Isolation of Actinomycetes from Soil Using Extremely High Frequency Radiation

A new method employing extremely high frequencies (EHFs) is proposed for the selective isolation of actinomycetes from soil. The pretreatment of soil suspensions with EHF wavelengths of 5.6 and 7.1 mm led to a nonselective isolation of actinomycetes. At the same time, the irradiation of soil suspensions within wavelength bands of 3.8–5.8 and 8–11.5 mm considerably augmented the total number of isolated actinomycetes and increased the fraction of the isolated rare genera by 2 and 7 times, respectively. The rare actinomycete genera were represented by Actinomadura, Microtetraspora, Nonomuraea, Micromonospora, Amycolatopsis, Pseudonocardia, Saccharotrix, and Streptosporangium.     

The application of succession analysis in combination with extremely high-frequency irradiation to the selective isolation of actinomycetes from soil

A new method employing succession analysis and extremely high-frequency (EHF) irradiation is proposed for the selective isolation of actinomycetes from soil. Total actinomycetes were efficiently isolated from soil suspensions irradiated in the wavelength band 4.6-5.8 mm on the 14th and 45th days of succession initiated by soil wetting and from soil suspensions irradiated in the wavelength band 8-11.5 mm on the 7th day of succession. The rare actinomycete genera Actinomadura, Micromonospora, Nonomuraea, Microbispora, Amycolatopsis, Pseudonocardia, Saccharothrix, Streptosporangium, Actinosynnema, Nocardioides, and Saccharopolyspora were isolated by either of the two approaches (succession analysis and EHF irradiation); however, the range of isolated rare actinomycetes was considerably wider when the combination of the two approaches was used. For instance, actinomycetes of the rare genera Actinocorallia, Promicromonospora, Actinoplanes, and Kibdelosporangium were isolated only when EHF irradiation was employed at the early stages of succession.     

The Application of Succession Analysis in Combination with EHF Irradiation to the Selective Isolation of Actinomycetes from Soil

A new method employing succession analysis and extremely high frequency (EHF) irradiation is proposed for the selective isolation of actinomycetes from soil. Total actinomycetes were efficiently isolated from soil suspensions irradiated in the wavelength band 4.6–5.8 mm on the 14th and 45th days of succession initiated by soil wetting and from soil suspensions irradiated in the wavelength band 8–11.5 mm on the 7th day of succession. The rare actinomycete genera Actinomadura, Micromonospora, Nonomuraea, Microbispora, Amycolatopsis, Pseudonocardia, Saccharothrix, Streptosporangium, Actinosynnema, Nocardioides, and Saccharopolyspora were isolated by either of the two approaches (succession analysis and EHF irradiation); however, the range of isolated rare actinomycetes was considerably wider when a combination of the two approaches was used. For instance, actinomycetes of the rare genera Actinocorallia, Promicromonospora, Actinoplanes, and Kibdelosporangium were isolated only when EHF irradiation was employed at the early stages of succession.     

广西北部湾茅尾海红树林生境放线菌分离培养基的比较

【背景】红树林生境拥有丰富的微生物资源,分离获得更多的纯培养放线菌为新型抗生素的发现提供菌种资源。【目的】使用新型放线菌分离培养基,发掘广西北部湾茅尾海红树林生境放线菌资源,评估新型放线菌分离培养基分离效果。【方法】设计出新型放线菌分离培养基——椰子汁-海藻糖培养基,对广西北部湾茅尾海红树林根际土壤和红树林根皮放线菌进行分离。基于分子生物学鉴定方法,对获得的纯培养放线菌进行多样性分析。【结果】从红树林根际土壤样品中分离获得65株放线菌,分布在5个目10个科15个属,使用YIM171培养基分离到7个属,MA培养基分离到5个属,椰子汁-海藻糖培养基分离到11个属,其中包括4株潜在新种;从红树林根皮样品中分离获得19株放线菌,分布在4个目7个科10个属,使用YIM171和椰子汁-海藻糖培养基分别分离到7个属,包括4株潜在新种;使用椰子汁-海藻糖培养基在红树林生境两类样品中获得3株潜在新种。【结论】广西北部湾茅尾海红树林根际土壤和根皮中放线菌多样性丰富,使用椰子汁-海藻糖培养基分离放线菌效果较佳,可用作放线菌的分离培养基。     

Use of the method of enriching of soil samples with calcium carbonate for isolation of Actinomyces

Possible application of a modified procedure for treatment of soil samples with calcium carbonate under humid conditions to isolation of actinomycetes was studied. The procedure proved to be rather efficient in regard to increasing the number of the isolates including representatives of rare genera as compared with the routine methods. The number of microorganisms isolated from the soil samples treated with calcium carbonate under humid conditions was 2 orders higher than that from untreated soil samples. 1033 actinomycete cultures were isolated from the soil samples treated in accordance with the above procedure and only 597 cultures were isolated from the untreated soil samples. The antibiotic activity of the isolates was studied and their taxonomic position at the genus level was determined. The preliminary treatment of soil samples equally stimulated development of actinomycetes belonging to different genera. It is advisable that the described procedure for isolation of actinomycetes from soil samples in screening of cultures producing new antibiotics be used in combination with other selective methods of isolation.     

A modified sucrose fractionation procedure for the isolation of frankiae from actinorhizal root nodules and soil samples

Summary The isolation and pure culture of the symbiotic nitrogen-fixing frankiae has always been difficult. In the past the isolation of these actinomycetes directly from soil samples has proven impossible and isolations from root nodules of many genera has been only poorly successful. We report here a modified sucrose fractionation procedure which increased the success of isolations from root nodules and which permitted the isolation ofFrankia directly from soil samples. Crushed nodule suspensions or soil suspensions were incubated briefly in 0.7% phenol (carbolic acid) just before application to a sucrose density gradient. This phenol incubation decreased the number of contaminating eubacteria and fungi but more importantly increased the number ofFrankia developing on the isolation plates. If the phenol incubation was used solely without sucrose fractionation noFrankia were isolated, suggesting the death of the organisms due to phenol toxicity. The use of selective nitrogen-deficient media proved important for the isolation of frankiae from soils.     

Isolation of antibiotic-producing Actinomycetes from soil samples exposed to UV light

The use of nonroutine means in isolation of microorganisms from natural substrates extended the possibilities of detecting new cultures which often appear to be producers of previously unknown antibiotics. A new procedure for isolating actinomyces of definite groups was developed. It implies preliminary exposure of soil suspensions to UV light. With the use of the procedure, 2539 strains of actinomycetes belonging to different genera were isolated. There was a marked decrease after the irradiation in isolation of cultures belonging to Streptomyces, a genus most widely distributed in nature and studied in detail while isolation of cultures belonging to other genera, promising as sources of novel antibiotics, increased. Micromonospora, Amycolatopsis and Nocardia proved to be the most stable to the effect of UV light. With the use of the procedure it is possible to increase 2-3-fold isolation of cultures belonging to Micromonospora, a genus known as a producer of many antibiotics including those used clinically.     

Selective medium with nalidixic acid for isolating antibiotic-producing Actinomyces

The majority of actinomycetes belonging to various genera proved to be resistant to nalidixic acid concentrations having an inhibitory effect on bacteria with trailing growth i.e. B. subtilis and B. mycoides. The bacteria prevented isolation of actinomycetes as pure cultures. The use of a selective medium with nalidixic acid for isolation of soil actinomycetes resulted in 20 per cent increase in the number of the actinomycetes isolated as pure cultures. Preliminary treatment of the soil samples with calcium carbonate under moist conditions followed by the inoculation to the medium with nalidixic acid made it possible to increase isolation of actinomycetes at most 100-fold. With this complex method 495 actinomycete cultures were isolated, their antibiotic properties were studied and their taxonomic position at the genus level was determined. The complex method including the preliminary treatment of soil samples with calcium carbonate followed by inoculation to the selective medium with nalidixic acid is efficient and may be recommended for screening organisms producing new antibiotics.     

辐射污染区土壤中放线菌的分离及多样性

从辐射污染区采集42份土样, 分别采用6种分离培养基进行放线菌的分离, 共获得152株放线菌。经形态、核糖体DNA扩增片段限制性内切酶(Amplifed ribosomal DNA restriction analysis, ARDRA)分析比较, 选取其中的60株进行16S rRNA基因测序。通过序列比对、聚类分析, 60株菌分布在放线菌纲中的12个属, 链霉菌属占大多数, 有大量稀有放线菌, 这表明辐射污染区具有较丰富的放线菌属种多样性。     

超声波处理土样分离放线菌

【目的】探索超声波处理土壤悬液,增加稀有放线菌的类群。【方法】将西双版纳热带雨林的混合土样,制成土壤悬液,用超声波分别处理0-120s,用平板稀释法分离放线菌,得到纯菌落后测定其16S rRNA基因序列,进行系统发育分析,将分离菌株鉴定到属;用超声波处理已经鉴定到种且常见的10种链霉菌0-5min,后进行培养,测定其存活率。【结果】土壤悬液经超声波处理不同时间,放线菌的数量和种类逐渐增加。超声波处理已知链霉菌1-5min,对链霉菌的数量没有明显影响。【结论】用超声波处理土壤悬液40s,可以大大增加放线菌的出菌总数,明显增加稀有放线菌的种类,是一种经济且简便易行的方法。     

八门湾红树林土壤小单孢菌的分离及其多样性

采用平板稀释法从海南八门湾红树林潮间带、木果榄和海莲红树根际土壤样品中分离和筛选小单孢菌科放线菌,对其分离方法进行了评价和比较,并通过16S rRNA基因测序探索了八门湾红树林小单孢菌科放线菌的多样性。采用4种预处理方法和5种分离培养基,经过排除重复菌株后共得到115株放线菌。16S rRNA基因测序结果显示,这些放线菌分布在5个科、9个属。其中小单孢菌科的菌株约占全部菌株的90%(105株),主要分布于3个属:Micromonospora(85%),Jishengella(9.5%)和Verrucosispora(5.5%),与其亲缘关系最近的菌株的相似性分布于98.5%~100%之间。     

Isolation of new actinomycete strains for the screening of new bioactive compounds

In order to facilitate the discovery of novel bioactive compounds from microorganisms, various techniques for isolation of new actinomycete strains have been attempted. Studies of the vertical distribution of actinomycetes in soil, isolation of actinomycetes from desert soils or fallen leaves, selective isolation of Kitasatospora strains using novobiocin or Actinoplanes strains using the chemotactic method, and the use of gellan gum as a solidifying agent were carried out. We discovered 9 novel bioactive compounds from actinomycete strains isolated under unusual conditions, and proposed two new genera, five new species and one new subspecies.     

Use of polyvalent phage for reduction of streptomycetes on soil dilution plates.

An isolation method was developed in which prior to inoculation soil suspensions were exposed to suspensions of polyvalent phage isolated to Streptomyces spp. The phage susceptibility of streptomycetes provided a selective means of reducing streptomycetes on isolation plates subsequent to inoculation, and this reduction was persistent after long incubation periods. The efficiency and applicability of the method developed were checked with different samples from a range of sources. The increased chances of development of other genera after the reduction of streptomycetes on soil dilution plates were assessed.     

云南若干地区土壤放线菌区系及资源考察 X.滇东南地区

从云南省文山、红河等地采集不同植被、不同海拔高度的土壤样品,用不同方法分离高温、中温放线菌,按国内外通用的方法进行鉴定。滇东南地区的土壤放线菌种类丰富,分离到了13个菌属,稀有放线菌的数量所占比例较高。原始森林中的放线菌种属最丰富。本文对该地区放线菌的生态分布进行了探讨。     

Humic acid-vitamin agar,a new medium for the selective isolation of soil actinomycetes

A new medium, designated HV agar, containing soil humic acid as the sole source of carbon and nitrogen was developed.The HV agar was superior to other currently used media, including colloidal chitin agar, glycerol-arginine agar and starch-casein-nitrate agar, for the isolation and enumeration of soil actinomycetes: It allowed the growth of the largest numbers of actinomycete colonies belonging to each genus of Streptomyces, Micromonospora, Microbispora, Streptosporangium, Nocardia, Dactylosporangium, Microtetraspora and Thermomonospora on the plate, while restricting the development of true bacteria. The HV agar supported adequate growth and good sporulation for these actinomycetes.Even when spore suspensions were used as the inoculum, the HV agar produced remarkably larger numbers of actinomycetes, especially strains of the genera Micromonospora, Microbispora, Streptosporangium, Dactylosporangium and Saccharomonospora, than did glycerol-arginine agar. It was found that the spores of these actinomycetes were activated upon germination by treatment at 20°C for 30 min with a O.2% solution of humic acid prior to incubation.     

温室黄瓜叶部微生物区系

采用稀释分离法和消毒叶片研磨液培养法对温室黄瓜叶围和内生微生物进行了分离,共分离到248个菌株,初步鉴定出13个属的叶围真菌,其中链格孢属(Alternaria)和青霉属(Penicillium)真菌为优势类群;鉴定出4个属的内生真菌,其中曲霉属(Aspergillus)真菌为优势类群;10个属的叶围细菌,其中芽孢杆菌属(Bacillus)和黄单胞菌属(Xanthomonas)细菌为优势类群;6个属的内生细菌,其中芽孢杆菌属和假单胞菌属(Pseudomonas)细菌为优势类群;6个属的叶围酵母菌,其中隐球酵母属(Cryptococcus)为优势类群;已鉴定出2个属的叶围放线菌,分别为链霉菌属(Streptomyces)和小多孢菌属(Micropolpspora).未分离到内生酵母菌和放线菌.     

Diversity of Soil Actinomycetes in Yunnan, China

Since 1978, about 4,200 soil samples have been collected from 22 selected areas of various vegetational and climatic types throughout the province of Yunnan. Actinomycetes of 29 genera were isolated by the methods employed. The correlations between diversity and climate were grouped into tropical, subtropical plateau, cool temperate mountain, and snowy mountain types. Actinomycete populations of the first two types were more complex than were the other ones. Correlations between actinomycete diversity and vegetation were also attempted. Six types of vegetation were compared. The diversity of actinomycetes was greatest in soil samples of a primeval forest, with an average of 9.0 genera isolated, followed by secondary forest and vegetable farmland samples, with averages of 6.7 and 6.5 genera isolated, respectively. The upper limit for the occurrence of thermophilic actinomycetes is about 3,500 m above sea level in Yunnan. Psychrophilic actinomycetes were isolated at up to the same altitude. In addition, the drier and poorer the soil was and the cooler the climate was, the lower the count of actinomycetes was and the higher the percentage of streptomycetes observed was. The genus Streptomyces appears to be the most important in ecological function. It represents up to 90% of all soil actinomycete diversity in Yunnan and is likely an important characteristic of the soil actinomycete population.     

Use of polyvalent phage for reduction of streptomycetes on soil dilution plates

D.I. KURTBÖKE, C.F. CHEN AND S.T. WILLIAMS. 1992. An isolation method was developed in which prior to inoculation soil suspensions were exposed to suspensions of polyvalent phage isolated to Streptomyces spp. The phage susceptibility of streptomycetes provided a selective means of reducing streptomycetes on isolation plates subsequent to inoculation, and this reduction was persistent after long incubation periods. The efficiency and applicability of the method developed were checked with different samples from a range of sources. The increased chances of development of other genera after the reduction of streptomycetes on soil dilution plates were assessed.     

三江源地区不同退化程度草地的土壤放线菌区系

用不同分离方法,对三江源地区不同退化程度草地土壤放线菌的数量和多样性进行了比较.从5份土样中共分离放线茵178株,根据表型特征和16S rRNA基因序列分析,分别归入7个已知属:小单孢菌属(Micromonospora)、原小单孢菌属(Promicromonospora)、诺卡氏菌属(Nocardia)、假诺卡氏菌属(Pseudonocardia)、游动放线菌属(Actinoplanes)、韩国生工茵属(Kribbella)和链霉菌属(Streptomyces).其中链霉菌属分离菌株可归入7个表型类群.发现轻度退化高寒草原的土壤放线菌数量,丰度和多样性高于重度退化高寒草原;针茅高寒草原的土壤放线菌数量和多样性高于蒿草高寒草甸,而其中链霉菌的种类低于后者.表明高寒草地的退化程度与其中土壤放线茵的数量和多样性呈负相关.     

川滇四区森林土壤纯培养放线菌多样性及生物活性

【目的】为了从放线菌发现新的药物先导化合物,研究了川滇4个地区的放线菌多样性及其生物活性。【方法】采集250份土样,用4种培养基分离放线菌;从中选择98株代表菌进行了初步分类鉴定;采用琼脂扩散法,检测了169株放线菌对4种细菌和7种真菌的抑菌活性;利用特异性引物扩增法,测定了它们产生的聚酮合酶(PKSI、PKSⅡ)基因、非核糖体多肽合成酶(NRPS)基因和多烯类化合物合成酶(CYP)基因。【结果】黄荆老林的放线菌有13个属,峨眉山、青城山仅5个属,九寨沟9个属,西双版纳达20个属;不同地区的放线菌具有抗菌活性的菌株平均约占10%;有27%-36%的菌株产生PKSI、II、NRPS、CPY化合物合成基因。【结论】在采集样品的地区中,人类干扰越少,放线菌的多样性越高。分离放线菌时,使用"极端"条件,虽然分离到的放线菌数量可能不多,但获得未知菌的比例较大。添加抑制剂可减少革兰氏阴性细菌和真菌,有利于分离放线菌。