两种栽培型广藿香内生真菌群落组成变化

为探索内生真菌与广藿香互作间对宿主活性成分形成机制的影响,该研究以成分差异较大的牌香和湛香为对象,采用传统形态学方法对所获菌株归类,通过真菌通用引物ITS1/ITS4扩增菌株rDNA-ITS序列,鉴定其分类地位并研究其多样性。结果表明：(1)用PDA和LBA培养基对苗期、分枝期和成株期广藿香茎叶组织块进行内生真菌分离,共获得3 070株菌株,其中牌香(PX)分离出1 624株,鉴定出1 319株,分属于36属;湛香(ZX)分离出1 446株,鉴定出994株,分属于33属。牌香分离出7种特有内生真菌,分别为香柱菌(Epichloe typhina)、盘长孢状刺盘孢菌(Colletotrichum gloeosporioides)、座腔孢菌(Botryosphaeria sp.)、丝核菌(Rhizoctonia sp.)及截盘多毛孢菌(Truncatella sp.),并首次分离到疫霉菌(Phytophthora sp.)和指疫霉菌(Sclerophthora sp.),这2种菌属于卵菌门内生菌。湛香分离出拟青霉菌(Paecilomyces sp.)和尾孢菌(Cercospora sp.)...     

北细辛内生真菌的分离鉴定及代谢产物的生物活性

采用表面消毒法分别从3种北细辛中分离获得10株形态特征不同的优势内生真菌,经形态学和18S rDNA ITS分子分类学分析鉴定为小丛壳属(Glomerella sp.)、叶点霉属(Phyllosticta sp.)、柄孢壳菌属(Po-dospora sp.)、刺盘孢属(Colletotrichum sp.)和镰孢属(Fusarium sp.)。对北细辛优势内生真菌的发酵产物进行体外抗肿瘤和抗菌活性检测,结果表明:除菌株E3、E4和E10外,其余菌株均有不同程度的抗肿瘤和抗菌活性;镰孢属(Fusarium sp.)菌株E9对A549、MDA-MB-231和PANC-1肿瘤细胞抑制率达75%以上;小丛壳属(Glomerella sp.)菌株E1和叶点霉属(Phyllosticta sp.)菌株E2靶向FabI的抗菌活性较强,抑制率达59%。     

思茅松毛虫肠道产脂肪酶菌株的筛选鉴定及酶学性质初步研究

利用纯培养和筛选培养,从思茅松毛虫幼虫肠道中分离得到7株产脂肪酶的菌株.通过提取基因组DNA并进行16S rDNA序列测定,构建产酶菌株的系统发育树,初步鉴定结果显示:菌株D2、D12、D19属于假单胞菌属(Pseudomonas sp.),菌株D7、D17属于芽胞杆菌属(Bacillus sp.),菌株D9、D16属于克雷伯氏菌(Klebsiella sp.).初步研究所产脂肪酶的酶学性质,确定这些酶的最适作用温度30～40℃、最适作用pH值8.0～9.0,为中温碱性脂肪酶.     

糖多孢菌属中新种的鉴定

从云南省昆明市郊水田土样中分离到Y84—4001和Y84一4003两株菌。其特点是不抗酸,气丝形成孢子链,菌落中心的基丝断裂,胞壁IV型,应置于糖多孢菌属。根据形态、培养特征和生理生化特性的研究,将菌株Y84—4001定名为橙黄糖多孢菌(Saccharopolyspora auranti-aca sp. Nov.),菌株Y8-4003定名为橙黄糖多孢菌昆明亚种(Saccharopolyspora aurantiacasubsp．Kunmingcnsis subsp. Nov.)。     

尾孢菌属五个新种

本文报道尾孢菌属五个新种,即美人蕉尾孢(Cercospora cannae Bai,Liu et Guo,sp.nov.),败酱尾孢(C.patriniae Liu et Guo,sp.nov.),骆驼蓬尾孢(C.pegani Liu et Guo,sp.nov.),旌节花尾孢(C.stachyuricola Liu et Guo,sp.nov.)和安息香尾孢(C.styracicola Liuet Guo,sp.nov.)。文中对各个新种作了汉文描述和附图,并有拉丁文特征简介。模式标本收藏于中国科学院真菌标本室。     

棒孢菌属四个新种

本文报道了棒孢菌属四个新种,即女贞棒孢(Corynespora ligustri Guo,sp.nov.),茉栾藤棒孢(C.merremiae Guo,sp.nov.)鸡血藤棒孢(C.millettiae Guo sp.nov.)和蔓荆子棒孢(C.viticis Guo,sp.nov.)。文中对各个新种作了汉文描述和附图,并有拉丁文描述和特征简介。模式标本收藏于中国科学院微生物研究所真菌标本室。     

水稻秸秆还田时间对土壤真菌群落结构的影响

为揭示水稻秸秆还田对土壤真菌群落结构的长期影响,采用荧光定量PCR和PCR-DGGE技术分析了秸秆还田90,180,270 d和360 d的土壤真菌基因丰度和群落结构组成演变趋势,并利用冗余分析(RDA)研究土壤真菌群落结构变化与环境因子的关系。结果表明:随着秸秆还田时间的增加,土壤真菌群体数量和多样性指数(H、R和E)显著增加,在360 d时达到最高。对DGGE图谱的特征条带进行胶回收、测序,系统进化分析表明,土壤真菌主要种群包括:接合菌(Zygomycete sp.)、盐腐霉菌(Pythium salinum)、肉盘菌(Uncultured Sarcosomataceae)、牛粪盘菌(Ascobolus stercorarius)、大链壶菌(Lagenidium giganteum)、青霉菌(Penicillium sp.)、曲霉属真菌(Aspergillus sp.)和疏绵状丝孢菌(Thermomyces lanuginosus)、灰绿曲霉菌(Aspergillus glaucus)、禾谷多粘菌(Polymyxa graminis)和枝顶孢霉菌(Acremonium sp.),其中青霉菌(Penicillium sp.)、曲霉属真菌(Aspergillus sp.)和枝顶孢霉菌(Acremonium sp.)具有纤维素降解能力,而枝顶孢霉菌(Acremonium sp.)在90 d时成为新的优势菌群。RDA分析表明,90 d和180 d秸秆还田与对照土壤的真菌群落结构较为类似,270 d和360 d的秸秆还田与对照土壤的真菌群落结构发生了明显变化。土壤有机碳、pH和速效磷是引起土壤真菌群落结构及多样性变异的主要因素。     

链霉菌S01菌株几丁质醇对植物病原真菌的拮抗作用

纯化后的链霉菌Ｓ０１菌株几丁质酶用环柱法在ＰＤＡ平板上对杨树腐烂病菌、葡萄孢菌（Ｂｏｔｒｙｔｉｓ ｓｐ．ＡＳ３．２６１６）、黄瓜黑腥病菌、辣椒疫病菌、棉花黄萎病菌、立枯丝核菌等植物病原真菌及产黄青霉、啤酒酵母的生长具有明显的抑制作用。在６株植物病原真菌的液体培养物中加入几丁质酶后,菌丝出现扭曲变形、细胞质聚集、外溢等异常现象。在高渗缓冲液中,经几丁质酶处理后,啤酒酵母、葡萄孢菌和产黄青霉的细胞壁受     

连作花生田根际土壤优势微生物的分离和鉴定

【目的】从不同连作年限的花生田根际土壤中分离优势微生物并进行鉴定,为研究花生连作后优势微生物的变化奠定基础。【方法】采用土壤稀释分离法从不同连作年限花生根际土壤中分离优势细菌、真菌和放线菌,结合菌株形态特征、培养性状、生理生化特征及16S rDNA序列分析对细菌、放线菌进行鉴定,通过形态特征、培养特征和分子鉴定方法对优势真菌进行鉴定。【结果】从连作花生田根际土壤中分离鉴定出7种优势细菌、7种优势真菌和7种优势放线菌。7种优势细菌分别为Leifsonia xyli、氯酚节杆菌(Arthrobacterchlorophenolicus)、黄色微杆菌(Microbacterium flavescens)、鞘氨醇单胞菌属(Sphingomonas sp.)、巴斯德菌属(Pasteurella sp.)、简单芽孢杆菌(Bacillus simplex)和巨大芽孢杆菌(Bacillus megaterium)。7种优势真菌分别为枝状枝孢菌(Cladosporium cladosporioides)、产紫青霉(Penicillium purpurogenum)、哈茨木霉有性型(Hypocrea lixii)、Exophiala pisciphila、微紫青霉(Penicillium janthinellum)、曲霉(Aspergillus sp.)和大丽轮枝菌(Verticillium dahliae)。7种优势放线菌分别为紫红链霉菌(Streptomyces violaceoruber)、华丽黄链霉菌(Streptomyces flaveus)、Streptomyces panaciterrae、不产色链霉菌(Streptomyces achromogenes)、假浅灰链霉菌(Streptomyces pseudogriseolus)、纤维素链霉菌(Streptomyces cellulosae)和金色链霉菌(Streptomyces aureus)。【结论】本研究是第一次系统的从连作花生根际土中分离鉴定优势微生物,种植花生后根际土壤中优势微生物的种类发生了明显变化,但变化没有规律。     

基于高通量测序的3种竹子小枝真菌群落

为了解感染丛枝病时竹子真菌群落的特征,以丛枝病侵染的斑竹(Phyllostachys bambusoides f. lacrima-deae)、毛竹(Ph. edulis)和早竹(Ph. praecox)小枝为研究材料,运用高通量测序技术、DNA条码技术和生物信息学技术,分析了感染丛枝病的斑竹、毛竹、早竹真菌群落的物种组成和多样性,探讨了竹子丛枝病病原菌与其他竹子真菌之间的相关性。结果表明:(1) 3种竹子小枝共鉴定到706个最终的OTUs,所有样品中均存在的有48个; 3种感病竹子真菌群落中相对丰度较高的菌群包括:针孢麦角菌属(Aciculosporium)、竹黄属(Shiraia)、漆斑菌属(Myrothecium)、棘壳孢属(Pyrenochaeta)、刺盘孢/炭疽菌属(Colletotrichum)、镰刀菌属(Fusarium)、弯梗孢属(Camptophora)和假酵母状菌属(Pseudozyma)。(2)3种感病竹子真菌群落间的Chao1指数和Shannon指数差异不显著(P0.05)。(3) 3种感病竹子的病原菌为竹针孢座囊菌(Aciculosporium take),该菌与13种竹子真菌之间存在负相关,其中9种的相关性达到显著水平(P0.05);该菌与2种竹子真菌之间存在较弱的正相关。(4)鉴定的竹子真菌中存在绿色木霉(Trichoderma virens)、枝孢霉属真菌(Cladosporium sp.)、竹黄属真菌(Shiraia sp.)、弯孢霉属真菌(Curvularia sp.)、青霉属真菌(Penicillium sp.)和枝顶孢属真菌(Acremonium sp.),这些真菌是竹子病害潜在的生防菌。     

降解菊粉担子菌的分离鉴定及其酶的产生

从菊芋地的腐木上分离到一株在以菊粉为唯一碳源和能源的培养基上生长良好,具有较高菊粉酶活性的担子菌菌株,经鉴定为采绒革盖菌（Ｃｏｒｉｏｌｕｓｖｅｒｓｉｏｌｏｒ）。该菌的菊粉酶大部分是胞外酶,此酶对菊粉的专一性高,其Ｉ／Ｓ比值在发酵过程中不断变化。菊粉酶活性平行地随菌体生长而增加。该酶的合成受菊粉诱导,受果糖抑制。当果糖浓度大于２．７ｍｇ／ｍｌ时,菊粉酶活性为零。菌体的匀质化可使生长加快从而获得大量菊粉酶。     

Molecular and biochemical characterization of a new endoinulinase producing bacterial strain of Bacillus safensis AS-08

Microbial inulinases are an important class of industrial enzymes, which are used for the production of fructooligosaccharides and high-fructose syrup. Endoinulinase producing bacterial strains were isolated from soil samples taken from the vicinity of Asparagus sp. root tubers. All the bacterial strains were screened for inulinase activity. The primary screening was carried out based on hydrolytic zone on agar plates containing inulin-based medium and Lugol’s iodine solution. Thus 30 inulinase producing bacterial strains were isolated. Out of 30 strains, 5 bacterial strains were found endoinulolytic, whereas 25 were exoinulolytic on the basis of action pattern of the enzyme. In tertiary screening, the bacterial isolate AS-08 was found to be most efficient for inulinase activity. Morphological, biochemical and physiological characteristics of the bacterial isolate AS-08 confirmed it as Bacillus sp. Furthermore, species-specific identification by 16S rDNA sequencing and phylogenetic analysis revealed the isolate as Bacillus safensis. Bacillus pumilus SH-B30 was found to be the nearest homolog. The strain showed maximum inulinase activity (12.56 U/mL) after 20 h of incubation at 37°C.     

Partial purification and characterization of exoinulinase produced from Bacillus sp.

Inulinase are industrial food enzymes which have gained much attention in recent scenario. In this study, Inulinase producing eight bacterial colonies were isolated and screened from three different plant root tubers soil sample. Among 8 inulinase producing colonies, the higher yielding colony was selected with 25.10?U/mL for further studies. The best inulinase producing colony was identified by partial 16S rRNA gene sequence as Bacillus sp. The crude inulinase was purified by using ammonium sulphate precipitation, dialysis and ion exchange chromatography on DEAE – sephacel and obtained 1.9 purification fold with total activity 293 U. The purified enzyme was subjected to characterization studies and it was found to be stable at 30–60?°C and optimum temperature was at 55?°C. The enzyme was stable at pH 3.0–7.0 and optimum pH was at 6.5. The K_m and V_max value for inulinase was found to be 0.117?mg/mL and 4.45?μmol?min?mg^?1 respectively, demonstrate its greater affinity. Hence, this enzyme can be widely used for the production of fructose, and fructooligosaccharides, which are important ingredients in food and pharmaceutical industry.     

克鲁维酵母Y-85菊粉酶的纯化和性质

克鲁维酵母(Kluyveromyces sp.)Y-85产生的胞内菊粉酶(endocellular inulinase)和胞外菊粉酶(exocellular inulinase)粗酶液分别经PEG6000-磷酸盐缓冲液双水相抽提得部分纯化酶液。前者进一步用硫酸铵分级沉淀、Protein-PAK DEAE离子交换、Protein-PAK200SW凝胶过滤后得到两个菊粉酶组分EⅠ和EⅡ;后者采用DEAE-Sephacel离子交换、Sephadex G150凝胶过滤后得到菊粉酶Eexo。经Waters 650E蛋白纯化系统鉴定,三者均呈单一的对称峰;EⅠ和EⅡ达聚丙烯酰胺盘状凝胶电泳纯。EⅠ、EⅡ和Eexo的分子量分别为42kD、65kD和57kD;三者均为糖蛋白,多糖含量分别为30％、35％和25％;I/S(Inulinaseactivity/Sucrase activity)比值分别为0.086、0.078和0.072;三者均属外切菊粉酶。EⅠ、EⅡ和Eexo酶反应最适pH分别为4.6、4.5和4.6,最适温度分别为52℃、52℃和55℃;Ag^+、Hg^(2+)和PCMB对酶活性有强烈的抑制作用;三者水解菊芋粉糖液的产物均为果糖(86.5％)和葡萄糖(13.5％)。     

Production and properties of inulinase from Penicillium sp. NFCC 2768 grown on inulin-rich vegetal infusions

Inulinase production by Penicillium sp. NFCC 2768 isolated from the rhizosphere soil of dahlia was studied on media containing inulin-rich plant extracts. The maximum inulinase activity (64.54 nkat/ml) was observed with the tuber extract of dahlia (Dahlia pinnata). The fungus produced substantial inulinase activity on asparagus root powder (45.23 nkat/ml) and garlic extracts (41.32 nkat/ml). The apparent molecular weight of the purified inulinase was 68 kDa. The optimum pH and temperature for enzyme activity were 5.0 and 50°C, respectively. Mn²⁺ and Ca²⁺ were found to enhance the inulinase activity, while Hg²⁺ was found to be a strong inhibitor. Inulinase liberated fructose, glucose, sucrose, kestose (GF₂), nystose (GF₃), and inulooligosaccharides (IOS). This study suggested the use of dahlia tuber extract and asparagus root powder as suitable substrates for inulinase production by the newly isolated Penicillium sp. NFCC 2768, and its application in the generation of fructose and IOS.     

Production of novel alkalitolerant and thermostable inulinase from marine actinomycete Nocardiopsis sp. DN-K15 and inulin hydrolysis by the enzyme

An actinomycete strain Nocardiopsis sp. DN-K15 showing high inulinolytic activity was isolated from marine sediment of Jiaozhou Bay in China. Under optimal conditions, Nocardiopsis sp. DN-K15 produced 25.1 U/ml of inulinase within 60 h of fermentation at shake flask level, which was 2.7-fold higher than the level in the basal medium. The optimal pH and temperature of the inulinase from strain DN-K15 were determined to be 60 °C and pH 8.0, respectively. The inulinase was highly active over a wide pH range (5.0–11.0) and retained more than 81 % of residual activity after incubation at 60 °C for 1 h, indicating its alkali-tolerant and thermostable nature. Thin layer chromatography analysis revealed that fructose was the main product of inulin hydrolysis, indicating its exoinulinase activity. The high yield of extracellular inulinase combined with its novel enzymatic property made Nocardiopsis sp. DN-K15 a potential candidate in biotechnological and industrial applications.     

Penicillium subrubescens, a new species efficiently producing inulinase

Inulin is a reserve carbohydrate in about 15 % of the flowering plants and is accumulated in underground tubers of e.g. chicory, dahlia and Jerusalem artichoke. This carbohydrate consists of linear chains of β-(2,1)-linked fructose attached to a sucrose molecule. Inulinases hydrolyse inulin into fructose and glucose. To find efficient inulin degrading fungi, 126 fungal strains from the Fungal Biotechnology Culture Collection (FBCC) at University of Helsinki and 74 freshly isolated strains from soil around Jerusalem artichoke tubers were screened in liquid cultures with inulin as a sole source of carbon or ground Jerusalem artichoke tubers, which contains up to 19 % (fresh weight) inulin. Inulinase and invertase activities were assayed by the dinitrosalicylic acid (DNS) method and a freshly isolated Penicillium strain originating from agricultural soil (FBCC 1632) was the most efficient inulinase producer. When it was cultivated at pH 6 and 28 °C in 2 litre bioreactors using inulin and Jerusalem artichoke as a carbon source, inulinase and invertase activities were on day 4 7.7 and 3.1 U mL^?1, respectively. The released sugars analysed by TLC and HPLC showed that considerable amounts of fructose were released while the levels of oligofructans were low, indicating an exoinulinase type of activity. Taxonomic study of the inulinase producing strain showed that this isolate represents a new species belonging in Penicillium section Lanata-divaricata. This new species produces a unique combination of extrolites and is phenotypically and phylogenetically closely related to Penicillium pulvillorum. We propose the name Penicillium subrubescens sp. nov. (CBS 132785^T = FBCC 1632^T) for this new species.     

Effect of media supplements and culture conditions on inulinase production by an actinomycete strain

Streptomyces sp. GNDU 1 produced high levels of extra-cellular inulinase (0.552 IU/ml) after 24 h at pH 7.5, temperature 46 degrees C in the presence of 1% inulin. The optimum temperature and pH for enzyme activity were 60 degrees C and 5.5 respectively. Yeast extract as a nitrogen source was found to be most suitable one for inulinase production whereas ammonium ion was inhibitory to the enzymatic production. All these conditions make Streptomyces sp. GNDU 1, a potential candidate for industrial enzymatic production of fructose from inulin.     

克鲁维酵母Y-85合成菊粉酶最适条件的研究

采用响应面方法对克鲁维酵母Ｙ－８５产菊粉酶培养基成份进行了优选,和正交试验相比,该法选出的最适培养基的酶发酵水平提高２８％。用１５Ｌ自控发酵罐进行产酶条件控制试验,并在１０００Ｌ罐上进行了５批次酶发酵中试,平均菊粉酶活性达６８．９ｕ／ｍｌ。     

黑曲霉产菊粉酶菌株的选育及培养基优化

为获得高产菊粉酶的黑曲霉菌株,以Aspergillus niger YH-1为出发菌株,经过亚硝基胍(NTG)诱变,以高温高菊芋粉相结合的方式进行梯度驯化,选育出一株产菊粉酶菌株YH-3,并运用响应面实验方法对该菌株的培养基进行优化。确定了最佳培养基组成:菊芋粉25.2 g/L、豆饼粉40 g/L、蔗糖酯4.9 g/L、NaCl 5.5 g/L。发现内切菊粉酶活力(I)由60.9 U/mL提高到165.0 U/mL,比出发菌株提高了1.7倍。研究证明蔗糖酯对于黑曲霉YH-3发酵产菊粉酶是一种有效的促进剂。