地黄内生放线菌的分离及地黄轮纹病拮抗菌Streptomyces folium leaf-16的新种鉴定

药用植物内生放线菌具有合成天然活性化合物的潜力,放线菌新种是寻找新型抗生素先导化合物的一个重要来源。【目的】挖掘药用植物地黄内生放线菌资源,并对地黄轮纹病拮抗菌株leaf-16进行新种鉴定。【方法】本研究采用五步消毒法分离河南道地药材地黄的内生放线菌,以地黄轮纹病原真菌草茎点霉(Phoma herbarum)为指示菌,采用平板对峙法筛选对该病菌有抑制作用的菌株,16S rRNA基因测序发现一株抗地黄轮纹病的放线菌新种leaf-16。通过形态、生理生化、细胞壁化学组分和分子生物学等特征对菌株leaf-16进行多相分类学鉴定。【结果】经平板对峙实验得到8株抗地黄轮纹病的放线菌,其中菌株leaf-16经16S rRNA基因测序、形态比较、生理生化、化学组分和分子生物学以及DNA-DNA杂交分析,确定菌株leaf-16为1株链霉菌新种,并命名为Streptomyces folium。【结论】菌株leaf-16为1株链霉菌新种,具有抑制地黄轮纹病原真菌的活性,为进一步分离新型抗地黄轮纹病的生物制剂奠定物质基础。     

Inoculum Density and Population Dynamics of Suppressive and PathogenicStreptomycesStrains and Their Relationship to Biological Control of Potato Scab

SeveralStreptomycesstrains are capable of suppressing potato scab caused byStreptomyces scabies.Although these strains have been successful in the biocontrol of potato scab in the field, little is known about how populations of pathogenicStreptomycesin the potato rhizosphere are influenced by inoculation of the suppressive strains. The effects of inoculum densities of pathogenic and suppressiveStreptomycesstrains on their respective populations on roots and in rhizosphere soil were examined during the growing season. The relationships between inoculum density or rhizosphere population densities and disease severity were also investigated. Populations of suppressiveStreptomycesstrain 93 increased significantly on roots with increasing inoculum dose. At its highest inoculum dose, the suppressive strain reached a population density greater than 10⁶CFU/g root 14 weeks after planting. The ability of the suppressive strain to increase its populations with increasing inoculum density was hindered at high inoculum doses of the pathogen, suggesting that density-dependent competitive interactions may be occurring between the two antagonists. Strain 93 was most effective at preventing scab early in the growing season (8 weeks after planting), when tubers were most susceptible to the scab disease. Population densities of the suppressive strain in soil were more highly negatively correlated with scab severity than were populations on roots, suggesting that rhizosphere soil rather than potato roots may be the primary source of inoculum of the suppressive strain for tubers.     

Biological control of maize seed pathogenic fungi by use of actinomycetes

The effectiveness of twoStreptomyces spp. strains to controlpathogenic fungi was studied in stored maizegrain. The treatments included seeddisinfection and inoculation withStreptomyces spp. strains previously isolatedfrom maize rhizosphere. Actinomycete inoculumconsisted of filtered suspension and totalsuspension of fermentor-producedStreptomyces spp. strains biomass. Treatmentswith Streptomyces spp. strains aloneeffectively suppressed the development ofAspergillus spp., Curvularia lunata, andDrechslera maydis and significantly(p < 0,05) reduced the incidence ofFusarium subglutinans and Cephalosporiumacremonium. Among the inoculation treatments,nondisinfested seed inoculated with filteredsuspension was the only treatment that did notsuppress the development of Penicilliumspp. Maize seed inoculation with totalsuspension of strains was the most effectivetreatment to control the incidence of seedpathogenic fungi. The development of theDiplodia maydis was only suppressed by thecombination of seed disinfection andinoculation with total suspension of strains.Although, the strain DAUFPE 11470 showed thegreatest effectiveness for controlling thefungi pathogenic to seed, root and shootdevelopment was reduced by treatment with thisstrain.The results indicate thatStreptomyces spp. strains reduce the incidenceof seed pathogenic fungi and have potential asa biological control agent. However, an efficient methodof seed treatment with the biological controlagent must be developed before it can become anagricultural practice.     

Suppression of root rot of mung bean (Vigna radiata L.) by Streptomyces sp. is associated with induction of peroxidase and polyphenol oxidase

Five strains of Streptomyces sp. were evaluated in vitro for their ability of inhibiting the mycelial growth of Macrophomina phaseolina, the causal agent of root rot of mung bean (Vigna radiata L.). Among the Streptomyces sp. strains tested, PDK showed the maximum in vitro inhibition of mycelial growth of M. phaseolina and recorded an inhibition zone of 21?mm. The strains CBE, MDU, SA and ANR recorded inhibition zones of 18, 16, 13 and 11?mm, respectively. These Streptomyces sp. strains were tested for their growth-promoting efficiency on mung bean seedlings. Among them, CBE and PDK recorded the maximum increase in shoot length, root length and seedling vigour compared with control, followed by MDU. Three Streptomyces sp. strains (CBE, MDU and PDK) that showed higher levels of inhibition of growth of M. phaseolina in dual culture assay and plant growth-promoting activity were tested for their biocontrol activity against root rot under greenhouse and field conditions. Seed treatment or soil application with powder formulation of Streptomyces sp. strains CBE, MDU and PDK was effective in controlling root rot disease; but, combined application through seed and soil increased the efficacy in both the greenhouse and field trials. Among the treatments, seed treatment plus soil application with powder formulation of Streptomyces sp. strain CBE proved to be most effective, which reduced the root rot incidence from 26.8% (with non-bacterised seeds) to 4.0% in Trial I and from 32.0 to 4.9% in Trial II. The above treatment recorded the highest yield in both the field trials, and the yield increase was 78 and 74% over control in Trial I and Trial II, respectively. Isozyme analysis of the Streptomyces sp.-treated plants indicates that seed treatment plus soil application strongly induce the activities of peroxidase (PO-1 and PO-2) and polyphenol oxidase (PPO-2 and PPO-3) in mung bean. Among the three strains tested, Streptomyces sp. strain MDU- treated plants showed higher levels of activities of PO and PPO. Based on the above findings, it can be concluded that both the direct inhibition of pathogen and induced resistance might be involved in the control of root rot of mung bean by Streptomyces sp.     

Isolation and characterization of actinomycetes antagonistic to pathogenic Vibrio spp. from nearshore marine sediments

Summary A total of 94 actinomycete strains were isolated from the marine sediments of a shrimp farm, 87.2% belonged to the genus Streptomyces, others were Micromonospora spp. Fifty-one percent of the actinomycete strains showed activity against the pathogenic Vibrio spp. strains. Thirty-eight percent of marine Streptomyces strains produced siderophores on chrome azurol S (CAS) agar plates. Seven strains of Streptomyces were found to produce siderophores and to inhibit the growth of Vibrio spp. in vitro. Two of them belonged to the Cinerogriseus group, the most frequently isolated group of Streptomyces. The results showed that streptomycetes could be a promising source for biocontrol agents in aquaculture.     

一株链霉菌21-1的分离鉴定及其对禾谷镰刀菌的抑菌活性

【背景】由禾谷镰刀菌(Fusarium graminearum)引起的小麦赤霉病严重威胁我国的小麦生产。【目的】筛选对禾谷镰刀菌具有拮抗能力的链霉菌菌株,为生防菌剂开发提供理论基础。【方法】利用平板对峙法筛选对禾谷镰刀菌具有拮抗能力的链霉菌;通过形态特征、生理生化特征和16S rRNA基因序列分析对其进行鉴定;通过病原菌菌丝生长、孢子产生及萌发抑制试验分析其发酵液的抑菌活性;利用人工接种试验测定该菌株发酵液的防病效果。【结果】筛选到一株对禾谷镰刀菌具有较强拮抗活性的链霉菌21-1,抑菌率为59.5%。依据形态特征、生理生化特性和16S rRNA基因序列分析,将该菌株鉴定为黄三素链霉菌(Streptomycesflavotricini)。菌株21-1发酵液能够抑制禾谷镰刀菌的菌丝生长、孢子产生及萌发过程,而且可以降低禾谷镰刀菌菌丝中可溶性蛋白质的含量,并增加丙二醛的含量。菌株21-1可以产生蛋白酶及纤维素酶。菌株21-1菌液10倍稀释液对小麦赤霉病的防效最佳,为70.1%。此外,菌株21-1发酵液对其他8种植物病原菌均有较好的抑制作用。【结论】菌株21-1对禾谷镰刀菌有较好的抑菌活性,具...     

Rapid detection and high-resolution discrimination of the genus Streptomyces based on 16S–23S rDNA spacer region and denaturing gradient gel electrophoresis

As the leading source of antibiotics, Streptomyces species are the subject of widespread investigation. Many approaches have been tried to aid in the classification of Streptomyces isolates to the genus, species, and strain levels. Genetic methods are more rapid and convenient than classification methods based on phenotypic characteristics, but a method that is universal in detecting all Streptomyces yet selective in detecting only Streptomyces is needed. The highly conserved nature of the 16S rRNA gene (16S rDNA) combined with the need to discriminate between closely related strains results in analyses of ribosomal intergenic spacer (RIS) regions being more productive than analyses of 16S rRNA genes. PCR primers were designed to amplify the RIS region as well as a sufficient length of the 16S rRNA gene to enable phylogenetic analyses of Streptomyces. Improved selectivity and specificity for the amplification of RIS sequences from Streptomyces with environmental samples was demonstrated. The use of RIS–PCR and denaturing gradient gel electrophoresis (DGGE) was shown to be a convenient means to obtain unique genetic “fingerprints” of Streptomyces cultures allowing them to be accurately identified at species, and even strain classification levels. These RIS–PCR and DGGE approaches show potential for the rapid characterization of environmental Streptomyces populations.     

<Emphasis Type="Italic">Streptomyces nanningensis</Emphasis> sp. nov. a novel Streptomycete from forest soil

A strain YIM 33098^T (= CCTCC AA001027^T = DSM 41831^T) was isolated from a forest soil sample collected from Nanning in Guangxi Province, China, in the course of screening for producers of new drug lead compounds. This strain was identified by using a polyphasic approach. The results showed that it should be assigned to the genus Streptomyces. An almost complete 16S rRNA gene sequence of the strain was determined and compared with those of representative Streptomyces species. Strain YIM 33098^T was clustered in the same subclade with Streptomyces tendae ATCC19812^T and Streptomyces eurythermus ATCC14975^T. Similarities of strain YIM 33098^T with the two strains were 97.35% and 97.42%, respectively. Based on the phenotypic and genotypic evidence, it is therefore proposed that strain YIM 33098^T should be classified in the genus Streptomyces as a new species under the name of Streptomyces nanningensis sp. nov.     

Screening and Identification of the Phospholipase D-producing Streptomyces

About 2,000 strains of streptomycetes and 200 strains of molds were tested for egg yolk-degrading activity. A strain isolated from soil was found to produce the strong activity in the culture medium.

The egg yolk-degrading substance was found to be phospholipase D and the producing strain was identified as Streptomyces hachijoensis. It was found out that the strains producing egg yolk-degrading substance appeared with high frequency in whirl-forming species in genus Streptomyces.     

高产杀粉蝶菌素的链霉菌鉴定及其拮抗水稻白叶枯菌活性研究

【目的】为保证农业生产可持续性发展,研发和使用环境友好的生物农药受到全社会的高度重视。微生物代谢产物农药是我国目前应用最广的生物农药,也是未来发展绿色农药的一个重要方向。【方法】利用包含水稻白叶枯菌(Xanthomonas oryzae pv. oryzae, Xoo) PXO99A的NA培养基琼脂平板,从水稻根际土壤中筛选能抑制Xoo生长的链霉菌。通过高效液相色谱和质谱分析活性代谢产物的化学结构;采用剪叶法接种Xoo到水稻叶片后,再喷施杀粉蝶菌素溶液(0.1 g/L),2周后测定叶枯症状;采用响应面分析法优化高产杀粉蝶菌素的发酵培养基;采用PacBio SMRT测序平台+Illumina HiSeq X Ten平台开展全基因组测序。平均核苷酸一致性(average nucleotide identity,ANI)用于比较HSW2009与其他链霉菌在全基因组水平的亲缘关系。【结果】分离到一株对Xoo生长有强抑制活性的链霉菌HSW2009,其活性代谢产物为杀粉蝶菌素A1(piericidin A1,简称PIE);喷施PIE可以减轻Xoo在水稻叶片内的侵染;优化HSW2009高产PIE的发...     

Protoplast Fusion in Streptomyces sp. for Increased Production of Laccase and Associated Ligninolytic Enzymes

Biodegradation of lignin by Streptomyces spp. results in the production of value-added chemicals such as Acid Precipitable Polymeric Lignins (APPLs), low molecular weight phenols, etc. To hasten the conversion metabolism through genetic manipulation, a preliminary attempt was made to standardize the methodology for isolation and regeneration of protoplasts. Protoplast fusion recombinants were developed and assayed for their ligninolytic activity, production of ligninolytic enzymes viz., peroxidase, laccase, polyphenol oxidase and crude protein. In comparison with the mutants and wild strain, fusion recombinant F₄ showed higher laccase activity and lower peroxidase activity. This attribute can be positively used for polymerization of free phenolics to polycondensates related to humic acids in soil or composting environments. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of Actinomycetes Producing Phospholipase D with High Transphosphatidylation Activity

Previously we isolated six actinomycetes strains, 9-4, 10-1, 10-2, 10-3, 10-6, and 21-4, that produce phospholipase D (PLD) with high transphosphatidylation activity. In this study, we identified these strains, and the PLD activities were compared with those of reference strains. 16S rDNA sequences and DNA–DNA hybridization tests indicated taxonomic affiliations of strain 9-6 with Streptomyces senoensis, strains 10-1 and 10-6 with S. vinaceus, and strains 10-2 and 10-3 with S. racemochromogenes. Strain 21-4, though identified as a Streptomyces sp., could not be identified with any known species. Meanwhile, most of the culture supernatants of reference strains demonstrated no or very weak PLD activity, while those of our strains exhibited significantly higher activity. All of the strains in this study were identified as Streptomyces species. The PLD activity of our strains exceeded most of the reference Streptomyces strains. The findings in this study imply that the Streptomyces strains, although they are members of the same species, can produce different quantities of PLD enzyme.     

Solubilisation and mineralisation of [14C]lignocellulose from wheat straw by Streptomyces cyaneus CECT 3335 during growth in solid-state fermentation

Nine Streptomyces strains were screened for their ability to solubilise and mineralise ¹⁴C-labelled lignin during growth in solid-state fermentation. Streptomyces viridosporus was confirmed as an active lignin-degrading organism along with a new isolate, Streptomyces sp. UAH 15, further classified as Streptomyces cyaneus CECT 3335. This organism was able to solubilise and mineralise the [¹⁴C]lignin fraction of lignocellulose (44.96 ± 1.77% and 3.41 ± 0.48% respectively) after 21 days of incubation. Cell-free filtrates from Streptomyces sp. grown in solid-state fermentation were capable of solubilising up to 20% of the [¹⁴C]lignin after 2 days incubation, with most of the product detected in the acid-soluble rather than in the water-soluble fraction. Identification of the extracellular enzymes produced during growth of S. cyaneus CECT 3335 revealed that extracellular peroxidase and phenol oxidase activities were present, with the activity of phenol oxidase being 100 times greater than peroxidase activity. The activity of these two enzymes was found to correlate with both solubilisation and mineralisation rates. This is the first report of phenol oxidase activity produced by a Streptomyces strain during growth in solid-state fermentation. A role for the enzyme in the solubilisation and mineralisation of lignocellulose by S. cyaneus is suggested. Received: 12 May 1997 / Accepted: 19 May 1997     

Isolation and characterization ofPenicillium funiculosum mutants with enhanced glucose oxidase production

After the mutagenesis ofPenicillium funiculosum with UV light andN-nitroso-N-methylurea, 83 of 2237 grown colonies were surrounded with increased zones of glucose oxidase diffusion. Analysis of the glucose oxidase activity of selected mutant strains grown in submerged cultures allowed 18 mutant strains to be obtained whose glucose oxidase activity was 5–153% higher (in a medium with glucose) and 4–83% higher (in a medium with sucrose) than that of the parent strain. Two of these mutant strains, UV6.31 and NMU95-132, possessed high glucose oxidase activity when grown in media with glucose or sucrose and produced large amounts of mycelia. The active and morphologically stable mutantP. funiculosum NMU95-132 was chosen for further selection work.     

Expandase-like activity mediated cell-free conversion of ampicillin to cephalexin by Streptomyces sp. DRS I

Cell-free extracts of Streptomyces sp. DRS I converted ampicillin to cephalexin, presumably due to the activity of the enzyme, expandase. The extract was fractionated and characterized by colorimetric and chromatographic measurements coupled with disc-agar diffusion bioassay against an ampicillin-resistant, cephalexin-sensitive E. coli strain. Though expandase could not be identified, the presence of a hitherto unreported expandase in Streptomyces sp. DRS I is suggested.     

Extracellular L-glutamate oxidase ofStreptomyces sp. Z-11-6: Obtainment and properties

Mutagenesis induced with nitrous acid and subsequent selection allowed a genetically stable mutant strain,Streptomyces sp. Z-11-6, to be obtained, whose L-glutamate oxidase activity was 40-fold higher than that of the original natural isolate and was as great as 1.6–1.8 units/ml of culture liquid. A procedure for the isolation and purification of the enzyme was developed; the biochemical properties of the enzyme were studied. Out of 20 amino acids tested (including D-glutamate), the glutamate oxidase fromStreptomyces sp. Z-11-6 was active only with L-glutamate. This allows the concentration of L-glutamate to be determined in the presence of other amino acids. Calcium chloride at a concentration of 0.1–0.5% promoted the secretion of the extracellular glutamate oxidase.     

Recovery of soil streptomycetes from arid habitats in Jordan and their potential to inhibit multi-drug resistant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> pathogens

A total of 161 different Streptomyces isolates were recovered from 5 soil samples representing the driest habitats of Jordan. These were then characterized and assessed for their antagonistic activity against four clinical multi-drug resistant Pseudomonas aeruginosa test pathogens. Results indicated that only 3 strains out of 139 and 6 out of 22 isolated at 27°C and 45°C, respectively, were active against at least three strains of pathogenic Pseudomonas. However, three Streptomyces strains (J_2b, J₄, and J₁₂) that were isolated at 45°C inhibited all of the tested pathogens with an inhibition zone ranging between 5 and 16 mm in diameter. Data obtained from comparing the inhibition activity of these unique Streptomyces strains toward multi-resistant Pseudomonas pathogens with standard used antibiotics revealed that these isolates produce possible different inhibitory bioactive compounds other than the standard antibiotics.     

Bacterial cholesterol oxidases are able to act as flavoprotein-linked ketosteroid monooxygenases that catalyse the hydroxylation of cholesterol to 4-cholesten-6-ol-3-one

A new metabolite of cholesterol was found in reaction mixtures containing cholesterol or 4-cholesten-3-one as a substrate and extra- or intracellular protein extracts from recombinant Streptomyces lividans and Escherichia coli strains carrying cloned DNA fragments of Streptomyces sp. SA-COO, the producer of Streptomyces cholesterol oxidase. The new metabolite was identified as 4-cholesten-6-ol-3-one based on comparisons of its high-performance liquid chromatography, gas chromatography/mass spectrometry, infrared and proton-nuclear magnetic resonance spectra with those of an authentic standard. Genetic analyses showed that the enzyme responsible for the production of 4-cholesten-6-ol-3-one is cholesterol oxidase encoded by the choA gene. Commercially purified cholesterol oxidase (EC 1.1.3.6.) of a Streptomyces sp., as well as of Brevibacterium sterolicum and a Pseudomonas sp., and a highly purified recombinant Streptomyces cholesterol oxidase were also able to catalyse the 6-hydroxylation reaction. Hydrogen peroxide accumulating in the reaction mixtures as a consequence of the 3β-hydroxysteroid oxidase activity of the enzyme was shown to have no role in the formation of the 6-hydroxylated derivative. We propose a possible scheme of a branched reaction pathway for the concurrent formation of 4-cholesten-3-one and 4-chotesten-6-ol-3-one by cholesterol oxidase, and the observed differences in the rate of formation of the 6-hydroxy-ketosteroid by the enzymes of different bacterial sources are also discussed.     

珠江口沉积物来源链霉菌SCSIO 40020中bafilomycins的鉴定及其生物合成基因簇的异源表达

【目的】从珠江口沉积物来源的菌株SCSIO40020中分离bafilomycins,并对其生物合成基因簇进行克隆和异源表达研究。【方法】通过分析菌株SCSIO 40020的16S rRNA基因序列并构建系统发育树以鉴定菌种,以柱层析法和制备色谱法对次级代谢产物进行分离纯化,借助波谱学手段完成单体化合物的结构鉴定,采用生物信息学分析定位bafilomycins的生物合成基因簇,通过筛选菌株SCSIO 40020基因组的细菌人工染色体文库和接合转移将bafilomycins生物合成基因簇导入3种链霉菌进行异源表达,利用高效液相色谱检测异源表达菌株的发酵产物。【结果】菌株SCSIO 40020被鉴定为链霉菌属菌株,从其发酵产物中分离鉴定了2个单体化合物bafilomycinsA1和D。克隆了链霉菌SCSIO40020中bafilomycins的生物合成基因簇并推导了其生物合成途径,在3种链霉菌中表达产生了bafilomycins。【结论】从珠江口环境中获得了一株产生bafilomycins的链霉菌SCSIO 40020,成功建立了该菌株次级代谢产物生物合成基因簇的异源表达体系,并首次在链霉菌...     

Srinivasan’s coagulation test in taxonomically classified streptomycetes

Srinivasan’s coagulation test was performed in 18 strains of the genusStreptomyces and one strain of the genusActinoplanes. The highest coagulation activity was detected in strains systematically classified in a series of streptomycetes with pink or red aerial mycelium:S. erythreus, Streptomyces sp. AJ/22,S. roseo-luteus andS. griseofuscus. With the exception ofS. griseofuscus these three cultures also exhibited the highest inhibitory activity againstB. subtilis. When using hemoglobin as substrate it was possible to detect acid, neutral and alkaline proteinases with the highest proteolytic activity at pH 3.0 to 4.0 in the most active strain ofS. erythreus.