【作者】 刘小莉；

【导师】 董明盛；

【作者基本信息】 南京农业大学 ， 食品科学， 2007， 博士

【摘要】 内生真菌是一类生长于活体植物组织中的特殊的真菌,研究相对较少。从生长于独特环境中、具有药用价值、树龄长或有地域特色的植物中一般都能分离出新的内生菌,而新种或罕见菌种又能为寻找新型生物活性物质提供前景。银杏是我国特色珍稀植物,具有“活化石”之美称。银杏在生长期间很少感染病虫害,寿命极长,因此具有很好的挖掘新型内生真菌的潜力。本研究采集江苏省泰兴市、邳州市古银杏枝条和树叶,采用组织分离法分离其中的内生真菌,筛选具有抗菌活性的菌株并通过形态特征或内转录间隔区（ITS）序列分析进行初步鉴定;对其中一株抗菌活性显著且稳定的不产孢菌进行形态学研究和分子解析,对培养条件和培养基组成进行优化,进而分离其发酵产物中的抗菌活性物质并进行结构鉴定;并研究子座的抗氧化活性,为进一步工业化生产应用提供工作基础和依据。主要研究结果如下:1.采用水琼脂平板和孟加拉红培养基从银杏枝条、树叶样品中分离得到55株内生真菌,其中29株为孟加拉红法分离所得,大部分为产孢真菌,包括1株交链孢霉、6株曲霉、10株青霉、1株毛霉、1株镰孢霉、9株不产孢真菌和1株酵母;26株为水琼脂法所得,包括1株青霉、3株交链孢霉、3株简梗孢霉和19株不产孢真菌。2.通过平板孔阱试验筛选具有抗菌活性的内生真菌,共得到23株具有不同程度抗菌活性的菌株,占总分离株数的41.82%。水琼脂法分离所得菌株的活性明显高于孟加拉红方法。3.对用水琼脂法分离所得的11株活性菌株进行初步鉴定。根据分生孢子形态对其中4株产孢菌进行鉴定,YX-2属于简梗孢霉属（Chromosporium sp.）、YX-9属于小单头孢霉属（Acremoniela sp.）、YX-30和YX-45均为交链孢霉（Alternaria spp.）。通过对内转录间隔区（ITS）进行序列分析,另外7株不产孢活性菌株中YX-16、YX-44与细交链孢霉、YX-13与砖红镰孢霉、YX-27、YX-28、YX-41与炭角菌、YX-47与一株分离自Picea glauca的未鉴定的内生菌分别具有较高的相似性,结果与形态特征比较的结果一致。4.对抗菌活性显著且稳定的不产孢真菌YX-28进行深入的形态和分子鉴定。YX-28在多种培养基上产生黑色炭化菌落和指状子座组织,但经过各种诱导产孢处理都不能产生有性或无性孢子。对18S rDNA和ITS片段进行分子解析,构建系统进化树,可将YX-28菌株归属为木内炭角菌Xylaria hypoxylon的近缘种。5.通过Custom Desigll设计,研究适宜Xylaria sp.YX-28子座生长的条件为棉籽壳:麦麸基质固液比为30:70（W/V）、以塑料膜封口、黑暗培养富集菌丝、弱散射光照射刺激子座产生,在该条件下子座产量平均为441.96g/m²。6.采用单因素试验法确定了Xylaria sp.YX-28液体摇瓶培养的最佳生长条件,该菌最适生长温度为25℃,适宜生长温度范围为5℃～37℃,高于37℃或低于5℃该菌停止生长;适宜生长的pn范围为3～12,且在pH为6时,菌丝生物量和产物活性最高;摇瓶培养的最适装液量为150mL/500mL,接种量为2.5g菌丝/150mL。7.采用析因设计和中心组合设计对Xylaria sp.YX-28的液体培养基组成进行优化,蛋白胨和硫酸锌的浓度是影响Xylaria sp.YX-28发酵产物抗菌活性的关键因子,优化所得的最优培养基组合为1.500%葡萄糖、0.200%NH₄Cl、0.500%黄豆粉、0.158%蛋白胨和0.168%ZnSO₄,Xylaria sp.YX-28发酵产物对金黄色葡萄球菌和大肠杆菌的抗菌活性与优化前相比分别提高了1.44和2.02倍。8.Xylaria sp.YX-28的大规模发酵液通过膜分离处理,抗菌活性最大的组分以乙酸乙酯萃取,浸膏通过薄层层析、柱层析、制备液相技术等分离纯化,得到的纯化组分经光谱手段鉴定为7-氨基-4-甲基香豆素。9.抗菌试验结果表明,7-氨基-4-甲基香豆素对受试的10株细菌、1株酵母和2株霉菌均表现出良好的抑制效果,与阳性对照相比活性相当,且具有更广的抗菌谱.10.研究了Xylaria sp.YX-28子座提取物的抗氧化能力,提取物中总酚、总黄酮含量与抗氧化活性之间都存在明显的正相关性,相关系数R²分别为0.7336、0.9392,甲醇提取物具有最高的总酚、总黄酮含量和最强的清除自由基能力。在β-胡萝卜素/亚油酸实验中,400μg·mL^-1的Xylaria sp.YX-28甲醇粗提物具有72.9%的抑制活性,显著高于阳性对照二丁基羟基甲苯（BHT）和抗坏血酸（Asc A）;而在自由基清除实验中则显示中等活性。11.通过GC/MS对Xylaria sp YX-28子座的甲醇提取物进行组成分析,鉴定出了多种具有抗氧化活性的酚类物质,包括2-肼基-8-羟基-4-苯基喹啉、3,4-二甲氧基-苯酚、2,4-二（1,1-二甲基乙基）-苯酚、3,4-二氢-8-羟基-3-甲基-异香豆素和弥罗松酚.更多还原

【Abstract】 Endophytic fungi,microorganisms that reside in the tissues of living plants,are relatively unstudied.Those plants growing in unique environmental settings,having ethnobotanical uses,having extreme age or interesting endemic locations generally contain novel endophytic microorganisms.Novel taxonomy of an endophyte or the acquisition of one that is only rarely seen,generally offers a prospect for finding novel bioactive natural products.Ginkgo biloba is a precious and rare plant endemic to China,named as "a living fossil". It is scarcely infected with plant pathogens and insect pests,and has a long life,so it has potential to isolate novel endophytic fungi.In our study,branches and leaves were collected from old Ginkgo biloba in Taixing and Pizhou,Jiangsu province.Endophytic fungi were isolated,and those with antimicrobial activity were screened and subsequently identified by morphological features or ITS sequence similarity analysis.Mycelia sterilia YX-28 with significant and stable antimicrobial activity was identified on the basis of morphological features and molecular analysis.Growth conditions and medium compositions were optimized,and compound（s） with antimicrobial activity was/were isolated and elucidated. In addition,conditions of solid fermentation for the growth of the stromata of the strain and its antioxidant activity were studied.The main results were given as follows:1.55 endophytic fungi were obtained from the branches and leaves of Ginkgo biloba. Among them,29 strains,mostly spore-producing fungi,were isolated on plates containing tetrachlorotetraiodo fluorescein di sodium（TFDS）,including 1 Alternaria sp.,6 Aspergillus spp.,10 Penicillium spp.,1 Mucor sp.,9 mycelia sterilia and 1 yeast.26 strains were obtained on water agar,including 1 Penicillium sp.,3 Alternaria spp.,3 Chromosporium spp.and 19 Mycelia Sterilia.The results indicated that water agar was more suitable for the isolation of endophytic fungi.2.Endophytic fungi with antimicrobial activity were screened using agar diffusion method,resulting in 23 bioactive strains,mostly mycelia sterilia,accounting for 41.82%of the total.The amount of strains with antimicrobial activity by water agar method was highly more than that by TFDS method.3.11 bioactive strains isolated on water agar plate were identified preliminarily.Among them,4 spore-producing strains were identified by the morphological features,resulting in YX-2 as Chromosporium sp.,YX-9 as Acremoniella sp.,and YX-30 and YX-45 as Alternaria spp.The other 7 mycelia sterilia were conducted sequence similarity analysis of ITS1-5.8S rDNA-ITS2,indicating that YX-13 has a high similarity with Fusarium lateritium,YX-16 and YX-44 with Alternaria tenuissima,YX-27,YX-28 and YX-41 with Xylaria,and YX-47 with a foliar endophyte from Picea glauca,respectively.The results of sequence analysis were in agreement with those of morphological features.4.Nonspore-producing fungus YX-28 with great and stable antimicrobial activity was further studied.YX-28 produced black and carbon-like colony on agar plates,and finger-like stromata on different media.However,neither sexual nor asexual spores produced through different spore promoting methods.According to molecular analysis of 18S rDNA and ITS1-5.8S rDNA-ITS2 sequences,YX-28 could be determined to be a similarity species of Xylaria hypoxylon.5.Custom Design was used to determine the conditions for the growth of stromata of Xylaria sp.YX-28.The results showed that the stromata grow well when 15g cotton seedcoat-wheat bran（3:1） as solid media with broth at a ratio of 30:70（W/V）,sealed the bottle with plastics,growing mycelia in the dark,and then in lightness.The production of the stomata was 441.96g/m².6.One-factor-at-a-time method was used to determine the suitable conditions for the growth and antimicrobial activity of Xylaria sp.YX-28.The optimal temperature is 25℃, while YX-28 can grow at a range from 5℃to 37℃,or stop growing beyond the range. YX-28 can grow at pH 3-12,with the optimal original pH 6.Medium volume of 150 mL in a 500 mL flask and inoculation of 2.5 g wet mycelia were suitable for Xylaria sp.YX-28.7.Fractional Factorial Design and Response Surface Methodology were used for the medium optimization of Xylaria sp.YX-28.Peptone and ZnSO₄ were key factors for the antimicrobial activity of Xylaria sp.YX-28.The optimized medium compositions were 1.500%glucose,0.200%NH₄Cl,0.500%soybean powder,0.158%peptone and 0.168% ZnSO₄,leading to an enhancement of the antimicrobial activity by 1.44-fold against S.aureus and 2.02-fold against E.coli,respectively.8.Membrane filtration was used to deal with large scale of fermentation broth of Xylaria sp.YX-28.The filtration part with the greatest antimicrobial activity was extracted by ethyl acetate.The extract was isolated through thin layer chromatography,column chromatography and preparative high performance liquid chromatography.A compound obtained was elucidated as 7-amino-4-mythylcourium.9.The antimicrobial test indicated that 7-amino-4-mythylcourium showed good inhibitory against the tested 10 bacteria,1 yeast and 2 moulds.Compared to the positive controls,7-amino-4-mythylcourium showed comparative activity,and had broader inhibitory spectrum.10.Antioxidant activity of the extracts of Xylaria sp.YX-28 was evaluated.A positive relation existed between the total phenolics and flavonoids content and the antioxidant activity,with the coefficients R² 0.7336 and 0.9392,respectively.The methanol extract had the highest total phenolics and flavonoids content and the DPPH scavenging activity.In theβ-carotene/linoleic assay,the methanol extract of Xylaria sp.YX-28 had a 72.9%bleaching activity at a concentration of 400μg·mL^-1,significantly higher than those of the positive controls BHT and Asc A,while exhibited moderate activity in DPPH scavenging assay.11.GC/MS were used to analyze the compositions of the methanol extract of Xylaria sp. YX-28.Several phenolics with antioxidant activity were identified,including 2-hydrazino-8-hydroxy-4-phenylquinoline,3,4-dimethoxy-phenol,2,4-bis（1,1-dimethyl ethyl）-Phenol,3,4-dihydro-8-hydroxy-3-methyl-isocoumarin,and ferruginol.更多还原