一株具有脱胶功能大麻内生真菌的分离和鉴定

采用组织块法对大麻(Cannabis sativa)根、茎、叶等组织中的内生真菌进行分离,利用平板透明圈法筛选具有脱胶功能的菌株,对获得的脱胶菌菌株进行形态学鉴定和分子生物学鉴定。结果表明:(1)从大麻根、茎、叶的组织部位共分离得到内生真菌16株,茎部分离到9株真菌,叶部5株,根部2株。(2)编号为DM6的内生真菌具有较强的果胶分解能力,其透明圈直径为2.49cm。(3)形态学鉴定发现,内生真菌DM6不能产生孢子,菌丝较为粗壮、分支较少、有明显的隔;分子学鉴定表明,内生真菌DM6与Phoma aliena(KC311486)序列的相似性最高,为99%,并且在系统发育树上位于同一分支上。因而内生真菌DM6可以鉴定为茎点霉属一种(Phomasp.)。     

益母草内生真菌分离与初步鉴定

利用平板分离法从药用植物益母草(Leonurus heterophyllus Sweet)中分离到15株内生真菌,来源于根、茎、叶分别有2,6,7株。经形态学观察和染色等方法,初步鉴定了益母草内生真菌有3个属,曲霉属(Aspergillus sp.)2株、头孢霉属(Cephalosporiumsp.)3株和囊孢霉属(Capsule sp.)10株。益母草内生真菌生物多样性较为单一,但不同部位内生菌的数量、种类及分布存在明显差异。     

罗汉果内生真菌多样性研究

该文采用传统形态学方法结合r DNA-ITS序列分析,对我国重要药用植物罗汉果中的内生真菌进行了鉴定并研究其多样性。结果表明:采用组织培养法从罗汉果健康植株中共分离得到150株内生真菌,包括罗汉果中雌株的内生真菌96株、雄株的内生真菌54株。122株内生真菌经形态学结合r DNA-ITS序列分析鉴定为9个属,均归属为子囊菌门,包含座囊菌纲(Dothideomycetes)和子囊菌纲(Sordariomycetes)。其中,座囊菌纲(Dothideomycetes)真菌包含3科、3属;子囊菌纲(Sordariomycetes)真菌包含6科、6属。优势属为刺盘孢属(Colletotrichum)和镰刀菌属(Fusarium)。罗汉果雌、雄植株不同组织中内生真菌的定殖率及分离率的变化规律均不相同,雌株中以根中内生真菌的定殖率和分离率最高,叶片中的最低;在雄株中以叶片中的定殖率和分离率最高,根中的最低。不同菌株在雌、雄两种植株的不同组织中的分布均不同,结合内生真菌群落组成的相似性比较结果,表明部分内生真菌对罗汉果雌株和雄株,以及同一植株中的不同组织均具有偏好性。不同组织中内生真菌的多样性指数在0.11~0.69的范围,其中雌株根部的内生真菌多样性指数最高。以上研究结果为后期探究内生真菌与罗汉果互作关系奠定了基础。     

黄花蒿内生菌的分离与初步鉴定

利用平板分离法从药用植物黄花蒿(Artemisia annua Linn.)的根、茎和叶中共分离内生菌80株,其中内生真菌37株、细菌40株、放线菌3株.经菌种形态观察和染色等,初步鉴定了黄花蒿内生真菌具有5个属,包括囊孢菌(Capsule)、头孢霉(Cephalosporium)、弯孢霉(Curvularia)、曲霉...     

内生真菌对姜黄素的微生物转化

目的:调查姜黄根茎内生真菌对姜黄素的微生物转化,以期获得一些姜黄素的结构类似物或衍生物。方法:利用表面消毒法分离内生真菌;采用薄层层析和高效液相色谱(HPLC)技术筛选生物转化姜黄素的内生真菌;利用硅胶柱层析和制备型HPLC分离纯化生物转化产物;应用波谱技术解析转化产物的化学结构;通过形态学特征和内转录间隔区(ITS)序列分析对内生真菌进行初步鉴定。结果:从姜黄根茎中分离出18株内生真菌,经筛选发现其中1株丝状真菌能转化姜黄素,其产物分别为去甲基姜黄素和二去甲基姜黄素。初步鉴定该内生真菌属于Diaporthe sp.。结论:内生真菌Diaporthe sp.能对姜黄素进行去甲基化修饰,推测它可能具有O-去甲基化酶系。     

飞龙斩血内生菌种群分布及抑菌活性检测

【目的】研究药用植物飞龙斩血内生菌的种群组成及其抑菌活性,以了解飞龙斩血内生菌的种群分布状态和得到抗菌活性菌株。【方法】采用严格的表面消毒程序、添加抑菌剂的方法分离、培养内生菌株。利用表型和分子技术相结合的方法对内生菌进行分类鉴定。纸片扩散法对分离获得的内生菌进行抑菌活性试验。【结果】从飞龙斩血植株内分离得到了3株内生细菌,1株内生放线菌和82株内生真菌。分类鉴定为14目16科27属,镰孢属(Fusarium)、拟盘多毛孢属(Pestalotiopsis)、曲霉属(Aspergillus)为飞龙斩血内生真菌中的优势种群。通过对30株病原指示菌的抑菌活性检测,18株内生菌对多种指示菌有明显的抑制作用。16株是内生真菌,分属11属。【结论】本试验研究了飞龙斩血内生菌的种群分布,获得了一些具有抗菌活性的内生菌,为飞龙斩血内生菌资源的开发利用提供了基础。     

飞龙斩血内生菌种群分布及抑菌活性检测

摘要:【目的】研究药用植物飞龙斩血内生菌的种群组成及其抑菌活性,以了解飞龙斩血内生菌的种群分布状态和得到抗菌活性菌株。【方法】采用严格的表面消毒程序、添加抑菌剂的方法分离、培养内生菌株。利用表型和分子技术相结合的方法对内生菌进行分类鉴定。纸片扩散法对分离获得的内生菌进行抑菌活性试验。【结果】从飞龙斩血植株内分离得到了3株内生细菌,1株内生放线菌和82株内生真菌。分类鉴定为14目16科27属,镰孢属(Fusarium)、拟盘多毛孢属(Pestalotiopsis)、曲霉属(Aspergillus)为飞龙斩血内生真菌中的优势种群。通过对30株病原指示菌的抑菌活性检测,18株内生菌对多种指示菌有明显的抑制作用。16株是内生真菌,分属11属。【结论】本试验研究了飞龙斩血内生菌的种群分布,获得了一些具有抗菌活性的内生菌,为飞龙斩血内生菌资源的开发利用提供了基础。     

新疆特色植物恰玛古块根内生真菌的分离与鉴定

植物体的内生真菌与其自身生长及生物防御均有密切关系,因此对健康恰玛古块根组织进行内生真菌分离与纯化,利用形态学结合分子鉴定方法对内生真菌群落多样性进行了初步研究。从恰玛古块根中共分离到可培养内生真菌29株,鉴定结果分属于8个属的11个种,其中支顶孢属(Acremonium sp.)和青霉属(Penicillium sp.)为优势属,分别占总菌株数的34.48%和27.59%。为国内外首次对新疆特色药用植物恰玛古内生真菌进行分离研究,并对其分类地位进行了确定。     

云南八角莲内生真菌分离、鉴定的初步探索

通过对云南八角莲(D.aurantiocaulis)内生真菌的分离、鉴定,获得87株内生真菌,并对87株进行分离、鉴定发现内生真菌在宿主植物中具有明显的多样性,无孢类群(22株)是优势菌,其次为镰孢菌属(10株)毛壳菌属(10株)、青霉属(9株)、小齿梗霉属(7株)、丛梗孢属(4株),首次报道云南八角莲内生真菌的类群和区系特点.     

毛泡桐内生真菌的分离、拮抗细菌菌株的筛选和鉴定

为寻找新的能产生抗生素的植物内生真菌,采用3种培养基对毛泡桐进行内生真菌的分离,以大肠埃希菌、枯草芽胞杆菌、金黄色葡萄球菌、青枯假单胞菌为指示菌,利用琼脂块法和滤纸片扩散法筛选能抑制细菌的菌株;通过形态特征和ITS序列分析鉴定高活性菌株。结果从毛泡桐的根、茎、叶中共分离得到46株内生真菌,至少能抑制一种指示菌的有9株,其中菌株KLBMP-Pt630、KLBMP-Pt675和KLBMP-Pt686活性较强,鉴定结果显示:KLBMP-Pt630为三线镰刀菌、KLBMP-Pt675为棒曲霉、KLBMP-Pt686属于肉座菌目真菌。     

Novel autotrophic arsenite-oxidizing bacteria isolated from soil and sediments

Arsenic oxidation is recognized as being mediated by both heterotrophic and chemoautotrophic microorganisms. Enrichment cultures were established to determine whether chemoautotrophic microorganisms capable of oxidizing arsenite As(III) to arsenate As(V) are present in selected contaminated but nonextreme environments. Three new organisms, designated as strains OL-1, S-1 and CL-3, were isolated and found to oxidize 10 mM arsenite to arsenate under aerobic conditions using CO2-bicarbonate (CO2/HCO3-) as a carbon source. Based on 16S rRNA gene sequence analyses, strain OL-1 was 99% most closely related to the genus Ancylobacter, strain S-1 was 99% related to Thiobacillus and strain CL-3 was 98% related to the genus Hydrogenophaga. The isolates are facultative autotrophs and growth of isolated strains on different inorganic electron donors other than arsenite showed that all three had a strong preference for several sulfur species, while CL-3 was also able to grow on ammonium and nitrite. The RuBisCO Type I (cbbL) gene was positively amplified and sequenced in strain CL-3, and the Type II (cbbM) gene was detected in strains OL-1 and S-1, supporting the autotrophic nature of the organisms.     

Molecular analysis of 16S–23S spacer regions ofAcetobacter species

16S-23S rDNA internal transcribed spacer regions (ITS) similarities were determined in 8 Acetobacter and 1 Gluconacetobacter strains. ITS-PCR amplification of the 16S-23S spacers showed 2 products of similar size in 7 strains; only 1 product of similar size was found in the 2 remaining strains. Analysis of the PCR products using restriction endonucleases HaeIII, HpaII and AluI revealed 3 different restriction groups of A. pasteurianus for AluI and HaeIII, and 4 restriction groups for HpaII. ITS nucleotide sequences of all studied strains exhibited a 52-98% similarity.     

Stereoselectivity and stereospecificity of the alpha,beta-dihydroxyacid dehydratase from Salmonella typhimurium.

1. In addition to the known 2R,3R- and 2R, 3S-2,3-dihydroxy-3-methylpentanoic acids (DHI), the 1S,3S- and sS,DR-isomers were prepared. 2S-2,3-Dihydroxy-3-methylbutanoic acid (DHV) was also prepared in addition to the known 2R-isomer. 2. The six dihydroxy acids were examined for their ability to promote the growth of isoleucine-valine (ilv)-requiring strains of Salmonella typhimurium and to serve as substrates for the alpha,beta-dihydroxyacid dehydratase of the same organism. 3. Only 2R,3R-2,3-dihydroxy-3-methylpentanoic and 2R-2,3-dihydroxy-3-methylbutanoic acids supported growth of the ilv strains of S. typhimurium. 4. alpha,beta-Dihydroxyacid dehydratase utilized the three isomers with the 2R-configuration as substrates but not those with the 2S-configuration. 5. In an additional growth study that utilized the 3R- and 3S-isomers of 3-methyl-2-oxopentanoic acid, the alpha-keto acid analogue of isoleucine, only the 3S-isomer supported growth. 6. It is concluded that the mechanism of action of the dehydratase is stereospecific in that the proton that is attached to C-3 of the substrate occupies the same steriochemical position as the departing hydroxyl group (Fig. 6).     

Characterization of Paenibacillus popilliae rRNA operons

The terminal 39 nucleotides on the 3' end of the 16S rRNA gene, along with the complete DNA sequences of the 5S rRNA, 23S rRNA, tRNA(Ile), and tRNA(Ala) genes were determined for Paenibacillus popilliae using strains NRRL B-2309 and Dutky 1. Southern hybridization analysis with a 16S rDNA hybridization probe and restriction-digested genomic DNA demonstrated 8 copies of the 16S rRNA gene in P. popilliae strains KLN 3 and Dutky 1. Additionally, the 23S rRNA gene in P. popilliae strains NRRL B-2309, KLN 3, and Dutky 1 was shown by I-CeuI digestion and pulsed-field gel electrophoresis of genomic DNA to occur as 8 copies. It was concluded that these 3 P. popilliae strains contained 8 rrn operons. The 8 operon copies were preferentially located on approximately one-half of the chromosome and were organized into 3 different patterns of genes, as follows: 16S-23S-5S, 16S-ala-23S-5S, and 16S-5S-ile-ala-23S-5S. This is the first report to identify a 5S rRNA gene between the 16S and 23S rRNA genes of a bacterial rrn operon. Comparative analysis of the nucleotides on the 3' end of the 16S rRNA gene suggests that translation of P. popilliae mRNA may occur in Bacillus subtilis and Escherichia coli.     

Molecular characterization of Serratia marcescens strains by RFLP and sequencing of PCR-amplified 16S rDNA and 16S-23S rDNA intergenic spacer

AIMS: To establish the specific DNA patterns in 16S rDNA and 16S-23S rDNA intergenic spacer (IGS) regions from different kinds of Serratia marcescens strains using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequences analysis. METHODS AND RESULTS: Two pairs of primers based on the 16S rDNA and 16S-23S rDNA IGS were applied to amplify the rrn operons of two kinds of S. marcescens strains. About 1500 bp for 16S rDNA and four fragments of different sizes for 16S-23S rDNA IGS were obtained. PCR-amplified fragments were analysed by RFLP and sequence analysis. Two distinct restriction patterns revealing three to five bands between two kinds of strains were detected with each specific enzyme. According to the sequence analysis, two kinds of strains showed approximately 97% sequence homology of 16S rDNA. However, there was much difference in the sequences of IGS between the two kinds of strains. Intercistronic tRNA of strains H3010 and A3 demonstrated an order of tRNA of 5'-16S-tRNA(Ala)-tRNA(Ile)-23S-3', but strain B17 harboured the tRNA of 5'-16S-tRNA(Glu)-tRNA(Ile)-23S-3'. CONCLUSIONS: The method was specific, sensitive and accurate, providing a new technique for differentiating different strains from the same species. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provided the first molecular characterization of 16S rDNA and 16S-23S rDNA IGS from S. marcescens strains.     

EPR characterization of soluble fragments of succinate dehydrogenase from mutant strains of Bacillus subtilis

Succinate dehydrogenase is a membrane-bound metallo-flavo-enzyme containing a bi- (S-1), a tri- (S-3) and a tetranuclear (S-2) iron-sulfur cluster. The catalytic portion of the enzyme contains two distinct subunits designated Fp and Ip. Using concentrated extracts from mutant strains of Bacillus subtilis it was demonstrated, by using low temperature EPR, that cluster S-2 can be assembled in a soluble succinate dehydrogenase. In a mutant with a truncated Ip subunit which lacks 7 of the 11 conserved cysteine residues, cluster S-1 lacked the spin relaxation properties attributable to an adjacent cluster S-2. These data are consistent with a model where one or more cysteine residues from the middle set of 4 conserved cysteines in the Ip subunit are ligands to the tetranuclear cluster.     

Ultrastructure ofDigitalis lanata tissue cultures

Summary The anatomy of twoDigitalis lanata tissue culture strains, S-1 and S-2, has been studied. The cardenolide accumulating cell aggregates consisted of highly vacuolated cells with very lobed nuclei in the periphery and a central part of meristematic cells. Sieve tube elements and companion cells and tracheids were observed in some of the cultures. The effect of gibberellic acid (GA₃) and SAN 9789 on the ultrastructure of the cultures was most apparent as regards the plastids and the amounts of mitochondria and ER. The content of starch seemed to be highest in the cardenolide accumulating strain S-2 grown in darkness (S-2 D) with or without GA3, but considerable amounts were also found in S-1 grown in darkness (S-1 D) with or without GA₃, which did not accumulate cardenolides. The amount of plastoglobuli was increased in S-1 by SAN treatment. It was also higher in S-2 D and S-2 D+GA₃ than in S-1 D and S-1 D+GA₃; i.e., it was high in tissues with blocked carotene synthesis. Many large plastoglobuli were also observed in apparently degenerating cells. The amount of ER seemed relatively high in cardenolide producing cultures. The amount of mitochondria was highly variable, but no correlation with cardenolide accumulation could be found.Abbreviations D dictyosome - ER endoplasmic reticulum - M mitochondrion - N nucleus - P plastid - PG plastoglobule - S starch grain - SC sieve cell - V vacuole - W cell wall - GA₃ gibberellic acid - S-1D strain S-1 cultured in darkness - S-1 L strain S-1 cultured in light - S-2 D strain S-2 cultured in darkness - S-2L8 strain S-2 previously cultured in darkness followed by 8 days in light in the present study - SAN SAN 9789 (Norflurazon) 4-chloro-5-(methylamino)-2-(,,,-trifluoro-m-tolyl)-3(2H)-pyridazinone     

Herminiimonas fonticola gen. nov., sp. nov., a Betaproteobacterium isolated from a source of bottled mineral water

Several yellowish-pigmented bacteria with an optimum growth temperature of about 30 degrees C, were recovered from the source (borehole) of bottled mineral water in the Serra da Estrela in Eastern Portugal. Phylogenetic analyses of the 16S rRNA gene sequence of strains S-94T , S-97, S-99 and S-92 indicated that these organisms represent a new species of the Betaproteobacteria that is not closely related to any other known species. The major fatty acids of the strains are 16:1 omega7c and 16:0. Ubiquinone 8 is the major respiratory quinone. The new isolates are strictly organotrophic and aerobic. The new strains only assimilated organic acids, glycine and alanine. Casamino acids and a mixture of all natural amino acids are not used as sole carbon and nitrogen sources; these are used as nitrogen source in the presence of organic acids. On the basis of the phylogenetic analyses, physiological and biochemical characteristics, we are of the opinion that strains S-94T, S-97, S-99 and S-92 represent a new species of a novel genus for which we propose the name Herminiimonas fonticola gen. nov., sp. nov.     

Improvement of the fatty acid composition of an oil-producing filamentous fungus, Mortierella alpina 1S-4, through RNA interference with delta12-desaturase gene expression

An oleaginous fungus, Mortierella alpina 1S-4, is used commercially for arachidonic acid production. Delta12-Desaturase, which desaturates oleic acid (18:1n-9) to linoleic acid (18:2n-6), is a key enzyme in the arachidonic acid biosynthetic pathway. To determine if RNA interference (RNAi) by double-stranded RNA occurs in M. alpina 1S-4, we silenced the Delta12-desaturase gene. The silenced strains accumulate 18:2n-9, 20:2n-9, and Mead acid (20:3n-9), which are not detected in either the control strain or wild type strain 1S-4. The fatty acid composition of stable transformants was similar to that of Delta12-desaturation-defective mutants previously identified. Thus, RNAi occurs in M. alpina and could be used to alter the types and relative amounts of fatty acids produced by commercial strains of this fungus without mutagenesis or other permanent changes in the genetic background of the producing strains.     

混合培养提高菌株解磷解钾能力的探讨

沈阳农业大学的微生物实验室保藏菌种中得到互相不拮抗的根瘤菌S-2、溶磷菌P-3和硅酸盐细菌K-5,对这3株菌进行两两复合及三菌复合,分别测试其溶磷、解钾能力。结果表明:3株菌在第10天时溶磷、解钾能力最强。溶磷能力:两两复合培养的溶磷能力比各菌单独培养溶磷能力要提高25.50%、51.54%、26.99%,并且三菌复合培养具有1+1+13的溶磷效果。解钾能力:两两复合培养时S-2与P-3组合溶解钾长石的能力增强,但S-2与K-5、P-3与K-5的组合并无明显的促进作用,三菌复合虽较各菌株单独培养时高,但未表现出1+1+13解钾效果。