隐板裸刺蛇尾共附生真菌的分离鉴定及其代谢产物的抑菌活性研究

为更好地开发和利用蛇尾共附生微生物资源,本研究对隐板裸刺蛇尾(Gymnolophus obscura)可培养共附生真菌及其代谢产物抑菌活性进行研究。实验采用3种培养基分离隐板裸刺蛇尾共附生真菌,通过Blast分析ITS rDNA基因序列鉴定真菌,并运用96孔板法评估真菌的乙酸乙酯提取物的抑菌活性。实验结果发现,从隐板裸刺蛇尾分离的共附生真菌有格孢腔菌目(Pleosporales)真菌7株和肉座菌目(Hypocreales)真菌4株。两株真菌(GXIMD02021和GXIMD02022)与已报道菌株的ITS rDNA序列相似度小于97%,是潜在新菌种,占已鉴定菌株数量的18.2%。所有共附生真菌代谢产物对至少一种指示菌有抑制活性。除GXIMD02019、GXIMD02020、GXIMD02022和GXIMD02023外,其他菌株代谢产物均抑制表皮葡萄球菌(Staphylococcus epidermidis);4株菌株(GXIMD02018、GXIMD02020、GXIMD02021和GXIMD02022)代谢产物抑制耐甲氧西林金黄色葡萄球菌(Methicillin-resistant ...     

青海柯柯盐湖16株细菌的ARDRA筛选及系统发育初步分析

 从青海柯柯盐湖泥样中分离的16株细菌,用2种限制性内切酶酶切进行ARDRA分析,其中12株菌的酶切带谱有较大差异.根据这12株菌的16SrDNA序列进行系统发育分析结果表明,12株菌有11株分布于芽孢杆菌科中的Halobacillus,Gracibacillus,Amphibacillus,Virgibacillus,Bacillus,Salibacillus和1个潜在的新属中,另1株属于Proteobacteria的Gamma亚群Halomonadaceae科,Halomonas属.而且16SrDNA系统发育分析提示这些菌株中存在3个新种和1个新属的可能.本研究得出的结论为:应用ARDRA来初步筛选菌株是可行的;青海柯柯盐湖这一极端环境中存在较丰富的新的芽孢菌种类.     

Illumina高通量测序分析百合内生真菌群落多样性

百合具有较高的药用食用价值,研究发现它的许多内生菌代谢产物有进一步开发利用的潜能,深入了解百合内生真菌群落结构与多样性,有助于发现新菌种以及具有特定功能的内生菌代谢产物。以ITS区的rDNA为靶标序列,采用Illumina MiSeq测序技术分别对百合叶片(bhy417)、百合茎秆(bhj417)和百合地下鳞茎(bhl417)三个样品的内生真菌群落结构组成和多样性进行测定分析。经过分析有以下结果:三个样品共得到用于后续样品分析的436394条有效序列和122205709个碱基;样品bhy417的内生真菌群落丰富度最大;三个样品之间的内生真菌群落多样性差异不显著、群落结构不同;球腔菌属Mycosphaerella、外囊菌属Taphrina、炭疽菌属Colletotrichum、散斑壳属Lophodermium、壳二孢属Ascochyta、Zymoseptoria属、Catenulostroma属、Phaeophleospora属、Endoconidiophora属、Hormonema属、乌氏霉属Uwebraunia、Capnobotrylla属、丛赤壳属Neonectria、长毛盘菌属Trichophaea等在样品中丰度较高。FUNGuild功能预测结果显示,百合内生真菌包括以下功能菌群:植物病原菌、动物病原菌、腐生菌、未定义腐生菌、内生菌、木材腐生菌、地衣寄生菌等。研究结果表明百合有丰富的内生真菌定殖,其中的植物病原菌以及一些目前还未鉴定出来的菌将可能对百合生防菌以及具特定功能代谢产物的研发提供帮助。     

滇藏地区8种鹅膏菌的ITS序列分析

 对滇藏地区8种鹅膏菌的核糖体DNA(rDNA)内转录间隔区(ITS)进行PCR扩增和测序,用ClustalX(version 1.81)软件对所测的ITS序列进行比对分析.结果表明,除5.8S rDNA(其长度为160 bp)比较保守外,8个不同种的ITS序列在长度和碱基位点上都存在较大差异,其中ITS1区间的片段长度为206～297 bp,ITS2区间的片段长度为191～238 bp.这说明鹅膏属的种间具有较高的遗传多样性.利用MEGA(version 3.1)软件进行聚类,8个鹅膏菌在D=0.14处分成3组,与传统分类有出入.这为鹅膏菌的进一步分类及研究提供了分子依据.     

Hydrothermal synthesis of pyrrhotite (Fex-1S) nanoplates and their antibacterial, cytotoxic activity study

The aim of this study was to synthesize and characterize Fe_(x-1)S 2D-nanostructures with pyrrhotite phase,as well as to explore their biological(antibacterial and cytotoxic)properties,namely the expression of reactive oxygen species(ROS)in the exposure of cells and bacteria.Based on hydrothermal synthesis,the characterization of asprepared 2D-nanostructures was performed by XRD,SEM,EDS,and TEM,in which the single-crystalline pyrrhotite phased Fe_(x-1)S nanoplate morphology was observed.The antibacterial activities of Fe_(x-1)S nanoplates against human pathogenic strains such as Staphylococcus aureus,Escherichia coli,and Enterococcus faecalis were tested.Minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)were determined following the broth microdilution method.Cytotoxicity and expression of intracellular ROS of pyrrhotite nanoplates on Human Gingival Fibroblast(HGF),Human Pulp Cells(HPC)and Human Osteoblast(HBC)were calculated.Cell viability was determined by the MTT method.All experiments were performed of three independent experiments and data were analyzed by Kruskal-Wallis and Mann-Whitney tests,also Pearson′s Correlation was performed.The nanoplates exhibited good bactericidal effect.All types of cells tested showed slight cytotoxicity.It was found that intracellular ROS is produced when cells and bacteria tested are exposed to pyrrhotite nanoplates in presence of both air and peroxide hydrogen.ROS production levels were higher in the bacteria than the cells exposed to these nanoplates.     

Antiviral activities of extracts from Hong Kong seaweeds

We extracted six Hong Kong brown seaweed species with hot water for their antiviral properties. The cytotoxicity and antiviral activity of these extracts were tested by MTT [3-（4,5-dimethylthiazol-2-yl）-2,5-diphenlytetrezolium bromide] method, cytopathic effect reduction assay, and plaque reduction assay. The antiviral effect was further determined by flow cytometric analysis. The results showed that most of these extracts inhibited the propagation of herpes simplex virus types 1 and 2 （HSV-1 and HSV-2） standard strains with very low cytotoxicity to the host cells. The extracts ofHydroclathrus clathratus and Lobophora variegata showed more potential anti-HSV activities than the extracts of the other four seaweeds. They also had moderate antirespiratory syncytial virus （RSV） activities but could not inhibit influenza A virus. Hydroclathrus clathratus was further extracted by diluted acid and alkali and the antiviral effects of the extracts were also detected. The result showed that the hot water extract contained the main carbohydrate components that exhibited the antiviral activities against various strains of HSV, including the acyclovir-resistant strain. HI-3, a compound fractionated from this hot water extract, showed a dose-dependent anti-HSV activity in flow cytometric analysis and plaque reduction assay.     

高效木质素降解菌的筛选及降解性能研究

【目的】筛选纯化及鉴定木质素降解菌,对其降解性能进行初步研究。【方法】从某制浆造纸厂的造纸废水污泥中,筛选获得2株具有木质素降解能力的细菌LD-1和LD-2。通过形态观察和16SrRNA序列分析对菌株进行鉴定;采用苯胺蓝平板脱色法及以TC为指标分别定性定量测定2株细菌的木质素降解能力。【结果】LD-1和LD-2分别被鉴定为Sphingobacterium属和Bacillus属的菌株,命名为Sphingobacterium sp.LD-1和Bacillus sp.LD-2。通过苯胺蓝平板脱色实验,发现LD-1和LD-2均具有良好的木质素降解能力。经测定,两株菌种发酵7d对木质素的降解率分别达到52.23%和50.36%。【结论】成功筛选到2株能高效降解木质素的菌株LD-1和LD-2。     

Biological control of aflatoxin contamination of crops

Aflatoxins produced primarily by two closely related fungi, Aspergillus flavus and Aspergillus parasiticus, are mutagenic and carcinogenic in animals and humans. Of many approaches investigated to manage aflatoxin contamination, biological control method has shown great promise. Numerous organisms, including bacteria, yeasts and nontoxigenic fungal strains of A.flavus and A. parasiticus, have been tested for their ability in controlling aflatoxin contamination. Great successes in reducing aflatoxin contamination have been achieved by application of nontoxigenic strains of A. flavus and A. parasiticus in fields of cotton, peanut, maize and pistachio. The nontoxigenic strains applied to soil occupy the same niches as the natural occurring toxigenic strains. They, therefore, are capable of competing and displacing toxigenic strains. In this paper, we review recent development in biological control of aflatoxin contamination.     

牛大力内生细菌的多样性及其促生特性研究

为研究牛大力Callerya speciose内生细菌多样性组成及其促生特性,从经表面消毒处理的牛大力根茎组织中分离内生细菌,采用16SrDNA序列分析法对内生细菌的多样性进行系统发育分析,并以固氮、溶磷性、产铁载体、产IAA能力等为指标,对内生细菌进行促生特性检测。从牛大力根和茎中共分离到59株内生细菌,包括16属20种,芽孢杆菌属是牛大力的优势菌群;在20种分离菌株中,分别有11,17,7株具有潜在的固氮、溶磷性、产铁载体能力,有13株菌可以合成IAA。其中RH17-2、RH17-5、GR2-1 3株菌同时具备固氮、溶磷性、产铁载体、产IAA 4种促生特性。从牛大力中分离得到的内生细菌具有丰富的物种多样性和促生特性,在未来的农业生产上具有潜在的应用价值。     

新疆羊肚菌rDNA的ITS序列分析

根据生境和形态学特征与大型真菌图鉴对照,从形体上挑选了4个羊肚菌子实体（其中3个来自哈密巴里坤,1个来自乌鲁木齐南山）,同时从四川绵阳某研究所购得一株高羊肚菌菌种做为实验阳性对照,为了确定收集的羊肚菌分类学地位,采用PCR技术,以rDNA—ITS为分子指标,对子实体和菌丝进行了ITS序列测定与分析,结合系统进化树确定品种归属。     

白虎头红树林区海泥可培养细菌分离鉴定及其活性研究

【目的】研究北海白虎头红树林区海泥可培养细菌多样性及其代谢产物的生物活性。【方法】采用纯培养法和分子生物学方法对样品中细菌多样性进行研究,利用滤纸片法和卤虫致死法测试其生物活性。【结果】从海泥中分离获得相关可培养细菌17株,选取9个代表性菌株采用16SrRNA基因序列的系统发育分析后发现,其中5株菌属于Firmicutes门,其余4株菌为Proteobacteria门。5株细菌的代谢产物有较好的抑菌活性,2株菌的代谢产物对卤虫致死活性明显,其中1株菌兼具两种活性。【结论】北海白虎头红树林区海泥中的细菌多样性较为丰富,并且部分细菌具有一定的生物活性。     

柳珊瑚Anthogorgia caerulea相关可培养共生放线菌多样性及其生物毒活性研究

【目的】研究斜阳岛附近海域柳珊瑚Anthogorgia caerulea可培养共生放线菌多样性及其发酵液代谢产物的生物毒活性。【方法】采用纯培养法和基于16SrRNA基因序列的系统发育分析对从广西斜阳岛附近海域采集的柳珊瑚Anthogorgia caeru[ea可培养共生放线菌多样性进行研究,利用卤虫致死法测试其生物毒活性。【结果】从柳珊瑚Anthogorgia caeru[ea中分离获得相关可培养放线菌23株,采用16SrRNA基因序列的系统发育分析后发现,这些菌株属于2个亚纲9个科9个属23种。绝大部分菌株都具有一定的生物毒活性,其中10株菌株有较强生物毒活性,l株有显著毒活性。【结论】广西斜阳岛附近海域柳珊瑚Anthogorgia caeru[ea中存在较为丰富的放线菌多样性,部分菌株具有较强的生物毒活性。     

无机离子对嗜盐放线菌株YIM 90615代谢产物多样性的影响

为了研究NaCl浓度、金属离子(Cr7+、Mg2+及K+)、氯化钪及卤素(I-、Br-及F-)对嗜盐放线菌株YIM 90615液体发酵过程中次级代谢产物多样性的影响,作者采用HPTLC(high performance thin layer chromatography)技术,结合紫外(254 nm)检测、碘及硫酸-茴香醛显色方法,对该菌代谢产物的HPTLC图谱进行了定性分析.结果显示,NaCl添加质量浓度为100 g/L时该菌株发酵效果最佳,有利于其代谢产物的多样性及量的积累;Cr7+和K+有利于其次生代谢产物的产生;1 g/L的氯化钪能增加其次生代谢产物的丰富性;F-对其次生代谢产物多样性有较好的促进作用.试验结果表明外源添加一定浓度和种类的盐离子有助于提升YIM 90615液体发酵过程中次生代谢产物多样性,为新型抗生素及其它有用化合物的产生创造合适条件,同时有助于嗜盐放线菌发酵培养基的筛选工作.     

北部湾广西海域海洋真菌多样性及其生物活性研究

本研究从北部湾广西海域采集的海绵、珊瑚、沉积物等样品中分离真菌,旨在挖掘北部湾的海洋真菌资源,筛选出具有潜在抗肿瘤或抗菌活性的菌株。采用稀释涂布法和基于内转录间隔区基因序列(ITS)的系统发育树法分析海洋真菌的多样性信息,并通过滤纸片琼脂扩散法和MTT比色法评价菌株的抗菌、抗肿瘤活性。分离得到45株海洋真菌,隶属17属。其中青霉属(Penicillium)和曲霉属(Aspergillus)为优势种群,分别占总菌株种类的26.7%和24.4%。此外,获得一株潜在的新种菌株Myrothecium gramineum GXIMD01018。活性筛选结果发现菌株Aspergillus japonicus GXIMD01014、Penicillium oxalicum GXIMD01021、Talaromyces purpureogenus GXIMD01024等6株真菌的次级代谢产物对人结直肠癌细胞具有较显著的细胞毒活性,菌株T.purpureogenus GXIMD01024的代谢粗提物具有一定的抗耐甲氧西林金黄色葡萄球菌(Methicillin-Resistant Staphylococc...     

金针菇rDNA-ITS序列分析

为研究金针菇rDNA-ITS序列特征,提取了不同来源金针菇子实体的基因组DNA,对其rDNA的内转录间隔区进行PCR扩增和序列测定,并提交NCBI获得登录号;将不同产地的金针菇ITS序列与本研究获得的金针菇ITS序列进行对比分析,利用ITS序列分析技术研究其遗传多样性,并构建了NJ系统发育树。结果表明,所有供试材料的ITS全长在710～719　bp范围内,G+C含量在49.3　%～49.9　%,所有序列共有34个变异位点,16个信息位点,ITS区遗传距离变化范围为0～0.016,即不同产地的金针菇ITS序列在进化过程中存在差异。此结果可为真菌分类鉴定等研究提供重要的资料和依据。     

高良姜与混淆品rDNA ITS序列的分析与鉴别

用PCR直接测序法测定和分析不同产地野生和种植品种的高良姜(Alpinia officinarum Hance),以及山姜（A. japonica（Thunb.） Miq.）、华山姜（A. chinensis (Retz.) Rosc.）和大高良姜(A. galanga (L.) Willd )等三种混淆品的rDNA ITS区序列,为高良姜种质资源研究和真伪鉴别提供分子数据。经测序、比对和排序得到812bp序列,包括18S3’端部分序列,ITS1、5.8S、ITS2全部序列和26S5’端部分序列。序列分析结果显示,高良姜种内序列高度一致,同源性达100%,测序结果中发现杂合位点,而广西样品杂合位点的两个碱基各占的比例与其它地方样品的不同,显示了高良姜种内rDNA ITS序列的细微差异。高良姜和混淆品的812bp序列中共有61个变异位点,60个是信息位点,同源性达96.32%,其中ITS1和ITS2中的11个位点在高良姜和混淆品中差别明显,可以鉴别高良姜和混淆品,因此rDNA ITS序列是高良姜真伪辨别的有效标记。基于DNA序列的高良姜和混淆品的系统分类结果与形态学的分类结果不完全一致,有待进一步的研究探讨。     

斜阳岛珊瑚礁区海泥可培养细菌分离鉴定及活性研究

【目的】研究广西斜阳岛珊瑚礁区海泥可培养细菌多样性及其代谢产物的生物活性。【方法】采用纯培养法和基于16SrRNA基因序列的系统发育分析细菌多样性,利用滤纸片琼脂扩散法和卤虫致死法研究其生物活性。【结果】从海泥中分离获得相关可培养细菌53株,这些菌株代表27个物种,其中20株菌属于厚壁菌门(74%),7株菌为变形菌门(26%)。活性研究发现,X-15(Erythrobacter citreus)的抑菌活性和生物毒活性都较强,具有开发新型食品防腐剂和抗肿瘤药物的潜力。【结论】广西斜阳岛海泥中的细菌多样性较为丰富。     

入侵植物紫茎泽兰叶斑真菌多样性及其致病性研究

 从云南、广西等12个地区的紫茎泽兰和其他8种植物叶片上共分离得到47个真菌菌株.根据菌落、菌丝以及孢子形态,结合18SrRNA基因限制性酶切片段长度多态性(RFLP)分型和序列测定分析,47个菌株共分为了19个类群,系统发育关系与Aspergillusniger,Cladosporiumcladosporioides,Sporisoriumreilianum,Colleto-trichumlupini,Alternaria sp.,Sporobolomycesroseus,Dothideomycetesp.,Phoma sp.,Filobasidiumelegans,Aureobasid-iumpullulans,Rhodotorulaslooffiae,R.glutinis,Penicilliumdecumbens,Pestalotiopsismaculans,Cryptococcusaureus,Bimurianovae-zelandiae,Xylariaceae sp.类似.这些菌株中仅有5个对紫茎泽兰离体叶片表现致病性,在系统发育树上分别和Myrothecium sp.,C.lupini,Alternaria sp.,C.cladosporioides,Dothideomycete sp.聚类.有2个R.gluti-nis菌株对马铃薯和白菜有致病作用,但对紫茎泽兰没有致病力.     

Bioactivity and constituents of several common seaweeds

Bioactivity and constituents of 8 common seaweeds from Dalian intertidal zone of northern Yellow Sea were investigated. In the anti-methicillin-resistant Staphylococcus aureus (MRSA) test, Symphyocladia latiuscula and Enteromorpha intestinalis showed obvious activities with MICs much lower than 1.0 mg·mL-1 . In the DNA damage repair test (DDRT), Chondrus ocellatu showed selective inhibitory activity against the DNA repair-defective E. coli strain vs. the wild-type E. coli strain; while Sym. latiuscula, Enteromorpha intestinalis and Sar. kjellmanianum showed significant anti-E. coli activity with MICs of 64-128 g·mL-1 . In the anti-Pyricularia oryzae test, Sym. latiuscula and Rh. confervoides strongly inhibited the germination of the spores of P. oryzae on agar plate. In the brine shrimp larvicidal test, Sym. latiuscula, Rh. confervoides and Sar. kjellmanianum exhibited potent toxicity against brine shrimp larvae, with LC50 much lower than 1 mg·mL-1 . The HPTLC analysis revealed their diversified secondary metabolites. The HPLC-DAD-MS analysis of the strongest species Sym. latiuscula and database searching showed that it can produce quite diversified metabolites, including halogenated ones, some of which may be new natural products. The results demonstrated the potentials of these seaweeds in the development of new antibiotics, antitumor drugs, agricultural fungicides and pesticides.     

乌头内生真菌YLAC-3抗菌活性及其鉴定

为了筛选产乌头碱的内生真菌,采用组织分离法从白喉乌头根中分离内生真菌,用纸片扩散法检测内生真菌对4种指示菌的抑菌作用,通过薄层层析法（TLC）和高效液相色谱法（HPLC）检测真菌次生代谢产物中乌头碱,最后基于真菌形态特征和rRNA基因内转录间隔区（ITS）序列分析对菌株进行鉴定.结果显示：从白喉乌头根部共分离得到20株具有抑菌活性的内生真菌,其中菌株YLAC-3发酵液对大肠杆菌、枯草芽孢杆菌、金黄色葡萄球菌和白色念球菌均有较强的抑菌活性,且其发酵液中发现类似于乌头碱的化合物.说明菌株YLAC-3发酵液有较为广泛的抑菌活性,它可能产生乌头碱.菌株YLAC-3菌落、菌丝体和分生孢子的形态特征类似于曲霉菌属,与已报道的塔宾曲霉菌ITS序列（KP196359）的相似度为99%.初步鉴定该菌株为曲霉菌属有效发表种Aspergillus tubingensis的一个菌株.