杜仲内生真菌资源和活性菌株生物学的初步研究
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摘要
内生真菌普遍存在于植物体中，其重要性正日益受到关注，不同植物体内的内生真菌种类与数量也不相同，自然界中存在大量的内生真菌资源有待开发。本研究选取杜仲Eucommia ulmoides Oliv．为研究材料，进行其内生真菌资源的研究工作。结果从不同地区杜仲组织中共分离到254株内生真菌菌株，分属于蜜孢霉 Sphacelia、刺孢壳 Chaetomella、交链孢 Alternaria、壳明单隔孢属 Diplodina、刺盘孢 Colletotrichum、匐柄霉 Stemphylium、头孢霉 Cephalosporium、球座菌 Guignardia和青霉菌 Penicillium等真菌属中。从两个采集地点来看，南京地区内生真菌类群比西安地区类群较丰富、优势种群分离率较高；从植株部位上看，叶片组织内生真菌类群较枝条和树皮丰富，且分离率高；此外，一些菌株还表现出一定的组织特异性。在所研究的杜仲内生真菌类群中，刺孢壳比较普遍存在，且分离率较高，属优势种群之一。生物活性研究表明：刺孢壳 Chaetomella菌株Z18的发酵代谢产物具有较广谱的抗菌作用和较高的抗氧化活性，TLC与HPLC检测显示它的抗氧化活性成分中可能含有与芦丁相同或相似的黄酮类化合物。刺孢壳 Chaetomella活性菌株Z18在不同的固体和液体培养时，不同碳源、氮源、培养温度、pH值、装液量以及生长因子等条件对其生长有不同的影响。在马铃薯葡萄糖培养基和马丁氏培养基中生长较好；比较适宜的碳氮比、培养温度、pH值、装液量和转速分别为10：1，25℃，pH 6，150mL／250mL和200rpm；VB_1、VB_6和叶酸等维生素以及Ca~(2+)、Fe~(2+)、Mg~(2+)等无机离子均能不同程度地对其产生促生长作用。利用随机引物扩增多态性(RAPD)分析优势的刺孢壳 Chaetomella种群表明，其DNA多态性与宿主来源和分离部位存在一定相关性，同一地区的菌株之间亲缘关系较近，分离自同一部位的菌株之间亲缘关系也比较近。菌株表现出一定的地理分化和宿主组织分化。
Endophytic fungi exist widely in the body of plants. The people have been paying more attention to them. There are different kinds and quantity of endophytic fungi in different plants. Majority of the resources are to be tapped. In our study on Endophytic fungi from Eucommia ulmoides Oliv., 254 strains of endophytic fungi were isolated from that plant in different regions. They are identified as Sphacelia, Chaetomella, Alternaria, Diplodina. Colletotrichum, Stemphylium, Cephalosporium, Guignardia and Penicillium based on morphological taxonomy. It is found out that endophytic fungi group in Nanjing has larger quantity of group and higher isolation ratio than in Xi'an. It is also true in the leaf than in the tress and bark. Chaetomella sp. exist more widely in that plants in different regions and the isolation ratio is more higher. It is one of the dominants of the endophytic fungi from Eucommia ulmoide Oliv.. In the study of bioactivity it is proved that the fermentation liquid of the strain of Z18 {Chaetome
lla sp.) can inhibit the growth of much bacteria and some pathogenic fungi and it has high antioxidative activity. It is indicated by TLC and HPLC that its antioxidative components may include flavone-like compounds which are same or very similar with rutin. The cultural characteristics of the strain of Z18 (Chaetomella sp.) was also studied. It is showed that it could grow well in potato dextrose liquid medium and Martin's medium. The optimum C/N, temperature, pH, loadage of conical flask and rotate speed for mycelium growth are 10:1, 25 C, pH 6, 100mL media volume in 250mL flasks and lOOrpm in speed respectively. The vitamin such as Vitamin B1, B6, FH4 and inorganic ions such as Ca2+ Fe2+ and Mg2+ could promote the growth rate of Z18 in different degree. Group of Chaetomella which is dominant is analyzed by the meathod of RAPD (Randomly Amplified Polymorphic DNA). It is proved that the cluster results of strains used in the study were certainly associated with host's geographic regions and the part of the
host where strains were isolated from. The strains in the same region showed a higher genetic relativity with higher similarity indexes among them and it is also true in the case of strains isolated from the same part of the plant.
Endophytic fungi exist widely in the body of plants. The people have been paying more attention to them. There are different kinds and quantity of endophytic fungi in different plants. Majority of the resources are to be tapped. In our study on Endophytic fungi from Eucommia ulmoides Oliv., 254 strains of endophytic fungi were isolated from that plant in different regions. They are identified as Sphacelia, Chaetomella, Alternaria, Diplodina. Colletotrichum, Stemphylium, Cephalosporium, Guignardia and Penicillium based on morphological taxonomy. It is found out that endophytic fungi group in Nanjing has larger quantity of group and higher isolation ratio than in Xi'an. It is also true in the leaf than in the tress and bark. Chaetomella sp. exist more widely in that plants in different regions and the isolation ratio is more higher. It is one of the dominants of the endophytic fungi from Eucommia ulmoide Oliv.. In the study of bioactivity it is proved that the fermentation liquid of the strain of Z18 {Chaetome
lla sp.) can inhibit the growth of much bacteria and some pathogenic fungi and it has high antioxidative activity. It is indicated by TLC and HPLC that its antioxidative components may include flavone-like compounds which are same or very similar with rutin. The cultural characteristics of the strain of Z18 (Chaetomella sp.) was also studied. It is showed that it could grow well in potato dextrose liquid medium and Martin's medium. The optimum C/N, temperature, pH, loadage of conical flask and rotate speed for mycelium growth are 10:1, 25 C, pH 6, 100mL media volume in 250mL flasks and lOOrpm in speed respectively. The vitamin such as Vitamin B1, B6, FH4 and inorganic ions such as Ca2+ Fe2+ and Mg2+ could promote the growth rate of Z18 in different degree. Group of Chaetomella which is dominant is analyzed by the meathod of RAPD (Randomly Amplified Polymorphic DNA). It is proved that the cluster results of strains used in the study were certainly associated with host's geographic regions and the part of the
host where strains were isolated from. The strains in the same region showed a higher genetic relativity with higher similarity indexes among them and it is also true in the case of strains isolated from the same part of the plant.
引文
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[2] Fisher P J, Petrini O, Sutton B C. A comparative study of fungal endophytes in leaves, xylem and bark of Eucalyptus nitens in Australia and England[J]. Sydowia, 1993, 45:338-345
[3] Strobel G, Stierle A, Stierle D. Taxol and taxane production by Taxomycetes andreanae, an endophytic fungus of Pacific Yew[J]. Science, 1993, 260(5105): 214-216
[4] Hyde K D et al. Diversity of ascomycetes on palms in the tropics. In: Hyde K D ed. Biodiversity of Tropical Microfungi Hong Kong: Hong Kong University Press. 1997, 141-156.
[5] Anorid A E et at. Are tropical fungal endophytes hyperdiverse.[J] Ecol Lett, 2000, 3:267-274
[6] 邹文欣，谭仁详．植物内生真菌研究新进展[J]．植物学报，2001，43(9)：881-892
[7] Strobel G, et al. Taxomuces andreanae, A proposed new Taxon for a Bubilliferous hyphomycete associated with pacific yew (Taxus brevifolia) [J]. Mycotaxon, 1993,Ⅹ Ⅳ Ⅱ: 71-80
[8] Strobel G, et al. Cryptocandin, a potent antimycotic from the endophytic fungus Cryptosporiopsis cf quercina[J]. Microbiology, 1999, 145: 1919-1926
[9] Glenn A E, Bacon C W, Price R, et al. Molecular phylogeny of Acremonium and its taxonomic implications[J]. Mycologia, 1996, 88: 369-383
[10] Al-Raddad A M. Distribution of different Glomus species in rainfed areas in Jordan[J]. Disrasat-Series B, Pure and Applied Science, 1993, 20(2): 165-182
[11] 王发园，刘润近．生物因子对AM真菌多样性的影响[J]．生态学报，2002，(22)3：403-408
[12] 张英，郭良栋，刘润进．都江堰地区丛枝菌根真菌多样性与生态研究[J]．植物生态学报，2003，27(4)：537-544
[13] 李建平，李涛，张之伟．金沙江干热河谷(元谋段)从枝菌根真菌多样性研究[J]．菌物系统，2003，22(4)：604-612
[14] Zhang M Q, Wang Y S, Xin L J. The relationship between the distribution of AM uingiand environmental factors[J]. Mycosytema, 1999, 18(1): 25-29
[15] Schardl, C. L. et al. Molecular phylogenetic relationships of nonpathogenic grass mycosymbionts and clavicipitacarous plant pathogens[J]. Plant Syst. Evol., 1991, 178,: 27-41
[16] Haemmerli U A, BRandle U E, Petrini O, et al. Differentiation of isolates of Discula umbrinella (teleomorph: Apirgnornonia errabunda) from beech, chestnut and oak using RAPD markers[J]. Mot Plant-Microbe Interactions, 1992, 5: 479-483
[17] 郭良栋．内生真菌研究进展[J]．菌物系统，2001，20(1)：148-152
[18] Fisher P J, Petrini O, Petrini L E, et al. Fungal endophytes from the leaves and twigs of Quercus
ilex L. from England, Marjorca and Switzerland[J]. New Phytol, 1994, 127: 133-137
[19] Southcott K A, Johnson J A. Isolation of endophytes from two species of palm, from Bermuda[J]. Can J Microbiol, 1997, 43: 789-792
[20] James T Blodgett, et al. Species composition of endophytic fungi in Amaranthus bybridus leaves, petioles, stems, and toots[J]. Microbiogia, 2000, 92(5): 853-850
[21] Petrini O. Ecological and physiologica aspects of host specificity in endophytic fungi. In: Redlin S C, Carris L M eds. Endophytic Fungi In Grassed and Woody Plants: Systematics, Ecology, and Evolution. St. Paul, Minnesota: APS Press. 1996, 87-100
[22] Fisher P J, Petrini O, Sutton B C. A comparative study of fungal endophytes in leaves, xylem and bark of Eucalyptus nitens in Australia and England. Sydowia, 1993, 45: 338-345
[23] Strobel G, Stierle A, Stierle D. Taxol and taxane production by Taxomycetes andreanae, an endophytic fungus of Pacific Yew[J]. Science, 1993,260(5105): 214-216
[24] Strobel G, Yang X, Sears J, et al. Taxol from Peatalotiopsis microspore, an endophytic fungus of T axus wallachiana[J]. Microbiology, 1996, 142: 435-440
[25] 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，1999，38(3)：116-118
[26] 李桂玲，王建锋，黄耀坚，等．植物内生真菌抗肿瘤活性菌株的筛选[J]．菌物系统，2001，20(3)：387-391
[27] 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
[28] Caruso M, Colombo A L, Fedeli L, et al. Isolation of endophytic fungi and actinomycetes taxane producers[J]. Ann Microbiol, 2000, 50: 3-13
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