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。     

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。     

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.     

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。     

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。     

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。     

聚酮类化合物生物合成途径基因阳性菌株生物多样性研究

从中国云南省采集土样,采用GPY培养基、淀粉_酪素琼脂培养基和甘油天门冬酰胺琼脂培养基分离得到了876株细菌放线菌菌株。经聚酮类化合物基因筛选得到75株Ⅰ型和Ⅱ型聚酮类化合物生物合成基因双阳性的菌株,经抗菌活性、形态、生理等结果分析比较,选取其中的10株进行了16S rDNA序列分析。在物种多样性方面,分离到的菌分布至少有7个科,8个属。其中有链霉菌科的链霉菌属,分枝杆菌科的分枝杆菌属,链孢囊菌科的链孢囊菌属和野野村菌属,诺卡氏菌科的诺卡氏菌属,微球菌亚目的一新属、产碱杆菌科的无色杆菌属和β_亚纲中与紫色杆菌属紧邻的一革兰氏阴性菌新属。综合其表型、基因型特征,初步确定其中8株为新物种。研究表明利用新的思路和程序从自然环境分离、鉴定未知菌是微生物资源开发利用的关键。     

罗布泊盐湖可培养放线菌多样性及PKS Ⅱ功能基因筛选

目的:挖掘新疆罗布泊盐湖可培养放线菌的资源和探索该环境放线菌产生Ⅱ型聚酮合成酶(PKS)基因的潜能。方法:从新疆罗布泊盐湖采集到10份样品,采用不同盐浓度的9种选择性培养基分离放线菌,并通过PCR技术扩增其16S r DNA序列、聚酮合成酶(PKS)基因分子筛选,测序后进行系统发育分析和BLAST比较研究。结果:试验结果共得42株放线菌,包括6个属,链霉菌属(Streptomyces)和拟诺卡氏菌属(Nocardiopsis)的类群最多,糖霉菌属(Glycomyces)、放线多孢菌属(Actinopolyspora)、微球菌(Micrococcus)和栖白蚁菌属(Isoptericola)的菌株相对较少。研究获得新的放线菌分类单元,其16S r DNA基因序列与已知菌株的序列相似性低于97%。42株放线菌中共有29株产生PKSⅡ基因,主要是链霉菌属和拟诺卡氏菌属。结论:研究结果表明罗布泊盐湖放线菌具有丰富的资源多样性和较强合成抗生素的潜在能力。     

1株具有抗根结线虫活性的红树林土壤放线菌的分离与鉴定

采用平板稀释法,从海南省东寨港红树林根际土壤中分离得到110株放线菌;利用24孔板液体筛选模型,筛选出具有抗线虫活性的放线菌菌株HA10401。该菌株发酵液在稀释20倍和40倍时,抗根结线虫校正死亡率分别为58.2%和52.6%。通过16S rDNA基因序列分析,菌株HA10401与链霉菌属菌株Strept omyces variabilis同源性最高(99.8%),在系统发育树上聚在同一分支内,亲缘关系最近。二者在形态特征、培养特征以及生理生化特征方面也基本一致,因此,鉴定菌株HA10401为变异链霉菌(Streptomyc es variabilis)。本研究首次报道了其抗根结线虫活性,值得进行更为深入的研究。     

诺卡氏菌形放线菌与有关分类单位的枝菌酸和磷酸类脂分析

通过对14株胞壁为I型和IV型的诺卡氏菌形放线菌与有关分类单盘的枝菌酸和磷酸类脂的分析,发现胞壁IV型原名为空腔诺卡氏菌N. coeliaca KCC A-0007菌株不含枝菌酸,磷酸类脂不属于任何已知类型(除含PE外,还含有PC),萘醌类型为MK-8(H4)。指出这株菌应从诺卡氏菌属转入无枝菌酸菌属,改定为空腔无枝菌酸菌Amycolata coeliaca[(Gray)Waksrnanet Henrici] comb. Nov. Liu ＆ Ruan,1986。另外还提出,区分类诺卡氏菌与链霉菌时,磷酸类脂类型比形态断裂特征更为重要。为此,将一株原定名为阿勒泰类诺卡氏菌Nocardioides al-taiensis (Wang,1982),依其磷酸类脂分析结果,与链霉菌(Ⅱ型)相同,而与类诺卡氏菌(I型)不同,改定为阿勒泰链霉菌Streptomyces altaiensis (Wang) comb．Nov．Liu ＆ Ruan, 1986。     

The nematicidal potential of local Bacillus species against the root-knot nematode infecting greenhouse tomatoes

Bacillus spp. were isolated from Iranian tomato fields and evaluated for their efficacy against root-knot nematodes, Meloidogyne javanica. The 52 spore-forming bacterium isolates were obtained from tomato rhizosphere following heat treatment. Eight isolates were chosen based on their potency in prevention of M. javanica egg hatch and juvenile mortality and production of proteolytic enzymes in Petri plates. Their ability to form biofilms were also determined in pot experiments. According to phenotypic traits and 16s rDNA sequencing, all selected isolates belonged to Bacillus spp. including B. cereus and B. pumilus. Treatment with bacterial culture filtrates in vitro caused juvenile mortality of 72 to 99% after 48 h. After four days, the percentage of egg hatch ranged from 1.6 to 59% depending on the isolate. Bacillus pumilus (ToIr-MA) and Bacillus sp. (ToIr-10) were found to have significant ability to produce extracellular proteases and to form maximum biofilm, considerably reducing the number of egg masses and root gall index (P = 0.05) in comparison to untreated plants. Application of ToIr-MA and ToIr-10 enhanced the fresh and dry weights of shoot and root systems. There was significant enhancement in dry root weight (45 and 50%) and shoot weight (67 and 75%). Results suggest that these two Bacillus spp. have potential as biocontrol agents against root-knot disease in tomato production.     

Actinomycetes and fungi isolated from plant-parasitic nematode infested soils: screening of the effective biocontrol potential,indole-3-acetic acid and siderophore production

Root-knot nematodes are serious pathogens that severe damage to major crops. They damage plant root system that caused significant yield losses. Moreover, the predisposition of nematode-infected plants is secondary infection from fungal plant pathogen that additional adverse effects on plant growth. Our target is to find the antagonist for control nematode, and secondary infection agents and stimulate plant growth. Twenty-three plant-parasitic nematode infested soils were taken from some provinces in the northern and center of Thailand and actinomycetes and fungi were isolated. Eighty-three isolates belong to actinomycete and 67 isolates were fungi. The predominant actinomycete taxa was Streptomyces (97.6%). The predominant fungal taxa were Penicillium (37.3%) and Fusarium (32.8%). All actinomycete and fungal isolates were subjected for primary screening in vitro for their effects on egg hatching and juvenile mortality of Meloidogyne incognita. Secondary screening was evaluated for antagonist effect on plant pathogenic fungi collected from nematode-infected plant, plant growth hormone (indole-3-acetic acid; IAA) and siderophore production. From primary screening, 7 actinomycete and 10 fungal isolates reduced egg hatching and kill juveniles of M. incognita after 7 days incubation. In secondary screening, 10 nematophagous microbes produced IAA and 9 isolates produced hydroxamate siderophore. Streptomyces sp. CMU-MH021 was selected as a potential biocontrol agent. It reduced egg hatching rate to 33.1% and increased juvenile mortality rate to 82% as contrasted to the control of 79.6 and 3.6%, respectively. This strain had high activity to against tested fungi and high ability on IAA (28.5 μg ml⁻¹) and siderophore (26.0 μg ml⁻¹) production.     

Isolation of Actinomycetes synthesizing proteases with thrombolytic activity

Proteases with the thrombolytic activity were studied in 212 strains of actinomycetes isolated from different soils of the Soviet Union. The cultures belonged to the genera Micromonospora, Nocardia and Streptomyces. Proteases were synthesized by 41% of the studied actinomycetes and some of their strains completely dissolved in vitro artificially obtained blood thrombi within 120-240 min. In the Streptomyces genus, more active strains were found in the groups Flavus, Fradia and Globisporus. The groups Olivaceus, Violaceus and Viridis had less active strains.     

Fungi and actinomycetes associated with Meloidogyne spp. eggs and females in China and their biocontrol potential

A survey was conducted to determine the microflora on eggs and females of Meloidogyne spp. collected from plant roots and infested soil in China. A total of 455 fungal isolates belonging to 24 genera and 52 isolates of actinomycetes were obtained from 28 samples from greenhouses and fields in Hainan, Yunnan, Fujian, Hebei, Shandong, and Beijing. The predominant fungal species were Paecilomyces lilacinus (49.3% of the isolates), Fusarium spp. (7.9%), Pochonia chlamydosporia (6.9%), Penicillium spp. (5.7%), Aspergillus spp. (3.2%), and Acremonium spp. (2.8%). Actinomycetes were frequently encountered (10.3%) as well. A total of 350 isolates of nematophagous fungi and actinomycetes were evaluated for their parasitism of eggs and effects on egg hatch and juvenile mortality in vitro. Pathogenicity varied among isolates, and 29.1% of isolates parasitized over 90% eggs 4 days after inoculation. Results also show that seven isolates of fungi and actinomycetes reduced egg hatch rates to less than 10% contrasted to the control of 65.8%, and three isolates killed all hatched juveniles after 7 days. Seventeen fungal isolates and four actinomycete isolates with high pathogenicity in vitro were selected to test biocontrol efficacy in the greenhouse. They reduced tomato root gall index by 13.4-58.9% compared to the no treatment control.     

A plant growth promoting rhizobacterium, Paenibacillus polymyxa strain GBR-1, suppresses root-knot nematode

Exposure of root-knot nematode, Meloidogyne incognita to various concentrations (5-100%) of culture filtrate of Paenibacillus polymyxa GBR-1 under in vitro conditions significantly reduced egg hatch and caused substantial mortality of its juveniles. The increase in the exposure durations of juveniles to culture filtrate and its concentrations increased the mortality rate. Similarly, higher concentrations increased its inhibitory effect on egg hatch. In higher concentrations (25-100%) egg hatch was inhibited by 84-91% after 2 days of exposures as compared to control in sterile distilled water. Application of various concentrations of culture filtrate extract or bacterial suspension of P. polymyxa GBR-1 into potting soil infested with 2000 J2 of M. incognita, reduced the root galling and nematode populations and increased tomato plant growth and root-mass production compared with untreated control (P< or = 0.05). The beneficial effect of P. polymyxa GBR-1 into potted soil increased exponentially with the increase in dose concentrations. Root gall index was reduced from 4.8 to 1.4 and 1.8 when potting soil was treated with 10% concentrations of culture filtrate extract and bacterial suspension, respectively, compared with untreated control. Application of bacterial suspension of P. polymyxa GBR-1 into potted soil at 3 day pre-inoculation of nematode was the most effective followed by simultaneously and at 2 days post-inoculation; as root galling was reduced by 62.5%, 58.3% and 50.0%, respectively, compared with untreated control.     

Isolation of Microorganisms Able To Metabolize Purified Natural Rubber

Bacteria able to grow on purified natural rubber in the absence of other organic carbon sources were isolated from soil. Ten isolates reduced the weight of vulcanized rubber from latex gloves by >10% in 6 weeks. Scanning electron microscopy clearly revealed the ability of the microorganisms to colonize, penetrate, and dramatically alter the physical structure of the rubber. The rubber-metabolizing bacteria were identified on the basis of fatty acid profiles and cell wall characteristics. Seven isolates were strains of Streptomyces, two were strains of Amycolatopsis, and one was a strain of Nocardia.     

Plant growth-promoting rhizobacteria, Paenibacillus polymyxa and Paenibacillus lentimorbus suppress disease complex caused by root-knot nematode and fusarium wilt fungus

Aim:  To screen and evaluate the biocontrol potential of Paenibacillus strains against disease complex caused by Meloidogyne incognita and Fusarium oxysporum f. sp. lycopersici interactions.
Methods and Results:  Paenibacillus strains were collected from rotten ginseng roots. The strains were tested under in vitro and pots for their inhibitory activities, and biocontrol potential against disease complex caused by M. incognita and F. oxysporum f. sp. lycopersici on tomato. In in vitro experiments, among 40 tested strains of Paenibacillus spp., 11 strains showed antifungal and nematicidal activities against F. oxysporum f. sp. lycopersici and M. incognita, respectively. Paenibacillus polymyxa GBR-462; GBR-508 and P. lentimorbus GBR-158 showed the strongest antifungal and nematicidal activities. These three strains used in pot experiment reduced the symptom development of the disease complex (wilting and plant death), and increased plant growth. The control effects were estimated to be 90–98%, and also reduced root gall formation by 64–88% compared to the untreated control.
Conclusion:  The protective properties of selected Paenibacillus strains make them as potential tool to reduce deleterious impact of disease complex plants.
Significance and Impact of the Study:  The study highlights biocontrol potential of Paenibacillus strains in management of disease complex caused by nematode-fungus interaction.     

四川阿坝不同生境绿绒蒿内生放线菌多样性

【背景】目前国内外对绿绒蒿内生放线菌的研究较为鲜见,利用可培养和免培养的方法可以丰富绿绒蒿内生放线菌资源。【目的】药用植物绿绒蒿生长于高海拔地区,而四川阿坝州生态环境适合绿绒蒿的生长,本研究旨在探究绿绒蒿内生放线菌的多样性及群落结构。【方法】从阿坝州6个不同地理位置采集的药用植物绿绒蒿,经严格的表面消毒后,用可培养和PCR-DGGE技术相结合的方法对其内生放线菌进行分析。【结果】可培养方法分析表明不同器官放线菌数量为根叶茎花;16S r DNA限制性内切酶片段长度多态性(RFLP)分析将供试菌株分为7个遗传群,系统发育分析表明,绿绒蒿内生放线菌可分为3簇遗传群落,链霉菌属(Streptomyces)为优势菌群,占总数的50%,还包括北里孢菌属(Kitasatospora)和厄氏菌属(Oerskovia),其中茎分离的放线菌多样性最好。不同地理位置的绿绒蒿经过PCR-DGGE技术分析表明,采样点松潘尕力台的根、茎、叶多样性指数最好,红原安曲乡的花多样性指数最好,而同一地理位置绿绒蒿内部组织之间总体来看茎和花的多样性指数最好,通过测序条带系统发育发现绿绒蒿内生放线菌可分为8簇遗传群落,红球菌属(Rhodococcus)为优势菌群,占总数的70%,还包括棒杆菌属(Corynebacterium)、诺卡氏菌属(Nocardia)、微球菌属(Micrococcus)、栖霉菌属(Mycetocola)、微杆菌属(Microbacterium)、假诺卡氏菌属(Pseudonocardia)和链霉菌属(Streptomyces)。【结论】绿绒蒿根、茎、叶、花组织中具有丰富的多样性,能够为以后活性物质和药用功效的研究奠定基础。