藏东南地区土壤放线菌的生态分布及活性研究

从藏东南原始森林、高山草甸、沼泽、粮田与保护地等不同植被、从2970~4590m不同海拔高度采集土样50份,用多种培养基分离中、低温放线菌,并对放线菌的数量、组成、生理生化特性以及它们的拮抗性等进行了研究。按放线菌的形态学特征进行了鉴定。结果表明:①从藏东南各种土壤中放线菌分离到9个属,其中束丝菌属在国内未见报道。以粮田中放线菌的数量和种类最多。②原始森林中拮抗性放线菌数量最多,提供了从原始森林土壤中可以筛选到更多拮抗性放线菌的重要信息。③抗革兰氏阳性细菌的放线菌菌株数较抗革兰氏阴性细菌的多,拮抗真菌的放线菌菌株数比拮抗细菌的多。④藏东南土壤链霉菌具有许多酶活性。     

Distribution of actinomycetes,their antagonistic behaviour and the physico-chemical characteristics of the world’s largest tidal mangrove forest

We examined the relationship between distribution of actinomycetes and antagonistic behaviour with the physico-chemical characteristics of the Sundarbans, off the Bay of Bengal, India. Soil/sediment samples were collected from three regions: near to the sea, intertidal regions and mangrove forests. For the enumeration of actinomycetes, four treatments combining dilution with distilled or sea water with or without heating followed by plating onto starch-casein, glycerol-arginine and starch-nitrate media were done. Dilution with seawater, heating and plating onto starch-casein yielded maximum number of actinomycetes. The highest number of actinomycetes was isolated from an intertidal region having alluvial soil and the lowest from a site containing sandy sediments. Antimicrobial activity was dependent upon seawater. Antimicrobial score of an actinomycetes strain was calculated allotting maximum points to the isolate showing activity against all the test bacteria, next lower point to the isolate showing activity against one less the total and so on. The antagonistic potential (AP) of a sampling site was the ratio of total antimicrobial score of the isolates and their number. The high AP sites were influenced by tides, while the low AP sites were not. Pearson's correlation between soil chemical parameters and microbiological parameters revealed soil nitrogen as the key factor determining the antagonistic activity.     

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.     

Study on the communities of endophytic fungi and endophytic actinomycetes from rice and their antipathogenic activities in vitro

The populations of endophytic fungi and actinomycetes from four rice cultivars in the Panyu district (Site 1) and Wushan district (Site 2) in Guangdong province, South China, were studied. The preponderant endophytic fungi and actinomycetes isolated belonged to Fusarium and Streptomyces respectively. The incidence of Streptomycetes griseofuscus ranged from 36.1 to 69% out of all the different rice cultivars from the two sites. It is the commonest population of endophytic actinomycetes, and constituted the greatest part of all the antagonistic communities. The distributions of endophytic fungi and actinomycetes in roots and leaves were different, endophytic fungi from leaves were diverse, some were organ-specific. More diverse endophytic actinomycetes were isolated from roots than from leaves. The endophytic fungi isolated from rice in Site 2 were more diverse than that in Site 1. The diversity of the endophytic actinomycetes, however, was less than that in Site 1. Acid soil in Site 2 is ideal for the growth and colonization of fungi while the alkaline soil in Site 1 is better for the growth and colonization of actinomycetes. The results suggested that differences in the chemical composition of soil could influence the endophytic microbial communities of rice plants. The endophytic fungi and actinomycetes isolated from poor-growing seedlings and susceptible rice cultivars were more abundant than that the disease-resistant counterparts. In the dual culture and activity detection of the metabolites, 41.2% of all the isolated endophytic fungi showed antagonism to rice pathogens. Fifty percent of all the isolated endophytic actinomycetes were antagonistic to those pathogens. The percentage of Streptomyces griseofuscus and hygroscopicus reached 55.4 and 21.4% of all the active actinomycetes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of glyphosate on the microbial activity of two Romanian soils

Glyphosate applied to soils potentially affect microbial activity. A series of field and laboratory experiments assessed the effect of this herbicide on soil microorganisms. The aim of experiments was to evaluate the effect of glyphosate application on the soil microbial community structure, function and their activity. We studied "in vitro", changes in the microbial activity of typical Chernozem and Gleysol soils, with and without applied glyphosate. The herbicide was applied at a rate of 2, respectively 4 mg kg(-1) of soil and microbial activity were measured by fluorescein diacetate (FDA) hydrolysis. We found an increase of 9 to 13% in FDA hydrolyses in the presence of glyphosate in rate of 2 mg kg (-1) compared with the same type of soil which had never received herbicide. The double quantity of glyphosate decrease soil microbial activity; the amount of hydrolyzed fluorescein is lower than the addition of 2 ppm. The greater decrease was observed in the Gleysol type where the fluorescein hydrolyzed is with 4, 85% lower than version control without glyphosate. Chemical characters of soil, influence soil biological activity when herbicide is added. In Chemozem case, rich in humus, whose predominant micro flora is represented by actinomycetes through glyphosate treatment these organisms growths of as major producers of antibiotics actinomycetes determine an inhibitory effect on eubacteria and micromycetes growth, which is highlighted by estimating a relatively small number of them. After 10 days, once with decreasing of glyphosate content in soil, decreases the number of active actinomycetes, therefore we are witnessing to a numerical growth of bacterial population. In Gleysol type the indigenous micro flora is represented by eubacteria, so when the glyphosate is added it was registered a high growth of these organisms fraction.     

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。     

库木塔格沙漠土壤细菌多样性及具有群体感应抑制活性放线菌筛选

【背景】群体感应抑制剂(quorum sensing inhibitor,QSI)作为抗生素潜在替代品,可有效降低致病菌传染性和毒性。沙漠土壤蕴藏着丰富的放线菌资源,是挖掘群体感应抑制剂的重要来源。【目的】解析库木塔格沙漠土壤细菌群落多样性,筛选并挖掘群体感应抑制活性放线菌资源。【方法】采用Illumina Nova Seq高通量测序技术揭示库木塔格沙漠土壤细菌群落组成,利用可培养方法进行土壤放线菌分离和鉴定;选用紫色杆菌CV026模型筛选群体感应抑制活性放线菌,并对其功能特性进行初步评价。【结果】Illumina Nova Seq高通量测序结果显示,样品土壤细菌涉及23门96目150属,优势菌门为变形菌门(Proteobacteria,61%)、放线菌门(Actinobacteria,28%),其中分枝杆菌属(Mycobacterium)为放线菌门最优势菌属(87.3%),其次为红球菌属(Rhodococcus,6.8%)和丙酸杆菌属(Cutibacterium,0.9%)。可培养方法共分离到108株放线菌,归属9科10属,其中优势菌属为链霉菌属(Streptomyces),占65....     

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.     

A new preferential medium for enumeration and isolation of desert actinomycetes

In order to facilitate the discovery of novel actinomycetes from the Egyptian deserts, which can be useful as new sources for bioactive metabolites, different media for enumeration and isolation of desert actinomycetes have been tested. For this purpose, 30 soil samples from different six sites representing the Western and Eastern deserts of Egypt were collected. The two deserts are considered hyper-arid and the soil characteristics were determined. The media used were glucose–yeast extract agar, soil extract agar and a new minimal medium (MM) containing glucose, yeast extract and mineral salts. The effects of the soil characteristics on the total viable actinomycete counts on the three media were evaluated. The results showed that the highest actinomycete count in samples from five out of six sites was obtained on MM. Also MM was more selective for actinomycetes and significantly decreased the number of fungal colonies and to a lower extent the number of bacterial colonies. Moreover, it supported the development of different and diverse groups of actinomycetes. From the results obtained in this study, MM is a new useful medium for enumeration and selective isolation of actinomycetes from the desert soils.     

青海湖水及湖滨盐化土壤放线菌的分类及耐盐性

采用3种含盐培养基(70g/L NaCl)分离青海湖水、湖泥、湖滨土壤及盐化荒地土壤中的中低温放线菌,纯化后按常规方法鉴定,并测定其耐盐性。结果表明:①湖泥、湖滨土壤及盐化荒地土壤中存在一定数量的中、低温耐盐放线菌。湖滨荒地土壤中的放线菌数量最高,其次为湖滨土壤及浅层湖水底淤泥。②鉴定出的耐盐性放线菌均为链霉菌(占供试菌总数的92.6%),且全部为白孢类群。③47.6%、19.0%和33.3%的供试放线菌的最高耐盐度分别为70、100和150g/L。     

Isolation of putative polyene-producing actinomycetes strains via PCR-based genome screening for polyene-specific hydroxylase genes

Polyene antibiotics, which include nystatin, pimaricin, amphotericin and candicidin, include a family of very promising antifungal polyketide compounds that are typically produced by soil actinomycetes. The presence of similar cytochrome P450 hydroxylase (CYP) genes in the biosynthetic gene clusters for these polyenes have been previously reported. Using this polyene, more than 200 independently isolated actinomycetes strains were screened by CYP-specific PCR. Four strains were isolated based on the presence of the expected size of the PCR-amplified DNA fragment in the chromosome. The nucleotide sequencing of the PCR-amplified DNA fragments showed that each of the four actinomycetes strains contained a highly homologous polyene-specific CYP gene. Each of the culture extracts from these four strains showed a typical polyene-like high-pressure liquid chromatography (HPLC) chromatogram profile, and strong antifungal activity against Candida albicans. This suggests that the polyene-specific PCR-guided genome screening approach is an efficient method for isolating potentially valuable polyene-producing actinomycetes.     

青海沙珠玉人工植被系统土壤放线菌生态分布及拮抗性

为探索人工植被重建对土壤放线菌的影响,采用稀释平板法及琼脂块法对青海沙珠玉地区植被重建后沙土中放线菌的生态分布及拮抗性进行了研究.结果表明：随着高寒干旱沙地植被重建和覆盖度的提高：土壤放线菌数量显著增加,其中退耕还草地土壤中放线菌总数较沙地提高145.4%,小单孢菌数量约为沙地的6倍;拮抗性放线菌平均筛出率大幅度增加,退耕还草地土壤中拮抗性放线菌、天然草地土壤中抗细菌放线菌及林地土壤中抗病原真菌放线菌的平均筛出率约分别为沙地的2、3.2及1.5倍;地表植被盖度与土壤养分对放线菌数量以及拮抗性放线菌数量有很大影响,其中有机质、碱解氮含量以及鲜草产量与放线菌总数的相关性达到极显著水平(P<0.01),有机质含量、鲜草产量与拮抗性放线菌株数的相关性达到极显著水平(P<0.01).此外,植被盖度、土壤全氮、全磷、全钾、全盐以及速效钾含量与放线菌总数、链霉菌数、小单孢菌数的相关性也达到显著水平(P<0.05).     

来自西双版纳3种有毒植物的免培养与纯培养放线菌多样性及生物活性

【背景】由于土壤放线菌中获得新化合物日益困难,抗生素滥用使致病菌耐药性不断增加,人们转向研究植物内生放线菌以期发现新化合物。【目的】探究西双版纳热带雨林有毒植物内生放线菌的多样性,为开发新药提供具有潜在生物活性的菌株。【方法】通过Illumina Hi Seq高通量测序和纯培养方法分析箭毒木、八角枫、马缨丹3种有毒植物的内生放线菌群落结构组成,利用纸片扩散法筛选抑菌活性,通过PCR扩增检测7类化合物合成基因。【结果】高通量测序的多样性分析和群落结构分析得出:3种有毒植物在门分类水平检测出古菌域的2个门、细菌域的18个门和暂定的Rsa HF231、WD272门;在属分类水平检测出30个属的放线菌,八角枫和马缨丹的微生物群落结构比箭毒木更丰富。纯培养分离获得11个属34株菌,分离自箭毒木的菌株比八角枫和马缨丹的菌株更多,而且大多数高通量检测出的菌种不能通过纯培养获得。抑菌活性检测结果显示:抗菌活性作用明显的菌株以链霉菌属为主。链霉菌属的NRPS和PKS基因的检出率明显高于其他化合物合成基因。【结论】有毒植物内生放线菌多样性非常丰富。有毒植物内生放线菌具有合成次生代谢产物的潜力,可以为生物农药及抗生素开发提供丰富的菌种资源。     

Comparative study of actinomycete communities associated with Lasius niger and Formica cunicularia ants and their nests

A comparative analysis of the abundance and biodiversity of actinomycete communities isolated from living ants Lasius niger and Formica cunicularia, as well as their anthills, has been carried out for the first time. The number of actinomycetes detected in L. niger ants is close to the number of actinomycetes in their anthills and one order is higher than that for F. cunicularia. Actinomycete communities of anthills and the intact soil are characterized by a high diversity, equitability, absence of severe dominants, and the presence of common species and differ by number and the range of species. Biodiversity of actinomycetes associated with living ants is considerably lower than in their nests and the surrounding soil and the range of actinomycetes is specific for both the species of ants.     

Antagonistic activity of soil acidophilic actinomycetes

It has been shown that soil acidophilic actinomycetes (mycelial prokaryotes with a growth optinum between pH 3 and 7) markedly differ from neutrophilic actinomycetes in antimicrobial activity: the former are more active against fungi and yeasts, whereas the latter effectively suppress Gram-positive bacteria. Acidophilic streptomycetes actively inhibit the growth of phytopathogenic fungi, especially on acidic media.     

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。     

Actinomycetes from solitary wasp mud nest and swallow bird mud nest: isolation and screening for their antibacterial activity

The aim of this study was to isolate and screen actinomycetes from solitary wasp and swallow bird mud nests for antimicrobial activity. The actinomycetes were isolated from soil of nests of solitary wasp and swallow bird, and identified on the basis of morphological characteristics and molecular biological methods. A total of 109 actinomycetal isolates were obtained from 12 soil samples (6 from each habitat) using two media. The highest number of actinomycetes were recovered on Humic acid vitamin agar media (65.13%, n = 71) as compared to actinomycetes isolation agar media (34.86%, n = 38). The antimicrobial activity of actinomycetes isolates was determined using the agar plug method. Among 109 isolates, 51 isolates (46.78%) showed antibacterial activity by agar plug assay. The morphological and molecular characteristics confirmed that the most of active isolates in both sample belonged to the genus Streptomyces, the other potential genera like Streptosporangium, Actinomadura, Saccharopolyspora, Thermoactinomycetes and Nocardia were also recovered, but in a low frequency. The isolates designated as 8(1)*, BN-6, MN 2(6), MN 2(7) and MN 9(V) showed most promising activity against various drug resistant bacterial pathogens. It seems that the promising isolates from these unusual/unexplored habitats may prove to be an important step in development of drug for treating multi-drug resistant bacterial pathogens.     

Microbiological differences between limed and unlimed soils and their relationship with cavity spot disease of carrots (Daucus carota L.) caused by Pythium coloratum in Western Australia

Application of lime (4000 kg ha^-1) to a soil used for commercial carrot production (pH 6.9) significantly (p<0.05) reduced the incidence of cavity spot disease of carrots compared to unlimed soil (pH 5.1). It significantly (p<0.01) increased soil microbial activity as measured by the hydrolysis of fluorescein diacetate and arginine ammonification. The application of lime resulted in a significant (p<0.01) increase in the total numbers of colony forming units (efu) of aerobic bacteria, fluorescent pseudomonads, Gram negative bacteria, actinomycetes and a significant (p<0.01) decrease in the cfu of filamentous fungi and yeasts compared to unlimed soil. Liming also increased the cfu of non-streptomycete actinomycetes rarely reported in similar studies. These non-streptomycete actinomycetes were estimated and isolated using polyvalent Streptomyces phages and the dry heat technique to reduce the dominance of streptomycetes on isolation plates. The non-streptomycete actinomycetes isolated included species of Actinoplanes, Micromonospora, Streptoverticillium, Nocardia, Rhodococcus, Microbispora, Actinomadura, Dactylosporangium and Streptosporangium. The numbers of actinomycetes antagonistic to Pythium coloratum, a causal agent of cavity spot disease of carrots increased in soil amended with lime. Application of lime also reduced the isolation frequency of P. coloratum from asymptomatic carrot roots grown in soil artificially infested with the pathogen, 3, 4 and 5 weeks after sowing.     

西藏仲巴五彩沙漠放线菌资源勘探及生物活性筛选

【背景】细菌耐药性问题日益严峻,新抗生素的研发速度远远落后于临床需要,从特殊生境中挖掘微生物药物资源有望解决以上问题。【目的】勘探西藏仲巴五彩沙漠土壤放线菌多样性并进行生物活性筛选,为发现药用放线菌资源、开发新型抗生素奠定基础。【方法】采用8种分离培养基,通过平板稀释涂布法分离放线菌;根据分离菌株的16S r RNA基因序列同源性分析放线菌多样性;采用PCR技术对分离的放线菌菌株进行II型聚酮合酶(PKS-II)酮缩酶结构域KS、非核糖体多肽合成酶(NRPS)腺苷酸化结构域A、安莎类抗生素生物合成前体3-氨基-5-羟基-苯甲酸合酶(AHBA)保守区、黄素腺嘌呤二核苷酸卤化酶(Halo)保守区抗生素生物合成基因检测;对生物合成基因检测阳性的菌株进行液体发酵,发酵液经乙酸乙酯萃取、菌体经丙酮浸提,获得提取浓缩物样品进行抑菌活性和抗氧化活性筛选。【结果】从4份土样中分离纯化到231株放线菌,分布于7个属,其中链霉菌为优势菌属。68株放线菌的生物合成基因分析显示至少具有1种生物合成基因簇,其中6株同时具有4种生物合成基因簇;进一步的抑菌活性检测显示所有检测的菌株至少表现为对1株检定菌具有抑菌活性,其中8株具有广谱抗菌活性;抗氧化活性筛选结果为13株显示总抗氧化能力阳性,10株具有较好的羟自由基清除能力,3株显示较强的氧自由基清除能力。【结论】西藏仲巴五彩沙漠土壤中含有较丰富的放线菌药用资源,具有从中发现放线菌新菌种和开发新抗生素的潜力。     

沙化对玛曲高寒草甸土壤微生物数量及土壤酶活性的影响

对不同沙化程度高寒草甸土壤物理性质和土壤酶活性及微生物结构进行调查分析,结果表明:(1)随沙化程度的加剧,玛曲高寒草甸土壤粘粒成分、含水量、蔗糖酶活性、脲酶活性、中性磷酸酶活性以及过氧化氢酶活性均呈现出下降趋势,而土壤细砂粒成分和pH值逐渐增大,土壤粗砂粒、细菌数量、真菌数量、放线菌数量以及微生物总数量均出现了先增大后减小的趋势。(2)在发生沙化的高寒草甸土壤中,细菌占土壤微生物的主要组分,然后依次是放线菌和真菌;细菌百分比在中度沙化的样地土壤中达到最大值,放线菌和真菌百分比在轻度沙化的样地土壤中达到最大值。(3)在玛曲高寒草甸沙化过程中,驱使土壤微生物数量变化的主导因子是土壤孔隙度和含水量;驱使土壤酶活性变化的主导因子,在沙化初期是土壤含水量、颗粒组成、动植物和pH,在沙化发生后是土壤微生物、颗粒组成、含水量及动植物。