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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.

In the past studies of Lyophlium shimeji, it was reported that the quantity of sufficient starch used as a carbon source was able to supply the factor that allows successful fruit-body formation without raising osmotic pressure in the medium. Glucoamylase are exo Glucosyl hydrolase, which catalyze the release glucose from the nonreducing ends of amylose, amylopectin, and other polysaccharides. Glucoamylase genes are found in many prokaryotic and eukaryotic microbes that use starch as a carbon source. It was believed to be important in the utilization of starch by the basidiomycetous fungus. Glucoamylase activity in the medium increased markedly during fruit-body formation. So study of the characteristic of glucoamylase in Pholiota nameko will provide the basis for P .nameko fruit body formation. In this research, in order to confirm the presence of glucoamylase gene in P. nameko genome, the genomic DNA was prepared from P. nameko NGW19-6 strain and was used as template to amplify the glucoamylases gene (PnGlu1). To prepare genomic DNA from the P. nameko NGW19-6 strain, the mycelium was grown on 10 ml of PD liquid medium (potato 200 g/l ,Glucose 20 g/l) prepared with tap water in a 100 ml Erlenmeyer flask and at 25°C for 7 days. Genomic DNA fragment encoding the glucoamylase protein (PnGlu1) were amplified by PCR with degenerate primer F15-GP2-AF/F15-GP2-BR. The primer pair was designed based on the amino acid sequences GLGEPKF and FDLWEEI, respectively, which are conserved in the glucoamylase protein of Laccaria bicolor. This produce fragments of approximately 400 bp. Next, to amplify the whole genomic clone of PnGlu1, oligonucleotide primer PnGP2F/ PnGP2R were designed based on the nucleotide sequence of DNA fragments amplified by cassette PCR method. The produced fragment has significant homology with glucoamylase of L. bicolor. To investigate the relationship between different composition of medium and glucoamylase expression, we checked the expression level of glucoamylase gene by realtime RT-PCR and measurement of glucoamylase enzyme activity.

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 the Pinaceae. Rhizopogon roseolus produced a fruiting body with a basic globose to subglobose shape. Basidiospores were encompassed in a glebal chamber in the fruiting body. However, little is known about basidiosporogenesis and nuclear behavior after karyogamy. We treated R. roseolus glebar chambers with Gimsa acid and observed their hymenium microscopically to characterize nuclear behavior and basidiosporogenesis. Our observations revealed the following five characteristics: ⅰ) meiosis and postmeiotic mitosis took place in the basidium; ⅱ) meiosis occurred in the center of the basidium; ⅲ) the sterigma appeared when the first meiotic division occurred; ⅳ) the center of the basidium constricted slightly when the second meiotic division occurred; ⅴ) after postmeiotic mitosis, asynchronous nuclear migration from the basidium to the basidiospores took place, producing eight uninucleate basidiospores. However, unusual nuclear behavior was frequently observed, indicating that regulations of the timing and the way of nucleus entering to the spores were not exact in R. roseolus.

[Object] Gamma-Amino Butyric Acid (GABA), a four-carbon non protein amino acid, is widely distributed in nature and acts as the major suppressive neurotransmitter in the mammalian central nervous system. Recently, GABA has been reported to have several physiological functions such as antihypertensive, diuretic, relaxing and antidiabetic effects. Due to these unique biological functions, GABA has been used as functional ingredients for functional foods. Glutamic Acid Decarboxylase (GAD), which catalyzes decarboxylation of L-glutamic acid to GABA, has been purified from mammals, higher plants, and some microorganisms and their properties have already been reported. On the other hand, mushrooms are commonly appreciated as healthy food due to their nutritional properties, such as low calorie, rich in fiber, vitamin and mineral. L-Glutamic acid and GABA are also commonly distributed in edible mushrooms. The fact that mushrooms accumulate GABA suggests the existence of GAD. However, the enzyme property of mushroom GAD has not been reported. In the present study, we tried to evaluate the property of Flammulina veltipes GAD, and the purification and characterization of enzyme is also investigated. [Methods and Results] Mushroom (fruit-body of Flammulina veltipes) used in this study was purchased in local market. GAD activity was determined by formation of GABA from L-glutamic acid in the presence of pyridoxal-5’- phosphate (PLP). The activity of enzyme was determined semi-quantitatively by color intensity of GABA on TLC analysis. Fruit-body was crushed and then centrifuged to separate supernatant and precipitate. To investigate the location of GAD, both supernatant and precipitate were subjected to enzyme reaction after dialysis. The enzyme activity of precipitate is stronger than that of supernatant. The formation of GABA was observed between pH 4 and 6 and the maximum color intensity of GABA was observed at pH 6. However, GAD activity was lost after dialysis for overnight against buffer of pH 6-11. These results suggest that GAD from Flammulina veltipes is stable at pH 4-5 in spite of its optimum pH for GABA production is around 6.

[Objective] The color is one of the important factor for commercial value of edible mushroom. Pleurotus cornucopiae is edible mushroom, naturally distributed in the northern part of Japan. Due to its beautiful yellowish color, P. cornucopiae is sometimes called as “Golden-Shimeji”. The unique flavor of this mushroom also help to enhacement its commercial value and the consumption of this mushroom has gradually increased. The yellow color of this mushroom is resistant against high temperature because the color is not diminished after deep-frying. These facts suggest that the structure and biosynthesis of this yellow color is very interesting. However, the report concerning about these profiles are none at all. In the present study, we tried to isolate yellow pigment from P. cornucopiae fruit body and some properties of the pigments were also investigated. [Methods and Results] The fruit body of P. cornucopiae was kindly given from Tanaka Machinery Corporation and kept in the freezer (-20°C) until use. The fruit body was homogenized with twice amount of ice cold distilled water with a mixer and subsequently filtered to be crude pigment solution. According to the result of ultra filtration by CentriPrep YM-10, molecular weight of the pigment was under 10,000. The resultant filtrate of YM-10 was concentrated by lyophilization and subsequently subjected to size exclusion chromatography (SEC) by Sephadex LH-20. Two different pigment fractions, brown and yellow, were obtained from SEC separation. According to the retention time of HPLC analysis with TSKgel G2500PWXL, the molecular weights of these two pigments are larger than size exclusion limit (Ca. 5,000). Moreover, detectable spot with ninhydrin reagent on thin layer chromatography (TLC) analysis suggest that these pigments are guessed as peptide compounds. In addition, no color changes were observed after heating for 20 min at 100℃.

When we consider that most Flammulina velutipes are foreign varieties, and the international disputes of genetic resources increase, the development and distribution of domestic varieties are very important. Flammulina velutipes, which belongs to the Basidiomycot, have abundant vitamins, and is one of popular mushrooms. 6 varieties of Flammulina velutipes were collected, the selection of desirable isolates and characterization were conducted after monosporous isolation and breeding. After isolation of 1,200 monospores from 6 varieties, the final 10 monospores from each isolate were selected, and 250 isolates were obtained in 15 breeding combinations. Through an investigation of fruiting body, 29 lines were selected at first, then 6 lines at secondary step, and the last 3 lines. The biological characteristics was that mycelia growth was 0.6~2.0cm below 15 ℃, and 2.3~3.8cm at 20~25℃ showing the best result, on the other hand, 1.1~2.4cm at 30℃. There were no differences in pH, but the mycelia density of E-2-8 line seemed to be lower. Mycelia growth and density in some kinds of media such as MCM and MEM were most favorable, but worst in Wa medium. In D-1-10 and C-3-5 of selection lines, yield of fruiting body per bottle(850cc) was 120∼125g, number of effective stem was 299, and stipe length was 93∼95mm showing the best growth.

This study investigated physiological characteristics and genetic relationship of 30 strains of Lentinula edodes, collected from the Europe, Asia, North America and preserved in the Forest Mushroom Research Institute(FMRI). In physiological characteristics, Papua New Guinea strain was excellent mycelium growth in 25℃ for 7 days on PDA media. For all strains, the optimal temperature for mycelial growth, their tunicate and color of hypha were observed. It surveyed their mycelial growth on oak sawdust media in test tube and independence with inter-strains and cultivar developed in FMRI by strain's anastomosis culture. It was carried by RAPD using operon primers as molecular genetic methods, investigated genetic relationships among strains using UPGMA in NYSYSpc(2.1) according to the presence or absence of bands. <This research was supported by Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries>

Ionizing treatments were applied at 5Gy, 10Gy, 50Gy, 100Gy and 500Gy to mushroom mycelium (Lentinula edodes) in order to assesss the effect of the gamma-ray radiation. We mutated by gamma irradiation, 12 strains were isolated. 9 strains of 12 strains were appeared antagonistic interaction on solid medium. Growth rate of mycelium in JMIR-3 and JMIR-6 strains were similar to their control. But, the growth rate of strain JMIR-1, JMIR-2, JMIR-4, JMIR-5, JMIR-7, JMIR-8, JMIR-9, JMIR-10, JMIR-11 and JMIR-12 had generally different compared to control. In the sequence of ITS region of selected strains, it was revealed that the total length ranged from 696 to 780bp. The reciprocal homologies of the ITS region sequences in 5Gy irradiation group (JMIR-5, JMIR-6, JMIR-7 and JMIR-8) and 10Gy irradiation group (JMIR-9, JMIR-10, JMIR-1 and JMIR-12) indicated 99%. The reciprocal homologies of the ITS region sequences in 500Gy irradiation group (JMIR-1, JMIR-2, JMIR-3 and JMIR-4) indicated from 95 to 99%. It seemed that mycelium growth and ITS sequence could be changed the irradiation dose of the gamma-ray radiation.

‘Gongi-2ho’a new variety of oyster mushroom, fitting for the bag culture, was bred and by mating between monokaryons isolated from GMPO35338 and Jangpug. In the major characteristics of fruit body, the pilei were thick and dark-gray and the stipes were thick and long with softness. It was great in elasticity and cohesivness of tissue as compare to Suhan-1ho. The optimum temperature for the mycelial growth was around 26~29℃ and that for the pinheading and growth of fruitbody was around 14~18℃. In the bag culture, it was required around 20 days in incubation period and 5 days in primordia formation. The fruit body was grew vital and uniform. The yield were shown by 323.3g/1kg bag. This variety has high yielding capacity, cultivation stability and the resistance to the bacterial brown blotch disease.

This study was conducted to develop superior hybrids of Pleurotus ostreatus with di-mono and mono-monoka-ryon crosses. Random Amplified Polymorphic DNA-PCR (RAPD-PCR) was used to compare its mitochondrial DNA profiles of hybrids using specific primers designed from microsatellite markers of Pleurotus salmoneo-stramineus. A total of fifty-six dikaryon-monokaryon hybrids were sampled for RAPD-PCR experiments and the results show that twenty-four hybrids were dikaryon and thirty-one hybrids were monokaryon. Interestingly, one hybrid was an intermediate form with the DNA profiles that are different from those of its parents. The DNA profiles from eighty-eight monokaryon-monokaryon hybrids were also analyzed by RAPD-PCR. The results of mitochondria DNA profiles show that seventy-one hybrids are the same to one of their parents, but seventeen hybrids show DNA profiles of both parents.

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.

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).

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.

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.

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.

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.

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

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.

[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.

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.

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.

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.

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.

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 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 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.

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.

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.