왕송이버섯의 안전 인공 생산기술 확립
Tricholoma gigantium, an edible mushroom, belongs to Tricholomataceae of Tricholoma and is distributed at Jeju-Do in Korea. It is also well-known as the medicinal mushroom in Taiwan. The cultivation method using the compost was developed in Korea in 1995. To develop a mass cultivation method and a superior strain, four strains were collected and tested. To find simple preservation method, the mycelium of f giganteum (MKACC 50852) and Pleurotus ostreatus (Chunchu No. 2) were stored on six different media in two kinds of storage vessels (tube and vial) for a period of 1-12 months at 4℃, 15℃, and 25℃ storage temperatures. At 4℃ storage condition, mycelia were not regenerated in all agar media, but in the sawdust medium after 3 months. At 15℃ storage, mycelial activity was maintained up to six months. On the other hand, P. ostreatus produced well-regenerated mycelia both at 4℃ and 15℃ storage up to 12 months. In conclusion, it is estimated that the mycelial storage condition of T. giganteum must be done at 15℃ and subcultured within six months. To establish the optimal cultivation conditions, various examinations were accomplished. Bag cultivation was more effective than box cultivation and the optimal relative humidity was more than 80%, Although the mycelial growth was tested in the substrate supplemented with different additives, such as rice bran and wheat bran, there's no significant difference between them. It suggested that the environmental conditions were more important than the substrate additives for cultivation. To develop the superior strains, monospores of MKACC 50852 and MKACC S0853 were isolated and crossed. All of the dikaryotic strains derived from the reciprocal crossing between them were cultivated to examine their productivities and morphological characteristics. The results showed that W-149, W-206 and W-305 were the best superior strains.
느타리버섯 수집계통의 유연관계 분석 및 재배특성 구명
1) 수집계통 특선검정 및 유연 관계 구명
Chapter 1. Identification of cultural characteristics and genetic variability by RAPD analysis on collected strains of oyster mushroom.
We used to research cultural characteristics and genetical variability by RAPD(random Amplified Polymorphic DNA) analysis on strains of oyster mushroom collected from the inside and outside of the country. For cultural characterization, fifty collected strains were cultivated in a bottle. As a result of cultural characterization, such as pinheading induction time, size, shape and color of stipe and pileus, the number of fruit body, yield and so on, thirty-two strains showed similar type to Chunchu 2ho variety and had a soft tissue and high yield. RAPD analysis of fifty collected strains of oyster mushroom using the URP, OPA and OPC primers demonstrated that they were divided into three classes: Chunchu strains having a pileus of deep funnel type and tough tissue, Wonhyung strains having a pileus of half-hemisphere type and breakable tissue and the other strains unlike usual form such as yellow and pink oyster mushroom. Consequently, Seven superior strains were selected and promoted to varieties through establishing a method of cultivation.
Chapter 2. Cultural characteristics of collected oyster mushrooms in bottle and bag cultivation using four kinds of mixed media.
Cultural characteristics of the strains and effects of environmental conditions on development of fruiting body were investigated. Oyster mushrooms which collected indoor and abroad were cultured on media composed of four kinds of mixed media : 1) Sawdust + Beet pulp + Cottonseed hulls (SBC, 50:30:20, v/v), 2) Sawdust + Beet pulp + Rice bran + Cottonseed hulls (SBRC, 50:40:8:2, v/v), 3) Corn cob + Beet pulp + Cottonseed hulls (CBC, 50:30:20, v/v), 4) Saw dust + Cotton husk + cottonseed hulls (SCC, 50:30:20, v/v).
SBRC media was good as culture medium even though there were some variations among species. In the SBRC media, pH , total nitrogen and C/H ratio were 5.4, 1.27% and 42:1, respectively. Compared to other media, incubation period and the primordia formation period were reduced about 2days and 3days, and the yield of fruit body was the highest.
Chapter 3. Cultural characteristics of collected oyster mushrooms in bottle and bag culture on temperatures.
These studies were carried out to investigate optimal growth temperature of strains in SBRC media chosen in the previous chapter. Five strains (KME20047, 20094, 20128, 20153, 20160) of Pleurotus ostreatus grew well at 15℃ and KME20063 (P. salmoneostramineus) and KME20064 (P. citrinopileatus), high temperature adaptable mushrooms, were good at 20'C . In general, development of fruit body was delayed and color of pilei became dark at the temperature lower than 15℃. But over 15℃, they showed the tendency of opposition. The optimal temperature for yield of Pleurotus ostreatus was ranged from 15 to 18℃ in bottle and bag cultivation. Temperature for high temperature adaptable mushrooms (KME20063, 20064) was 20~23℃.
Chapter 4. Functional properties of edible mushrooms
The object of this study was to investigate the physiological functionality of water extracts from six cultivated mushroom. We determined their antioxidant activities, total phenolic contents, ACE inhibiting activities, fibrinolytic activities. Total phenolic contents of P. citrinopileatus and Grifola frondosa were relatively higher than the others. Grifola frondosa also showed the strongest antioxidant ability and fibrinolytic activity, whereas P. citrinopileatus had the highest ACE inhibiting activity. This study suggested that both P. citrinopileatus and Grifola frondosa could be used as candidates for the development of new biofunctional food.
Chapter 5. Development of a new strain using collected oyster mushooms.
This studies were carried out to develop new strains using collected oyster mushooms. Several superior strains were selected as parents to further improve their superiority. The 80 cross combinations with monokaryons isolated from the parents produced 156 individuals. Significant variations were observed in growth characteristics, morphology, and yield of fruit body Based on color of pileus, shape of stripe and yield of fruit body, four superior inaviduals were screened.
'KME35242(C05137)', a new variety of oyster mushroom, fitting for the bag cultivation was bred by mating between monokaryons isolated from KME20094 and Chunchu 2ho. In the major characteristics of this mushroom, the pilei were thick and gray and the stripes were thick and long with hardness. The optimum temperature for the primordia and growth of fruit body was around 15~16℃. Incubation period was around 25 days for the bag cultivation. The yield was shown about 122.4g/850cc bottle and 151.7g/1kg bag.
'KME35243(C06107)', a new variety of oyster mushroom, fitting for bottle cultivation was bred by mating between monokaryons isolated from C05086 and Heuphyung. In the characteristics of fruit body, the pilei were infundibuliform and grey and the stripes were slender and long with elasticity. The optimum temperature for the primordia and growth of fruit body was around 15~16℃. Incubation period was required around 18 days for the bag cultivation. The yield was shown about 138.6g/8S0cc bottle and 121.6g/1kg bag.
'Sunjung', a new variety of golden oyster mushroom, was selected by mating between monokaryons isolated from KME20064 and KME20066. The optimum temperature for the mycelial growth was about 26~ 28"C and that for the primordia and growth of fruit body was about 18~23'C . Compared to the control which took 25 days of incubation period, Sunjung required 21 ~22days. Its Primordia were formed after a lump of mycelia was developed. In the characteristics of fruit body, pileus was infundibuliform and yellow and the stripes was slender, long and white. The yield was 112.9g/850cc bottle and 275.5g/2kg P.P bag.
'Jeakdan' was selected by mating between monokaryons isolated from KME20063 and KME20193. The major characteristics of the mushroom showed a lot of pinheadings, the pink and infundibuliform pileus. The optimum temperature for the mycelial growth was around 26~30℃ and that for the primordia and growth of fruit body was around 18~23℃ . Primordia and young mushrooms were bright pink but became pale while the mushroom were mature. Incubation period was about 20 days in bottle cultivation and 21 days in bag cultivation. The number of valid stipes was 31.5/850cc bottle and 47.0/2kg bag. The yield was 134.5g/850cc bottle and 350.0g/2kg bag.
2-2. 느타리 유통품종의 재배특성 검정 및 품종판별법 개발
The genus Pleurotus are widely distributed throughout the world, the worldwide production and consumption have increased and over 40% of the total amount of the mushroom production in Korea and cultivated the most. As this study analyzed molecular phylogenetic relationships of the genus Pleurotus. It was performed with 81 strains of 8 species in the genus Pleurotus, and included studies on morphogenetic characteristics and the molecular genetic methods. Physiological characteristics examination of the genus Pleurotus showed their optimal medium was MEA for incubation and their optimal temperature was 30℃. Generally P. ostreatus strains showed good mycelial growth in range of temperature from 25℃ to 30℃ in PDA media, while P. nebrodensis and P. cystidiosus strains did not. Analysis of phylogenetic relationships of the genus Pleurotus was performed by the URP-PCR, AFLP, and rDNA sequencing analysis, This analysis indicated that there must be the same strains, even having different names, in current commercial strains. To develop the discrimination methods on commercial varieties, several specific bands related to Weonhyeong, Suhan and Chunchu-2 were cut out from the gel of RAPD products. They were cloned to pGEM vector and sequenced. Based on those sequence, new potential primers were designed and applied to all current commercial strains. Most of them, however, did not show the expected results except S-OPO5 primer set. This new SCAR marker produced a band of 1,500bp, accompanied by 2180, 2183, 2240, 2595, 2594, 2725, 2072, and 2016 which used to produce the greyish brown and hemispherical fruiting bodies typical to Weonhyeong. According to the results of ITS sequencing in rDNA region, 40 Pleurotus strains were identified as 8 species and divided into 6 clades (clade I ~cladeVI). Each clade was composed of representative species and its similar species. P. ostreatus and P. fiorida belonged to clade 1, P. nebrodensis and P. eryngii to clade Ⅱ, P. saior-caiu to clade Ⅲ, P. incarnatus to clade IV, P. cornucopiae to clade V, P. cystidiosus to clade Vl. These results were identical to RFLP analysis of ITS region. Analysis of ITS region sequencing and RFLP polymorphism were very useful in taxonomy.
잎새버섯 인공재배법 개발 연구
신령버섯 우량균주 수집 선발 및 생산성 향상 연구
This study was conducted to collect and select superior strains and improve productivity in Agaricus brasiliensis Murill which is known as an edible and medicinal mushroom. The results are as follows.
Three strains, ASI 1181, ASI 1202, ASI 1203 out of collected 9 ones were good in mycelial growth with 75~80mm/15days. Their mycelial growth was compact. The optimum media for mycelial growth and preservation were PDA and MEA. For the growth of spawn, wheat grain medium was better than the others.
As a result of medium selection for the Agaricus brasiliensis shelf cultivation system, mycelia grew well at all different media but sugar cane bagasse was the best for the shelf system. The period for the mycelial growth was 13 days in the sugar cane bagasse substrate and 16 days in the rice straw one. Ddays from spawn inoculation to development of fruit bodies were 34~36 days.
Although product quality was low, yields were high of 34.5, 33.0, 26.3 kg/3.3㎡ at the media of 'rice straw + cotton waste', 'sugar cane bagasse +cottonseed hulls', 'sugar cane bagasse + cottonseed hulls' respectively. In the media of rice straw, sugar cane bagasse, rice straw + sugar cane bagasse, yields were 33.2, 30.3, 23.8 kg/3.3㎡ respectively. Especially, the yield of sugar cane bagasse was a little bit low but product quality was better. To mechanize composting, it was expected to be substituted for rice straw.
In the selection of casing material, the casing with only peat moss for button mushroom was not proper for Agaricus brasiliensis in terms of product's quantity and quality. However the casing with clay loam and peat moss 50% + clay loam 50% showed good results .
만가닥버섯의 품종선발 및 재배기술 개발
The polymerase chain reaction using ITSI, ITS4 primers against genomic DNA of Lyophyllum ulmarium 44 strains produced a band of about 700bp. Its size was identical in all strains. When the PCR products of all strains were digested with Sau3A I, the DNA pattern of two strains (lane 3 and 10) was different from the other strains. This result indicated that these strains were genetically distinguished from the other strains. When digested with Xsp I restriction enzyme, the DNA pattern was divided into four parts.
The content of total polyphenolic compound in Hypsizigus marmoreus was 506.71 mg per 100 g and total antioxidant activity was 609.2 AEAC (Ascorbic acid Equivalent Antioxidant Capacity per 100 g mushroom). EDA (Electron donations ability) of extracted material from Hypsizigus marmoreus was 34.6% and the metal ion removing activity of extracts using hydrothermal method was 61.6%. Effect of tyrosinase inhibition was 17% and that of angiotensin converting enzyme inhibition was 84% in Hypsizigus marmoreus.
Forty-four Lyopyllum ulmarium strains received from NIAST (National Institute of Agricultural Science and Technology) were investigated to select good strains, and to use breeding source. Eleven strains (MKACC-51989, 51990, 51998, 51999, 52001, 52004, 52006, 52007, 52009, 52010, 52012) were selected first based on mycelial growth on PDA media. The best condions for mycelial growth of Lyopyllum ulmarium were in MCM as a culture medium, at 25'c (temperature), and at 7.0(pH). The yield of MKACC-52007 ,the best of them, was 136g per 850m1 bottle. The optimum conditions for fruit body production were in sawdust of Douglas pine+beat pulp+rice bran (60:20:20) as a substrate, in water content 65±2%, and by post-incubation condition of 20 days at 5℃.
For breeding of Lyopyllum ulmarium, 808 dikaryons were obtained by 15 mixed matings First of all 120 out of 808 dikaryons were grown in MCM medium for 12 days. For 1757 strain, mycelial incubation period was 34.7day and yield was 107.1g per 850m1 PP bottle in sawdust of Doglus pine and rice bran(8:2) combination.
노루궁뎅이버섯 재배법 개발
These experiments were conducted to develop the cultural practice using diverse cultivation type using bottle, plastic bag and short cut log and the variety by good strain selection of Hericium erinaceus from 2003 to 2007. The number of tested H. erinaceus strains was 18, composed of 13 strains from KACC, 4 strains from china and one strain from japan. In general, the optimum temperature of mycelia growth was 26"C and mycelia grew well at Hamada, CDA, MCM medium. The fruitbody type of 18 strains could be classfied into 4 types such as long spin (MKACC 51863, 51869, 51872, 51875, Chinese 14, Japanese 18), short spin (MKACC 51864, 51866, 51867, 51868, Chinese 15, 16), sponge (MKACC 51865, 51874) and coral type (MKACC S1870, S1871, 51873, Chinese 17). The strains showing a good fruitbody quality and a high yield were MKACC 51863, 51867, 51869, 51870, S1875, and Chinese 14. Among them, MKACC 51875 was selected as the best strain and given a name of "NORU 1HO". Its optimum temperature for mycelia growth was 23'c at Hamada medium, fruitbody had long spin of 4.2mm, incubation period was 15 days, and average yield was 126g/1,100cc in bottle cultivation. The optimum media composition for H. erinaceus culture was oak sawdust 70% + rice bran 15% + wheat bran 9% + sugar cane molasses 6%, and the average yield was 150g/1,100cc in this media. "NORU2HO" was bred by the croosing between monokaryons derived from NORU 1HO. It took 30days this variety to harvest from inoculation because its mycelia grew very vital and uniform. The optimum plastic bag size for bag culture was 20 cm in diameter and in 2kg weight, and media composition was oak sawdust 80% + rice bran 20%, In this culture method, the average yield was 138.8g/2kg bag. In the log culture for H. erinaceus, the contamination rate of sawdust inoculum was lower than that of liquid inoculum. In conclusion, the most effective cultural practice for H. erinaceus was the bottle culture method.
곡물배지를 이용한 곤충기생버섯 재배기술 확립
This study was carried out to analyse the genetic relationships within the genus entomopathogenic fungi by RAPD. Also cultural conditions for artificial fruiting body production and the fibrinolytic activity and fibrin plate assay of the entomopathogenic fungi were studied.
In the analysis of relationships within the genus entomopathogenic fungi based on molecular biological data, the sizes of RAPD amplified by PCR were from 1,000bp to 10kb in all isolates. The results of UPGMA analysis showed homology of 96.5~ 100% in the KACC 50810, 51090, 52339 and JenBug I, 87.7~91.2% in KACC 51130, 50161, and 50171, 38.g~84.2% in KACC 52338, S2333 and JenBug Ⅱ
The grain media for artificial fruiting of Cordyceps militariscontained contained the brown rice and black purple rice. Mycelial growth and density of Cordyceps militaris were the highest in the medium of malt extract 0.7% and pH 5.0 ~5.5.
The optimal conditions of fruiting bodies formation were temperature of 17℃, light intensity of 600 lux, C0₂ of 500ppm and humidity intensity of 95%.
The fibrinolytic activity of the fruiting body of Cordyceps militaris grown on black sesame grain media was 0.16 plasmin unit/mg protein based on fibrin plate assay. Fibrin hydrolysis enzyme acted on a-chain after 30 min then β, γ-chain after 10 min and clearly dissolved 3 hours later on SDS-PAGE.
양송이 우량균주선발 및 안전생산 기술개발
The white button mushroom, Agaricus bisporus(Lange) Imbach, is the most important commercially cultivated mushroom species in the world. To develop a new button mushroom cultivar, several experiments were conducted. The optimal culture medium for mycelium growth was compost extraction medium. The optimal carbon source of the media was sucrose and the nitrogen source was malt extraction and peptone. It took 19-21 days for ASI 1171 and the foreign. strains such as A-15, X-21 and X-22 to reach their full growth In wheat grain substrates. Mycelial clumping was to be observed in ASI 1090 and 1171, In temperature growth test, some high temperature adaptable strain such as ASI 1171, 1151, 1152 1065 were selected and they were able to grow at 25~30℃. In casing soil, ASI 1151, 1090 and 1138 showed better mycelial growth than others and days for first pinhead formation were varied between 36 days (ASI 1151, 1152) and 50 days (ASI 1109). The optmal C/N ratio for mycelial growth of ASI 1090, 1105, 1108, 1109 and 1151 was 10, that of ASI 1088 and X-22 was 20, and that of the others was 10~30. On casing soil sterilization method, high temperature(100℃) casing soil sterilization was inferior to non sterilization method. high temperature sterilization led to the results that, days for first pinhead formation was prolonged and overall fruitbody yield was decreased compared to non sterilization treatment and fungicide applying method. There is much room for improvement in casing soil sterilization method. When spent mushroom compost by oyster mushroom was used as compost material, mycelium growth was hampered, but the overall fruitbody yield and individual fruitbody weight were not different compared to rice straw compost. Our results showed that, the weight and size of wild strains of Agaricus spp. were inferior to the commercial strains, but they had some advantages in mycelium growth, high temperature growing ability and pathogen resistance. To introduce them into breeding program, development of mating method and single spore isolation must be prerequisite. Phylogenetic relationship of genus Agaricus was studied by comparing the nuclear ribosomal intergenic spacer(IGS) I region and structural characteristics were analyzed. Sequence of IGS I region on 5 species(A. bisporus, A. edulis, A. bitorquis, A. campestris, and A. potobella) of genus Agaricus were from 1,323bp(A. bisporus ASI 1056) to 1,491bp(A. potobella ASI 1210) in length and GC contents of them were from 47%(A. bisporus) to 49%(A. potobella). Size differences in IGS I region among Agaricus species were caused by sequence insertion in 3'end region. Especially A. potobella showed higher GC content than other species did had repeated GG/GC patterns at inserted sequence. This study indicated that sequence analysis of IGS region can be a good tool in phylogenetic study of basidiomycetes.
큰느타리버섯 생력재배기술개발
Pleurotus erygii is one of the most popular edible mushrooms and belongs to the genus Pleurotus and the family Pleurotaceae. Commercial production of this species began in Italy in the mid 1970s and is now made in over a dozen other countries. In Korea, since commercial production began in the late 1990s, production increased rapidly Total production increased from 6,842 t in 2002 to over 43,230 t in 2005. The increasing popularity of Pleurotus erygii among consumers is due to its unique favor, texture and shelf life. However, most of them were cultivated using polypropylene bottle. In this study, we investigated suitable materials, its mixing ratio, inoculation method, pot diameter and shape, substrate weight, technique to induce the primordia of mushroom, carbon dioxide concentration, and cultivation method having high quality in pot cultivation of P. eryngii. The proper substrate for P. eryngii cultivation was douglas fir sawdust and poplar sawdust with rice bran, wheat bran, and dry bean curd at the ratio of 60 : 10 : 10 : 10 : 10 (%, v/v), respectively. Suitable pot diameter and substrate weight in pot cultivation were estimated about 16cm and 1,000 - 1,200 g/pot, respectively. When the substrate was fully grown by the mycelia, the bags were simply opened and moved to the growing room. Simultaneously, covering the newspapers on the top of bag were able to facilitate the formation of primordia in P. eryngii. An increase of carbon dioxide concentration resulted in decreasing cap sizes and increasing length of stipes. This result was quite similar to the effect of carbon dioxide concentration in Pleurotus ostreatus. The optimum carbon dioxide concentration was about 2,000 ppm in the pot cultivation of P. eryngii. After the formation of primordia in the side of bag, it was desirable to remove the top half of the bag for harvesting the mushroom with high quality.
큰갓버섯 인공재배 기술 개발
Collected wild M. procera had characteristics of fruiting body that the size of pilei was 11.1~27.8cm and the length of stipes was 16.1~9.9cm. Soil at its habitat contained a little more organic matters and effective phosphoric acids than general soil did. The experiment of mycelial biomass on media supplemented phosphoric acid and casein had little significance in that there was no difference compared to PDA, and the more supplementation resulted in the less biomass. Mycelial growth was the best at 25℃ and incubation period was the shortest of 18 days. Mycelia grew well at pH 5.0~6.0 of the media but were getting worse at below pH 4.5. Isolation of pure culture from the collected mushrooms used to be successful and had nothing to do with collecting time, region and isolated tissue of them. Biomass and density of the mycelia were the best at 25℃. The experiment of fruiting condition on this mushroom indicated that fruiting body was able to be produced in Sep. ~Oct. and spawns inoculated after changing temperature in Jul. ~Aug. were effective to make fruiting bodies.