Isolation of Endophytic Streptomyces Strains from Surface-Sterilized Roots

When the roots of 28 plant species were surface sterilized and incubated on agar medium, endophytic actinomycetes in the root cortex were observed by direct microscopic observation and pure culture techniques.     

一株海洋放线菌的分离鉴定及活性研究

从大连海域20~30 m深处生长的海绵中分离到一株有很高抗菌活性的链霉菌D164。根据培养和显微形态、生理生化数据、16S rRNA基因序列数据分析,菌株D164鉴定为娄彻氏链霉菌(Streptomyces rochei)。对菌株D164发酵产物进行抗农业病原菌、杀虫和除草活性检测,结果表明,菌株D164发酵产物具有很高的抗农业病原菌活性,同时又具有很高的杀虫和除草活性,其活性化合物值得进一步研究。     

热带药用植物根际放线菌的分离、鉴定及生物活性分析

从海南热带植物园采集12种药用植物的根际土样,采用选择性分离方法,分离得到400株根际放线菌。使用5种活性筛选模型对分离菌株进行生物活性评价,154株放线菌在一个或多个活性筛选模型中显示为阳性,菌株初筛阳性率达38.5%;根据菌株形态特征并结合代谢产物的生物活性,从中挑选出28株菌进行16S rRNA基因序列分析,发现其分属于链霉菌属、诺卡氏菌属、小单孢菌属和野野村菌属。     

Non-Frankia Actinomycetes Isolated from Surface-Sterilized Roots of Casuarina equisetifolia Fix Nitrogen

Based on partial 16S sequences, we previously described a novel group of nonsymbiotic, acetylene reduction activity-positive actinomycetes which were isolated from surface-sterilized roots of Casuarina equisetifolia growing in Mexico. An amplified rRNA restriction analysis confirmed that these actinomycetes are distinct from Frankia, a finding substantiated by a 16S rRNA gene phylogenetic analysis of two of the Mexican isolates. Further support for these actinomycetes being separate from Frankia comes from the very low DNA-DNA homology that was found. Nevertheless, the Mexican isolates may be diazotrophs based not only on their ability to grow in N-free medium and reduce acetylene to ethylene but also on the results from ¹⁵N isotope dilution analysis and the finding that a nifH gene was PCR amplified. A comparison of the nifH sequences from the various isolates showed that they are closely related to nifH from Frankia; the similarity was 84 to 98% depending on the host specificity group. An analysis of complete 16S rRNA gene sequences demonstrated that the two strains analyzed in detail are most closely related to actinobacteria in the Thermomonosporaceae and the Micromonosporaceae.     

Isolation of antimicrobial producing Actinobacteria from soil samples

Emergence of multidrug resistant bacteria has made the search for novel bioactive compounds from natural and unexplored habitats a necessity. Actinobacteria have important bioactive substances. The present study investigated antimicrobial activity of Actinobacteria isolated from soil samples of Egypt. One hundred samples were collected from agricultural farming soil of different governorates. Twelve isolates have produced activity against the tested microorganisms (S. aureus, Bacillus cereus, E. coli, K. pneumoniae, P. aeruginosa, S. Typhi, C. albicans, A. niger and A. flavus). By VITEK 2 system version: 07.01 the 12 isolates were identified as Kocuria kristinae, Kocuria rosea, Streptomyces griseus, Streptomyces flaveolus and Actinobacteria. Using ethyl acetate extraction method the isolates culture’s supernatants were tested by diffusion method against indicator microorganisms. These results indicate that Actinobacteria isolated from Egypt farms could be sources of antimicrobial bioactive substances.     

广西山口红树林内生放线菌的分离、筛选及初步鉴定

从广西山口国家红树林生态自然保护区采集8种真红树植物和5种半红树植物的根、茎、叶及胚轴等组织样品共计41份。经过表面消毒后粉碎,分别以几丁质、腐殖酸、木聚糖等作为主要营养及能量来源设计了8种分离培养基,从植物组织中分离得到118株放线菌。使用6种新药筛选模型对分离菌株进行了生理活性筛选,77株放线菌的发酵液样品在一个或多个药物筛选模型中显示为阳性,初筛阳性率为65.3%。对77株筛选阳性菌株进行的初步分类研究结果显示,其中44株属于链霉菌属,25株为小单孢属的菌株,3株属于糖丝菌属,3株为诺卡氏属菌株,1株是拟诺卡氏属菌株,1株是伦茨氏菌属的菌株。对一株在抗多重耐药检定菌HH22和降血脂模型上都显示出良好活性的菌株I07A-01824进行表型和基因型分析,确定I07A-01824为黄白链霉菌(Streptomyces albidoflavus)菌株。该研究显示,红树植物内生环境不仅仅孕育了丰富多样的内生放线菌,而且红树植物内生放线菌也是新型天然生理活性次级代谢产物的有效来源。     

广西沿海地区红树林根系土壤中放线菌的分离与鉴定

本研究通过分离纯培养,从广西北海及防城港红树林根系土壤中分离出放线菌并提取其总DNA,用放线菌通用引物对获得菌株的16S rDNA进行PCR扩增,对获得的扩增产物进行DNA序列测定及菌株鉴定.研究结果表明,从红树林根系土壤样品中分离出15株典型放线菌菌株.16S rDNA测序比对鉴定结果显示,15株典型放线菌菌株中有12株属于链霉菌属(Streptomyces),是常见菌属;3株属于拟诺卡氏菌属(Nocardiopsis),为稀有放线菌.本研究分离纯化获得15株典型放线菌,初步揭示了广西沿海地区红树林土壤中放线菌的多样性.     

小麦NO3-转运蛋白基因TaNRT2.3的克隆和表达分析

用RT-PCR和RACE技术在NO3-诱导处理的小麦(Triticum aestivum L.)根中克隆到一个硝酸根转运蛋白基因的cDNA,命名为TaNRT2.3(GenBank登录号AY053452).序列分析表明,TaNRT2.3全长1 744 bp,其中含有1 521bp的ORF,编码507个氨基酸,具有12个跨膜区,属于MFS超基因家族中的NNP家族.TaNRT2.3与其他植物中已知的NRT2具有很高的同源性.Northern杂交表明:TaNRT2具有在根中表达的组织特异性,而在叶中未检测到.TaNRT2的表达受NO3-诱导,在含NH4 介质中不表达.NO3-在低浓度(5～200μmol/L)和高浓度(2.0 mmol/L)时均起作用.通过研究小麦在0.2 mmol/LNO3-条件下TaNRT2的表达水平及对NO3-的吸收效率,表明TaNRT2在小麦高效吸收NO3-方面起着重要的作用.分根实验表明植物中N循环本身可以作为吸收N的调节信号.     

Isolation and Identification of Vesicular-Arbuscular Mycorrhiza-Stimulatory Compounds from Clover (Trifolium repens) Roots

Two isoflavonoids isolated from clover roots grown under phosphate stress were characterized as formononetin (7-hydroxy,4′-methoxy isoflavone) and biochanin A (5,7-dihydroxy,4′-methoxy isoflavone). At 5 ppm, these compounds stimulated hyphal growth in vitro and root colonization of an undescribed vesicular-arbuscular mycorrhiza, a Glomus sp. (INVAM-112). The permethylated products of the two compounds were inactive. These findings suggest that the isoflavonoids studied may act as signal molecules in vesicular-arbuscular mycorrhiza symbiosis.     

东北地区保护地黄瓜根茎上真菌的分离与鉴定

对采自东北地区保护地内的38株黄瓜枯萎病病株及8株未发病黄瓜植株的根茎进行真菌分离与鉴定,共得到真菌11个属18个种,其中威尔士棒孢(Corynespora cambrensis)为中国新记录种。镰孢属(Fusarium)为优势属,分离到8个种及变种。     

Isolation,Classification and Antagonistic Properties of Alkalitolerant Actinobacteria from Algerian Saharan Soils

Abstract

The Sahara, one of the most extreme environments on Earth, constitutes an unexplored source of alkalitolerant actinobacteria. In this work, we studied the diversity of alkalitolerant actinobacteria in various soils collected from different regions of the Algerian Sahara. A total of 29 alkalitolerant actinobacterial strains were isolated by using a complex agar medium. The diversity of these actinobacteria was evaluated using a polyphasic approach, which included morphological, chemotaxonomic, physiological (numerical taxonomy) and 16S rRNA gene analyses. The isolates which were assigned to the genus Nocardiopsis, shared relatively low 16S rRNA gene sequences similarities compared to closely related species suggesting that they belonged to putatively new species. All of the strains were tested for antibiotic activity against a broad range of microorganisms and screened for genes encoding polyketide synthases and non-ribosomal peptide synthetases and found to have the potential to produce secondary metabolites. Consequently, the study supports the view that extreme environments contain many novel actinobacteria, which represent an unexplored source for the discovery of biologically active compounds.     

桉树根系共生真菌的分离与鉴定

南方土壤缺磷现象较为严重,菌根真菌等共生真菌对植物吸收磷等养分具有重要的促进作用。该研究采集尾叶桉(Eucalyptus urophylla)、窿缘桉(Eucalyptus exserta)和尾巨桉(E.urophylla×E.grandis)3种华南地区主要造林树种根系,采用组织分离法进行真菌分离,通过形态特征和核糖体18SrDNA基因ITS序列分析进行鉴定,经柯赫氏法则(Kochs Rule)确定桉树根系共生真菌,为桉树共生真菌理论研究和资源利用提供依据。结果表明:(1)3种桉树根系中,窿缘桉具有外生菌根(ectomycorrhizas,ECM)和丛枝菌根(arbuscular mycorrhizas,AM)结构,尾叶桉和尾巨桉同时具有AM结构、ECM结构和深色有隔内生真菌(dark septate endophytes fungi,DSE)结构。(2)3种桉树根系中分离鉴定出6种真菌:三色小皮伞菌(Marasmius tricolor)、黑柄裸脚伞(Gymnopus melanopus)、茎点霉属(Phomasp.)、镰刀霉属(Fusariumsp.)、二型伞霉(Umbelopsis dimorpha)和芒弗里亚拟盘多毛孢(Pestalotiopsis mangifolia)。(3)6种真菌回接巨桉(Eucalyptus grandis)组培苗,三色小皮伞菌和黑柄裸脚伞形成ECM结构,为ECM真菌;茎点霉属、镰刀霉属、二型伞霉和芒弗里亚拟盘多毛孢形成DSE典型的深色有隔菌丝和微菌核结构,推测为DSE;其中2种ECM真菌为桉树中首次报道。     

Isolation of Casparian Strips from Pea Roots

Casparian strips were isolated from roots of pea seedlings.The plasma membrane was firmly attached to the wall of the individualcells in the isolated Casparian strip. The outer electrondenselamella of the plasma membrane, which is characteristic of themembrane of the Casparian strip, was not removed from the isolatedCasparian strip by treatment with either 1 M NaCl or 2 M sodiumthiocyanate. Although treatment with 1% Triton X-100 disruptedthe lamellar structure of the membrane and removed most of theelectron-dense material of the outer lamella, no detectablepolypeptides were extracted by treatment of isolated Casparianstrips with 1% Triton X-100. Small amounts of electron-densematerial remained after treatment with Triton X-100 and thismaterial seemed to be removed almost entirely by subsequenttreatment of the same sample with 2% SDS. Since polypeptidesof 46, 30 and 20 kDa, which were detected in the extract obtainedby treatment with SDS, were not detected in an extract of totalcell walls of pea roots, it seems possible that these polypeptidesare specific to the Casparian strip and, therefore, that theymay be responsible singly or together for the adhesion of theplasma membrane to the cell wall at the Casparian strip. (Received January 30, 1992; Accepted April 23, 1992)     

Rational Approaches to Improving the Isolation of Endophytic Actinobacteria from Australian Native Trees

In recent years, new actinobacterial species have been isolated as endophytes of plants and shrubs and are sought after both for their role as potential producers of new drug candidates for the pharmaceutical industry and as biocontrol inoculants for sustainable agriculture. Molecular-based approaches to the study of microbial ecology generally reveal a broader microbial diversity than can be obtained by cultivation methods. This study aimed to improve the success of isolating individual members of the actinobacterial population as pure cultures as well as improving the ability to characterise the large numbers obtained in pure culture. To achieve this objective, our study successfully employed rational and holistic approaches including the use of isolation media with low concentrations of nutrients normally available to the microorganism in the plant, plating larger quantities of plant sample, incubating isolation plates for up to 16 weeks, excising colonies when they are visible and choosing Australian endemic trees as the source of the actinobacteria. A hierarchy of polyphasic methods based on culture morphology, amplified 16S rRNA gene restriction analysis and limited sequencing was used to classify all 576 actinobacterial isolates from leaf, stem and root samples of two eucalypts: a Grey Box and Red Gum, a native apricot tree and a native pine tree. The classification revealed that, in addition to 413 Streptomyces spp., isolates belonged to 16 other actinobacterial genera: Actinomadura (two strains), Actinomycetospora (six), Actinopolymorpha (two), Amycolatopsis (six), Gordonia (one), Kribbella (25), Micromonospora (six), Nocardia (ten), Nocardioides (11), Nocardiopsis (one), Nonomuraea (one), Polymorphospora (two), Promicromonospora (51), Pseudonocardia (36), Williamsia (two) and a novel genus Flindersiella (one). In order to prove novelty, 12 strains were characterised fully to the species level based on polyphasic taxonomy. One strain represented a novel genus in the family Nocardioides, and the other 11 strains were accepted as novel species. In summary, the holistic isolation strategies were successful in obtaining significant culturable actinobacterial diversity within Australian native trees that includes rare and novel species.     

Isolation and Characterization of a Boron-Polysaccharide Complex from Radish Roots

A boron-polysaccharide complex was purified from a Driselasedigest of cell walls of radish roots. The complex had a molecularweight of 7.5 KDa and contained boron (0.232%, w/w), uronicacid (52.3%, w/w) and neutral sugars (32.4%). ¹¹B-NMR spectroscopicanalysis suggested that the boron was present as a tetravalent1 : 2 borate-diol complex. ¹This work was supported by a Grant-in-Aid (no. 04660069) fromthe Ministry of Education, Science and Culture. ⁴Present address: Kasai Experimental Farm, Sumitomo ChemicalCo., Ltd. Kasai, Hyogo, 675-23 Japan.     

Isolation and Identification of Fructo-oligosaccharides in Roots of Asparagus (Asparagus officinalis L.)

Eight fructo-oligosaccharides were isolated from purified oligosaccharide fractions of the roots of Asparagus officinalis L. (Liliaceae). By examination of constituent sugars, gas-liquid-chromatographic analysis of methyl derivatives, and investigation of partial acid hydrolyzates and products of β-fructofuranosidase action, they were confirmed to be 1^F(1-β -fructofuranosyl)_n sucrose [n = 1 (1-kestose), 2 (nystose), and 3], 6^G (1-β-fructofuranosyl)_n sucrose [n = 1 (neokestose), 2, and 3], 1^F,6^G-di-β-fructofuranosyl sucrose, and a new pentasaccharide 1^F (1-β-fructofuranosyl)₂-6^G-β-fructofuranosyl sucrose.