• 한국균학회소식:학술대회논문집
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• 한국균학회 2014년도 추계학술대회 및 정기총회
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• pp.32-32
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• 2014

Meju is a brick of dried fermented soybeans and is the core material for Jang such as Doenjang and Ganjang. Jang is produced by addition of salty water to Meju and is considered the essential sauces of authentic Korean cuisine. Meju is fermented by diverse microorganisms such as bacteria, fungi and yeasts. It is known that fungi play an important role in the Meju fermentation and they degrade macromolecules of the soybeans into small nutrient molecules. In previous study, 26 genera and 0 species were reported as Meju fungi. However, it is not comprehensively examined where the fungi present on the Meju are originated. In order to elucidate the origin of the fungi present on the Meju, the mycobiota of 500 samples soybean kernels, 296 rice straw pieces and air samples of Jang factories was determined in 0, 2 and 7 Jang factories respectively. Forty-one genera covering 86 species were isolated from the soybeans and 33 species were identical with the species from Meju. From sodium hypochlorite untreated soybeans, Eurotium herbariorum, Eurotium repens, Cladosporium tenuissimum, Fusarium fujikuroi, Aspergillus oryzae/flavus and Penicillium steckii were the predominant species. In case of sodium hypochlorite-treated soybeans, Eurotium herbariorum, E. repens and Cladosporium tenuissimum were the predominant species. Of the 4 genera and 86 species isolated from soybeans, 3 genera and 33 species were also found in Meju. Thirty-nine genera and 92 species were isolated from the rice straws and 40 species were identical with the species from Meju. Fusarium asiaticum, Cladosporium cladosporioides, Aspergillus tubingensis, A. oryzae, E. repens and Eurotium chevalieri were frequently isolated from the rice straw obtained from many factories. Twelve genera and 40 species of fungi that were isolated in the rice straw in this study, were also isolated from Meju. Especially, A. oryzae, C. cladosporioides, E. chevalieri, E. repens, F. asiaticum and Penicillium polonicum that are abundant species in Meju, were also isolated frequently from rice straw. C. cladosporioides, F. asiaticum and P. polonicum that are abundant in low temperature fermentation process of Meju fermentation, were frequently isolated from rice straw incubated at $5^{\circ}C$ and $25^{\circ}C$, while A. oryzae, E. repens and E. chevalieri that are abundant in high temperature fermentation process of Meju fermentation, were frequently isolated from rice straw incubated at $25^{\circ}C$ and $35^{\circ}C$. This suggests that the mycobiota of rice straw have a large influence in mycobiota of Meju. Thirty-nine genera and 92 species were isolated from the air of Jang factories and 34 species were identical with the species from Meju. In outside air of the fermentation room, Cladosporium sp. and Cladosporium cladosporioides were the dominant species, followed by Cladosporium tenuissimum, Eurotium sp., Phoma sp. Sistotrema brinkmannii, Alternaria sp., Aspergillus fumigatus, Schizophyllum commune, and Penicillium glabrum. In inside air of the fermentation room, Cladosporium sp., Aspergillus oryzae, Penicillium chrysogenum, A. nidulans, Aspergillus sp., C. cladosporioides, Eurotium sp., Penicillium sp., C. tenuissimum, A. niger, E. herbariorum, A. sydowii, and E. repens were collected with high frequency. The concentrations of the genus Aspergillus, Eurotium and Penicillium were significantly higher in inside air than outside air. From this results, the origin of fungi present on Meju was inferred. Of the dominant fungal species present on Meju, Lichtheimia ramosa, Mucor circinelloides, Mucor racemosus, and Scopulariopsis brevicaulis are thought to be originated from outside air, because these species are not or are rarely isolated from rice straw and soybean; however, they were detected outside air of fermentation room and are species commonly found in indoor environments. However, A. oryzae, P. polonicum, E. repens, P. solitum, and E. chevalieri, which are frequently found on Meju, are common in rice straw and could be transferred from rice straw to Meju. The fungi grow and produce abundant spores during Meju fermentation, and after the spores accumulate in the air of fermentation room, they could influence mycobiota of Meju fermentation in the following year. This could explain why concentrations of the genus Aspergillus, Eurotium, and Penicillium are much higher inside than outside of the fermentation rooms.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2014년도 추계학술대회 및 정기총회
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• pp.25-25
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• 2014

Three species of Fusarium, F. fujikuroi, F. verticillioides and F. proliferatum, are known to be associated with bakanae disease of rice [1, 2]. F. fujikuroi infects rice flowers and survive in endosperm and embryo of the seeds. Infected seed is an important source of primary inoculum of pathogens [3]. Seeds of rice (Oryza sativa cv. Boramchan) collected from bakanae-infected field were found to be 96% infected with Fusarium sp., 52% with F. fujikuroi, 42% with F. verticillioides, and 12% with F. proliferatum as determined by incubation method and species-specific PCR assays. F. fujikuroi was detected at lemma/palea, endosperm and embryo whereas F. verticillioides and F. proliferatum were recovered only from lemma/palea by means of component plating test. Seed disinfection methods have been developed to control bakanae disease and prochloraz has been most widely used for rice seeds. Two chemicals formulated with prochloraz (PC 1) and prochloraz + hexaconazole (PC 2) that inhibit biosynthesis of ergosterol strongly reduced the incidence of Fusarium spp. on selective media to 4.7% and 2.0%, respectively. Disease symptoms of rice seedlings in nursery soil were alleviated by chemical treatment; seedlings with elongated leaves or wide angle between leaf and stem were strikingly reduced from 15.6 to 3.2% (PC 1) and 0 (PC 2), stem rots were reduced from 56.9 to 26.2% (PC 1) and 32.1% (PC 2), and normal seedling increased from 0.4 to 13.3% (PC 2). Prochloraz has some disadvantages and risks such as the occurrence of tolerant pathogens [4] and effects on the sterol synthesis in animals and humans [5]. For these reasons, it is necessary to develop new disinfection method that do not induce fungal tolerance and are safe to humans and animals. Chlorine dioxide ($ClO_2$), that is less toxic, produces no harmful byproducts, and has high oxidizing power, has been reported to be effective at disinfection of several phytopathogenic fungi including Colletotrichum spp. and Alternaria spp. [6]. Gaseous $ClO_2$ applied to rice seeds at a concentration of 20 ppm strongly suppressed mycelial growth of Fusarium fujikuroi, F. verticillioides and F. proliferatum. The incidence of Fusarium spp. in dry seed with 8.7% seed moisture content (SMC) tended to decrease as the concentration of $ClO_2$ increased from 20 to 40 ppm. Applying 40 ppm $ClO_2$ at 90% relative humidity, incidence was reduced to 5.3% and resulted in significant reduction of disease symptoms on MS media. In nursery soil, stem rot was reduced from 56.9 to 15.4% and the number of normal seedlings increased from 0.4 to 25.5%. With water-soaked seeds (33.1% SMC) holding moisture in the endosperm and embryo, the effectiveness of disinfection using $ClO_2$ increased, even when treated with only 20 ppm for four hours. This suggests that moisture was a key element for action of $ClO_2$. Removal of the palea and lemma from seeds significantly decreased the incidence of Fusarium spp. to 3.0%. Seed germination appeared to decrease slightly by water-soaking at $30^{\circ}C$ because of increased SMC and by physical damage of embryos from hulling. These results indicate that the use of gaseous $ClO_2$ was effective as a means to disinfect rice seeds infected with Fusarium spp. and that moisture around the pathogens in the seed was an important factor for the action of $ClO_2$. Further investigations should be conducted to ascertain the best conditions for complete disinfection of Fusarium spp. that infect deep site of rice seeds.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2018년도 춘계학술대회 및 임시총회
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• pp.27-27
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• 2018

The production scale of mushroom cultivation in Korea is approximately 600 billion won, which is 1.6% of the Korean gross agricultural output. Annually, ca. 190,000 tons of mushrooms are harvested in Korea. Although the numbers of mushroom farms and cultivators are constantly decreasing, the total mushroom yields are increasing due to the large-scale cultivation facilities and automation. The recent expansion of the well-being trend causes increase in mushroom consumption in Korea: annual per capita consumption of mushroom was 3.9kg ('13) that is a little higher than European's average. Thus the exports of mushrooms, mainly Flammulina velutipes and Pleurotus ostreatus, have been increased since the middle of 2000s. Recently, however, it is slightly reduced. However, Vietnam, Hong Kong, the United States, the Netherlands and continued to export, and the country has increased recently been exported to Australia, Canada, Southeast Asia and so on. Canned foods of Agaricus bisporus was the first exports of the Korean mushroom industry. This business has reached the peak of the sale in 1977-1978. As Korea initiated trade with China in 1980, the international prices of mushrooms were sharply fall that led to shrink the domestic markets. According to the high demand to develop new items to substitute for A. bisporus, oyster mushroom (Pleurotus ostreatus) was received the attention since it seems to suit the taste of Korean consumers. Although log cultivation technique was developed in the early 1970s for oyster mushroom, this method requires a great deal of labor. Thus we developed shelf cultivation technique which is easier to manage and allows the mass production. In this technique, the growing shelf is manly made from fermented rice straw, that is the unique P. ostreatus medium in the world, was used only in South Korea. After then, the use of cotton wastes as an additional material of medium, the productivity. Currently it is developing a standard cultivation techniques and environmental control system that can stably produce mushrooms throughout the year. The increase of oyster mushroom production may activate the domestic market and contribute to the industrial development. In addition, oyster mushroom production technology has a role in forming the basis of the development of bottle cultivation. Developed mushroom cultivation technology using bottles made possible the mass production. In particular, bottle cultivation method using a liquid spawn can be an opportunity to export the F.velutipes and P.eryngii. In addition, the white varieties of F.velutipes were second developed in the world after Japan. We also developed the new A.bisporus cultivar "Sae-ah" that is easy to grown in Korea. To lead the mushroom industry, we will continue to develop the cultivars with an international competitive power and to improve the cultivation techniques. Mushroom research in Korea nowadays focuses on analysis of mushroom genetics in combination with development of new mushroom varieties, mushroom physiology and cultivation. Further studied are environmental factors for cultivation, disease control, development and utilization of mushroom substrate resources, post-harvest management and improvement of marketable traits. Finally, the RDA manages the collection, classification, identification and preservation of mushroom resources. To keep up with the increasing application of biotechnology in agricultural research the genome project of various mushrooms and the draft of the genetic map has just been completed. A broad range of future studies based on this project is anticipated. The mushroom industry in Korea continually grows and its productivity rapidly increases through the development of new mushrooms cultivars and automated plastic bottle cultivation. Consumption of medicinal mushrooms like Ganoderma lucidum and Phellinus linteus is also increasing strongly. Recently, business of edible and medicinal mushrooms was suffering under over-production and problems in distribution. Fortunately, expansion of the mushroom export helped ease the negative effects for the mushroom industry.

• 한국균학회지
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• 제7권1호
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• pp.13-73
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• 1979

양송이 합성퇴비(合成堆肥) 배지(培地)의 제조(製造)에 있어서 탄소원(炭素原), 질소원(窒素源) 등(等) 영양원(營養源)과 물리적(物理的) 안정(安定)을 위(爲)한 보조재료(補助材料)의 선정(選定), 볏짚을 주재료(主材料)로 사용(使用)할 때의 퇴비재료(堆肥材料)의 배합(配合), 야외퇴적(野外堆積) 및 후발효(後醱酵), 볏짚 퇴비배지(堆肥倍地)에서의 유해생물(有害生物) 발생(發生) 및 방제(防除)에 관(關)한 연구(硏究)를 수행(遂行)한 바 그 결과(結果)를 요약(要約)하면 다음과 같다. 1. 합성퇴비배지(合成堆肥倍地)의 탄소원(炭素原)으로서 볏짚은 보리짚과 밀짚보다 발효(醱酵)가 신속(迅速)하고 퇴비(堆肥)의 질소함량(窒素含量)이 높으며 배지(培地)의 질(質)이 양호(良好)하여 양송이 자실체(字實體) 수량(收量)이 현저(顯著)히 높았다. 2. 한국(韓國)에서 생산(生産)되는 일본형(日本型) 벼와 통일품종(統一品種等) 두 계통(系統)의 볏짚은 초형(草型) 및 이화학적(理化學的) 성질(性質)이 달라서 퇴비(堆肥)의 발효상태(醱酵狀態)에 차이(差異)가 많았다. 통일(統一)볏짚은 발효(醱酵)가 빠르게 진행(進行)되므로 퇴적기간(堆積期間)을 단축(短縮)하고 수분공급량(水分供給量)을 감소(減少)시키며 물리성(物理成) 안정재(安定材)를 첨가(添加)하여야 한다. 3. 보릿짚 퇴비(堆肥)는 볏짚퇴비(堆肥)보다 생산성(生産性)이 낮으나 보릿짚과 볏짚을 50 : 50으로 혼용(混用)하면 볏짚과 대등(對等)한 수량(收量)을 얻을 수 있었다. 4. 퇴비배지(堆肥倍地)의 전질소(全窒素), 전유기물(全有機物) 질소(窒素) 및 Amino산태(酸態), Amide태(態) Amino당태(糖態) 질소(窒素)와 자실체(字實體) 수량간(收量間)에는 각각(各各) 높은 정(正)의 상관(相關)이 있으나 Ammonia태(態) 질소(窒素)는 균사생장 및 자실체(字實體) 형성(形成)에 심(甚)히 유해(有害)하였다. 5. 볏짚을 주재료(主材料)로 사용(使用)할 때 무기태(無機態) 질소원(窒素源)으로서 요소(尿素)가 가장 좋았고 유안(硫安)과 석회질소(石灰質素)는 부적당(不適當)하였다. 요소(尿素)는 3회(回) 분시(分施)할 때 손실(損失)이 감소(減少)되고 퇴비(堆肥)의 질소함량(窒素含量)이 증가(增加)하였다. 6. 유기태영양원(有機態營養源) 중(中) 들깻묵, 참깻묵, 밀기울, 계양(鷄養) 등(等)의 첨가(添加)는 퇴비(堆肥)의 발효(醱酵)를 양호(良好)하게 하고 자실체수량(字實體收量)을 증가(增加)시켰다. 7. 들깻묵, 밀기울 등(等) 유기태영양원(有機態營養源)은 장유박(醬油粕), 이분조미료폐비(泥粉調味料廢肥) 등(等) 공장폐엽물(工場廢葉物)로서 대체(代替)하여 재배(栽培)할 수 있었다. 8. 볏짚퇴비(堆肥) 제조시(製造時) 석고(石膏)와 Zeolite를 첨가(添加)하면 과습(過濕) 및 결착(結着) 등(等)으로 인(因)한 물리성(物理性)의 악화(惡化)가 방지(防止)되며, 자실체수량(字實體收量)이 증가(增加)하는데 그 효과(效果)는 일본형(日本型) 볏짚보다 통일(統一)에서 현저(顯著)하였다. 9. 볏짚을 주재료(主材料)로 퇴비재료(堆肥材料)를 배합(配合)할 때 계양(鷄養) 10%, 깻묵 5%, 요소(尿素) $1.2{\sim}1.5%$, 석고(石膏) 1%를 첨가(添加)하고 봄재배(栽培) 때는 발열촉진(發熱促進)을 위(爲)하여 미강(米糠)을 첨가(添加)하는 것이 좋았다. 10. 볏짚배지(培地)의 야외퇴적시(野外堆積時) 적산온도(積算溫度)와 퇴비(堆肥) 부열도간(腐熱度間)에는 r=0.97의 높은 상관(相關)이 이고 적산온도(積算溫度) $900{\sim}1000^{\circ}C$일 때 자실체(字實體) 수량(收量)이 가장 많았다. 11. 퇴적기간(堆積期間)이 길어질수록 퇴비(堆肥)의 부열도(腐熱度)가 높아지고 전질소함량(全窒素含量)이 증가(增加)하고 Ammonia태(態) 질소(窒素)는 감소(減少)하였는데, 볏짚배지(培地)의 퇴적기간(堆積期間)은 봄재배(栽培) $20{\sim}25$일(日), 가을재배(栽培) 15일(日)이 적당(適當)하였고 그때의 부열도(腐熱度)는 각각 19및 24%였다. 12. 퇴비(堆肥) 후발효시(後醱酵時) 수분함량(水分含量)이 높은 퇴비(堆肥)를 진압(鎭壓) 하여 입상(入床)할 때 공기유통(空氣流通)이 감소(減少)하여 Ammonia태(態) 질소(窒素)의 잔류량(殘溜量)이 증가(增加)하고 Methane과 유기산(有機酸) 등(等) 환원성(還元性) 물질(物質)의 생성(生成)이 많았다. r=-0.76, 휘발성(揮發性) 유기산(有機酸)과는 r=-0.73의 부(負)의 상관(相關)이 있었다. 13. 입상시(入床時) 퇴비(堆肥)의 수분함량(水分含量) $69{\sim}80%$ 범위(範圍)에서 자실체(字實體) 수량(收量)은 수분함량(水分含量)이 증가(增加)할수록 감소(減少)하였는데 (r=-0.78) 이것은 공극량(孔隙量)의 감소(減少)에 기인(基因)하는 것이었다. 입상시(入床時) 균상(菌床)의 적정 공극량(孔隙量)은 $41{\sim}45%$. 14. 후발효(後發效) 정열(頂熱)은 병해충 방제(防除) 뿐 아니고 Ammonia의 제거(除去)를 위(爲)해서 필수적(必須的) 과정(科程)이며 정열후(情熱後) 4일간(日間)의 발효(發效) 과정(科程)이 필요(必要)하였다. 15. 볏짚 퇴비배지(堆肥倍地)에서 양송이 균(菌)에 유해(有害)한 영향(影響)을 미치는 사장균 10종(種)이 동정(同定)되었는데 그 중(中) Diehliomyces microsporus, Trichoderma spp.,Stysanus stemoitis 등(等)은 발생빈도(發生頻度)가 높고 피해(被害)가 심(甚)하였다. 16. Diehliomyces는 재배사(栽培舍) 온도조절(溫度調節), Basamid와 Vapam처리(處理)로서 방제(防除)가 가능(可能)하며 Trichoderma spp.는 Bavistin과 Benomyl 철포(撤布)로서 방제(防除)되었다. 17. 퇴비중(堆肥中) 서식(棲息)하여 양송이를 가해(加害)하는 4종(種)의 선충과 5종(種)의 응애(類)는 퇴비(堆肥)를 $60^{\circ}C$에서 6시간(時間) 정열(頂熱)시키므로서 방제(防除)할 수 있었다.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2015년도 춘계학술대회 및 임시총회
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• pp.45-46
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• 2015

Clubroot disease of crucifers has occurred since 1957. It has spread to the whole China, especially in the southwest and nourtheast where it causes 30-80% loss in some fields. The disease has being expanded in the recent years as seeds are imported and the floating seedling system practices. For its effective control, the Ministry of Agriculture of China set up a program in 2010 and a research team led by Dr. Yueqiu HE, Yunnan Agricultural University. The team includes 20 main reseachers of 11 universities and 5 institutions. After 5 years, the team has made a lot of progresses in disease occurrence regulation, resources collection, resistance identification and breeding, biological agent exploration, formulation, chemicals evaluation, and control strategy. About 1200 collections of local and commercial crucifers were identified in the field and by artificiall inoculation in the laboratories, 10 resistant cultivars were breeded including 7 Chinese cabbages and 3 cabbages. More than 800 antagostic strains were isolated including bacteria, stretomyces and fungi. Around 100 chemicals were evaluated in the field and greenhouse based on its control effect, among them, 6 showed high control effect, especially fluazinam and cyazofamid could control about 80% the disease. However, fluzinam has negative effect on soil microbes. Clubroot disease could not be controlled by bioagents and chemicals once when the pathogen Plasmodiophora brassicae infected its hosts and set up the parasitic relationship. We found the earlier the pathogent infected its host, the severer the disease was. Therefore, early control was the most effective. For Chinese cabbage, all controlling measures should be taken in the early 30 days because the new infection could not cause severe symptom after 30 days of seeding. For example, a biocontrol agent, Bacillus subtilis Strain XF-1 could control the disease 70%-85% averagely when it mixed with seedling substrate and was drenching 3 times after transplanting, i.e. immediately, 7 days, 14 days. XF-1 has been deeply researched in control mechanisms, its genome, and development and application of biocontrol formulate. It could produce antagonistic protein, enzyme, antibiotics and IAA, which promoted rhizogenesis and growth. Its The genome was sequenced by Illumina/Solexa Genome Analyzer to assembled into 20 scaffolds then the gaps between scaffolds were filled by long fragment PCR amplification to obtain complet genmone with 4,061,186 bp in size. The whole genome was found to have 43.8% GC, 108 tandem repeats with an average of 2.65 copies and 84 transposons. The CDSs were predicted as 3,853 in which 112 CDSs were predicted to secondary metabolite biosynthesis, transport and catabolism. Among those, five NRPS/PKS giant gene clusters being responsible for the biosynthesis of polyketide (pksABCDEFHJLMNRS in size 72.9 kb), surfactin(srfABCD, 26.148 kb, bacilysin(bacABCDE 5.903 kb), bacillibactin(dhbABCEF, 11.774 kb) and fengycin(ppsABCDE, 37.799 kb) have high homolgous to fuction confirmed biosynthesis gene in other strain. Moreover, there are many of key regulatory genes for secondary metabolites from XF-1, such as comABPQKX Z, degQ, sfp, yczE, degU, ycxABCD and ywfG. were also predicted. Therefore, XF-1 has potential of biosynthesis for secondary metabolites surfactin, fengycin, bacillibactin, bacilysin and Bacillaene. Thirty two compounds were detected from cell extracts of XF-1 by MALDI-TOF-MS, including one Macrolactin (m/z 441.06), two fusaricidin (m/z 850.493 and 968.515), one circulocin (m/z 852.509), nine surfactin (m/z 1044.656~1102.652), five iturin (m/z 1096.631~1150.57) and forty fengycin (m/z 1449.79~1543.805). The top three compositions types (contening 56.67% of total extract) are surfactin, iturin and fengycin, in which the most abundant is the surfactin type composition 30.37% of total extract and in second place is the fengycin with 23.28% content with rich diversity of chemical structure, and the smallest one is the iturin with 3.02% content. Moreover, the same main compositions were detected in Bacillus sp.355 which is also a good effects biocontol bacterial for controlling the clubroot of crucifer. Wherefore those compounds surfactin, iturin and fengycin maybe the main active compositions of XF-1 against P. brassicae. Twenty one fengycin type compounds were evaluate by LC-ESI-MS/MS with antifungal activities, including fengycin A $C_{16{\sim}C19}$, fengycin B $C_{14{\sim}C17}$, fengycin C $C_{15{\sim}C18}$, fengycin D $C_{15{\sim}C18}$ and fengycin S $C_{15{\sim}C18}$. Furthermore, one novel compound was identified as Dehydroxyfengycin $C_{17}$ according its MS, 1D and 2D NMR spectral data, which molecular weight is 1488.8480 Da and formula $C_{75}H_{116}N_{12}O_{19}$. The fengycin type compounds (FTCPs $250{\mu}g/mL$) were used to treat the resting spores of P. brassicae ($10^7/mL$) by detecting leakage of the cytoplasm components and cell destruction. After 12 h treatment, the absorbencies at 260 nm (A260) and at 280 nm (A280) increased gradually to approaching the maximum of absorbance, accompanying the collapse of P. brassicae resting spores, and nearly no complete cells were observed at 24 h treatment. The results suggested that the cells could be lyzed by the FTCPs of XF-1, and the diversity of FTCPs was mainly attributed to a mechanism of clubroot disease biocontrol. In the five selected medium MOLP, PSA, LB, Landy and LD, the most suitable for growth of strain medium is MOLP, and the least for strains longevity is the Landy sucrose medium. However, the lipopeptide highest yield is in Landy sucrose medium. The lipopeptides in five medium were analyzed with HPLC, and the results showed that lipopeptides component were same, while their contents from B. subtilis XF-1 fermented in five medium were different. We found that it is the lipopeptides content but ingredients of XF-1 could be impacted by medium and lacking of nutrition seems promoting lipopeptides secretion from XF-1. The volatile components with inhibition fungal Cylindrocarpon spp. activity which were collect in sealed vesel were detected with metheds of HS-SPME-GC-MS in eight biocontrol Bacillus species and four positive mutant strains of XF-1 mutagenized with chemical mutagens, respectively. They have same main volatile components including pyrazine, aldehydes, oxazolidinone and sulfide which are composed of 91.62% in XF-1, in which, the most abundant is the pyrazine type composition with 47.03%, and in second place is the aldehydes with 23.84%, and the third place is oxazolidinone with 15.68%, and the smallest ones is the sulfide with 5.07%.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2016년도 춘계학술대회 및 임시총회
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• pp.19-20
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• 2016

Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.