in Pleurotus eryngii and Pleurotus ferulae. This technique is useful in those species where microsatellite loci are rare in the mitochondria.

Hypsizygus marmoreus, belongs to Tricholomataceae of Agaricales, is widely cultured as the Beech mushroom in Japan. “Haemi” is the first variety developed by intra-specific crossing in Korea. It was improved with hybridization between monokaryotic strain derived from MKACC51987-8 and MKACC51988- 12. The optimum temperature of mycelial growth and fruiting body development were 23-25℃ and 11~17℃, respectively. The color of fruitingbody was grayish brown and cap type was umbrella. It suggest that 'Haemi' is new commercial variety for consumer who concern over healthy and tasty food

[Introduction] Matsutake mushroom (Tricholoma matsutake), an ectomycorrhizal fungus has low activity for polysaccharide hydrolyzing enzymes such as amylase, cellulase and hemicellulase. Therefore, only glucose and a few other monoand di-saccharides can be used to grow this fungus. Then, we examined the possibility for the mass production of the mycelia using the saccharified rice bran as growth substrate. [Methods and Results] For the selection of Kouji-mold, 32 strains of Aspergillus spp. were tested. These fungi were cultivated in potato dextrose liquid (PDL) medium added rice bran and amylase activities were assayed. As a result, Kouji-mold from Isesou Mugi Kouji (Asp. oryzae) was selected to use for saccharification of rice bran. When the saccharified rice bran was used as the growth substrate, the mycelical growth was enhanced. In this mushroom, the dry weight mycelia increased at about 3.0 times (control : 50mg, saccharified rice bran : 160mg, 60days, 24℃) by using saccharified rice bran compared with that of the control without saccharified rice bran. From there results, we see that saccharified rice bran is a useful culture material for the mass production of matsutake mycelia. Chemical analysis in saccharified rice bran solution was done with TLC and HPLC. The large amounts of glucose and maltose were detected in the saccharified solution. Now, we are trying to hydrolyze of cellulose and hemicelluloses components in rice bran using Asp. kawachii or Asp. saitoi isolated from Shouchu kouji.

[Introduction] Larix kaempferi (Larch), rich biomass in Hokkaido, is available as an inexpensive medium for mushroom production. We have previously developed a variety of Hypsizygus marmoreus with high ability to utilize larch sawdust without watering, Hm 219 (Marbure 219) with high fruit-body yield and high score of sensory evaluation, was selected. In this study, we investigated the effects of supplements to larch sawdust-based medium on taste components of fruit-bodies of Hm 219. [Materials and methods] The strain of H. marmoreus used in this study was Hm 219, the stock culture of Hokkaido Research Organization Forest Products Research Institute. Each 850 ml plastic bottle containing 540 g of larch sawdust-based substrate, was used for cultivation. Moisture content of each medium was adjusted to 61% based on the fresh weight of the mixture of solid materials. The substrate substituted with 0-40% of supplements (wheat bran, soybean curd residue and soybean shell) for rice bran as a nutrient, were used for cultivation. Cultivation was conducted by the standard procedure reported earlier (Harada et al., 2004). The harvested fruit-bodies were freeze-dried to determine the chemical composition. Soluble sugars, free amino acids and 5’-nucleotides were extracted with hot water from freeze-dried powder. These soluble components were determined by using HPLC. [Results] As a result, fruit-body yields on the substrate substituted with 20% of soybean shell for rice bran as a nutrient, were about 20% higher than those on the substrate with rice bran only. According to the replacement rate of soybean curd residue increasing , morphological quality of fruit-body tended to decline. As major free amino acids in fruit-body, monosodium glutamate-like (MSG-like) components which gave the umami taste, including aspartic acid and glutamic acid, and sweet components including alanine, threonine and serine were detected. As flavor 5’-nucleotides, GMP and IMP were found. In fruit-body of H. marmoreus, with respect to major soluble sugars, mannitol and trehalose were mainly contained. Each taste component content indicated differences among the different supplements to larch sawdust-based medium. The equivalent umami concentration (EUC ) is the concentration of MSG equivalent to the umami intensity of that given by the mixture of MSG and the 5’-nucleotide (Lee, Yu-Ling et al., 2009). The EUC value in a cultivation condition was more 15% higher than that of control condition. [References] Harada, A. et al. (2004) Effects of strain and cultivation medium on the chemical composition of the taste components in fruit-body of Hypsizygus marmoreus. Food Chemistry, 84, 265-270. Lee, Yu-Ling et al. (2009) Composition and non-volatile components of Hypsizygus marmoreus, LWT- Food Science and Technology, 42, 594-598.

Essential oil from Chamaecyparis obtusa leaves and water extract from Pseudotsuga menziesii sawdust were examined for antifungal activity against Trichoderma spp. (Trichoderma aggressivum, Trichoderma atroviride, Trichoderm harzianum. Trichoderma koningii, Trichoder-ma viride). Generally, essential oil and water extract inhibited the growth of Trichoderma spp. by about 25% and 33%, respectively, at 1000 ppm. Antifungal activity of water extract was slightly higher than essential oil at 1000 ppm. Antifungal activity was increased with increasing concentration of essential oil and water extract. This study was carried out the investigation of the chemical composition of essential oil from Chamaecyparis obtusa leaves and water extract from Pseudotsuga menziesii sawdust. The essential oil was analyzed by GC-MS and water extract analyzed by GC-MS/HS. Thirty-eight components were identified, of which terpinyl acetate (13.41%), elemol (11.86%) and isobornyl acetate (7.89%) were the main compounds in essential oil. Twenty-four components were identified, of which 2-Isopropoxy-ethylamine (46.45%), Epifluorohydrin (8.62%) and Trans-2,3-Dimethyloxirane (7.78%) were the main compounds in water extract. Based on all results described above, we conclude that the essential oil and water extact may have a potent in vitro antifungal activity against Trichoderma spp.

This experiment was carried out to clarify the effect of light qualities on the growth characteristics and yield of fruiting body in the cultivation of Lyopyllum ulmarium. The intensity of illumination by type of light was in the order of white light(2,270Lux), yellow light(1,750Lux), blue light(460Lux) and red light(400Lux). An investigation of fruiting body showed these results that the pileus size and stipe diameter of fruiting body on CBM (Chungbuk mushroom)-1757 were much larger than Hypsizigus marmoreus, and an effect of yellow light seemed to be better than those of another light. In comparison with Hypsizigus marmoreus, the growth duration of CBM-1757 was shortened by 8 days which included 2 days for fungi culture, 1 day for first pinning requirement and 1 day for growth. The growth duration in yellow light illumination was about 70 days showing the tendency of 2~4 days reduction. There were no differences in results such as number of effective stem and fresh weight. The yield of fruiting body per bottle in CBM-1757(95.6g) was little higher than Hypsizigus marmoreus(94.8g). By a white light’s standard, the yields of blue and red light illumination were decreased by 2~9%, but that of yellow light illumination was increased by 8%. The chromaticity results showed that brightness, red and yellow coloration of CBM-1757 were higher than those of Hypsizigus marmoreus, and yellow light treatment was more effective than another light.

The Trichoderma disease, commonly referred to as green mold, has been previously considered as a problem in mushroom production, because it typically occurred in association with low-quality compost or poor hygiene. We studied growth of Trichoderma spp. (Trichoderma aggressivum, Trichoderma atroviride, Trichoderma harzianum, Trichoderma koningii, Trichoderma viride) is under the control of environment factors ; 15℃ - 35 ℃ of incubation temperature, pH 3 - pH 11 of medium and 55% - 75% moisture content of rice straw medium. At the temperature of incubation, Trichoderma spp. had the highest growth rate at 25 ℃ and had no growth at 35 ℃. At the pH of medium, Trichoderma spp. had the highest growth rate at pH 5 and had the lowest growth rate at pH 11. In the Moisture content of rice straw medium, Trichoderma spp. had the highest growth at 75% moisture content and had the lowest growth rate at 55% moisture content.

A harmful fungus occurred seriously in bed-log of shiitake(Lentinula edodes) in Jangheung-Gun, Korea. The fungus was identified as Bjerkandera adusta by its morphology and ITS(Internal Transcribed Spacer) analysis. The fungus was reported as causal agent of stem-rot of Populus euramericana in Korea, but not reported in bed-log of shiitake until this notification. Thus, studies were made to investigate inside condition of bed-log of shiitake damaged by B. adusta, physiological characteristics of B. adusta and antagonism between these two fungi. First of all, B. adusta is white-rotting fungus like shiitake and wood-rotting condition is similar to that of shiitake. But, there are a lot of small spots in damaged wood tissue under bark which are not seen in case of shiitake. Optimal temperature for mycelial growth of B. adusta is ca. 30℃ while that of shiitake is ca. 25 ℃. When confrontation cultures were made between these two fungi under 15℃, 20℃, 25℃ and 30℃, B. adusta has antagonistic ability against shiitake in all the temperatures. From the results of experiments, if the bed-logs of shiitake are exposed to high temperature, there should be mass propagation of B. adusta, and shiitake mycelia will be seriously injured by the fungus. Therefore, to prevent the damage by B. adusta, it is needed to grow the mycelia of shiitake fast in the bed-log, and to avoid exposure of the bed-log to high temperature in summer.

In order to develop of practical model of the sawdust cultivation of Lentinula edodes, we conducted experiments of the sawdust cultivation in different conditions of cultivation environment and shapes of sawdust medium. For different types of cultivation environments, we provided environmental control system facility, vinyl house and forest cultivation. We also chose the 2.6kg cylindrical type and the 1.5kg cylindrical type for shapes of medium. We decided cultivation on shelves for the 2.6kg cylindrical type sawdust medium and cultivation on the ground for the 1.5kg cylindrical type sawdust medium to cultivate the mushroom. The result showed that forest cultivation was the first place cultivated mushroom followed by environmental control system facility and cultivation vinyl house. Among 2 types of sawdust medium, the 1.5kg cylindrical type showed better quantity in terms of a fruit body of mushroom.

The microscopic characteristics of brown layers in oak sawdust cultivation bed of Lentinula edodes was investigated with scanning electron microscope. Parenchymatous cells became thinner and the middle layers of the cells degraded in the oak sawdust bed at a plastic cultivation shed in four months. L. edodes hyphae penetrated into the pit and profusely branched within the bassal tube cells. Pit pores of ray parenchymatous cell in the decomposed sawdust became larger than in the uncolonized cells and were circular or irregular. The brown layer producing fruit body was 0.34㎜ thick and 0.67㎏/㎠ in hardness with especially 0.20㎜ thick layer of highly dense hypahe. On the other hand the brown layer without fruit body was 1.17㎜ thick, 0.94㎏/㎠ in hardness, and 0.80㎜ thick with smooth surface of highly compact dense hypae. This nonfruiting brown surface was heavily colonized with contaminating fungal hyphae and bacterial cells. We conclude that physical and biological characteristics of brown layer can affect fruiting of L. edodes.

The effect of sterilization and incubation temperatures of fermented oak sawdust on Lentinula edodes hyphal growth was investigated. The pile of 33 tonnes of oak sawdust fermented in a plastic shed for 24 days. During the fermentation the acidity of the sawdust within the pile was lowered to pH 4.2 and temperatures increased to 58℃ in over 20cm depth. The sawdust samples collected at 20, 60, 80 and 100㎝ depth each and moistened to 65% water content were sterilized at 65, 100 and 121℃ each for an hour. The sterilized sawdust contained in 50㎖ test tubes was inoculated with L. edodes hyphae cultured on potato dextrose agar medium and then was incubated at 15, 20, 25℃ for four weeks. L. edodes hyphae grew faster, 94.6mm, on the sawdust collected from 60cm depth than other depths, and did at 25 incubation temperature after sterilization at 121℃. The hypahe grew only 9.9cm on the sawdust from 100cm depth. When the sawdust from 60cm depth was sterilized at 100℃, the hyphae grew best by 22.1cm at 15℃. However, on the sawdust sterilized at 65℃ the hyphae did not grow at all. Thus, we conclude that sawdust fermentation under 60㎝ depth and autoclaving it can improve L. edodes hyphal growth, but the sterilization of sawdust at 65℃ is not sufficient for the hyphal growth.

The price of mushrooms harvested by bottle cultivation is rapidly dropping and the income of farm households is also rapidly decreasing due to the increase of production cost. Although some of these mushroom farms have employed the systems of mass production, they are in financial difficulty because of the investment they need to do for facilities such as cultivation room and automated systems. In the other hands, some retailers want to buy a small volume of mushrooms of many different mushrooms produced by the small farms and these small farms was required to investigate the common cultural condition for mushroom production of many different mushrooms. In this study, we investigated the common cultural conditions for production of many different mushrooms (e.g., Pleurotus eryngii and Agrocybe cylindracea) at the bottle cultivation farms. The cultural period of Pleurotus eryngii and Agrocybe cylindracea was 30~35 days. The optimum temperature of the mycelial growth was 25~28℃ with the growth room being maintained about 20~23℃ with the consideration of the respiration heat of the mycelium. The temperature of mushroom growth room was 16~18℃ for all growth periods. In our results, Pleurotus eryngii and Agrocybe cylindracea produced the highest yields at the substrate formulation of sawdust 75%, rice brain 20%, soybean cake wastes 5%, water contents 70% and 850 ml P.P. In the long run, our results will result in the development of new automatic cultivation model and increase the income of small production farms.

This study was carried out to investigate characteristic pattern of fruiting body of Ganoderma lucidum and their antioxidant activity. Mycelia of all strains were firstly inoculated into potato dextrose agar(PDA) and then transfered to a media of saw dust which contained 20% rice bran. These mycelia of saw dust were then inoculated into oak tree in polyethylene bags which has been sterilized for 8h at 120℃. The polyethylene bags were sent to a growth room for growth of fruit bodies. Antioxidant activities of each fruiting body were investigated by DPPH method.

The effects of improved aeration in saw dust bag cultivation on the Lentinula edodes hyphal growth were measured by CO2 concentration within the bags, the hyphal growth and bag weight loss. The aeration of the bags was controlled with 10 kinds of pore sizes of the lid and with pores on one side only or both side on the lid. In the conventional bags with a cotton plug CO2 concentration was 3.83%, the hyphal growth 2.25 ㎜/day and bag weight loss 0.84g, while in the bags with a lid 20.4㎜ diameter pore the CO2 concentration was 4.00%, the hyphal growth 2.68㎜/day and bag weight loss 1.3g. The bag with a lid pored on both top and bottom side was lower in CO2 concentration by 0.50%, higher hyphal growth rate by 0.04 ㎜/day and lower weight loss by 0.26g than the bag with a lid pored bottom side only. We concluded that the most effective lid for bag cultivation of L. edodes was the one with 20.4㎜ pores on both side of the lid.