抗菌活性植物内生菌筛选及其发酵条件优化
|
摘要
植物内生菌是一个庞大的具有普遍存在性的微生物特殊类群。近年来,研究表明植物中内生菌能产生抗肿瘤、杀菌等特殊活性物质,这使得植物内生菌的应用范围不断扩展,同时也成为药物研究一个新的热点。
本论文是在抗结核分枝杆菌的植物内生菌筛选的基础上,根据“一菌多筛”的原则,扩展为具有抑菌活性的植物内生菌筛选及其活性菌株发酵优化。论文进行了植物样本表面消毒、内生菌分离纯化、发酵及发酵产物预处理、抗菌活性菌株筛选、活性菌株鉴定及其发酵条件优化等研究。获得的主要结果如下: 1植物内生菌的分离纯化
采用已优化的表面消毒方法对30种植物样本进行处理,分离得到46株内生菌。但9株(3株真菌,6株细菌)不能在体外培养,另有7株内生真菌在本实验特定的人工培养条件下不产生有性分生孢子。
2抗菌活性植物内生菌的筛选
①得到菌丝预处理丙酮样本37份,乙酸乙酯样本37份;采用酒精沉淀法对发酵液进行预处理,得到样本37份。②采用定量的二倍稀释法(培养基采用改良罗氏斜面培养基)对111份预处理样本进行初筛,得到来源于商陆(Phytolacca acinosa)的EP-S23,狭叶十大功劳(Mahonia fortunei)EM-L40、香樟(Cinnamomum camphora (Linn.)Presl)EC-S44的3株活性内生真菌,其发酵液提取物浓度为10mg/ml时对人型结核标准菌株(H37Rv)有抑制作用。其中,EC-S44的发酵液提取物浓度在15mg/ml时对人型结核耐药菌株有抑制生长作用。③采用KB纸片法对以上3株菌进行复筛,得到香樟内生菌EC-S44发酵液提取物浓度为10mg/ml时对金黄色葡萄球菌、枯草芽孢杆菌、铜绿假单孢菌、大肠杆菌均有不同程度抑制作用,其中对金黄色葡萄球菌的抑菌圈到达16mm,同时用琼脂二倍稀释法测定最小抑菌浓度(MIC值)为6.5mg/ml。
3活性菌株的鉴定和发酵条件优化
①通过初步形态学鉴定:香樟(Cinnamomum camphora (Linn.)Presl)EC-S44为镰孢霉属(Fusarium),将其命名为Fusarium sp.菌株EC-S44。
②对Fusarium sp.菌株EC-S44发酵条件进行优化,得到其在本实验中的最优发酵条件:土豆汁培养基(3%的葡萄糖、0.3%的牛肉膏),4%发酵接种量,20%的装液量,8.0的初始pH值,26℃培养温度,150rpm转速。在此条件下,菌株对结核分枝杆菌耐药株的抑制能力增强,MIC值降低到12.5mg/ml。
Endophytes are one kind of large, ubiquitous fungus group. Recent years, studies on Endophytes show that some antitumor and antibacterial products made by Endophytes. This allows applications of Endophytes enlarge more and more, and Endophytes become the one new hot point of medicine research.
Based on searching for anti-Mycobacterium tuberculosis Endophytes, according to the principle of using much more Tested bacteria for searching active Endophytes, this study is extended to be as‘Screening for antibacterial Endophytes’. Such the following items were carried through: plant sample surface sterilization, Endophytes isolation and purification, pretreatment for Endophytes fermentation products, screening for antibacterial Endophytes, identification and ferment optimization of active strain. The results obtained are following:
1 Isolation and purification of Endophytes
30 sorts of plant were dealt by the optimal surface sterilization method, and 46 strains were isolated. But 9 of them (3strains fungi, 6strains bacterium) can’t be cultivated in vitro. And 7 of the left don’t have fruiting body under our experimental condition.
2 Screening for antibacterial Endophytes
①the Endophytes fermentation pretreatment samples,37 acetone samples of hypha,37 ethyl acetate samples of hypha,and 37 fermentation broth samples dealt by ethanol deposition method were gotten.②Using the double dilution method (Rhodes incline solid medium) to deal with 111 samples, the anti-Mycobacterium tuberculosis Endophyts are obtained that are Strain EP-S23 deriving from Phytolacca acinosa, Strain EM-L40 deriving from Mahonia fortunei, and Strain EC-S44 deriving from Cinnamomum camphora (Linn.) Presl, which can inhibit the growth of Mycobacterium tuberculosis standard strain (H37Rv) with the fermentation compound density of the 10mg/ml. And strain EC-S44 deriving from Cinnamomum camphora (Linn.) Presl with the fermentation compound density of the 15mg/ml can inhibit the growth of Mycobacterium tuberculosis drug-resistance strain.③Using the Agar dilution method to screen the former three active Endophytes again, the extraction compound solution of Cinnamomum camphora (Linn.) Presl strain EC-S44 can inhibit 5 Tested pathogens with different density, and the inhibition zone of active strain EC-S44 against Staphylococcus aureus (ATCC 25923) is 16mm, and MICs is 6.5mg/ml.
3 Identification and ferment optimization of active strain
①Active Strain EC-S44 is identified by morphology as the one of Fusarium, named as Fusarium sp. Strain EC-S44.
②The optimum ferment condition of Fusarium sp. strain EC-S44 is: PDA (3% glucose, 0.3% beef extract), 4% fermentation inoculation amount, 8.0 initial pH,26℃, 150rpm.Under this condition, for Fusarium sp. strain EC-S44, the capability of anti- Mycobacterium tuberculosis drug-resistance strain enhances, and the MICs is 12.5 mg/ml.
本论文是在抗结核分枝杆菌的植物内生菌筛选的基础上,根据“一菌多筛”的原则,扩展为具有抑菌活性的植物内生菌筛选及其活性菌株发酵优化。论文进行了植物样本表面消毒、内生菌分离纯化、发酵及发酵产物预处理、抗菌活性菌株筛选、活性菌株鉴定及其发酵条件优化等研究。获得的主要结果如下: 1植物内生菌的分离纯化
采用已优化的表面消毒方法对30种植物样本进行处理,分离得到46株内生菌。但9株(3株真菌,6株细菌)不能在体外培养,另有7株内生真菌在本实验特定的人工培养条件下不产生有性分生孢子。
2抗菌活性植物内生菌的筛选
①得到菌丝预处理丙酮样本37份,乙酸乙酯样本37份;采用酒精沉淀法对发酵液进行预处理,得到样本37份。②采用定量的二倍稀释法(培养基采用改良罗氏斜面培养基)对111份预处理样本进行初筛,得到来源于商陆(Phytolacca acinosa)的EP-S23,狭叶十大功劳(Mahonia fortunei)EM-L40、香樟(Cinnamomum camphora (Linn.)Presl)EC-S44的3株活性内生真菌,其发酵液提取物浓度为10mg/ml时对人型结核标准菌株(H37Rv)有抑制作用。其中,EC-S44的发酵液提取物浓度在15mg/ml时对人型结核耐药菌株有抑制生长作用。③采用KB纸片法对以上3株菌进行复筛,得到香樟内生菌EC-S44发酵液提取物浓度为10mg/ml时对金黄色葡萄球菌、枯草芽孢杆菌、铜绿假单孢菌、大肠杆菌均有不同程度抑制作用,其中对金黄色葡萄球菌的抑菌圈到达16mm,同时用琼脂二倍稀释法测定最小抑菌浓度(MIC值)为6.5mg/ml。
3活性菌株的鉴定和发酵条件优化
①通过初步形态学鉴定:香樟(Cinnamomum camphora (Linn.)Presl)EC-S44为镰孢霉属(Fusarium),将其命名为Fusarium sp.菌株EC-S44。
②对Fusarium sp.菌株EC-S44发酵条件进行优化,得到其在本实验中的最优发酵条件:土豆汁培养基(3%的葡萄糖、0.3%的牛肉膏),4%发酵接种量,20%的装液量,8.0的初始pH值,26℃培养温度,150rpm转速。在此条件下,菌株对结核分枝杆菌耐药株的抑制能力增强,MIC值降低到12.5mg/ml。
Endophytes are one kind of large, ubiquitous fungus group. Recent years, studies on Endophytes show that some antitumor and antibacterial products made by Endophytes. This allows applications of Endophytes enlarge more and more, and Endophytes become the one new hot point of medicine research.
Based on searching for anti-Mycobacterium tuberculosis Endophytes, according to the principle of using much more Tested bacteria for searching active Endophytes, this study is extended to be as‘Screening for antibacterial Endophytes’. Such the following items were carried through: plant sample surface sterilization, Endophytes isolation and purification, pretreatment for Endophytes fermentation products, screening for antibacterial Endophytes, identification and ferment optimization of active strain. The results obtained are following:
1 Isolation and purification of Endophytes
30 sorts of plant were dealt by the optimal surface sterilization method, and 46 strains were isolated. But 9 of them (3strains fungi, 6strains bacterium) can’t be cultivated in vitro. And 7 of the left don’t have fruiting body under our experimental condition.
2 Screening for antibacterial Endophytes
①the Endophytes fermentation pretreatment samples,37 acetone samples of hypha,37 ethyl acetate samples of hypha,and 37 fermentation broth samples dealt by ethanol deposition method were gotten.②Using the double dilution method (Rhodes incline solid medium) to deal with 111 samples, the anti-Mycobacterium tuberculosis Endophyts are obtained that are Strain EP-S23 deriving from Phytolacca acinosa, Strain EM-L40 deriving from Mahonia fortunei, and Strain EC-S44 deriving from Cinnamomum camphora (Linn.) Presl, which can inhibit the growth of Mycobacterium tuberculosis standard strain (H37Rv) with the fermentation compound density of the 10mg/ml. And strain EC-S44 deriving from Cinnamomum camphora (Linn.) Presl with the fermentation compound density of the 15mg/ml can inhibit the growth of Mycobacterium tuberculosis drug-resistance strain.③Using the Agar dilution method to screen the former three active Endophytes again, the extraction compound solution of Cinnamomum camphora (Linn.) Presl strain EC-S44 can inhibit 5 Tested pathogens with different density, and the inhibition zone of active strain EC-S44 against Staphylococcus aureus (ATCC 25923) is 16mm, and MICs is 6.5mg/ml.
3 Identification and ferment optimization of active strain
①Active Strain EC-S44 is identified by morphology as the one of Fusarium, named as Fusarium sp. Strain EC-S44.
②The optimum ferment condition of Fusarium sp. strain EC-S44 is: PDA (3% glucose, 0.3% beef extract), 4% fermentation inoculation amount, 8.0 initial pH,26℃, 150rpm.Under this condition, for Fusarium sp. strain EC-S44, the capability of anti- Mycobacterium tuberculosis drug-resistance strain enhances, and the MICs is 12.5 mg/ml.
引文
[1] 李宓.各国积极应对结核病防治新挑战[R].北京:中华人民共和国卫生部.2005
[2] 曾松荣,王海坤,徐成东,等.抗癌药用植物内生真菌的潜在应用价值[J].韶关学院学报(自然科学版). 2001,9(22):46-48
[3] G.A.Strobel, B.Daisy, U.Castillo, et al. Natural products from endophytic microorganisms[J]. J. Nat. Prod. 2004, 67: 257-268.
[4] J.Y.Li, G.A.Strobel. Jesterone and hydroxy-jesterone antioomycete cyclohexenone epoxides from the endophytic fungus Pestalotiopsis jesteri [J]. Phytochemistry. 2001, 57: 261-265.
[5] H.Y, L.Chaomin, B.Kulkarni, et al. Exploring chemical diversity of epoxyquinoid natural products: synthesis and biological activity of jesterone and related molecules[J]. Org. Lett. 2001, 3:1649-1652.
[6] J.Y.Li, J.K.Harper, D.M.Grant, et al. Ambuic acid, a highly functionalized cyclohexenone with antifungal activity from Pestalotiopsis spp. and Monochaetia sp. [J] Phytochemistry. 2001, 56:463-468.
[7] X.Zou, J.C.Meng, H. Lu, et al. Metabolites of Colletotrichum gloeosporioides, an endophytic fungus in Artemisia mongolica [J]. J Nat Prod.2000, 63: 1529-1530.
[8] R.G.Shu, F.W.Wang, Y.M.Yang, et al. Antibacterial and xanthine oxidase inhibitory cerebrosides from Fusarium sp. IFB-121, an endophytic fungus in Quercus variabilis [J]. Lipids. 2004, 39: 667-673.
[9] Y.M.Ma, Y.Li, J.Y.Liu, et al. Anti-Helicobacter pylori metabolites from Rhizoctonia sp. Cy064, an endophytic fungus in Cynodon dactylon [J]. Fitoterapia. 2004, 75: 451-456.
[10] J.Y.Liu, Y.C.Song, Z.Zhang, et al. Aspergillus fumigatus CY018, an endophytic fungus in Cynodon dactylon as a versatile producer of new and bioactive metabolites [J]. J Biotechnol.2004, 114: 279-287.
[11] P.Kongsaeree, S.Prabpai, N.Sriubolmas, et al. Antimalarial dihydroisocoumarins produced by Geotrichum sp., an endophytic fungus of Crassocephalum crepidioides [J]. J Nat Prod. 2003, 66: 709-711.
[12] V.Hellwig, T.Grothe, A. Mayer-Bartschmid, et al. Altersetin, a new antibiotic from cultures of endophytic Alternaria spp. Taxonomy, fermentation, isolation, structure elucidation and biological activities [J]. J Antibiot. 2002, 55: 881-892.
[13] F.Pelaez, A.Cabello, G.Platas, et al. The discovery of enfumafungin, a novel antifungal compound produced by an endophytic Hormonema species biological activity and taxonomy of theproducing organisms [J]. Syst Appl Microbiol. 2000, 23: 333-343.
[14] S.Kim, D.S.Shin, T.Lee, et al. Periconicins, two new fusicoccane diterpenes produced by an endophytic fungus Periconia sp. with antibacterial activity [J]. J Nat Prod.2004, 67: 448-450.
[15] S.F. Brady, J.Clardy. CR377, a new pentaketide antifunga agent isolated from an endophytic fungus [J]. Jnat Prod.2000, 63:1447-1448
[16] 郭良栋.内生真菌研究进展[J].菌物系统. 2001,20 (1 ): 148-152,
[17] 李强,刘军,周东坡,等.植物内生菌的开发与研究进展[J].生物技术通报.2006,(3):33-36
[18] J.K. Stone, C.E. Bacon, J.F.Jr White. An overview of endophytic microbes: endopytism defined [J]. Microbial Endophytes. 2000:3-29.
[19] W. X.Zou, R.X.Tan. Biological and chemical diversity of endophytes and their potential application[J]. Advances in Plant Sciences. 1999: 2: 183-190
[20] G.A.Strobel, B.Daisy.Bioprospecting for Microbial Endophytes and Their Natural Products [J]. Microbiology and Molecular Biology Reviews. 2003, 4 (67): 491-502
[21] 何红,蔡学清,洪永聪.辣椒内生细菌的分离及拮抗菌的筛选[J].中国生物防治. 2002,18(4):171-175
[22] 袁军, 孙福在.防治马铃薯环腐病有益内生细菌的分离和筛选[J].微生物学报. 2002,42(3):270-274
[23]何红,蔡学清等.内生菌BS-2菌株的抗菌蛋白及其防病作用[J].植物病理学报. 2003,33(4):373-378
[24] Y.M.Ma, Y.Li, J.Y.Liu, et al. Anti-Helicobacter pylori metabolites from Rhizoctonia sp. Cy064, an Endophytic fungus in Cynodon dactylon [J]. Fitoterapia.2004, 75: 451-456.
[25] U.Castillo, J.K.H, G.A.Strobel.Kakadumycins novel antibiotics from treptomyces sp. NRRL30566, an endophyte of Grevillea pteridifolia [J]. FEMS Microbiolog Letters.2003, 224:183-190
[26] 邹文欣,谭仁祥.植物内生菌研究新进展[J].植物学报.2001, 43(9): 881-892.
[27] 李桂玲,王建锋,黄耀坚,等.几种药用植物内生真菌抗真菌活性的初步研究[J].微生物学通报. 2001,28(6):42-46
[28] 彭小伟,杨丽源,陈有为,等.黄花夹竹桃内生真菌抗病原细菌的初步研究[J]菌物研究. 2003,1(1):33-36
[29] 李能章,彭远义.一株芦荟抗菌内生细菌的分离鉴定及生物学性质研究[J].生物技术通讯2004,2(15):89-92
[30] 孙力军,陆兆新.植物内生菌抗菌活性物质研究进展[J].食品与发酵工业-综述与专题评论. 2005, 2(31):51-57
[31] G.A.Strobel, R.V.Miller, C.Miller, et al. Cryptocandin, a potent antimycotic from theendophytic fungu Cryptosporiopsis cf. quercina[J]. Microbiol.1999, 145:1919-1926
[32] J.Y.Li, G.A. Strobel, J.Harper ,et al. Cryptocin, a potent tetramic acid antimycotic from the endophytic fungu Cryptosporiopsis cf. quercina [J]. Org Lett. 2000:2767-770
[33] S.F.Brady, J.Clardy. CR377, a new pentaketide antifunga agent isolated from an endophytic fungus [J]. Jnat Prod. 2000, 63:1447-1448
[34] U.F.Castillo, G.A.Strobel, E.J.Ford, et al. Munumbicins, wide spectrum antibiotics produced by Streptomyces (NRRL30562) endophytic on Kennedianigriscans [J]. Microbiology. 2002, 148:2675-2685
[35] 黄午阳.植物内生真菌的抗菌活性研究[J]. 南京中医药大学学报. 2005. 1(21): 16-23
[36] F.U.Castillo, G. A. Strobel, E.J.Ford, et al. Munumbicins, wide-spectrum antibiotics produced by Streptomyces NRRL 30562,endophytic on Kennedia nigriscans [J]. Microbiology. 2002, 148:2675-2685
[37] 徐叔云,卞如濂,陈修.药理实验方法学[M].北京:人民卫生出版社, 2001.1712-1714
[38] 马绪荣,苏德模.药品微生物学检验手册[M].北京:科学出版社.2000:222
[40] 林爱玉,邢晓科,郭顺星.4种药用半红树植物内生真菌的分离及其抗菌活性研究[J].中国药学杂志.2006, 41(12):892-894
[41] H.Lu, W.XZou, J.C.Meng, et al. New bioactive metabolites produced by Colletotrichum sp., an endophytic fungus in Artemisia annua [J]. Plant Science. 2000,151: 67-73
[42] C.U.Castillo, A.K. Harper, G.A.Strobe, et al. Kakadumycins, novel antibiotics from treptomyces sp. NRRL 30566, an endophyte of Grevillea pteridifolia [J].FEMS Microbiology Letters. 2003, 224 :183-190
[43] K.A.Southcott, J.A.Johnson. Isolation of endophytes from two species of palm, from Bermuda [J]. Can J Microbial. 1997, 43: 789-792
[44] 杨文博.微生物学实验[M].北京:化学工业出版社.2004,7:217-218
[45] 杨正强,张耀兮,陈小静,等.华重楼内生菌SS02的分离与抗菌活性的初步研究[J]. 微生物学通报.2006年33(2):54-57
[46] C.H.Liu, W.X.Zou, H.Lu, et.al. Antifungal activity of Artemisia annua endophyte cultures against phytopathogenic fungi[J]. Journal of Biotechnology. 2001,88:277-282
[47] 徐叔云,卞如濂,陈修.药理实验方法学[M].北京:人民卫生出版社(3版).2001:1712-1713
[48] 徐任生,陈仲良.中草药有效成分提取与分离(第二版)[M].上海:上海科学技术出版社. 1989,12:9-10
[49] 马绪荣,苏德模.药品微生物学检验手册[M].科学出版社.北京:2000,3.211
[50] U. F. Castillo, G. A. Strobel, E. J. Ford, et al. Munumbicins, wide-spectrum antibiotics produced by Streptomyces NRRL 30562,endophytic on Kennedia nigriscans [J]. Microbiology. 2002,148:2675-2685
[51] 张正,岳志红. WST248-2005.厌氧菌的抗微生物药敏感试验方法[S].北京.中华人民共和国卫生部:11-12
[52] 马绪荣,苏德模.药品微生物学检验手册[M].科学出版社.北京:2000,3:214
[53] 苏印泉,朱红薇,马希汉,等.杜仲内生真菌的抑菌活性筛选[J].西北植物学报. 2005,25(6):1153-1157
[54] 李萌.蔬菜内生放线菌的分离和生物活性初探[D]. 中国科学院研究生院(大连化学物理研究所). 2003:23-26
[55] 朱红薇.杜仲内生真菌活性物质的研究[D]. 西北农林科技大学:2005.31
[56] 霍娟.杜仲内生真菌资源和活性菌株生物学的初步研究[D]. 南京师范大学:2004.17-18
[57] 杨文博.微生物学实验[M].北京:化学工业出版社,2004.7:127
[58] 李能章.植物抗菌内生菌A11的分离鉴定及其活性物质的初步研究[D]. 西南农业大学.2004:36-52
[59] 邵力平,沈瑞祥,张素轩,等.真菌分类学[M]. 北京:中国林业出版社.1984.354-460
[60] 沈瑞祥,刘碧荣译.真菌—高级论文集第四卷 A.B 册(检索部分).北京:1981
[61] 魏景超.真菌鉴定手册[M].上海:上海科技出版社.1979:611-640
[62] 魏景超.真菌鉴定手册[M].上海:上海科技出版社.1979:611-632
[63] 杨文博.微生物学实验[M].北京:化学工业出版社.2004.7:217-223
[64] Poulev, O’Neal J M, L. S. Elicitation.a new window into plant chemodiversity and phytochemical drug discovery [J] Med Chem.2003.46(12):2542-2547
[65] 曾松荣 徐成东 王海坤 张玲琪.药用植物内生真菌及其具宿主相同活性成分的机制初探[J].中草药.2000, 31(4):65-72
[66] 刘志民.药用植物治疗各种结核病俄语文献50年的研究与进展[J]. 中药研究与信息.2000,2(9):44-50
[67] 孙力军,陆兆新,别小妹.株抗菌植物内生菌EJH-2菌株的分离和鉴定[J].中国微生态学杂志.2006,18(1):23-26.
[68] 纪丽莲.芦竹内生真菌F0238细胞生长及其代谢调控[J].微生物学通报.2005,32(4):51-56
[69] 徐庆妍.两株红树植物内生真菌代谢物及抗菌抗肿瘤活性研究[D]. 厦门大学.2004:82
[70] 邓墨渊,王伯初,杨再昌,等.分子生物学技术在植物内生菌分类鉴定中的应用[J].氨基酸和生物资源 2006,28(3):9-14
[71] 孙奎.杜仲和无花果内生真菌的初步研究[D].西北农林科技大学.2004:35
[72] 孙建华,宇克莉,路福平,等.丝状真菌Sr18发酵过程中的形态学研究[J].天津科技大学学报. 2005.20(4):1-5
[2] 曾松荣,王海坤,徐成东,等.抗癌药用植物内生真菌的潜在应用价值[J].韶关学院学报(自然科学版). 2001,9(22):46-48
[3] G.A.Strobel, B.Daisy, U.Castillo, et al. Natural products from endophytic microorganisms[J]. J. Nat. Prod. 2004, 67: 257-268.
[4] J.Y.Li, G.A.Strobel. Jesterone and hydroxy-jesterone antioomycete cyclohexenone epoxides from the endophytic fungus Pestalotiopsis jesteri [J]. Phytochemistry. 2001, 57: 261-265.
[5] H.Y, L.Chaomin, B.Kulkarni, et al. Exploring chemical diversity of epoxyquinoid natural products: synthesis and biological activity of jesterone and related molecules[J]. Org. Lett. 2001, 3:1649-1652.
[6] J.Y.Li, J.K.Harper, D.M.Grant, et al. Ambuic acid, a highly functionalized cyclohexenone with antifungal activity from Pestalotiopsis spp. and Monochaetia sp. [J] Phytochemistry. 2001, 56:463-468.
[7] X.Zou, J.C.Meng, H. Lu, et al. Metabolites of Colletotrichum gloeosporioides, an endophytic fungus in Artemisia mongolica [J]. J Nat Prod.2000, 63: 1529-1530.
[8] R.G.Shu, F.W.Wang, Y.M.Yang, et al. Antibacterial and xanthine oxidase inhibitory cerebrosides from Fusarium sp. IFB-121, an endophytic fungus in Quercus variabilis [J]. Lipids. 2004, 39: 667-673.
[9] Y.M.Ma, Y.Li, J.Y.Liu, et al. Anti-Helicobacter pylori metabolites from Rhizoctonia sp. Cy064, an endophytic fungus in Cynodon dactylon [J]. Fitoterapia. 2004, 75: 451-456.
[10] J.Y.Liu, Y.C.Song, Z.Zhang, et al. Aspergillus fumigatus CY018, an endophytic fungus in Cynodon dactylon as a versatile producer of new and bioactive metabolites [J]. J Biotechnol.2004, 114: 279-287.
[11] P.Kongsaeree, S.Prabpai, N.Sriubolmas, et al. Antimalarial dihydroisocoumarins produced by Geotrichum sp., an endophytic fungus of Crassocephalum crepidioides [J]. J Nat Prod. 2003, 66: 709-711.
[12] V.Hellwig, T.Grothe, A. Mayer-Bartschmid, et al. Altersetin, a new antibiotic from cultures of endophytic Alternaria spp. Taxonomy, fermentation, isolation, structure elucidation and biological activities [J]. J Antibiot. 2002, 55: 881-892.
[13] F.Pelaez, A.Cabello, G.Platas, et al. The discovery of enfumafungin, a novel antifungal compound produced by an endophytic Hormonema species biological activity and taxonomy of theproducing organisms [J]. Syst Appl Microbiol. 2000, 23: 333-343.
[14] S.Kim, D.S.Shin, T.Lee, et al. Periconicins, two new fusicoccane diterpenes produced by an endophytic fungus Periconia sp. with antibacterial activity [J]. J Nat Prod.2004, 67: 448-450.
[15] S.F. Brady, J.Clardy. CR377, a new pentaketide antifunga agent isolated from an endophytic fungus [J]. Jnat Prod.2000, 63:1447-1448
[16] 郭良栋.内生真菌研究进展[J].菌物系统. 2001,20 (1 ): 148-152,
[17] 李强,刘军,周东坡,等.植物内生菌的开发与研究进展[J].生物技术通报.2006,(3):33-36
[18] J.K. Stone, C.E. Bacon, J.F.Jr White. An overview of endophytic microbes: endopytism defined [J]. Microbial Endophytes. 2000:3-29.
[19] W. X.Zou, R.X.Tan. Biological and chemical diversity of endophytes and their potential application[J]. Advances in Plant Sciences. 1999: 2: 183-190
[20] G.A.Strobel, B.Daisy.Bioprospecting for Microbial Endophytes and Their Natural Products [J]. Microbiology and Molecular Biology Reviews. 2003, 4 (67): 491-502
[21] 何红,蔡学清,洪永聪.辣椒内生细菌的分离及拮抗菌的筛选[J].中国生物防治. 2002,18(4):171-175
[22] 袁军, 孙福在.防治马铃薯环腐病有益内生细菌的分离和筛选[J].微生物学报. 2002,42(3):270-274
[23]何红,蔡学清等.内生菌BS-2菌株的抗菌蛋白及其防病作用[J].植物病理学报. 2003,33(4):373-378
[24] Y.M.Ma, Y.Li, J.Y.Liu, et al. Anti-Helicobacter pylori metabolites from Rhizoctonia sp. Cy064, an Endophytic fungus in Cynodon dactylon [J]. Fitoterapia.2004, 75: 451-456.
[25] U.Castillo, J.K.H, G.A.Strobel.Kakadumycins novel antibiotics from treptomyces sp. NRRL30566, an endophyte of Grevillea pteridifolia [J]. FEMS Microbiolog Letters.2003, 224:183-190
[26] 邹文欣,谭仁祥.植物内生菌研究新进展[J].植物学报.2001, 43(9): 881-892.
[27] 李桂玲,王建锋,黄耀坚,等.几种药用植物内生真菌抗真菌活性的初步研究[J].微生物学通报. 2001,28(6):42-46
[28] 彭小伟,杨丽源,陈有为,等.黄花夹竹桃内生真菌抗病原细菌的初步研究[J]菌物研究. 2003,1(1):33-36
[29] 李能章,彭远义.一株芦荟抗菌内生细菌的分离鉴定及生物学性质研究[J].生物技术通讯2004,2(15):89-92
[30] 孙力军,陆兆新.植物内生菌抗菌活性物质研究进展[J].食品与发酵工业-综述与专题评论. 2005, 2(31):51-57
[31] G.A.Strobel, R.V.Miller, C.Miller, et al. Cryptocandin, a potent antimycotic from theendophytic fungu Cryptosporiopsis cf. quercina[J]. Microbiol.1999, 145:1919-1926
[32] J.Y.Li, G.A. Strobel, J.Harper ,et al. Cryptocin, a potent tetramic acid antimycotic from the endophytic fungu Cryptosporiopsis cf. quercina [J]. Org Lett. 2000:2767-770
[33] S.F.Brady, J.Clardy. CR377, a new pentaketide antifunga agent isolated from an endophytic fungus [J]. Jnat Prod. 2000, 63:1447-1448
[34] U.F.Castillo, G.A.Strobel, E.J.Ford, et al. Munumbicins, wide spectrum antibiotics produced by Streptomyces (NRRL30562) endophytic on Kennedianigriscans [J]. Microbiology. 2002, 148:2675-2685
[35] 黄午阳.植物内生真菌的抗菌活性研究[J]. 南京中医药大学学报. 2005. 1(21): 16-23
[36] F.U.Castillo, G. A. Strobel, E.J.Ford, et al. Munumbicins, wide-spectrum antibiotics produced by Streptomyces NRRL 30562,endophytic on Kennedia nigriscans [J]. Microbiology. 2002, 148:2675-2685
[37] 徐叔云,卞如濂,陈修.药理实验方法学[M].北京:人民卫生出版社, 2001.1712-1714
[38] 马绪荣,苏德模.药品微生物学检验手册[M].北京:科学出版社.2000:222
[40] 林爱玉,邢晓科,郭顺星.4种药用半红树植物内生真菌的分离及其抗菌活性研究[J].中国药学杂志.2006, 41(12):892-894
[41] H.Lu, W.XZou, J.C.Meng, et al. New bioactive metabolites produced by Colletotrichum sp., an endophytic fungus in Artemisia annua [J]. Plant Science. 2000,151: 67-73
[42] C.U.Castillo, A.K. Harper, G.A.Strobe, et al. Kakadumycins, novel antibiotics from treptomyces sp. NRRL 30566, an endophyte of Grevillea pteridifolia [J].FEMS Microbiology Letters. 2003, 224 :183-190
[43] K.A.Southcott, J.A.Johnson. Isolation of endophytes from two species of palm, from Bermuda [J]. Can J Microbial. 1997, 43: 789-792
[44] 杨文博.微生物学实验[M].北京:化学工业出版社.2004,7:217-218
[45] 杨正强,张耀兮,陈小静,等.华重楼内生菌SS02的分离与抗菌活性的初步研究[J]. 微生物学通报.2006年33(2):54-57
[46] C.H.Liu, W.X.Zou, H.Lu, et.al. Antifungal activity of Artemisia annua endophyte cultures against phytopathogenic fungi[J]. Journal of Biotechnology. 2001,88:277-282
[47] 徐叔云,卞如濂,陈修.药理实验方法学[M].北京:人民卫生出版社(3版).2001:1712-1713
[48] 徐任生,陈仲良.中草药有效成分提取与分离(第二版)[M].上海:上海科学技术出版社. 1989,12:9-10
[49] 马绪荣,苏德模.药品微生物学检验手册[M].科学出版社.北京:2000,3.211
[50] U. F. Castillo, G. A. Strobel, E. J. Ford, et al. Munumbicins, wide-spectrum antibiotics produced by Streptomyces NRRL 30562,endophytic on Kennedia nigriscans [J]. Microbiology. 2002,148:2675-2685
[51] 张正,岳志红. WST248-2005.厌氧菌的抗微生物药敏感试验方法[S].北京.中华人民共和国卫生部:11-12
[52] 马绪荣,苏德模.药品微生物学检验手册[M].科学出版社.北京:2000,3:214
[53] 苏印泉,朱红薇,马希汉,等.杜仲内生真菌的抑菌活性筛选[J].西北植物学报. 2005,25(6):1153-1157
[54] 李萌.蔬菜内生放线菌的分离和生物活性初探[D]. 中国科学院研究生院(大连化学物理研究所). 2003:23-26
[55] 朱红薇.杜仲内生真菌活性物质的研究[D]. 西北农林科技大学:2005.31
[56] 霍娟.杜仲内生真菌资源和活性菌株生物学的初步研究[D]. 南京师范大学:2004.17-18
[57] 杨文博.微生物学实验[M].北京:化学工业出版社,2004.7:127
[58] 李能章.植物抗菌内生菌A11的分离鉴定及其活性物质的初步研究[D]. 西南农业大学.2004:36-52
[59] 邵力平,沈瑞祥,张素轩,等.真菌分类学[M]. 北京:中国林业出版社.1984.354-460
[60] 沈瑞祥,刘碧荣译.真菌—高级论文集第四卷 A.B 册(检索部分).北京:1981
[61] 魏景超.真菌鉴定手册[M].上海:上海科技出版社.1979:611-640
[62] 魏景超.真菌鉴定手册[M].上海:上海科技出版社.1979:611-632
[63] 杨文博.微生物学实验[M].北京:化学工业出版社.2004.7:217-223
[64] Poulev, O’Neal J M, L. S. Elicitation.a new window into plant chemodiversity and phytochemical drug discovery [J] Med Chem.2003.46(12):2542-2547
[65] 曾松荣 徐成东 王海坤 张玲琪.药用植物内生真菌及其具宿主相同活性成分的机制初探[J].中草药.2000, 31(4):65-72
[66] 刘志民.药用植物治疗各种结核病俄语文献50年的研究与进展[J]. 中药研究与信息.2000,2(9):44-50
[67] 孙力军,陆兆新,别小妹.株抗菌植物内生菌EJH-2菌株的分离和鉴定[J].中国微生态学杂志.2006,18(1):23-26.
[68] 纪丽莲.芦竹内生真菌F0238细胞生长及其代谢调控[J].微生物学通报.2005,32(4):51-56
[69] 徐庆妍.两株红树植物内生真菌代谢物及抗菌抗肿瘤活性研究[D]. 厦门大学.2004:82
[70] 邓墨渊,王伯初,杨再昌,等.分子生物学技术在植物内生菌分类鉴定中的应用[J].氨基酸和生物资源 2006,28(3):9-14
[71] 孙奎.杜仲和无花果内生真菌的初步研究[D].西北农林科技大学.2004:35
[72] 孙建华,宇克莉,路福平,等.丝状真菌Sr18发酵过程中的形态学研究[J].天津科技大学学报. 2005.20(4):1-5