海南西海岸红树林伴生植物内生放线菌多样性及其延缓衰老活性初筛

为寻找新型抗衰老药物,该文以海南西海岸红树林伴生植物为研究对象,采用9种不同培养基从7种伴生植物21份样品中分离纯化放线菌,通过PCR扩增,16S rRNA基因序列分析已纯化放线菌的多样性,利用秀丽隐杆线虫(Caenorhabditis elegans)模型筛选菌株来进行延缓衰老活性研究。结果表明:(1)从7种伴生植物21份样品中共分离到26株海洋放线菌,隶属于9科15属,分别为拟诺卡菌属、短状杆菌属、短小杆菌属、Demequina、戈登氏菌属、类诺卡氏菌属、Lysinimicrobium、细杆菌属、假诺卡氏菌属、微球菌属、原小单孢菌属、拟无枝酸菌属、Yimella、北里孢菌属和链霉菌属,其中链霉菌属为优势菌属。(2)经秀丽隐杆线虫模型筛选,发现有2株海洋放线菌的发酵粗提物具有延缓秀丽隐杆线虫衰老的作用。综上结果说明海南西海岸红树林伴生植物中含有丰富多样的药用放线菌资源,为海洋放线菌抗衰老研究奠定了基础。     

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。     

河北九莲城淖尔可培养放线菌多样性及抗菌活性筛选

【目的】勘探干涸的九莲城淖尔土壤放线菌多样性并进行活性筛选,以期发现药用微生物资源,为新抗生素的发现奠定基础。【方法】采用15种分离培养基,以稀释涂布法分离放线菌;根据分离菌株的16S rRNA基因序列同源性分析放线菌多样性;发酵液经乙酸乙酯萃取,菌丝体经丙酮浸提,获得提取浓缩物样品;样品通过纸片扩散法进行抗菌活性初筛;抗菌阳性菌株采用PCR技术进行Ⅰ型聚酮合酶(PKS I)KS域、Ⅱ型聚酮合酶(PKS II)KS域和非核糖体多肽合成酶(NRPS)A结构域抗生素生物合成基因的检测。【结果】从11份盐湖土壤样品中分离纯化到251株放线菌,其分布于放线菌纲的10个目15个科31个属,其中优势菌属为链霉菌属和拟诺卡氏菌属;251株放线菌中包括57株耐(嗜)盐放线菌,其优势菌属为拟诺卡氏菌属(22株)和涅斯捷连科氏菌属(15株)。基于16S r RNA基因序列的系统发育分析显示,菌株J11Y309为糖霉菌科潜在新属,菌株J12GA03为分枝杆菌科潜在新种。96株放线菌活性检测结果显示,56株至少对1株检定菌具有抗菌活性,阳性率为58.3%;56株有活性的放线菌中,47株至少含有1种抗生素生物合成基因,其中17株同时具有3种抗生素生物合成基因。【结论】干涸的九莲城淖尔土壤中含有较为丰富的药用放线菌资源,具有从中发现放线菌新物种和新抗生素的潜力。     

三七内生放线菌的分离及拮抗三七根腐病原活性研究

挖掘云南文山三七内生放线菌菌种资源以及对分离到的放线菌菌株进行三七根腐病病原菌的抗菌活性评价，为生物防治三七根腐病的发生、传播、有效治理及解决三七连作障碍和后续研究三七内生放线菌的代谢产物提供菌种资源。采用三种分离方法、六种分离培养基对文山两个产地三七全株植物进行内生放线菌分离，分离到的菌株通过16S rRNA基因扩增测序鉴定到属，通过琼脂扩散法对菌株发酵液进行抗菌活性初筛。共分离到56株内生放线菌，经形态特征初步排重后，选22株代表菌株进行16S rRNA基因扩增测序，并鉴定到属，其分属于链霉菌属(Streptomyces)、放线产孢菌属(Actinomycetospora)、拟诺卡氏菌属(Nocardiopsis)、黄英菌属(Yinghuangia)四个属。对23株菌株发酵液进行抗菌活性初筛，14株菌株(60.87%)具有不同程度的抗菌活性，其中菌株S004、S006、S014、S016、S022、S042、S047、S053对三七根腐病病原菌有较好的抗菌活性，具备进一步深入研究的价值。本研究初步探索出适合三七内生放线菌分离的分离方法，获得一批三七内生放线菌菌种资源，并筛选出8株对...     

诺卡氏菌属中的两个新种

由土壤中分离的两株诺卡氏菌形放线菌A-100菌株和186菌株．经鉴定,其形态和细胞壁化学组分均属诺卡氏菌属,但培养特征和生理生化特性与该属中的已知种不同。因此认为这两株菌是诺卡氏菌属中的两个新种,并分别命名为鲜黄诺卡氏菌Nocardia galba n.sp和绛红色诺卡氏菌Nocardia purpurea n. sp.。     

东乡野生稻内生放线菌分离及菌株S123次级代谢产物分析

【目的】从东乡野生稻(Oryza rufipogon)中分离和鉴定内生放线菌,对其进行抗菌活性筛选,并分析高抗菌活性菌株S123的次级代谢产物。【方法】采用S培养基对东乡野生稻内生放线菌进行分离、纯化,并构建16S rRNA基因序列系统发育进化树进行菌株鉴定。以琼脂扩散法和菌丝生长速率法进行抗菌活性筛选,同时设计简并引物检测菌株I型聚酮合酶(PKS-I)基因。对具广谱抗菌活性的菌株S123进行分批大量发酵,运用多种色谱方法对发酵产物进行分离、纯化,利用MS和NMR分析鉴定化合物的结构。【结果】从东乡野生稻中共分离到11株内生放线菌,分别属于链霉菌属(8株)和假诺卡氏属(3株)。其中有8株具有抗菌活性,8株呈现I型PKS阳性。从高抑菌活性菌株S123中分离到化合物Nigericin和17-O-demethylgeldanamycin,其中Nigericin对金黄色葡萄球菌、枯草芽孢杆菌及水稻纹枯病菌均有抑制活性。【结论】对东乡野生稻内生放线菌进行了分离、鉴定和抗菌活性筛选,并从中分到两种与I型PKS基因相关活性的化合物Nigericin和17-O-demethylgeldanamycin,为研究东乡野生稻内生放线菌的多样性和次级代谢产物的分离提供依据。     

青海盐碱环境中具抗肿瘤活性放线菌的筛选和多样性研究

从我国青海省采集盐碱土样或泥样,用添加1.0~3.0mol/L NaCl的GPY琼脂培养基和ISP2琼脂培养基分离到145株典型放线菌菌株。采用6种肿瘤细胞株对分离菌株的发酵产物进行体外筛选,得到26株抗肿瘤活性阳性菌株(17.9%),19株为拟诺卡氏属(Nocardiopsis)菌株,7株为链霉菌属(Streptomyces)菌株。在抗肿瘤活性、形态特征、生理生化特性和全细胞水解物氨基酸组分分析等实验结果的基础上,选取差异较大的8株抗肿瘤活性阳性菌株进行16S rRNA基因序列测定和系统发育多样性分析。结果表明,2株属于链霉菌属(Streptomyces)的1个已知物种和1个潜在新种;6株属于拟诺卡氏菌属(Nocardiopsis),可能代表该属的4个新种。研究表明青海盐碱环境中存在产生抗肿瘤活性物质的重要放线菌资源,也提示其中蕴藏着较丰富的微生物多样性。     

蜚蠊肠道抗真菌活性放线菌的筛选与鉴定

对美洲大蠊肠道具有抗真菌活性的内生放线菌进行筛选和分类鉴定。以白色念珠菌、红色毛癣菌、黑曲霉和烟曲霉4种人体致病真菌为研究对象,采用牛津杯法和菌株对峙共培养法对159株蜚蠊肠道内生放线菌进行体外抗真菌活性筛选,采用16S rDNA PCR、Blast同源性比对和构建系统发育树等方法对菌株进行分子生物学鉴定。研究结果表明,45株内生放线菌具有抗真菌活性,诸多菌株抗真菌谱较广,有10株内生放线菌对4种受试真菌均有拮抗作用。其中,链霉菌属为优势菌属有32株,占71.11%;戈登氏菌属有6株,微杆菌属有2株,分枝杆菌属、无色杆菌属、小单孢菌属、纤维微菌属和壤霉菌属各有1株。说明蜚蠊肠道含有丰富的抗真菌活性放线菌资源。     

连翘和水茄内生放线菌的多样性、活性及生物合成基因的PCR筛查

从采自成都地区的中药植物连翘Forsythia suspense和水茄Solanum torvum的根部分离到14株内生放线菌。活性筛选表明,10株菌的发酵粗提物具有不同程度的抗肿瘤活性,占全部菌株的71%;3株菌具有抗细菌活性,其中菌株A263具有较强的细胞毒活性和广谱抗细菌活性。基于16S rRNA基因部分序列的相似性分析表明,菌株A275属于克里贝拉菌属Kribbella,其余13株属于链霉菌属Streptomyces。多种生物合成基因的筛查实验表明,5株菌同时具有PKS-I、PKS-II、NRPS型基因,其中A255和A263还具有3,5-AHBA合酶基因,但仅A275具有oxyB基因。结果可以推测,链霉菌是这2种中药植物根部的优势内生放线菌,生物合成基因的PCR筛查能极大地弥补传统活性筛选模型的不足,内生放线菌具有产生丰富生物活性化合物的巨大潜力。     

宁夏白芨滩自然保护区苦豆子内生放线菌多样性及其分布

【目的】探索宁夏干旱荒漠区苦豆子内生放线菌的多样性及区系组成,为苦豆子内生放线菌的合理开发和利用提供理论依据。【方法】从宁夏白芨滩自然保护区不同植被和土壤类型的6个样区采集健康苦豆子样品30份,采用组织匀浆法从苦豆子植株的根部、茎部、叶部和种子中分离内生放线菌,根据培养性状、菌落、孢子等的形态特征和16S rRNA基因序列分析对分离菌株进行鉴定;根据苦豆子内生放线菌的相对频率、物种多样性指数、丰富度指数和相似性系数分析其区系组成特点。【结果】从30份苦豆子样品中,共分离得到内生放线菌111株,这些菌株分属于链霉菌属(Streptomyces)、拟诺卡氏菌属(Nocardiopsis)、普劳斯氏菌属(Prauserella)、Actinophytocola、微杆菌属(Microbacterium)、束丝放线菌属(Actinosynnema)、嗜热油菌属(Thermoleophilum)、糖霉菌属(Glycomyces)和糖丝菌属(Saccharothrix) 9个属,其中链霉菌属(Streptomyces)和拟诺卡氏菌属(Nocardiopsis)为优势属。苦豆子内生放线菌的分布具有一定的组织特异性,根和种子中的内生放线菌数量高于茎和叶。植被类型中,沙生植被草原(样区Ⅳ)分离的苦豆子内生放线菌物种多样性指数最高,而荒漠草原(样区Ⅴ)最低。荒漠草原(样区Ⅴ)和林地(样区Ⅵ)内生放线菌群落有密切的相似性。【结论】苦豆子体内含有丰富的内生放线菌资源,其内生放线菌具有很高的宿主特异性,而且其分布受生境影响。     

The rhizospheres of traditional medicinal plants in Panxi, China, host a diverse selection of actinobacteria with antimicrobial properties

Actinobacteria are a prolific source of antibiotics. Since the rate of discovery of novel antibiotics is decreasing, actinobacteria from unique environments need to be explored. In particular, actinobacterial biocontrol strains from medicinal plants need to be studied as they can be a source of potent antibiotics. We combined culture-dependent and culture-independent methods in analyzing the actinobacterial diversity in the rhizosphere of seven traditional medicinal plant species from Panxi, China, and assessed the antimicrobial activity of the isolates. Each of the plant species hosted a unique set of actinobacterial strains. Out of the 64 morphologically distinct isolates, half were Streptomyces sp., eight were Micromonospora sp., and the rest were members of 18 actinobacterial genera. In particular, Ainsliaea henryi Diels. hosted a diverse selection of actinobacteria, although the 16S ribosomal RNA (rRNA) sequence identity ranges of the isolates and of the 16S rRNA gene clone library were not congruent. In the clone library, 40% of the sequences were related to uncultured actinobacteria, emphasizing the need to develop isolation methods to assess the full potential of the actinobacteria. All Streptomyces isolates showed antimicrobial activity. While the antimicrobial activities of the rare actinobacteria were limited, the growth of Escherichia coli, Verticillium dahliae, and Fusarium oxysporum were inhibited only by rare actinobacteria, and strains related to Saccharopolyspora shandongensis and Streptosporangium roseum showed broad antimicrobial activity.     

Isolation and identification of actinobacteria from surface-sterilized wheat roots

This is the first report of filamentous actinobacteria isolated from surface-sterilized root tissues of healthy wheat plants (Triticum aestivum L.). Wheat roots from a range of sites across South Australia were used as the source material for the isolation of the endophytic actinobacteria. Roots were surface-sterilized by using ethanol and sodium hypochlorite prior to the isolation of the actinobacteria. Forty-nine of these isolates were identified by using 16S ribosomal DNA (rDNA) sequencing and found to belong to a small group of actinobacterial genera including Streptomyces, Microbispora, Micromonospora, and Nocardiodes spp. Many of the Streptomyces spp. were found to be similar, on the basis of their 16S rDNA gene sequence, to Streptomyces spp. that had been isolated from potato scabs. In particular, several isolates exhibited high 16S rDNA gene sequence homology to Streptomyces caviscabies and S. setonii. None of these isolates, nor the S. caviscabies and S. setonii type strains, were found to carry the nec1 pathogenicity-associated gene or to produce the toxin thaxtomin, indicating that they were nonpathogenic. These isolates were recovered from healthy plants over a range of geographically and temporally isolated sampling events and constitute an important plant-microbe interaction.     

定殖植物根内和根围放线菌的分离鉴定及其体外抑菌促生效应

【目的】旨在分离、筛选并鉴定体外具抑菌促生作用的定殖于植物根内和根围的放线菌,以期丰富放线菌种质资源,为研制植物病害生防菌剂提供技术依据。【方法】采用稀释涂布平板法分离盐碱地、湿地、工业污染土壤中优势植物根内及其根围中的放线菌;通过平板对峙试验筛选具有抑菌效应的菌株,进而采用Salkowski比色法、CAS平板检测法和无氮源培养法进一步检测抑菌菌株的促生作用;通过形态观测、生理生化特性检测及16SrRNA基因序列分析鉴定菌种。【结果】共分离到链霉菌属(Streptomyces)、诺卡氏菌属(Nocardia)和小单孢菌属(Micromonospora) 3属共283株定殖于植物根内和根围的放线菌,3个采样地中湿地数量最多,均为根围土根内;其中链霉菌属占总数的77%,可分为10个类群。经筛选获得7株抑菌活性和促生效应较强的菌株,其中菌株H6-1抑菌效应最大,其无菌发酵液对尖孢镰刀菌(Fusariumoxysporum)、禾谷镰刀菌(Fusariumgraminearum)、灰葡萄孢菌(Botrytis cinerea)、立枯丝核菌(Rhizoctoniasolani)、轮纹大茎点霉(Macrophomakawatsukai)和瓜类炭疽菌(Colletotrichumorbiculare)的抑制率分别为32.3%、42.6%、48%、72.2%、58.1%和60.5%;而D11-4菌株的促生作用最强,能产吲哚乙酸(22.3 mg/L)、产铁载体(晕圈直径25.2 mm)和固氮。经鉴定这7株放线菌是吸水链霉菌变种(Streptomyces angustmyceticus) H4-6、娄彻氏链霉菌(Streptomyces rochei) S2-2、浑圆链霉菌(Streptomycesglobosus)H6-1、(Streptomycesiakyrus)GD8-4、波卓链霉菌(Streptomyces bottropensis) GH8-6、(Streptomyces paradoxus) H8-2和(Streptomyces coralus) D11-4。【结论】三个生境中定殖于植物根内和根围的放线菌类群丰富且所筛选获得的7株放线菌具有生防潜力,值得进一步研发。     

川楝内生放线菌多样性及抗菌活性筛选

【目的】探究药用植物川楝内生放线菌多样性,从中挖掘出新的放线菌菌株,发现新的潜在农业生防和医药先导化合物。【方法】从四川境内的资阳、遂宁以及重庆万州采集川楝的根、茎、叶、果、皮,采用纯培养方法,用4种培养基共分离获得148株放线菌。通过形态学观察筛选出60株放线菌进行RFLP分析,选出代表菌株进行16S r RNA基因序列分析。以3株细菌和6株病原真菌作为指示菌株,检测初筛出的60株菌株的抗菌活性以及聚酮合酶(PKSⅠ、PKSⅡ)基因、非核糖体多肽合成酶(NRPS)基因和卤化酶(Halo)基因。【结果】基于16S r RNA-RFLP分析,60株放线菌被分成10簇,筛选出25株代表菌株分别属于7个属,包括Streptomyces、Micromonospora、Planotetraspora、Streptosporangium、Nocardiopsis、Prauseria、Microbispora,其中链霉菌占73.3%。供试的川楝内生放线菌对细菌、真菌有不同程度的抗菌活性;其中含有4类化合物合成基因的菌株占10%-55%。【结论】药用植物川楝内生放线菌具有丰富的多样性,且不同地区不同部位川楝组织中放线菌的种群存在差异;分离菌株广谱的抗菌活性证明,川楝内生放线菌在次生代谢产物合成方面具有巨大潜力,这为进一步的药物开发提供了丰富的菌种资源。     

A Study on actinobacterial diversity of Hampoeil cave and screening of their biological activities

Caves are oligotrophic, dark and less-explored environments and are considered as sources of promising microbial strains in biotechnology. Hampoeil Cave is located in massive dolomite with thin bedded limestone in northwestern of Iran. In an isolation and screening program, various samples from soil, water, floor, wall and ceiling of Hampoeil cave and its invertebrates were collected. Four various treatments and 10 different isolation media were used for the isolation of the actinobacteria. Screening of the isolates for antimicrobial activity against 10 bacteria and fungi, 5 hydrolytic enzymes production and resistance to 5 heavy metals have been performed. Among 33 various samples, 76 actinobacteria from various genera, including Streptomyces, Micromonospora, Micrococcus, Kocuria and Corynebacterium were isolated. Eighty percent of the strains had one of the studied hydrolytic enzyme activity. At least one type of antimicrobial activity was seen in 25.3% of the isolates. Resistance to one metal or more was seen in 26.32% of the isolates. The ratio of rare-actinobacteria in the oligotrophic samples to enriched samples is 20% more than Streptomyces. Percentage of strains with the highest activity in esterase, amylase, DNase, protease or lipase activity that were isolated from organic-rich environmental samples were 100, 100, 100, 82 and 82%, respectively. Also, 26.32% of the actinobacterial isolates resisted to heavy metals. It was concluded that Hampoeil cave is a good source in finding cave-living actinobacteria potent in producing hydrolytic enzymes and bioremediation.     

Isolation and characterization of endophytic streptomycete antagonists of Fusarium wilt pathogen from surface-sterilized banana roots

A total of 131 endophytic actinomycete strains were successfully isolated from surface-sterilized banana roots. These isolates belonged to Streptomyces (n=99), Streptoverticillium (n=28), and Streptosporangium (n=2) spp. The remaining 2 isolates were not identified. About 18.3% of the isolates inhibited the growth of pathogenic Fusarium oxysporum f. sp. cubense on banana tissue extract medium. The most frequently isolated Streptomyces sp. strain S96 was similar to Streptomyces griseorubiginosus. About 37.5% of the S. griseorubiginosus strains were antagonistic to F. oxysporum f. sp. cubense. The antagonism of strain S96 was lost when FeCl(3) was introduced into the inhibition zone. In vivo biocontrol assays showed that the disease severity index (DSI) was significantly (P=0.05) reduced and mean fresh weight increased (P=0.001) in plantlets treated with strain S96 compared to those grown in the absence of the biocontrol strain. These findings indicate the potential of developing siderophore-producing Streptomyces endophytes for the biological control of fusarium wilt disease of banana.     

塔里木盆地胀果甘草内生放线菌多样性及抗菌活性分析

【目的】发掘具有开发前景的放线菌资源,对分离自新疆胀果甘草的内生放线菌的多样性、抗菌活性和次级代谢产物合成相关基因进行研究。【方法】采用5种培养基和3种前处理方法,从胀果甘草中分离获得80株放线菌。基于菌株形态学特征,对36株代表菌株进行抗菌活性检测,通过特异性引物扩增方法,检测了PKS I、PKS II、NPRS和卤化酶基因,探究其合成天然产物的潜在能力。结合筛选结果,选取其中20株代表菌,经16S r RNA基因测序,对其进行系统发育分析。【结果】培养基E2和E3结合热处理的分离效果较好;86.1%的代表菌株对供试的细菌、病原真菌表现出了不同程度的抗菌活性,PKS I、PKS II、NRPS基因和卤化酶基因阳性检出率分别为16.7%、72.2%、25.0%和11.1%。具有活性功能的代表菌株经16S r RNA基因测序分析,分别属于链霉菌属(Streptomyces)、小单胞菌属(Micromonospora)、红球菌属(Rhodococcus)和游动放线菌属(Actinoplanes)4个属,其中链霉菌属(Streptomyces)为优势菌属,占60%以上。【结论】胀果甘草是我国传统的药用植物,其植株内部蕴藏着丰富的放线菌资源,并在次生代谢产物合成方面拥有巨大潜力,具有进一步开发的价值。     

Diversity of culturable actinobacteria isolated from marine sponge <Emphasis Type="Italic">Haliclona</Emphasis> sp.

This study describes actinobacteria isolated from the marine sponge Haliclona sp. collected in shallow water of the South China Sea. A total of 54 actinobacteria were isolated using media selective for actinobacteria. Species diversity and natural product diversity of isolates from marine sponge Haliclona sp. were analysed. Twenty-four isolates were selected on the basis of their morphology on different media and assigned to the phylum Actinobacteria by a combination of 16S rRNA gene based restriction enzymes digestion and 16S rRNA gene sequence analysis. The 16S rRNA genes of 24 isolates were digested by restriction enzymes TaqI and MspI and assigned to different groups according to their restriction enzyme pattern. The phylogenetic analysis based on 16S rRNA gene sequencing showed that the isolates belonged to the genera Streptomyces, Nocardiopsis, Micromonospora and Verrucosispora; one other isolate was recovered that does not belong to known genera based on its unique 16S rRNA gene sequence. To our knowledge, this is the first report of a bacterium classified as Verrucosispora sp. that has been isolated from a marine sponge. The majority of the strains tested belong to the genus Streptomyces and three isolates may be new species. All of the 24 isolates were screened for genes encoding polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). PKS and NRPS sequences were detected in more than half of the isolates and the different "PKS-I-PKS-II-NRPS" combinations in different isolates belonging to the same species are indicators of their potential natural product diversity and divergent genetic evolution.