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|標題:||牛樟內生真菌之多樣性與Colletotrichum sp. CKL005菌株之有效生物活性物質分析|
Diversity of fungal endophytes from Cinnamomum kanehirai and the analyses of bioactive materials of the Colletotrichum sp. isolate CKL005
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|摘要:||植物內生真菌 (fungal endophytes) 為一群生長於健康植物組織內之真菌，其多樣性與寄主本身的特異性和生活環境有關。許多研究報告指出內生真菌可產生與寄主相似甚至更多具生物活性的代謝產物，不僅能夠抑制有害微生物的入侵或生長，亦能擴大應用於其他工業、農業及生物醫學領域。牛樟樹 (Cinnamomum kanehirai Hay.; Syn. C. micranthum (Hay.), Icon. Pl. Form. ) 為台灣本土特有且稀有的闊葉樹種，具有多種生物活性物質，如脂肪醇、薄荷烷類、松香醇等。此外，牛樟樹在自然界為牛樟芝 (Taiwanofungus camphoratus; Syn. Antrodia cinnamomea and A. camphorata ) 之主要生長寄主，更顯出牛樟樹具有極高的經濟價值。本研究為調查牛樟樹內生真菌多樣性和分布與內在環境和外在環境之關係，並篩選具拮抗能力之菌株，分析有效物質成分與種類加以應用。99年4月至100年12月間自惠蓀林場內健康牛樟樹分離到189株內生真菌菌株，另自國立自然科學博物館內牛樟樹分離到35株。經形態與分子生物學鑑定，得知來自惠蓀林場牛樟樹之內生真菌菌株可分成13科22屬，其中以Phomopsis、Colletotrichum、Pestalotiopsis、Nigrospora、Xylaria及Guignardia等六屬為主要菌相；而分離自科博館牛樟樹之菌株可分為7科7屬，依分離率高低依次為Nigrospora、Colletotrichum、Phomopsis、Guignardia、Exserohilium、Torula及 Rhodotorula，顯示天然環境中所生長之牛樟樹內生真菌菌相，較人工環境生長之牛樟樹內生真菌菌相複雜。進一步分析惠蓀林場內牛樟樹之成熟與嫩綠枝條的內生真菌菌相分布，得知兩種枝條內的內生真菌菌相數目差異不大，然內生真菌中某些屬僅能自成熟枝條分離，而某些特定屬則只能自嫩綠枝條上分離，證實牛樟樹不同部位所分離出來的內生真菌族群具有差異性。另比較不同時期牛樟樹內生真菌菌相與氣候環境之關係，以惠蓀林場為調查點，於不同時間點採集林場內牛樟樹枝條進行分離，結果顯示除Pestalotiopsis、Colletotrichum、Nigrospora及Phomopsis等4屬可全年被分離外，其餘各時間所分離之菌相有明顯不同，證實氣候條件能影響內生真菌於牛樟樹內的族群分布與密度。
於篩選內生真菌菌株對植物病原菌拮抗能力試驗中，顯示224株內生真菌中，有6株對3種供試病原真菌具有抑制作用，進一步測試對8種植物真菌病原，一種卵菌病原及4種病原細菌之拮抗能力，顯示CKL005菌株抑制病原菌的效果最顯著。經以近鄰結合法 (NJ)分析CKL005菌株之所屬，指出CKL005菌株的LSU D1/D2與ACT基因序列和Colletotrichum屬的真菌較接近，然ITS rDNA序列卻顯示與Colletotrichum屬真菌不具明顯親緣關係。於形態學分析方面，指出CKL005菌株與分子親緣性較接近Colletotrichum屬種類於形態上相似。在碳素源的利用方面，顯示CKL005菌株可有效利用葡萄糖與蔗糖，於液態培養下可促進菌絲生長，而固態培養下則以葡萄糖與麥芽糖對拮抗能力之促進較其他碳素源佳，對芒果炭疽病菌絲生長抑制率分別為53.0及52.4%。在氮素源的利用方面，顯示供試氮素源無法有效促進CKL005菌株生長與提高拮抗活性，對芒果炭疽病菌絲生長抑制率介於20.0-40.9%。複合碳氮素源試驗結果顯示，以麥芽糖配合硫酸銨、尿素及硝酸銨對芒果炭疽病菌絲生長抑制結果較佳，抑制率依次為51.6、52.0及48.6%。本研究比較碳氮素源與PDA培養基之培養效果，得知馬鈴薯葡萄糖瓊脂培養基能促進CKL005菌株菌絲的生長，為最適培養基，且酸鹼值6-6.5與溫度28℃時生長良好。
以乙酸乙酯萃取CKL005菌株菌絲與液態培養液中的有效抗生物質，由初步試驗結果顯示，發酵液的萃取物質對抑制芒果炭疽病菌絲生長不顯著，EC50值大於500 mg/L；反之，菌絲萃取液的抑制效果顯著，對草莓灰黴病菌、十字花科黑斑病菌、芒果及白菜炭疽病菌的抑制效果最顯著，其EC50值分別為50.66±0.07、17.56±0.07、7.84±0.09及14.39±0.07 mg/L，然對茄科疫病菌的效果不顯著，EC50值為282.53±0.07 mg/L，另對三株不同寄主分化型Fusarium oxysporum之萎凋病菌與十字花科幼苗立枯病菌之EC50皆大於500 mg/L。將具抑制效果的菌絲萃取物經管柱層析之後，得到沖提液極性為90 %的乙酸乙酯與10 %的甲醇分離部，對芒果炭疽病菌株與白菜炭疽病菌菌絲生長皆有抑制效果，抑制率分別為42.5%與42.0%。經逆向高效能液相層析進一步純化，並選取具有活性的第4分離部進行NMR與GC/MS分析，得知主要成分包括β-胡蘿蔔素、單棕櫚甘油酸、乙酸去氫皮質醇、麥角固醇、蟾蜍他靈、皮質類固醇、洋地黃毒苷及吉勃素，其中以酯類蟾蜍他靈的相對含量最高。除拮抗物質，CKL005菌株亦能產生可抑制白菜炭疽病菌絲生長的揮發性氣體。將揮發性氣體以氣相質譜儀(GC/MS)進行分析比對，結果得知可能有抗菌作用的成分為二種BHT (Butylated hydroxytoluene) 類脂溶性抗氧化劑（2,5-Cyclohexadiene-1,4- dione,2,6-bis(1,1-dimethylethy)-與2,6-Bis(1,1-dimethylethyl)-4-methylphenol (BHT)）及 (3H)-one, 3,6,7-thimethoxy- isobenzofuran-1。於溫室測試CKL005菌絲萃取液，對不結球白菜種子發芽率影響與防治炭疽病之效果。結果證實萃取液對不結球白菜種子發芽率有提高的效果，但無顯著差異，處理4、8及12小時之發芽率分別為95.0、96.4及96.8%，而以水浸泡之發芽率則為93.2、93.0及93.0%。另測量發芽3日後60株幼苗總重量，處理4、8及12小時，分別為1.36、1.46及1.50克，對照組則為0.98、0.92及1.04克。於溫室測試防治白菜炭疽病的效果，得知只接種白菜炭菌病孢子懸浮液植株罹病度52.78 %，先施用10或100mg/L菌絲萃取液植株罹病度均為25.0%，後施用10或100 mg/L菌絲萃取液之植株罹病度分別為11.1%與19.4%，顯示只要施用菌絲萃取液，皆能有效降低罹病度。實驗最後以5L發酵槽進行CKL005菌株菌絲體增量測試，試驗結果顯示所獲得之菌絲量高於三角燒瓶搖瓶培養3.8倍，未來可以5L發酵槽進行更多防治試驗。|
Fungal endophytes, residing in healthy plants, show high diversities corresponding with host specificity and ecology. Previous studies indicated that fungal endophytes can produce metabolites showing same function with the metabolites produced by host plants. Moreover, the metabolites produced by fungal endophytes not only can use to control microorganisms, but also have been used industry, agriculture and biomedical. Cinnamomum kanehirai Hay. ( Syn. C. micranthum (Hay.), Icon. Pl. Form.) is an arbor indigenous in Taiwan and it can produce several compounds including fatty alcohol, trans-p-methane-1,4-diol, terpineol etc. In addition, C. kanehirai is the host of Taiwanofungus camphoratus ( Syn. Antrodia cinnamomea and A. camphorata ) in nature condition. The objectives of this study are investigating the diversity and distribution of fungal endophytes in C. kanehirai associated with environmental variations and examining the bioactivity against plant pathogens. Furthermore, the application to controll crop disease is constructed in greenhouse. A total of 224 fungal isolates were obtained from C. kanehirai , including 189 isolates from Hui-Sun Forest (HSF) and 35 isolates from National Museum of Natural Science (NMNS), during April 2010 to December 2011. Based on morphological and molecular characteristics, 189 isolates from HSF could be identified as 13 families 22 genera and 35 isolates from NMNS could be identified as 7 families 7 genera. Among 189 isolates from HSF, the Phomopsis, Colletotrichum, Pestalotiopsis, Nigrospora, Xylaria and Guignardia are dominant genera; meanwhile, isolation frequency of the 35 isolates from NMNS followed Nigrospora, Colletotrichum, Phomopsis, Guignardia, Exserohilum, Torula and Rhodotorula. The results showed that the fungal endophytes from the natural environment are more diverse than artificial environment. Comparing the distribution of fungal endophytes from mature or young twigs of C. kanehirai in HSF, the results revealed that number of isolates were not significantly different. However, certain genera could only exist in mature twigs and some genera could only be obtained from the young twigs. Analyzing the effect of collected timing of C. kanehirai on difference of fungal endophytes during 2010~2011, the results indicated that only Pestalotiopsis, Colletotrichum, Nigrospora and Phomopsis could be obtained throughout the year. Thus, the diversity of fungal endophytes could be affected by different climate. The bioactivities against plant pathogens showed that 6 of 224 isolates had ability to inhibit mycelial growth of three fungal pathogens. Among the 6 isolates, the CKL005 isolate was significantly against 8 fungal pathogens, one oomycetes and 4 bacterial pathogens. The phylogenetic analysis revealed that CKL005 isolate was closely related to Colletotrichum based on LSU D1/D2 rDNA and ACT gene sequences; however, the CKL005 isolate was distinguished from Colletotrichum based on ITS rDNA sequence. Comparing the morphology of CKL005 isolate with Colletotrichum species showed closely, and the results showed that certain morphological characteristics were similar with the Colletotrichum species. The effect of different carbon sources on mycelial growth of CKL005 isolate indicated that glucose and maltose could enrich the biomass of CKL005 isolate at liquid culture condition. Moreover, the two carbon sources could increase the bioactivities against C. gloeosporioides with higher inhibition rate of 53.0% and 52.4% at solid culture condition. The other side, the effect of different nitrogen sources on mycelial growth or bioactivity of CKL005 isolate was not significant. The activities against C. gloeosporioides were lower and the inhibition rate was 20.0% to 4.09%. In addition, the combination of maltose with ammonium sulfate or urea or ammonium nitrate had higher ability to inhibit mycelial growth of C. gloeosporioides than other combinations, and inhibition rates were followed 51.6%, 52.0% and 48.6%. In this study, the PDA was used to culture CKL005 isolate for further test because PDA is best for enriching biomass and bioactivity of CKL005 isolate than other carbon or nitrogen source at pH 6.0-6.5 and 28°C. Comparing the extracts of fermenter and mycelia of CKL005 on inhibiting C. gloeosporioides, the results demonstrated that mycelial extract by ethyl acetate had better efficacy than fermenter. The EC50 of fermenter extract to C. gloeosporioides was more than 500 mg/L. However, the EC50 of mycelial extract to different plant pathogens were lower than fermenter extract. The EC50 of mycelial extract to Botrytis cinerea, Althernaria brassicicola, C. gloeosporioides and C. higginsianum were 50.66±0.07 , 17.56±0.07,7.84±0.09 and 4.39±0.07 mg/L. The other side, the EC50 of mycelial extract to Phytophthora capsici was 282.53±0.07 mg/L. However, the EC50 of mycelial extract to three formae speciales of Fusarium oxysporium and Rhizoctonia solani were more than 500 mg/L. Based on HPLC analysis, the fraction 4 extracted by 90% ethyl acetate and 10 methanol had highest efficacy on inhibiting C. gloeosporioides and C. higginsianum. The further analysis by GC-MS, the major compounds of mycelial extract included β-carotene, glycerol 1-palmitate, prednisolone acetate, ergosterol, gammabufotalin, hydrocortilsoreacetate, digitoxin and gibberellic acid. In this study, the volatile compounds produced by CKL005 isolate showed inhibition on mycelial growth of C. higginsianum. The GC-MS analysis indicated that several compounds might associate with inhibition to C. higginsianum, including 2,5-Cyclohexadiene-1,4- dione,2,6-bis(1,1-dimethylethy)-, 2,6-Bis(1,1-dimethylethyl)-4-methylphenol (BHT) and (3H)-one, 3,6,7-thimethoxy- isobenzofuran-1. For test the efficacy of mycelial extract on controlling C. higginsianum in greenhouse, the results showed that spraying 10 or 100 mg / L of mycelial extract could reduced disease servity 27% at least. A preliminary test on the germination of cabbage seeds, results showed the germination rate were improved and the weight of seedlings were enhanced by soacking with 10 mg/L mycelial extraction of CKL005 isolate.
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