苦皮藤内生真菌农用活性成分的研究
|
摘要
本论文以一株编号为 2B 的苦皮藤内生真菌为出发菌株，应用形态学的方法鉴定了
其种属地位；研究了其代谢产物的农用生物活性，并对目标活性物质进行分离和结构解
析，采取诱变育种手段选育了高产菌株，并对高产菌株的发酵条件进行了优化。通过试
验结果的分析和讨论，取得了以下结论：
1 根据真菌的分类及鉴定方法，用光学显微镜观察 2B 菌株气生菌丝、大型分生孢
子、小型分生孢子及菌落性状，对比相关分类系统，将 2B 菌株鉴定为层出镰刀菌
(Fusarium proliferatum)。
2 在对 2B 菌株发酵产物生物活性研究中，发现 2B 菌株发酵产物甲醇提取物有明
显的抑菌活性，在 1000 μg·mL-1浓度下对番茄早疫病菌（Alternaria solani）、烟草赤星
病菌（Alternaria alternata）、小麦根腐病菌（Bipolaris sorokiniana）、玉米大斑病菌
（Exserohilum turcicum）4 种供试菌的菌丝生长和孢子萌发抑制率均大于 80%；在盆栽
试验中，对黄瓜霜霉病菌（Pseudoperonospora cubensis）的治疗和保护作用均在 70%以
上。
3 采用常压柱层析、HPLC 制备等方法，在生物活性追踪指引下，从 2B 菌株发酵
产物分离出 4 个化合物 2B-1，2B-2，2B-3，2B-4。经波谱技术( IR，1H NMR，13C NMR，
COSY，MS)，结合化学检验方法，并与相关文献对比，将分离得到的化合物分别鉴定
为 enniatin B，enniatin B1，enniatinA1，ergosterin。化合物 2B-1，2B-2，2B-3 在 100μg·mL-1
浓度下，对玉米大斑病原菌菌丝生长抑制率分别为 68.18%，87.49%，90.91%。
4 建立了恩镰孢菌素（enniatins）的 HPLC 检测方法。检测条件为色谱柱 (C18 柱，
0.9×30cm,10μm)；流动相(V∶V)甲醇∶水(88∶12)；流速 1 mL·min-1；检测波长
UV229nm；室温下 5μL 进样，该方法相对标准偏差 RSD< 1%，平均回收率在 98%以上。
5 采用紫外线（UV）,甲基磺酸乙酯（EMS），超声波+UV + EMS 3 种诱变方式进
行复合诱变，同时结合形态变异与产量之间的相关关系，挑选形态差异明显的单菌落，
然后通过摇瓶发酵筛选 enniatins 高产菌株，通过 3 轮诱变筛选大幅度提高了菌种的生
产能力，最后得到的突变株 UE152 代谢 enniatinns 总产量为 59.72 mg·100mL-1，是出发
菌株摇瓶效价 2.9 倍，且该突变株的高产遗传特性稳定。
6 通过单因子试验和多因子正交试验筛选获得 UE152 菌株最佳的摇瓶培养基配方
和发酵条件为葡萄糖 2%、土豆 20%、蛋白胨 0.5%、NH4Cl 0.3%、MgS040.1%和 CaCO3
0.1%，培养温度 26℃，接种量 10 块菌饼（每 50 mL 发酵液），初始 pH 值为 6.0~7.0，
装液量 50 mL（每 250 mL 三角瓶），转速 160rpm，发酵时间 120h。在上述实验条件下
enniatinns 总产量可达 88.68 mg·100mL-1，从正交试验结果分析当中看出，如果适当增加
通气量，产量可望更高。
Endophytic fungus of strain 2B isolated from Celastrus angulatus has been studied on the taxonomy,
agricultural bioactivities, isolation and identification of active components, breeding of high yield strains,
and optimization of fermentation conditions in this paper. The main results are as follows:
1 By means of observation of aerial mycelium, macroconidia, microconidia and colony morphology
characteristics of strain 2B using optics microscope, comparised with related classify system, strain 2B was
identified as Fusarium proliferatum.
2 The agricultural bioactivities of the strain 2B fermentation products were tested. The results showed
that the methanol extracts of mycelium had evident fungicidal activity. The extracts was tested against
Alternaria solani, Alternaria longipes, Bipolarissorokiniana shoem, Exserohilum turcicum, the results
indicated that the inhibiting rates against mycelium growth and spore germination were both more than
80% at the concentration of 1000 μg·mL-1. The results of pot test indicated that the methanol extracts of
mycelium exhibited more than 70% for both protective efficacy and therapeutic efficacy against
Pseudoperonospora cubensis disease at the same concentration.
3 By means of open column chromatography on silica gel and preparative HPLC, four compounds,
2B-1, 2B-2, 2B-3, 2B-4, were isolated from the methanol extracts of fermentation products of strain 2B.
On the basis of spectral technology (MS, FT-IR, H NMR, 1 13C NMR, COSY), chemical methods and
comparison with related reports of literatures, their structures were identified as enniatinB, enniatinB1,
enniatinA1 and ergosterin, respectively. At the concentration of 100 μg·mL-1, the inhibiting rates of
compound 2B-1, 2B-2, 2B-3 against the mycelium growth of Exserohilum turcicum were 68.18%, 87.49%,
90.91%, respectively.
4 The analytical method of HPLC for enniatins was set up. The operating conditions were chromatogram
column Hypersil ODS (C18, 300mm × 9mm i.d.，10 μm), inject volume 5μL at room temperature,
methanol-water (88:12, V/V) as mobile phase with a flow rate of 1.0mL·min-1and UV detection at 229nm.
The relative standard deviation (RSD) was less than 1%, and the average recovery was over 98%.
5 Using Endophytic fungus of strain 2B as starting strain, mutagenized with ultraviolet (UV) , EMS and
ultrasonic+UV + EMS in sequence, at the same time ,based on the relationship between the variation of
modality and the capcity of the mutants’ yield, the mutant strains which had great variation of modality
were selected. Through three times mutagenized and the shake flask fermentation test, a mutant UE-152
high-producing enniatins was obtained, which had much higher productive qualitied than its original strain.
Its enniatins yield reached 59.72 mg·100mL-1 as 2.9 times high as that of original strain, and the heredity
character of the high productivity was stable.
6 The optimum composition of culture medium and fermentation conditions of the strain UE152 were
demonstrated by the test of uni-factor and orthogonal design, respectively. The optimum culture medium
consisted of glucose 2%, potato 20%, peptone 0.5% , NH4Cl 0.3%, MgS04 0.1%, CaCO3 0.1% and original
pH6.0-7.0. Inoculum volume was 10 entries per 50mL medium. A 250mL flask containing 50mL medium
was cultivated at 26℃ for 120h and the rotatory velocity was 160 rpm. Under the best culture conditions,
the general yield of Enniatins was 88.68 mg·100 mL-1. The results of orthogonal test showed if increased
the aeration level, the yield could be higher.
其种属地位；研究了其代谢产物的农用生物活性，并对目标活性物质进行分离和结构解
析，采取诱变育种手段选育了高产菌株，并对高产菌株的发酵条件进行了优化。通过试
验结果的分析和讨论，取得了以下结论：
1 根据真菌的分类及鉴定方法，用光学显微镜观察 2B 菌株气生菌丝、大型分生孢
子、小型分生孢子及菌落性状，对比相关分类系统，将 2B 菌株鉴定为层出镰刀菌
(Fusarium proliferatum)。
2 在对 2B 菌株发酵产物生物活性研究中，发现 2B 菌株发酵产物甲醇提取物有明
显的抑菌活性，在 1000 μg·mL-1浓度下对番茄早疫病菌（Alternaria solani）、烟草赤星
病菌（Alternaria alternata）、小麦根腐病菌（Bipolaris sorokiniana）、玉米大斑病菌
（Exserohilum turcicum）4 种供试菌的菌丝生长和孢子萌发抑制率均大于 80%；在盆栽
试验中，对黄瓜霜霉病菌（Pseudoperonospora cubensis）的治疗和保护作用均在 70%以
上。
3 采用常压柱层析、HPLC 制备等方法，在生物活性追踪指引下，从 2B 菌株发酵
产物分离出 4 个化合物 2B-1，2B-2，2B-3，2B-4。经波谱技术( IR，1H NMR，13C NMR，
COSY，MS)，结合化学检验方法，并与相关文献对比，将分离得到的化合物分别鉴定
为 enniatin B，enniatin B1，enniatinA1，ergosterin。化合物 2B-1，2B-2，2B-3 在 100μg·mL-1
浓度下，对玉米大斑病原菌菌丝生长抑制率分别为 68.18%，87.49%，90.91%。
4 建立了恩镰孢菌素（enniatins）的 HPLC 检测方法。检测条件为色谱柱 (C18 柱，
0.9×30cm,10μm)；流动相(V∶V)甲醇∶水(88∶12)；流速 1 mL·min-1；检测波长
UV229nm；室温下 5μL 进样，该方法相对标准偏差 RSD< 1%，平均回收率在 98%以上。
5 采用紫外线（UV）,甲基磺酸乙酯（EMS），超声波+UV + EMS 3 种诱变方式进
行复合诱变，同时结合形态变异与产量之间的相关关系，挑选形态差异明显的单菌落，
然后通过摇瓶发酵筛选 enniatins 高产菌株，通过 3 轮诱变筛选大幅度提高了菌种的生
产能力，最后得到的突变株 UE152 代谢 enniatinns 总产量为 59.72 mg·100mL-1，是出发
菌株摇瓶效价 2.9 倍，且该突变株的高产遗传特性稳定。
6 通过单因子试验和多因子正交试验筛选获得 UE152 菌株最佳的摇瓶培养基配方
和发酵条件为葡萄糖 2%、土豆 20%、蛋白胨 0.5%、NH4Cl 0.3%、MgS040.1%和 CaCO3
0.1%，培养温度 26℃，接种量 10 块菌饼（每 50 mL 发酵液），初始 pH 值为 6.0~7.0，
装液量 50 mL（每 250 mL 三角瓶），转速 160rpm，发酵时间 120h。在上述实验条件下
enniatinns 总产量可达 88.68 mg·100mL-1，从正交试验结果分析当中看出，如果适当增加
通气量，产量可望更高。
Endophytic fungus of strain 2B isolated from Celastrus angulatus has been studied on the taxonomy,
agricultural bioactivities, isolation and identification of active components, breeding of high yield strains,
and optimization of fermentation conditions in this paper. The main results are as follows:
1 By means of observation of aerial mycelium, macroconidia, microconidia and colony morphology
characteristics of strain 2B using optics microscope, comparised with related classify system, strain 2B was
identified as Fusarium proliferatum.
2 The agricultural bioactivities of the strain 2B fermentation products were tested. The results showed
that the methanol extracts of mycelium had evident fungicidal activity. The extracts was tested against
Alternaria solani, Alternaria longipes, Bipolarissorokiniana shoem, Exserohilum turcicum, the results
indicated that the inhibiting rates against mycelium growth and spore germination were both more than
80% at the concentration of 1000 μg·mL-1. The results of pot test indicated that the methanol extracts of
mycelium exhibited more than 70% for both protective efficacy and therapeutic efficacy against
Pseudoperonospora cubensis disease at the same concentration.
3 By means of open column chromatography on silica gel and preparative HPLC, four compounds,
2B-1, 2B-2, 2B-3, 2B-4, were isolated from the methanol extracts of fermentation products of strain 2B.
On the basis of spectral technology (MS, FT-IR, H NMR, 1 13C NMR, COSY), chemical methods and
comparison with related reports of literatures, their structures were identified as enniatinB, enniatinB1,
enniatinA1 and ergosterin, respectively. At the concentration of 100 μg·mL-1, the inhibiting rates of
compound 2B-1, 2B-2, 2B-3 against the mycelium growth of Exserohilum turcicum were 68.18%, 87.49%,
90.91%, respectively.
4 The analytical method of HPLC for enniatins was set up. The operating conditions were chromatogram
column Hypersil ODS (C18, 300mm × 9mm i.d.，10 μm), inject volume 5μL at room temperature,
methanol-water (88:12, V/V) as mobile phase with a flow rate of 1.0mL·min-1and UV detection at 229nm.
The relative standard deviation (RSD) was less than 1%, and the average recovery was over 98%.
5 Using Endophytic fungus of strain 2B as starting strain, mutagenized with ultraviolet (UV) , EMS and
ultrasonic+UV + EMS in sequence, at the same time ,based on the relationship between the variation of
modality and the capcity of the mutants’ yield, the mutant strains which had great variation of modality
were selected. Through three times mutagenized and the shake flask fermentation test, a mutant UE-152
high-producing enniatins was obtained, which had much higher productive qualitied than its original strain.
Its enniatins yield reached 59.72 mg·100mL-1 as 2.9 times high as that of original strain, and the heredity
character of the high productivity was stable.
6 The optimum composition of culture medium and fermentation conditions of the strain UE152 were
demonstrated by the test of uni-factor and orthogonal design, respectively. The optimum culture medium
consisted of glucose 2%, potato 20%, peptone 0.5% , NH4Cl 0.3%, MgS04 0.1%, CaCO3 0.1% and original
pH6.0-7.0. Inoculum volume was 10 entries per 50mL medium. A 250mL flask containing 50mL medium
was cultivated at 26℃ for 120h and the rotatory velocity was 160 rpm. Under the best culture conditions,
the general yield of Enniatins was 88.68 mg·100 mL-1. The results of orthogonal test showed if increased
the aeration level, the yield could be higher.
引文
[1]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
[2]陈世苹，高玉葆，内生真菌与禾本科植物之间的关系及其生态学意义[J]，生态学杂志，2000，19
（5）：52-57
[3]李保军，郑晓红等，新疆部分禾草的植物内生真菌调查[J]，中国草地，1996，2：29-32.
[4]马天有，董兆麟，植物分离产紫杉醇的内生真菌的研究[J]，西北大学学报，1999，29（1）：47-49
[5]李海燕，王志军，张玲琪等，一种桃儿七内生真菌的分离初报[J]，云南大学学报(自然科学版)，
1999，21（3）：243
[6]王伟，贺雄雷，钟英长，南方红豆杉内生菌及紫杉烷类产物的初步鉴定[J]，中山大学学报(自然科
学版），1999，38（3）：116-118
[7]邱德有，黄美娟，方晓华等，一种云南红豆杉内生真菌的分离[J]，真菌学报，1994，13（4）：314-316
[8]王建锋，吕华鹰，黄耀坚等，一株新的紫杉醇产生菌及其抗肿瘤活性[J]，厦门大学学报(自然科学
版)，1999，18（4）：485-487
[9]郭良栋，内生真菌研究进展[J]，菌物系统，2001，20（1）：148-152
[10]高微微，郭顺星，三种内生真菌对铁皮石、金线莲生长影响的研究[J]，中草药，2002，33（6）：
543-545
[11]王剑文，夏忠豪，谭仁样，内生真菌 Colletotrichum sp. B501 的寡糖提取物对黄花蒿发根中青蒿
素生物合成的诱导[J]，植物学报，2002，44（10）：1233-1238
[12]梁宇，高玉葆，内生真菌对植物生长及抗逆性的影响[J]，植物学通报，2000，17（1）：52-59
[13]任安芝，高玉葆，李侠，内生真菌感染对黑麦若干抗旱生理特征的影响[J]，应用与环境生物学
报，2002，8（5）：535-539
[14]陈世苹，高玉葆，梁宇等，水分胁迫下内生真菌感染对黑麦草叶内游离脯氨酸和脱落酸含量的影
响[J]，生态学报，2001，21（12）：164-172
[15]梁宇，陈世苹，高玉葆等，内生真菌感染对干旱胁迫下黑麦草生长的影响[J]，植物生态学报，
2002，26（5）：621-626
[16]吴静萍，郑师章，密花石解菌根菌分离鉴定及其代谢产物的测定[J]，复旦学报，1994, 33（5）：
547-552
[0][17]张集慧，王春兰，郭顺星等，兰科药用植物的 5 种内生真菌产生的植物激素[J]，中国医学科
学学院学报，1999, 21（6）：460-465
[18]江东福等，龙血树真菌群及其对血竭形成的影响[J]，云南植物研究，1995，17（l）：79-81
[19]姜威等，东北红豆衫内生真菌代谢产物的化学研究[J]，中国抗生素杂志，1998，23（4）：263-266
[20]王建锋，吕华鹰，黄耀坚，郑忠辉，苏文金，一株新的紫杉醇产生菌及其抗肿瘤活性[J]，厦门
大学学报（自然科学版），1999，38（4）：485-487
[21]王建锋等，植物内生真菌产紫杉醇的研究[J]，微生物学通报，2000，27（1）：58-60
[22]邱观荣，紫杉醇植物内生真菌来源及其生物多样性[J]，国外医药—抗生素分册，1998，19（4）：
265-266
[23]郭波，李海燕，张玲琪，一种产长春碱真菌的分离[J]，云南大学学报（自然科学版），1998，20
（3）：214-215
42 苦皮藤内生真菌农用生物活性的研究
[24]张玲琪, 对鸢尾新鲜根状茎发酵生香的微生物研究[J]，云南大学学报（自然科学版），1997，19
（4）：362-365.
[25]张玲琪等，发酵产莺尾酮真菌的分离鉴定及产香特性的初步研究[J]，菌物系统，1999，18（1）：
49-54
[26]梁宗琦，真菌次生代谢产物多样性及其潜在应用价值[J]，生物多样性，1997，7（2）：145-150
[27]翟中和编，细胞生物学[M]，北京：高等教育出版社，1995，379-382
[28]王伯荪，李鸣光，彭少麟等，植物种群学[M]，广州：广东高等教育出版社，1995：294-296
[29]曾松荣等，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）：
306-308
[30]林永成主编，海洋微生物及其代谢产物[J]，北京：化学工业出版社，2003，406-408
[31]兰琪，硕士学位论文，西北农林科技大学，2002
[32]王拱辰，陈辉珍，促进镰刀菌产孢的培养基[J]，植物病理学报，1994，25（2）：165-166
[33]张纪忠，微生物分类学[M]，上海：复旦大学出版社，1990，358-361
[34]慕立义,吴文君,王开义，植物化学保护研究方法[M]，中国农业出版社，1994
[35]陈年春等，农药生物测定技术[M]，北京农业大学出版社，1991
[36]方中达，植病研究方法[M]，中国农业出版社，1998
[37]张玲琪，王海坤，邵华，美登木内生真菌产球毛壳甲素的分离与鉴定[J]，中国药学杂志，2002，
37（3）：172-175
[38]王美芬，韦荣芳，陈家源等，密花美登木抗癌成分的研究[J]，药学学报，1981，16（1）：59-62
[39]余卿，抗菌素菌种选育[J]，微生物杂志，1987，7（2）：75-83
[40]姚汝华，微生物工程原理[M]，广州：华南理工大学出版社，1997
[41]陶文沂，工业微生物生理与遗传育种学[M]，北京：中国轻工业出版社，1996
[42]工业技术资料汇编（第四辑），微生物的选种和育种[M]，上海：上海人民出版社，1972
[43]陈代杰，朱宝泉，工业微生物菌种选育与发酵控制技术[M]，上海：上海科学技术文献出版社，
1995
[44]周成，邵华等，植物内生真菌研究的应用潜力分析[J].天然产物研究与开发，2002，14（2）：69-72
[45]姜广策，林永成等，中国南海红树内生真菌 NO.1403 次级代谢物的研究[J]，中山大学学报（自
然科学版），2000，39（6）：68-72
[46]周东坡，平文祥，紫杉醇产生菌分离的研究[J]，微生物学杂志，2001，21（1）：18-20
[47]宁文，曹日强，真菌诱导物在植物次生代谢中的调节作用[J]，植物生理学通讯，1993，29（5）：
321-329
[48]杜道林.我国生物农药产业及其发展.海南师范学院学报（自然科学版）,2001，14（2）：36～37
[49]肖林，黄为一，尼日利亚菌素高产菌株的选育[J]，微生物学通报，2002，29（4）：59-62
[50]孟昭礼，吴献忠等，银杏提取液对四种植物病原菌的抑菌作用[J]，植物病理学报，1995，25（4）：
357-360
[51]周德庆，生物学实验方法[M]，上海：上海科学技术出版社，1986
[52]王琦，梅汝鸿，我国生物农药的发展现状[J]，中国微生态学杂志，2001，13（3）：177-178
[53]孙广宇，宗兆锋，植物病理学实验技术[M]，北京：中国农业出版社，2002
参考文献 43
[54]Stone J K, Bacon C W,White J F Jr.An overview of endophytic microbes:endophytism defined.Bacon
C W,White J F Jr.Microbial Endophytes.New York,Marcel Dekker, 2000,3:267-274
[55]Bacon C W,Porter J K,Robbins J D,et al.Epichloe typhina from toxic tall fescue grasses. Appl Environ
Mcrobiol,1977,34:576-581
[56]Glenn A,Bacon C W,Price R, et al. Molecular phylogeny of Acremonium and its taxono-mic
implications. Mycologia,1996,88:369-383
[57]Prestidge R A et al.An association of Lolium endophyte with ryegrass resistance to Argentine stem
weevil.Proc .N .Z.Weed Pest Control Conf.,1982,35:119-122
[58] Stierle A, Strobel G, Stierle D.Taxol and taxane production by Taxomyces andreana, an endophytic
fungus of Pacific yew. Science, 1993,260:214-216
[59] Zhang J Z, Zhang L H, Wang X H et al. Microbial transformation of 10-deacetyl-7-epitaxol and 1
B-hydroxybaccatin 1, by fungi from the inner bark of Taxus yunnanensis. J Nat Prod, 1998,61:
497-500
[60] Clay K. Effects of fungal endophytes on the seed and seedling biology of Lolium pernne and Festuca
arundinacea.Oecologia,1987,73:358-362
[61] Gasoni L,de Gurfinkel B S.The endophyte Cladorrhinum foecundissimum in cotton roots:phosphorus
uptake and host growth.Mycol Res,1997,101:867-870
[62]Marks S,Clay K. Physiological responses of Festuca arundinacea to fungal endophyte infection. New
Phytol,1996,133:727-733
[0][63] Cheplick G P,Clay K. Acquired chemical defense of grasses:the role of fungal endophytes.
Oikis,1988,52:309-318
[64]Funk C R, White R H, Breen J P. Importance of Acremvnium endophytes in turf-grass breeding and
management.Agric Ecosystem Environ, 1993,44: 215-232
[65]White Jr J F, Cole G T. Endophyte-host associations in forage grasses III .In vitro inhibition of fungi by
Acremvnium coenophialum. Mycologia, 1985, 77: 487-489
[66] Lu H,Zou W X, Meng J C,Hu J, Tan R X. New bioactive metabolites produce by Colletotrichum sp,an
endophytic fungus in Arte meisia annua. Plant Sci, 2000, 151:67-73
[0][67]Rowan D D. Lolitre ms,peramine and paxilline-mycotoxins of the ryegrass-endophyte interaction.
Agric Ecosyst Environ,1993,44:103-122
[0][68]Breen J P. Acremonium endophyte interactions with enhanced P1ant resistance to insects. Annv. Rev.
Etomol,1994,39:401-423
[69]Ju Y et al. Bioactive flavonoids from endophyte infected blue grass(Poa ampla).J Agric Food Chem,
1998,46:3785-3788
[70]Findlay J A, Li G, Penner P E, Miller J D.Novel diterpenoid insect toxin from a conifer endophyte. J
Nat Prod, 1995, 58:197-200
[71]Findlay J A, Buthelezi S, Lavoie R, Pena-Rodriguez L, Miller J D. Bioactive isocou marins and related
metabolites from conifer endophytes.J Nat Prod,1995,58:1759-1766
[72]Findlay J A, Li G, Johnson J A. Bioactive compounds from needles.Can J Chem, 1997, 75:716-719
44 苦皮藤内生真菌农用生物活性的研究
[73]Findlay J A, Buthelezi S, Li G, Seveck M, Miller J D. Insect toxins from an endophytic fungus from
wintergreen. J Nat Prod, 1997,60:1214-1215
[74]Strobel G A, Miller R V, Martinez-Miller C,Condron M M, Teplow D B[0],Hess W M.Cryptocandin,a
potent antimycotic from the endophytic fungus Cryptosporiopsis cf.
quercina.Mcrobiology,1999,145:1919-1926
[75]Li J Y,Strobel G A, Harper J,Lobkovsky E,Clardy J. Cryptocin,a potent tetramic acid antimycotic from
the endophytic fungus Cryptosporiopsis cf.quercina.Org Lett，2000,2:767-770
[76]Lu H,Zou W X, Meng J C,Hu J,Tan R X. New bioactive metabolites produced by
Colletotrichum sp.，an endophytic fungus in Artemisia annua.Plant Sci,2000,151:67-73
[77]Zou W X, Meng J C,Lu H,Chen G X, Shi G X, Zhang T Y,Tan R X. Metabolites of Colletotrichum
gloeosporioides,an endophytic fungus in Artemisia mongolica.J Nat Prod,2000,63:1529-1530
[78] Liang Y, Gao Y B, Chen S P, et a1. Effects of endophyte infection on photosythensis, transpiration and
water use efficiency of Lolium perenne under dought stress.Acta Phytoecologica Sinica, 2001，
26(5):537-543
[79]Wagenaar M M, Corwin J,Strobel G, Clardy J.Three new cytochalasins produced by an endophytic
fungus in the genus Rhinocladiella.J Nat Prod,2000,63:1692-1695
[80]Lee J C,Strobel G A,Lobkovsky E and C1ardy J.Torreyanic acid:a selectively cytotoxic quinone
dimmer from the endophytic fungus Pestalotiopsis microspora.Journal of Organic
Chemistry,1996,61(10):3232-3233
[81]TL Stanford, NP Stafford, LC Coelho, etc. Production and characterization of a thermostable
alpha-amylase from Nocardiopsis sp. endophyte of yam bean.Bioresour Technol, 2001,76 (2):137-41
[0][82]TL Stamford, NP Stamfod,LC Coelho, etc. Production and characterization of a thermostable
glucoamylase from Streptosporangium sp. endophyte of maize leaves.Bioresour. Technol. 2002,83(2):
105-109
[83]Gaumann E,et al.Enniatin, a new antibiotic active against Mycobacteria. Experientia，1947,9:202-203
[84]Hiroshi Tomoda，Hiroyuki Nishida，et al.New Cyclodepsipeptides, Enniatins D E and F produced by
Fusarium sp,FO-1305. J Antibiotics,1992,45:1207-1215
[0][85]Tawnya C,McKee,Heidi R, Bokesch,Jinping L.McCormick,et al.Isolation and Characteri- zation of
New Anti-HIV and Cytotoxic leads from Plants，Marine，and Microbial Organism.J Natural
Products,1997, 60(5):431-438
[0][86]Lin Y C,Wang j,Wu X Y,et al.A Novel Compound Enniatin G from the Mangrove fungus Fungus
Halosarpheia sp.(strain #732)from the South China Sea.Journal of Aust Chem,2002,55(3):225-227
[87]Burgess l W,Summerell B A,Bullock S,et al. Laboratory Manual for Fusarium Research.Sydney: The
university of Sydney press,1994
[88]Guo L D,Hyde K D,Liew E CY.A method to promote sporulation in palm endophytic fungi.Fungal
Diversity,1:109-113
[89]Fisher P J,Petrini O,Petrini L E,Sutton B C.Fungal endophytes from the leaves and twigs of Quercus
ilex L.from England,Marjorca and Switzerland.New Phytol,127:133-137
参考文献 45
[90]Guo L D,Hyde K D.Indentification of endophytic fungi from Livistona chinensis based on morphology
and rDNA sequences. New Phytol,147:617-630
[91]Audhya T K, Russell D W. Production of enniatins by Fusarium sambulinum: selection of high-yield
conditions from liquid surface culture.Journal of General Microbiology, 1974, 82:181-196
[92]Blackwell B A,Miller J D,Apsimin J W,et al.Isolation and characterijatim of enniatins from
Fusarium-Avenaceum DAOM 196490.Canadian Journal of Chemistry, 1992, 70: 5-10
[93]Sauriol F,Hynes R C,Xu X J, et al.Novel Quinajolinones and enniatins from Fusarium-Laterium Nees.
Canadian Journal of Chemistry, 1993, 71: 9-16
[94]Bishop D L,et al.Seedlborne fungal contamination:Consequences in spacegrown wheat.
Phytopathology,1997,87:1125-1133
[95]Brady S F,Clardy J.CR377,a new pentaketide antifungal agent isolated from an endophytic fungus.J
Nat Prod,2000,2:1447-1448
[96]Clay K,Holah J.Fungal endophyte symbiosis and plant diversity in successional
fields.Science,1999,285:1742-1745
[97]Guo B,Dai J R,et al.Cytonic acids A and B:novel tridepside inhibitors of hCMV protease from the
endophytic fungus Cytonaema species.J Nat Prod,2000,63:602-604
[98]Melissa M,et al.Dlicerandrols new antibiotic and cytotoxic dimmers produced by the fungus
Phomopsis longicolla isolated from an endan gered Mint.Journal of Natural
Products,2001,64:1006-1009
[99]Nisbet L J,Fox F M.The importance of microbiodiversity to biotechnology.The Biodiversity of
Microorganisms and Invertebrates:Its Role in Sustainable Agriculture,1991,229-244
[100]Singh M P,Janso J E,et al.Biological activity of guanacastepene,a novel diterpenoid antibiotic
produced by an unidentified fungus CR115.J antibiot,2000,53:256-261
[101]Sturz,A.V,et al.Biodiversity of endophytic bacteria which colonize red clover nodules,roots,stems and
foliage and their influence on host growth.Plant Pathology,1997,48:360-369
[102]Wilson D,Carroll G C.Avoidance of high-endophyte space by gall-forming
insects.Ecology,1997,78:2153-2165
[103] Masahiko I,et al.Phomoxanthones A and B,Novel xanthone dimmers from the endophytic fungus
Phomopsis species.Journal of Natural Products,2001,64:1015-1018
[104]James T,Blodgett ,et al.Species composition of endophytic fungi in Amaranthus bybridus
leaves,Petioles,stems and roots.Microbiogia,2000,92(5):853-859
[105]Clay K,Holah J.Fungal endophyte symbiosis and plant diversity in successional
fields.Science,1999,285:1742-1745
[106]Wilson D.Endophyte the evolution of tem ,and clarification of its, use and definition.
Oikos,1995,73:274-276
[2]陈世苹，高玉葆，内生真菌与禾本科植物之间的关系及其生态学意义[J]，生态学杂志，2000，19
（5）：52-57
[3]李保军，郑晓红等，新疆部分禾草的植物内生真菌调查[J]，中国草地，1996，2：29-32.
[4]马天有，董兆麟，植物分离产紫杉醇的内生真菌的研究[J]，西北大学学报，1999，29（1）：47-49
[5]李海燕，王志军，张玲琪等，一种桃儿七内生真菌的分离初报[J]，云南大学学报(自然科学版)，
1999，21（3）：243
[6]王伟，贺雄雷，钟英长，南方红豆杉内生菌及紫杉烷类产物的初步鉴定[J]，中山大学学报(自然科
学版），1999，38（3）：116-118
[7]邱德有，黄美娟，方晓华等，一种云南红豆杉内生真菌的分离[J]，真菌学报，1994，13（4）：314-316
[8]王建锋，吕华鹰，黄耀坚等，一株新的紫杉醇产生菌及其抗肿瘤活性[J]，厦门大学学报(自然科学
版)，1999，18（4）：485-487
[9]郭良栋，内生真菌研究进展[J]，菌物系统，2001，20（1）：148-152
[10]高微微，郭顺星，三种内生真菌对铁皮石、金线莲生长影响的研究[J]，中草药，2002，33（6）：
543-545
[11]王剑文，夏忠豪，谭仁样，内生真菌 Colletotrichum sp. B501 的寡糖提取物对黄花蒿发根中青蒿
素生物合成的诱导[J]，植物学报，2002，44（10）：1233-1238
[12]梁宇，高玉葆，内生真菌对植物生长及抗逆性的影响[J]，植物学通报，2000，17（1）：52-59
[13]任安芝，高玉葆，李侠，内生真菌感染对黑麦若干抗旱生理特征的影响[J]，应用与环境生物学
报，2002，8（5）：535-539
[14]陈世苹，高玉葆，梁宇等，水分胁迫下内生真菌感染对黑麦草叶内游离脯氨酸和脱落酸含量的影
响[J]，生态学报，2001，21（12）：164-172
[15]梁宇，陈世苹，高玉葆等，内生真菌感染对干旱胁迫下黑麦草生长的影响[J]，植物生态学报，
2002，26（5）：621-626
[16]吴静萍，郑师章，密花石解菌根菌分离鉴定及其代谢产物的测定[J]，复旦学报，1994, 33（5）：
547-552
[0][17]张集慧，王春兰，郭顺星等，兰科药用植物的 5 种内生真菌产生的植物激素[J]，中国医学科
学学院学报，1999, 21（6）：460-465
[18]江东福等，龙血树真菌群及其对血竭形成的影响[J]，云南植物研究，1995，17（l）：79-81
[19]姜威等，东北红豆衫内生真菌代谢产物的化学研究[J]，中国抗生素杂志，1998，23（4）：263-266
[20]王建锋，吕华鹰，黄耀坚，郑忠辉，苏文金，一株新的紫杉醇产生菌及其抗肿瘤活性[J]，厦门
大学学报（自然科学版），1999，38（4）：485-487
[21]王建锋等，植物内生真菌产紫杉醇的研究[J]，微生物学通报，2000，27（1）：58-60
[22]邱观荣，紫杉醇植物内生真菌来源及其生物多样性[J]，国外医药—抗生素分册，1998，19（4）：
265-266
[23]郭波，李海燕，张玲琪，一种产长春碱真菌的分离[J]，云南大学学报（自然科学版），1998，20
（3）：214-215
42 苦皮藤内生真菌农用生物活性的研究
[24]张玲琪, 对鸢尾新鲜根状茎发酵生香的微生物研究[J]，云南大学学报（自然科学版），1997，19
（4）：362-365.
[25]张玲琪等，发酵产莺尾酮真菌的分离鉴定及产香特性的初步研究[J]，菌物系统，1999，18（1）：
49-54
[26]梁宗琦，真菌次生代谢产物多样性及其潜在应用价值[J]，生物多样性，1997，7（2）：145-150
[27]翟中和编，细胞生物学[M]，北京：高等教育出版社，1995，379-382
[28]王伯荪，李鸣光，彭少麟等，植物种群学[M]，广州：广东高等教育出版社，1995：294-296
[29]曾松荣等，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）：
306-308
[30]林永成主编，海洋微生物及其代谢产物[J]，北京：化学工业出版社，2003，406-408
[31]兰琪，硕士学位论文，西北农林科技大学，2002
[32]王拱辰，陈辉珍，促进镰刀菌产孢的培养基[J]，植物病理学报，1994，25（2）：165-166
[33]张纪忠，微生物分类学[M]，上海：复旦大学出版社，1990，358-361
[34]慕立义,吴文君,王开义，植物化学保护研究方法[M]，中国农业出版社，1994
[35]陈年春等，农药生物测定技术[M]，北京农业大学出版社，1991
[36]方中达，植病研究方法[M]，中国农业出版社，1998
[37]张玲琪，王海坤，邵华，美登木内生真菌产球毛壳甲素的分离与鉴定[J]，中国药学杂志，2002，
37（3）：172-175
[38]王美芬，韦荣芳，陈家源等，密花美登木抗癌成分的研究[J]，药学学报，1981，16（1）：59-62
[39]余卿，抗菌素菌种选育[J]，微生物杂志，1987，7（2）：75-83
[40]姚汝华，微生物工程原理[M]，广州：华南理工大学出版社，1997
[41]陶文沂，工业微生物生理与遗传育种学[M]，北京：中国轻工业出版社，1996
[42]工业技术资料汇编（第四辑），微生物的选种和育种[M]，上海：上海人民出版社，1972
[43]陈代杰，朱宝泉，工业微生物菌种选育与发酵控制技术[M]，上海：上海科学技术文献出版社，
1995
[44]周成，邵华等，植物内生真菌研究的应用潜力分析[J].天然产物研究与开发，2002，14（2）：69-72
[45]姜广策，林永成等，中国南海红树内生真菌 NO.1403 次级代谢物的研究[J]，中山大学学报（自
然科学版），2000，39（6）：68-72
[46]周东坡，平文祥，紫杉醇产生菌分离的研究[J]，微生物学杂志，2001，21（1）：18-20
[47]宁文，曹日强，真菌诱导物在植物次生代谢中的调节作用[J]，植物生理学通讯，1993，29（5）：
321-329
[48]杜道林.我国生物农药产业及其发展.海南师范学院学报（自然科学版）,2001，14（2）：36～37
[49]肖林，黄为一，尼日利亚菌素高产菌株的选育[J]，微生物学通报，2002，29（4）：59-62
[50]孟昭礼，吴献忠等，银杏提取液对四种植物病原菌的抑菌作用[J]，植物病理学报，1995，25（4）：
357-360
[51]周德庆，生物学实验方法[M]，上海：上海科学技术出版社，1986
[52]王琦，梅汝鸿，我国生物农药的发展现状[J]，中国微生态学杂志，2001，13（3）：177-178
[53]孙广宇，宗兆锋，植物病理学实验技术[M]，北京：中国农业出版社，2002
参考文献 43
[54]Stone J K, Bacon C W,White J F Jr.An overview of endophytic microbes:endophytism defined.Bacon
C W,White J F Jr.Microbial Endophytes.New York,Marcel Dekker, 2000,3:267-274
[55]Bacon C W,Porter J K,Robbins J D,et al.Epichloe typhina from toxic tall fescue grasses. Appl Environ
Mcrobiol,1977,34:576-581
[56]Glenn A,Bacon C W,Price R, et al. Molecular phylogeny of Acremonium and its taxono-mic
implications. Mycologia,1996,88:369-383
[57]Prestidge R A et al.An association of Lolium endophyte with ryegrass resistance to Argentine stem
weevil.Proc .N .Z.Weed Pest Control Conf.,1982,35:119-122
[58] Stierle A, Strobel G, Stierle D.Taxol and taxane production by Taxomyces andreana, an endophytic
fungus of Pacific yew. Science, 1993,260:214-216
[59] Zhang J Z, Zhang L H, Wang X H et al. Microbial transformation of 10-deacetyl-7-epitaxol and 1
B-hydroxybaccatin 1, by fungi from the inner bark of Taxus yunnanensis. J Nat Prod, 1998,61:
497-500
[60] Clay K. Effects of fungal endophytes on the seed and seedling biology of Lolium pernne and Festuca
arundinacea.Oecologia,1987,73:358-362
[61] Gasoni L,de Gurfinkel B S.The endophyte Cladorrhinum foecundissimum in cotton roots:phosphorus
uptake and host growth.Mycol Res,1997,101:867-870
[62]Marks S,Clay K. Physiological responses of Festuca arundinacea to fungal endophyte infection. New
Phytol,1996,133:727-733
[0][63] Cheplick G P,Clay K. Acquired chemical defense of grasses:the role of fungal endophytes.
Oikis,1988,52:309-318
[64]Funk C R, White R H, Breen J P. Importance of Acremvnium endophytes in turf-grass breeding and
management.Agric Ecosystem Environ, 1993,44: 215-232
[65]White Jr J F, Cole G T. Endophyte-host associations in forage grasses III .In vitro inhibition of fungi by
Acremvnium coenophialum. Mycologia, 1985, 77: 487-489
[66] Lu H,Zou W X, Meng J C,Hu J, Tan R X. New bioactive metabolites produce by Colletotrichum sp,an
endophytic fungus in Arte meisia annua. Plant Sci, 2000, 151:67-73
[0][67]Rowan D D. Lolitre ms,peramine and paxilline-mycotoxins of the ryegrass-endophyte interaction.
Agric Ecosyst Environ,1993,44:103-122
[0][68]Breen J P. Acremonium endophyte interactions with enhanced P1ant resistance to insects. Annv. Rev.
Etomol,1994,39:401-423
[69]Ju Y et al. Bioactive flavonoids from endophyte infected blue grass(Poa ampla).J Agric Food Chem,
1998,46:3785-3788
[70]Findlay J A, Li G, Penner P E, Miller J D.Novel diterpenoid insect toxin from a conifer endophyte. J
Nat Prod, 1995, 58:197-200
[71]Findlay J A, Buthelezi S, Lavoie R, Pena-Rodriguez L, Miller J D. Bioactive isocou marins and related
metabolites from conifer endophytes.J Nat Prod,1995,58:1759-1766
[72]Findlay J A, Li G, Johnson J A. Bioactive compounds from needles.Can J Chem, 1997, 75:716-719
44 苦皮藤内生真菌农用生物活性的研究
[73]Findlay J A, Buthelezi S, Li G, Seveck M, Miller J D. Insect toxins from an endophytic fungus from
wintergreen. J Nat Prod, 1997,60:1214-1215
[74]Strobel G A, Miller R V, Martinez-Miller C,Condron M M, Teplow D B[0],Hess W M.Cryptocandin,a
potent antimycotic from the endophytic fungus Cryptosporiopsis cf.
quercina.Mcrobiology,1999,145:1919-1926
[75]Li J Y,Strobel G A, Harper J,Lobkovsky E,Clardy J. Cryptocin,a potent tetramic acid antimycotic from
the endophytic fungus Cryptosporiopsis cf.quercina.Org Lett，2000,2:767-770
[76]Lu H,Zou W X, Meng J C,Hu J,Tan R X. New bioactive metabolites produced by
Colletotrichum sp.，an endophytic fungus in Artemisia annua.Plant Sci,2000,151:67-73
[77]Zou W X, Meng J C,Lu H,Chen G X, Shi G X, Zhang T Y,Tan R X. Metabolites of Colletotrichum
gloeosporioides,an endophytic fungus in Artemisia mongolica.J Nat Prod,2000,63:1529-1530
[78] Liang Y, Gao Y B, Chen S P, et a1. Effects of endophyte infection on photosythensis, transpiration and
water use efficiency of Lolium perenne under dought stress.Acta Phytoecologica Sinica, 2001，
26(5):537-543
[79]Wagenaar M M, Corwin J,Strobel G, Clardy J.Three new cytochalasins produced by an endophytic
fungus in the genus Rhinocladiella.J Nat Prod,2000,63:1692-1695
[80]Lee J C,Strobel G A,Lobkovsky E and C1ardy J.Torreyanic acid:a selectively cytotoxic quinone
dimmer from the endophytic fungus Pestalotiopsis microspora.Journal of Organic
Chemistry,1996,61(10):3232-3233
[81]TL Stanford, NP Stafford, LC Coelho, etc. Production and characterization of a thermostable
alpha-amylase from Nocardiopsis sp. endophyte of yam bean.Bioresour Technol, 2001,76 (2):137-41
[0][82]TL Stamford, NP Stamfod,LC Coelho, etc. Production and characterization of a thermostable
glucoamylase from Streptosporangium sp. endophyte of maize leaves.Bioresour. Technol. 2002,83(2):
105-109
[83]Gaumann E,et al.Enniatin, a new antibiotic active against Mycobacteria. Experientia，1947,9:202-203
[84]Hiroshi Tomoda，Hiroyuki Nishida，et al.New Cyclodepsipeptides, Enniatins D E and F produced by
Fusarium sp,FO-1305. J Antibiotics,1992,45:1207-1215
[0][85]Tawnya C,McKee,Heidi R, Bokesch,Jinping L.McCormick,et al.Isolation and Characteri- zation of
New Anti-HIV and Cytotoxic leads from Plants，Marine，and Microbial Organism.J Natural
Products,1997, 60(5):431-438
[0][86]Lin Y C,Wang j,Wu X Y,et al.A Novel Compound Enniatin G from the Mangrove fungus Fungus
Halosarpheia sp.(strain #732)from the South China Sea.Journal of Aust Chem,2002,55(3):225-227
[87]Burgess l W,Summerell B A,Bullock S,et al. Laboratory Manual for Fusarium Research.Sydney: The
university of Sydney press,1994
[88]Guo L D,Hyde K D,Liew E CY.A method to promote sporulation in palm endophytic fungi.Fungal
Diversity,1:109-113
[89]Fisher P J,Petrini O,Petrini L E,Sutton B C.Fungal endophytes from the leaves and twigs of Quercus
ilex L.from England,Marjorca and Switzerland.New Phytol,127:133-137
参考文献 45
[90]Guo L D,Hyde K D.Indentification of endophytic fungi from Livistona chinensis based on morphology
and rDNA sequences. New Phytol,147:617-630
[91]Audhya T K, Russell D W. Production of enniatins by Fusarium sambulinum: selection of high-yield
conditions from liquid surface culture.Journal of General Microbiology, 1974, 82:181-196
[92]Blackwell B A,Miller J D,Apsimin J W,et al.Isolation and characterijatim of enniatins from
Fusarium-Avenaceum DAOM 196490.Canadian Journal of Chemistry, 1992, 70: 5-10
[93]Sauriol F,Hynes R C,Xu X J, et al.Novel Quinajolinones and enniatins from Fusarium-Laterium Nees.
Canadian Journal of Chemistry, 1993, 71: 9-16
[94]Bishop D L,et al.Seedlborne fungal contamination:Consequences in spacegrown wheat.
Phytopathology,1997,87:1125-1133
[95]Brady S F,Clardy J.CR377,a new pentaketide antifungal agent isolated from an endophytic fungus.J
Nat Prod,2000,2:1447-1448
[96]Clay K,Holah J.Fungal endophyte symbiosis and plant diversity in successional
fields.Science,1999,285:1742-1745
[97]Guo B,Dai J R,et al.Cytonic acids A and B:novel tridepside inhibitors of hCMV protease from the
endophytic fungus Cytonaema species.J Nat Prod,2000,63:602-604
[98]Melissa M,et al.Dlicerandrols new antibiotic and cytotoxic dimmers produced by the fungus
Phomopsis longicolla isolated from an endan gered Mint.Journal of Natural
Products,2001,64:1006-1009
[99]Nisbet L J,Fox F M.The importance of microbiodiversity to biotechnology.The Biodiversity of
Microorganisms and Invertebrates:Its Role in Sustainable Agriculture,1991,229-244
[100]Singh M P,Janso J E,et al.Biological activity of guanacastepene,a novel diterpenoid antibiotic
produced by an unidentified fungus CR115.J antibiot,2000,53:256-261
[101]Sturz,A.V,et al.Biodiversity of endophytic bacteria which colonize red clover nodules,roots,stems and
foliage and their influence on host growth.Plant Pathology,1997,48:360-369
[102]Wilson D,Carroll G C.Avoidance of high-endophyte space by gall-forming
insects.Ecology,1997,78:2153-2165
[103] Masahiko I,et al.Phomoxanthones A and B,Novel xanthone dimmers from the endophytic fungus
Phomopsis species.Journal of Natural Products,2001,64:1015-1018
[104]James T,Blodgett ,et al.Species composition of endophytic fungi in Amaranthus bybridus
leaves,Petioles,stems and roots.Microbiogia,2000,92(5):853-859
[105]Clay K,Holah J.Fungal endophyte symbiosis and plant diversity in successional
fields.Science,1999,285:1742-1745
[106]Wilson D.Endophyte the evolution of tem ,and clarification of its, use and definition.
Oikos,1995,73:274-276