Novel cyclic peptide, epichlicin, from the endophytic fungus, Epichloe typhina

The novel cyclic peptide, epichlicin, was isolated from Epichloe typhina, an endophytic fungus of the timothy plant (Phleum pretense L.). Its structure was determined by NMR studies and by mass spectrometry. Enantiomers of 3-amino tetradecanoic acid, a constituent amino acid of epichlicin, were synthesized as authentic standards. The stereochemistry of each amino acid was elucidated through a combination of the advanced Marfey method and chemical manipulation. Epichlicin showed inhibitory activity toward the spore germination of Cladosporium phlei, a pathogenic fungus of the timothy plant at an IC50 value of 22 nM.     

宁前胡内生真菌Fusarium tricinctum次生代谢产物研究

为研究宁前胡内生真菌Fusariumtricinctum次生代谢产物,采用液体发酵法发酵内生真菌,利用硅胶、凝胶、MPLC、制备液相等多种方法分离获得13个单体化合物。利用波谱方法分别鉴定为cyclo-(L-pro-L-pro)(1)、cyclo-(S-pro-S-leu)(2)、cyclo-(L-phe-L-phe)(3)、cyclo-(D-pro-L-phe)(4)、cyclo-(L-pro-L-phe)(5)、cyclo-(D-pro-L-leu)(6)、cyclo-(S-pro-S-leu)(7)、cyclo-[D-(4-hydroxyprolinyl)]-(L)-leucine(8)、cyclo-[L-(4-hydroxyprolinyl)]-(L)-leucine(9)、cyclo-(trans-4-hydroxy-L-prolyl-L-phenylalanine)(10)、cyclo-(D-cis-hyp-L-phe)(11)、苯甲酸苄酯(12)和苯乙酸(13),所有化合物均首次从内生真菌F.tricinctum中分离得到。体外抗肿瘤活性显示,化合物4、6、7对CAR、CAL27、SCC-4、SCC-9、HSC-3五种口腔癌肿瘤细胞有一定的细胞毒活性。     

一株产紫杉醇罗汉松内生真菌的分离和鉴定

[目的]紫杉醇是重要的抗癌药物,主要从罗汉松等植物中提取,为了保护罗汉松等种质资源,本文从罗汉松植株中分离产紫杉醇内生真菌,并对内生真菌所产紫杉醇的抗肿瘤活性进行了分析.[方法]采用组织块法自罗汉松的根、茎、叶等组织中分离内生真菌;通过四唑蓝(Methyl ThiazolylTetrazolium,MTT)比色法筛选有抗肿瘤活性的内生真菌菌株,通过薄层层析(Thin Layer Chro-matography,TLC)和高效液相色谱(High Performance Liquid Chromatography,HPLC)对内生真菌所产活性物质进行鉴定;采用抽提法抽提内生真菌所产紫杉醇,应用Vero细胞对抽提的紫杉醇的活性进行了分析.[结果]从罗汉松属(Podocrapus)植物中分离到155株内生真菌,其中28株内生真菌具有较高的抑癌活性.将其中一株菌株A2命名为EPTP-1,经形态学和分子分类学分析鉴定为烟曲霉(Aspergillus fumigatus).菌株EPTP-1中抽提的紫杉醇5.553μg/L～555.3 μg/L作用24h表现出明显的致细胞凋亡作用.菌株EPTP-1发酵5天时紫杉醇的产率为0.5578±0.0294 mg/L.[结论]从罗汉松中分离到了一株产紫杉醇内生真菌EPTP-1,可作为紫杉醇类药物工业化生产的候选菌株.     

木榄内生真菌菌株ZD6及其代谢产物的抑菌活性

根据形态学特征和ITS序列分析结果,将一株从木榄茎中分离到的内生真菌菌株ZD6鉴定为桔青霉Penicillium citrinum Thom.,该菌株在优化后的发酵培养基(1%麦芽糖,2%甘露醇,1%谷氨酸钠,0.5%蛋白胨,0.15%酵母膏,200g/L土豆汁,pH6.5)中28℃、160r/min振荡培养7d后的发酵液具有明显的抑菌活性。利用柱层析及重结晶等方法从该发酵液的乙酸乙酯和正丁醇提取物中分离得到4个化合物:大黄素、环(丙氨酸-甘氨酸)、赤藓醇和甘露醇,其中的大黄素和赤藓醇为首次在桔青霉中发现,它们对枯草芽孢杆菌Bacillus subtilis的生长具有明显的抑制作用,最低抑菌浓度(MIC)分别为25μg/mL和50μg/mL;大黄素对绿脓杆菌Pseudomonas aeruginosa的生长也具有一定的抑制作用,MIC为100μg/mL。     

极地真菌Geomyces pannorum SA3-2-YM次级代谢产物的研究

采用聚酰胺柱层析、硅胶柱层析、反相硅胶柱层析、凝胶柱层析和高效液相HPLC等色谱技术,对极地真菌Geomyces parmorum SA3-2-YM的发酵液提取物进行分离纯化,共得到13个化合物,通过NMR和MS等波谱数据确定结构为:cyclo-(L-Trp-L-Pro)(1)、cyclo-(L-Trp-L-Tyr)(...     

Antifungal Metabolites Produced by Chaetomium globosum No.04, an Endophytic Fungus Isolated from Ginkgo biloba

The fungal endophyte Chaetomium globosum No.04 was isolated from the medicinal plant Ginkgo biloba. The crude extract of the fungus fermentation were active in the agar-diffusion tests against the phytopathogenic fungi Rhizopus stolonifer and Coniothyrium diplodiella. Further bioassay-guided chemical investigation led to the isolation and purification of six alkaloids and three non-targeted compounds from 50 L fermentation of this endophytic fungus and their structures were elucidated as chaetoglobosin A, C, D, E, G, R (1-6), ergosterol, allantoin and uracil, by means of spectroscopic analysis. Compounds 1-6 showed significant growth inhibitory activity against R. stolonifer and C. diplodiella at a concentration of 20 μg/disc. We present here, for the first time, the potent antifungal activity of chaetoglobosins from endophytic fungi against two important phytopathogenic fungi R. stolonifer and C. diplodiella.     

Antifouling diketopiperazines produced by a deep-sea bacterium, Streptomyces fungicidicus

Modern antifouling coatings use heavy metals and toxic organic molecules to prevent biofouling, the undesirable growth of marine organisms on man-made substrata. In an ongoing survey of deep-sea microorganisms aimed at finding low toxic antifouling metabolites, an actinomycete bacterium was isolated from the Pacific sediment at the depth of about 5000 m. The bacterium was closely related to Streptomyces fungicidicus (99% similarity) according to 16S ribosomal RNA sequence information. The spent culture medium of this bacterium inhibited barnacle larval attachment. Bioassay-guided fractionation was employed to isolate antifouling compounds. The ethyl acetate extract was fractionated by using an open silica gel column. Active fractions were further purified on a HPLC C18 column. Five diketopiperazines, cyclo-(L-Leu-L-Pro), cyclo-(L-Phe-L-Pro), cyclo-(L-Val-L-Pro), cyclo-(L-Trp-L-Pro), and cyclo-(L-Leu-L-Val) were isolated for the first time from a deep sea bacterium, and the structures of the compounds were elucidated by nuclear magnetic resonance spectroscopy and mass spectrometry. The pure diketopiperazines were tested for antilarval activity using the barnacle Balanus amphitrite. Effective concentrations that inhibited 50% larval attachment (EC50) after 24 h ranged from 0.10- 0.27 mM. The data suggest that diketopiperazines and other compounds from deep-sea bacteria may be used as novel antifoulants.     

银杏内生真菌No.1028的培养条件及其代谢产物对作物真菌病害的抑制作用

通过大量筛选从银杏茎中分离到一株内生真菌(编号为No.1028),其代谢产物对重要作物真菌病害病原菌具有明显的抑制作用。结果表明:离体条件下内生真菌发酵液对番茄早疫病菌、黄瓜枯萎病菌、菜豆炭疽病菌、葡萄炭疽病菌、水稻纹枯病菌、黄瓜立枯病菌均有较强的抑制作用,对上述不同病原菌的抑制率分别为:66.7%、48.3%、64.6%、36.5%、57.1%和23%。同时也研究了不同碳源、氮源、无机离子对内生真菌No.1028生物学特性和生长的影响。结果发现玉米粉、黄豆饼粉比较适合其生长和抑菌代谢产物的合成,Na+对抑菌代谢产物的合成有较明显的促进作用。     

大黄鱼共生菌Bacillus coagulans LL1103的代谢产物研究

[目的]研究大黄鱼共生菌Bacillus coagulans LL1103的次生代谢产物及其抑菌活性。[方法]利用色谱层析技术对B.coagulans LL1103发酵液乙酸乙酯和正丁醇提取物进行分离纯化;运用波谱技术鉴定化合物的结构;采用微量肉汤稀释法对化合物进行抗菌活性测定。[结果]从大黄鱼内生菌B.coagulans LL1103发酵液中分离并鉴定9个环二肽类化合物,分别为:(1)环(4-羟基-脯氨酸-亮氨酸)、(2)环(异亮氨酸-丙氨酸)、(3)环(脯氨酸-缬氨酸)、(4)环(脯氨酸-苯丙氨酸)、(5)环(脯氨酸-亮氨酸)、(6)环(甘氨酸-丙氨酸)、(7)环(脯氨酸-甘氨酸)、(8)环(脯氨酸-丙氨酸)和(9)环(络氨酸-甘氨酸)。活性评价显示化合物3、7和9对大肠埃希菌(Escherichia coli)具有较强抑制作用,最低抑菌浓度(minimal inhibitory concentration,MIC)分别为8.0、4.0和16.0μg/mL。[结论]从大黄鱼共生菌B.coagulans LL1103的发酵液中分离得到3个对大肠埃希菌(E.coli)具有较好抑制活性的化合物。     

大戟内生菌的抑菌活性及其与宿主相关性研究

为了开发利用药用植物的内生菌资源,以大戟和其内生真菌作为材料,以一株乌桕来源的内生菌作为对照,研究自身具有抑菌作用的药用植物的内生菌在抑菌作用上和宿主的相关性.结果表明,大戟内生菌镰刀菌E5有抑制宿主受试病原菌的作用,并在活植物体内表现出对受试病原菌的拮抗作用;其抑菌能力受宿主成分刺激而加强,而宿主成分对受试病原菌无抑制作用,乌桕来源的内生菌链隔孢S12无抑制受试病原菌的作用.内生菌E5需要在氧气充足条件下培养才能产生抑菌作用,抑菌活性最大产生时间和生物量最大值不同时出现.内生菌E5的抑菌能力受到pH、温度、紫外线影响.大戟内生菌E以上特点,体现了其与宿主之间的互惠共生关系.     

芦竹内生真菌F0238对植物病原菌的拮抗作用

从黄海海岸低盐药用植物芦竹(Arundo donax L．)中分离得到木霉属的内生真菌F0238,对其进行拮抗植物病原菌活性及作用机制的试验研究。结果表明F0238发酵液对Botrytis cinerea,Sclerotium rolfsii等8种植物病原菌有强的抑菌活性及抑制孢子萌发作用,且这种作用在发酵原液被稀释了160倍后仍然存在;对峙培养试验表明F0238菌对植物病原菌有很强的拮抗作用,拮抗机制主要为重寄生作用、营养竞争作用及抗生作用等。     

铁皮石斛内生真菌次生代谢产物研究

为了解铁皮石斛(Dendrobium officinale)内生真菌Phyllosticta aristolochiicola的次生代谢产物,从该真菌中分离得到15个化合物,经波谱分析分别鉴定为N-methyl-2-pyrolidinone (1)、环-(甘氨酸-L-脯氨酸)(2)、环-(D-丙氨酸-L-脯氨酸)(3)、环-(L-缬氨酸-L-脯氨酸)(4)、环-(L-亮氨酸-L-脯氨酸)(5)、cyclo-(L-Leu-D-4-hydroxyprolinyl)(6)、环-(L-苯丙氨酸-L-脯氨酸)(7)、环-(L-苯丙氨酸-L-4-羟基脯氨酸)(8)、环-(L-酪氨酸-L-脯氨酸)(9)、环-(L-苯丙氨酸-L-亮氨酸)(10)、啤酒甾醇(11)、对羟基苯乙醇(12)、对羟基苯乙酸(13)、(2S,3R)-1-(4-羟基苯基)丁烷-2,3-二醇(14)和(2R,3S)-1-苯基丁烷-2,3-二醇(15)。采用MTS法检测抗肿瘤活性表明,化合物2、10和14对HL-60、A-549、SMMC-7721、MCF-7和SW-480细胞株具有一定的抑制活性。     

Five New Guaiane Sesquiterpenes from the Endophytic Fungus Xylaria sp. YM 311647 of Azadirachta indica

Five new guaiane sesquiterpenes, 1 – 5 , were isolated from the culture broth of the endophytic fungus Xylaria sp. YM 311647, isolated from Azadirachta indica A. Juss . The structures of these compounds were elucidated on the basis of spectroscopic analyses, and their inhibitory activities against five pathogenic fungi were evaluated. All guaiane sesquiterpenes showed moderate or weak antifungal activities in a broth microdilution assay.     

Effect of bacterial polysaccharides on the growth ofGaeumannomyces graminis var.tritici and wheat roots

Agrobacterium sp. and related species which in the soil and in the rhizosphere of wheat accompany the fungus Gaemannomyces graminis var. tritici and cause take-all of the wheat roots produced polysaccharides in pure cultures (glucans, mannoglucans and galactomannoglucans). These polysaccharides were utilized better by the mycelium of G. graminis than glucose and polysaccharides of plant origin that occurred on the surface of wheat roots (the so-called mucigel). At lower concentrations these bacterial polysaccharides stimulated growth of wheat roots, higher concentrations (more than 0.1%) were inhibitory. Bacteria inoculated on the surface of wheat first inhibited and then stimulated the development of the plants and their growth. Changes in the growth rate of wheat, the rhizosphere of which was colonized by bacteria simultaneously with the fungus G. graminis and also some changes in the course of the disease of wheat roots caused by the fungus can be explained by the inhibitory or stimulatory effect of polysaccharides of accompanying bacteria.     

滇牡丹内生真菌Alternaria sp.PR-14的代谢产物研究

从滇牡丹内生真菌交链孢霉属(Alternaria sp.)菌株PR-14的发酵液提取物中分离得到6个化合物,通过波谱技术分别鉴定为交链孢酚(1)、djalonensone(2)、脑苷酯B(3)、脑苷酯C(4)、(2S,3S,4R,2’R)-2-(2’-hydroxytetracosanoylamino)octadecane-1,3,4-triol(5)和甘露糖醇(6),以上化合物均为首次从滇牡丹内生真菌中分离得到。     

气相色谱-质谱定量检测水产品腐败菌群体感应DKPs信号分子

【目的】为了分析水产品腐败菌群体感应的新型信号分子二酮哌嗪(DKPs)化合物,建立一种简便、灵敏的气相色谱-质谱(GC-MS)定量检测方法。【方法】通过优化气相色谱和质谱条件、培养基和提取溶剂建立定量检测方法,确定Cyclo-(L-Pro-L-Gly)、Cyclo-(L-Pro-L-Leu)、Cyclo-(L-Leu-L-Leu)和Cyclo-(L-Pro-L-Phe)4种DKPs标准品的特征离子,并检测水产品腐败菌荧光假单胞菌(Pseudomonas fluorescens)和波罗的海希瓦氏菌(Shewanella baltica)中的DKPs。【结果】4种DKPs化合物在1-200 mg/L范围内线性良好,检测限为0.06、0.10、0.06和0.04 mg/L,定量限为0.16、0.18、0.14和0.12 mg/L,回收率为51.8%-88.5%,标准偏差为1.4%-8.3%。以LB培养基为细菌培养基,氯仿作为萃取溶剂,检测的DKPs含量较高。两种水产品腐败菌都检测到DKPs活性,主要种类为Cyclo-(L-Pro-L-Leu)和Cyclo-(L-Pro-L-Phe)。随着两种细菌的生长,培养上清中DKPs含量显著增加,在12 h达到最高。【结论】建立了检测细菌DKPs的GC-MS定量方法,具有较高精密度、准确度,能够准确定量分析4种DKPs的含量。为探究水产品特定腐败菌DKPs的调控机制奠定基础。     

丁香内生死亡谷芽孢杆菌DX78的分离鉴定及其抑菌成分

【背景】植物内生菌往往产生与植物相同、相似或新颖的次生代谢产物,丁香具有广谱优异的抗菌活性,可从中分离到强抑菌作用的内生细菌。【目的】筛选抑制姜瘟病菌的丁香内生细菌并分离其活性成分。【方法】牛津杯法筛选拮抗内生细菌;根据16S rRNA基因序列鉴定菌株;有机溶剂萃取、硅胶柱层析和薄层制备色谱分离活性成分;测定1HNMR、13CNMR和DEPT(135°)并对分离的活性成分进行结构鉴定;滤纸片法和菌丝生长速率法测定活性成分抑菌活性。【结果】共分离到112株丁香内生细菌,从叶中分离最多,占37.4%。其中17株对姜瘟病菌有抑制,DX78菌株抑制作用最好,经鉴定为死亡谷芽孢杆菌,并从其发酵液中追踪分离到邻苯二甲酸二丁酯(dibutyl phthalate, DBP)。DBP对姜瘟病菌、猕猴桃溃疡病菌的MIC分别为0.3 mg/disc、0.25 mg/disc;其还可抑制多种植物病原真菌,特别对苹果炭疽叶枯病菌、番茄灰霉病菌和苹果腐烂病菌的抑制EC50仅分别为3.751、18.568和22.019μg/mL。以苹果炭疽叶枯病菌为靶标菌,戊唑醇抑制毒力约为DBP抑制毒力的4.5倍,DBP抑制毒...     

Stimulatory effect of cooling tower biocides on amoebae.

Two species of amoebae were isolated from the cooling tower of an air-conditioning system and examined for effects of exposure to four cooling tower biocides, a thiocarbamate compound, tributyltin neodecanoate mixed with quaternary ammonium compounds, another quaternary ammonium compound alone, and an isothiazolin derivative. The amoebae isolated were Acanthamoeba hatchetti and a Cochliopodium species. Two other amoeba cultures, an A. hatchetti culture and Cochliopodium bilimbosum, were obtained from the American Type Culture Collection (ATCC) and were also tested. The cooling tower isolates were more resistant to most of the biocides than the ATCC isolates were. The isothiazolin derivative was the least inhibitory to all four amoeba isolates, and tributyltin neodecanoate mixed with quaternary ammonium compounds was the most inhibitory to three of the four isolates. After exposure to lower concentrations of the biocides, including for one strain the manufacturer's recommended concentration of one biocide, the cooling tower amoeba populations increased significantly compared with unexposed controls, whereas the ATCC isolates were not stimulated at any of the concentrations tested. In some cases, concentrations which stimulated cooling tower amoebae inhibited the growth of the ATCC isolates. These results suggest that cooling tower amoebae may adapt to biocides, underscoring the need to use freshly isolated cooling tower organisms rather than organisms from culture collections for testing the efficacy of such biocides. The stimulatory effect of biocides on amoeba populations is an alarming observation, since these organisms may be reservoirs for legionellae. Biocides used to control microbial growth may actually enhance populations of host organisms for pathogenic bacteria.     

番茄灰霉病拮抗内生细菌的筛选、鉴定及其活性

对安徽省淮北市番茄植株根、茎、叶中内生细菌及其数量进行了调查和筛选,并测定了其抑菌活性。结果表明,番茄根、茎和叶中的内生细菌的数量分别为5.69×105、5.16×105和2.83×105CFU.g-1鲜重。根据分离部位和表型特征,从健康番茄植株体内分离到267株内生细菌,通过对峙实验,筛选到11株对番茄灰霉病菌有拮抗作用的菌株,占所分离内生细菌总数的4.12%。来自茎组织中的菌株XF136的抑菌效果最佳,抑菌带宽度达32.2mm。根据形态特征、生理生化特性、(G+C)mol%和16SrDNA序列分析,将菌株XF136鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。室内测定菌株XF136发酵滤液对灰霉病菌菌丝生长及分生孢子萌发的抑制作用,结果表明,菌株XF136发酵滤液可以抑制灰霉病菌菌丝生长和分生孢子萌发,且浓度越高,抑制能力越强;当发酵滤液浓度为20%时则完全抑制灰霉病菌菌丝生长和分生孢子萌发。盆栽防效试验结果表明,10%菌株XF136发酵滤液对番茄灰霉病防治效果与50%多菌灵600倍液相当,20%发酵滤液对番茄灰霉病的防治效果高于50%多菌灵600倍液。本研究表明,菌株XF136是防治番茄灰霉病潜在的优良生防菌株,具有良好的开发应用价值。     

源于植物内生无花果拟盘多毛孢真菌的新色原酮类次生代谢产物研究

从植物内生真菌无花果拟盘多毛孢菌株(Pestalotiopsis fici;AS3.9138=W106-1)的放大发酵粗提物中分离得到4个异戊二烯基取代的色原酮类新结构次生代谢产物pestaloficiolsM-P(1-4),并应用质谱和核磁共振技术确定了上述化合物的结构。生物活性测试结果表明化合物2能够抑制HIV-1病毒在C8166细胞中的复制;化合物3和4对宫颈癌细胞(HeLa)具有细胞毒活性;另外,化合物3对烟曲霉Aspergillus fumigatus也具有较强的抑制活性。