冬青卫矛内生真菌筛选及其代谢产物的农药活性研究
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摘要
植物内生真菌能够产生结构新颖、功能特殊的次生代谢产物,是新活性化合物的潜在资源,在农业医药行业中具有重要的应用前景。本研究对冬青内生真菌进行分离并对其次生代谢产物进行了农用活性筛选。主要研究结果如下:
1.采用组织培养法从冬青卫矛(Euonymus japonicus)中分离到49株植物内生真菌,经初筛和复筛,筛选出一株具有杀菌活性的菌株M-1。
2.对M-1菌株发酵液进行了杀菌活性研究。M-1菌株发酵液对番茄早疫病菌( Alernaria solani )、西瓜枯萎病菌( Fusarium oxysporium )和苹果炭疽病菌(Colletotrichum gloesporioides)菌丝生长抑制率达到80.3%、78.5%和87.8%。M-1菌株发酵液对枯草芽孢杆菌、蜡状芽孢杆菌、金黄色葡萄球菌和大肠杆菌的抑菌圈直径分别为23.0mm、20.0mm、16.0mm和20.0mm。M-1发酵液的乙酸乙酯萃取物对苹果炭疽病菌的毒力最高, IC_(50)值为63.6μg/mL。Doskochilova溶剂系统鉴定表明M-1菌株的发酵液中含有硫藤黄菌素类抗生素。
3.通过单因子实验获得了冬青卫矛内生真菌菌株M-1摇瓶发酵的最佳培养条件。最佳培养基配方和发酵条件为20%胡萝卜、2%葡萄糖、0.4%酵母粉、0.2% MgSO_4和0.2% ZnSO_4,培养温度26℃,接种量:每100 mL发酵液18块菌饼,装液量为110/250mL,初始pH为7.5左右,发酵时间240h。
4.根据真菌的分类及鉴定方法,对比相关分类系统,将M-1菌株初步鉴定为半知菌亚门,链格孢属(Alternaria)真菌。
The plant endophyte can metabolize many intereting secondary metabolites with novel structure and distinctive activities, which is an important source of new bioactive moleculars. The aims of this study are to isolate endophytic fungus from Euonymus japonicus and screen the strains that produce agricultural antibiotics, then work about the isolation and identification of active components have been carried out。The main results are as follows:
1. Taking different separating culture medium, 49 strains of endophytic fungus were separated from Euonymus japonicu. The screening tests indicated that M-1 strain possessed potant fungicidal activities.
2.The fungicidal activites of M-1 strain has been studied. The inhibition rate of fermentation broth of M-1 strain against Alternaria solani, Fusarium oxysporium sp niveum, Colletotrichum gloesporioides were 80.3%,78.5% and 87.8%respectively. The result of antibacterial bioassay indicated that the inhabitation zone of M-1 strain against Bacillus cereus Bcillus subtills Staphyococcus aureus and Escherichia coli were 23.0mm 20.0mm 16.0mm and 20.0mm, respectively.The ethyl acetate extract of M-1 fermentation broth had higher antifungal activity against C. gloesporioides, and the IC_(50) was 63.6μg/mL. By the Doskochilova solvent system, fermentation broth of M-1 may include sulfurluteolin.
3. The optimum composition of culture medium and fermentation conditions of the strain M-1were demonstrated respectively. The optimum culture medium is about pH 7.5. Inoculum volume was 18 entries per 100mL medium. A 250mL flask containing 110mL medium was cultivated at 26℃for 240h. Under this culture conditions, the product and activity against Bacillus cereus were high than any conditions.
4. By means of observation on aerial mycelium, macroconidia, microconidia and colony morphology characteristics and comprised with related classify system, strain M-1 was preliminarily identified as Alternaria spp..
1.采用组织培养法从冬青卫矛(Euonymus japonicus)中分离到49株植物内生真菌,经初筛和复筛,筛选出一株具有杀菌活性的菌株M-1。
2.对M-1菌株发酵液进行了杀菌活性研究。M-1菌株发酵液对番茄早疫病菌( Alernaria solani )、西瓜枯萎病菌( Fusarium oxysporium )和苹果炭疽病菌(Colletotrichum gloesporioides)菌丝生长抑制率达到80.3%、78.5%和87.8%。M-1菌株发酵液对枯草芽孢杆菌、蜡状芽孢杆菌、金黄色葡萄球菌和大肠杆菌的抑菌圈直径分别为23.0mm、20.0mm、16.0mm和20.0mm。M-1发酵液的乙酸乙酯萃取物对苹果炭疽病菌的毒力最高, IC_(50)值为63.6μg/mL。Doskochilova溶剂系统鉴定表明M-1菌株的发酵液中含有硫藤黄菌素类抗生素。
3.通过单因子实验获得了冬青卫矛内生真菌菌株M-1摇瓶发酵的最佳培养条件。最佳培养基配方和发酵条件为20%胡萝卜、2%葡萄糖、0.4%酵母粉、0.2% MgSO_4和0.2% ZnSO_4,培养温度26℃,接种量:每100 mL发酵液18块菌饼,装液量为110/250mL,初始pH为7.5左右,发酵时间240h。
4.根据真菌的分类及鉴定方法,对比相关分类系统,将M-1菌株初步鉴定为半知菌亚门,链格孢属(Alternaria)真菌。
The plant endophyte can metabolize many intereting secondary metabolites with novel structure and distinctive activities, which is an important source of new bioactive moleculars. The aims of this study are to isolate endophytic fungus from Euonymus japonicus and screen the strains that produce agricultural antibiotics, then work about the isolation and identification of active components have been carried out。The main results are as follows:
1. Taking different separating culture medium, 49 strains of endophytic fungus were separated from Euonymus japonicu. The screening tests indicated that M-1 strain possessed potant fungicidal activities.
2.The fungicidal activites of M-1 strain has been studied. The inhibition rate of fermentation broth of M-1 strain against Alternaria solani, Fusarium oxysporium sp niveum, Colletotrichum gloesporioides were 80.3%,78.5% and 87.8%respectively. The result of antibacterial bioassay indicated that the inhabitation zone of M-1 strain against Bacillus cereus Bcillus subtills Staphyococcus aureus and Escherichia coli were 23.0mm 20.0mm 16.0mm and 20.0mm, respectively.The ethyl acetate extract of M-1 fermentation broth had higher antifungal activity against C. gloesporioides, and the IC_(50) was 63.6μg/mL. By the Doskochilova solvent system, fermentation broth of M-1 may include sulfurluteolin.
3. The optimum composition of culture medium and fermentation conditions of the strain M-1were demonstrated respectively. The optimum culture medium is about pH 7.5. Inoculum volume was 18 entries per 100mL medium. A 250mL flask containing 110mL medium was cultivated at 26℃for 240h. Under this culture conditions, the product and activity against Bacillus cereus were high than any conditions.
4. By means of observation on aerial mycelium, macroconidia, microconidia and colony morphology characteristics and comprised with related classify system, strain M-1 was preliminarily identified as Alternaria spp..
引文
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[2] St robel G A. Endophytes as sources of bioactive product s microbes and infection[J ] . Microbes and Infection ,2003 ,5 :535 - 544.
[3] Azevedo J L, Maccheroni J W, Pereira J O, et al. Endophytic microorganisms: a review on insect control and recent advances on tropicalplants[J].ElectronicJournalofBiotechnology, 2000,3(1):40-65.
[4] 何美仙. 植物内生真菌作为生防因子的研究进展[J]. 植物保护,2005, 31(1): 10-14.
[5] 郭良栋. 内生真菌研究进展[J]. 菌物系统, 2001, 20(1): 148-152.
[6] 周成, 邵华, 张玲琪, 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): 69-73.
[7] Strobel G A. Endophytes as sources of bioactive products[J].MicrobesInfect, 2003, 5(6): 535-544.
[8] 文才艺, 吴元华, 田秀玲. 微生物源生物化学农药的研究与开发进展[J]. 农药, 2004, 43(10): 438-441.
[9] Rowan D D. Lolitrems, peramine and paxilline-mycotoxins of the yegrass-endophyte interaction[J]. Agric Ecosyst Environ, 1993,44: 103-122.
[10] Blankenship J D, Spiering M J, Wilkinson H H, et al. Production ofoline alkaloids by the grass endophyte, Neotyphodium uncinatum,in defined media[J]. Phytochemistry, 2001, 58(3): 395-401.
[11] Strobel G, Daisy B, Castillo U, et al. Natural products from endophytic microorganisms[J]. J Nat prod, 2004, 67(2): 257-68.
[12] Findlay J A, Li G, Penner P E, et al. Novel diterpenoid insect toxin from a conifer endophyte[J]. J Nat Prod, 1995, 58: 197-200.
[13] Findlay J A, Li G, Johnson J A. Bioactive compounds from needles[J].Can J Chem, 1997, 75: 716-719.
[14] Findlay J A, Buthelezi S, Li G, et al. Insect toxins from an endophyte fungus from wintergreen[J]. J Nat Prod, 1997, 60: 1214-1215.
[15] Larry A C, John A F, Noamn J W, et al. Metabolites toxic to sprucebudworm from balsam fir needle endopgytes[J]. Mycol Res, 1992,96(4): 281-286.
[16] 兰琪, 姬志勤, 顾爱国, 等. 苦皮藤内生真菌中杀虫杀菌活性物质的初步研究[J]. 西北农林科技大学学报(自然科学版),2004, 32(1): 79-84.
[17] White Jr J F, Cole G T. Endophyte host associations in forage grass Ⅲ . In vitro inhibition of fungi by Acremoniumcoenophialum[J]. Mycologia, 1985, 77: 487-489.
[18] Wicklow D T. Patterns of fungal association within kernels harvested in North Carolina[J]. Pl Dis, 1988, (72): 113-115.
[19] 刘晓光, 高克祥, 谷建才. 毛白杨内生菌优势种毛壳ND35室内拮抗作用的研究[J]. 林业科学, 1999, 35(5): 7-61.
[20] 高克祥, 刘晓光, 郭润芳, 等. 木霉菌对五种植物病原真菌的重寄生作用[J]. 山东农业大学学报(自然科学版), 2002, 33(1): 37-42.
[21] 顾爱国. 苦皮藤内生真菌农用活性成分的研究(硕士论文)[D].杨凌: 西北农林科技大学, 2004.
[22] 吴雄宇, 李曼玲, 胡谷平, 等. 南海红树林内生真菌2508代谢物研究[J]. 中山大学学报(自然科学版), 2002, 41(3): 34-37.
[23] 李桂玲, 王建峰, 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): 64-68.
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