新疆棉花黄萎病菌拮抗细菌的分离、筛选与鉴定

【背景】棉花黄萎病是由大丽轮枝菌(Verticillium dahliae Kleb.)引起的一种世界性病害,近年来对该病害的生物防治因具有环境友好和人畜安全的特性而倍受关注。【目的】筛选棉花黄萎病高效拮抗细菌并对其进行鉴定,为棉花黄萎病的生物防治扩充菌种资源。【方法】采用稀释涂布平板法分离细菌,并进行拮抗细菌的初筛和复筛,通过形态特征、生理生化特征和16S rRNA基因序列分析对筛选到的细菌进行鉴定,确定其分类地位。【结果】初筛分离到535株对病原菌具有拮抗作用的细菌,并选取了108株拮抗细菌进行复筛,最终筛选到了4株优势拮抗细菌。通过形态观察、生理生化特征和16SrRNA基因序列分析,将菌株BHZ-29、SHT-15、SHZ-24和SMT-24分别鉴定为贝莱斯芽孢杆菌(Bacillusvelezensis)、枯草芽孢杆菌斯皮兹仁亚种(Bacillus subtilis subsp. spizizenii)、萎缩芽孢杆菌(Bacillus atrophaeus)和香草芽孢杆菌(Bacillus vanillea)。【结论】获得了4株高效拮抗细菌,并且首次报道了香草芽孢杆菌对棉花黄萎病菌具有抑制作用。     

高产铁载体棉田土壤细菌SS05的筛选与鉴定

【目的】研究从棉田土壤中筛选得到的高产铁载体细菌产铁载体能力、分类地位和抑菌活性。【方法】通过改良蔗糖-天冬氨酸培养基选择性筛选产铁载体细菌,通过分光光度计法测定铁载体活性,通过混菌法测定产铁载体细菌上清液对棉花枯萎病致病菌尖孢镰刀菌(Fusarium oxysporum)的抑菌效果,采用形态学、生理生化鉴定及16S rDNA序列系统发育分析对高产铁载体菌株进行鉴定。【结果】从棉田土壤中筛选到162株产铁载体细菌,30株产铁载体能力较强的细菌中21株具有较高产铁载体能力,菌株SS05的铁载体活性单位达到98.3%;在低铁条件下,SS05上清液对F.oxysporum具有显著的抑制作用;SS05与莫哈韦芽孢杆菌(Bacillus mojavensis)最为接近。【结论】SS05是高产铁载体菌株,与莫哈韦芽孢杆菌(Bacillus mojavensis)最为接近,在低铁培养条件下其上清液对F.oxysporum具有显著的抑制作用。     

黑麦草根际产铁载体细菌HMGY6B的筛选鉴定及对病原菌的拮抗作用

【目的】从石灰性土壤中分离筛选出铁载体合成能力强、抗病效果好的菌株。【方法】采用刃天青(Chrome azural S,CAS)平板检测法,定性、定量筛选产铁载体能力较强的菌株,通过菌株形态、生理生化特征、16S r RNA基因序列相似性和系统发育分析,鉴定细菌类型,然后采用平板对峙法研究菌株与病原菌的拮抗作用。【结果】从多年生黑麦草根际土壤中分离得到一株产铁载体能力很强的菌株HMGY6B,经鉴定,该菌属假单胞菌属Pseudomonas菌株,其产生的儿茶酚型铁载体对黄瓜灰霉病(Botrytis cinerea)有显著的拮抗作用,在低铁条件下(0.16、2、5、10μmol/L FeCl_3)对黄瓜灰霉病的生长抑制率高达91.2%,但在富铁条件下(50μmol/L FeCl_3)降为30.2%,100μmol/L FeCl_3抑制率仅为5.5%。【结论】菌株HMGY6B可用于今后复合型抗病生物菌肥的开发研制。     

土壤产铁载体细菌的筛选及其对铁氧化物的活化与利用

【背景】土壤中铁主要以难溶态铁氧化物的形式存在,有效性较低,产铁载体细菌对铁氧化物的活化是提高铁利用效率的有效途径。【目的】从林木土壤筛选产铁载体细菌,并观察菌株对难溶性铁氧化物的利用效应,可为土壤微生物资源开发及其在养分调控中的作用提供理论依据。【方法】通过CAS检测法从林木根系附近表层土壤中分离产铁载体细菌,借助生物培养实验分析温度和pH对微生物生长和铁载体产生的影响,通过振荡平衡实验,探究细菌产铁载体对铁氧化物的活化效应。【结果】通过CAS检测法从林木根系附近表层土壤中分离得到12株产铁载体细菌,16S rRNA基因扩增子测序初步鉴定结果显示筛选细菌均为假单胞菌属。选取铁载体产生能力和生长活性较高的两株细菌ARSB02和CNRSB01作为重点研究对象,结果显示,不同条件下CNRSB01的生物量和铁载体产量均高于菌株ARSB02,22 h时菌株ARSB02和CNRSB01的铁载体活性单位分别达到67.07%和84.60%。pH 5.0-8.0的范围内两株细菌可以保持较好的铁载体产生能力,菌株ARSB02和CNRSB01在pH7.0时铁载体产生能力最强,铁载体活性单位分别达到38.9...     

撕裂蜡孔菌(Emmia lacerata)SR5抑菌特性及生防潜力评价

【背景】撕裂蜡孔菌(Emmia lacerata)是一种在世界范围内广泛分布的白腐真菌,对植物病原真菌有较好的抑制作用,可作为生防真菌进行开发和利用。【目的】对撕裂蜡孔菌SR5的抑菌能力和胞外产铁载体能力进行测定,挖掘其生防潜力。【方法】采用平板对峙法检测SR5对9种植物病原真菌的抑菌能力,并通过不同浓度的发酵原液测定真菌胞外代谢物的抑菌效果;结合铬天青S(chrome azurol S, CAS)检测法测定真菌产铁载体能力,明确SR5抑菌特性。【结果】SR5以过度生长的方式快速竞争营养和生存空间,拮抗9种植物病原真菌,抑菌率为23.7%–62.7%,对可可毛色二孢(Lasiodiplodia theobromae)的拮抗等级为Ⅳ级,而对其余8种病原真菌的拮抗等级为Ⅲ级,其中对香港丽赤壳(Calonectria hongkongensis)和间座壳(Diaporthe sp.)抑菌效果最佳;CAS检测法表明SR5能产生分泌型铁载体,产铁载体能力中等,最高铁载体活性单位(siderophore unit, SU)为44.1%。【结论】SR5以过度生长方式快速竞争营养和生存空间,而且以分泌...     

铁对产铁载体的沼泽红假单胞菌光合色素与铁载体合成的影响

【目的】探求铁对1株能产生铁载体的不产氧光合细菌(Anoxygenic Phototrophic Bacteria,APB)光合色素和铁载体合成的影响。【方法】通用CAS法检测铁载体产生,Arnow、Csaky和Shenker法检测铁载体类型;吸收光谱法和HPLC法分析光合色素的组分和含量。【结果】Rhodopseudomonas palustris CQV97能够产生异羟肟酸型铁载体,未添加FeCl3时,铁载体含量最高,铁载体的产生与生长并非关联型。随FeCl3浓度升高,菌体生长潜伏期缩短,生长速率、最终生物量以及细菌叶绿素(Bacteriochlorophyll,BChl)a和类胡萝卜素(Carotenoid,Car)含量均提高,而检测到的游离铁载体含量降低;菌体积累的BChl a的组成和相对含量未见明显变化,但主要的Car组分由Spirilloxanthin转化为Rhodopin,菌体中积累Car组分的平均共轭体系降低,Car组成的改变与色素提取液的Car特征性光谱蓝移现象相吻合。【结论】首次报道APB能够产生铁载体,CQV97菌株能够产生异羟肟酸型铁载体。阐明了铁对CQV97生长、铁载体产生和光合色素合成的影响规律,这些研究结果为深入开展APB铁载体分离纯化以及生物功能研究奠定了基础。     

植物根际促生菌Bacillus mycoides Gnyt1铁载体分泌相关功能基因的挖掘

【目的】为挖掘优良促生菌(PGPR)菌株,分析和定位功能基因,发现其科学价值和工业化开发,为农业生产服务。【方法】以Bacillus mycoides Gnyt1菌株为材料,采用二代和三代测序技术相结合的研究体系,对菌株进行全基因组测序研究,分析菌株核基因组可能存在的功能基因和菌株分泌铁载体相关的基因。【结果】本研究基因组序列拼接后总长度为5597907 bp,GC百分含量为35.57%,该菌株中与铁载体分泌相关的基因共9条,其中2条基因主要存在于Porphyrin metabolism途径,与细胞内铁的运输代谢有关。【结论】通过全基因组测序,最终确定了与铁载体分泌相关的功能基因为GYT1和GYT2,为下一步基因功能验证奠定了基础。     

百合枯萎病拮抗细菌的筛选、鉴定及其抑菌物质研究

【目的】筛选对百合枯萎病具有抑菌活性的拮抗细菌,对其抑菌活性物质进行初步分离纯化分析。【方法】以强致病力的百合尖孢镰刀菌(Fusarium oxysporum)为靶菌,采用系列稀释法和平板对峙法初筛拮抗细菌,并通过产铁载体能力、水解酶活性、土壤定殖力等多种生防特性指标进行复筛,结合形态学特征、生理生化指标和16S rRNA基因序列比对鉴定其分类地位;利用百合尖孢镰刀菌作为靶菌进行活性追踪,结合酸沉淀、快速柱色谱、HPLC等分离纯化手段,对菌发酵液中的抑菌活性物质进行纯化分析。【结果】在64株百合根际细菌和386株海洋细菌中进行初筛,得到9株对百合镰刀菌具有较强拮抗活性的菌株,最后筛选了1株拮抗活性较强且产铁载体能力和水解酶活性、土壤定殖能力较高的菌株11B91,鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),其抑菌活性物质初步推测可能为iturin和fengycin脂肽类化合物。【结论】菌株11B91在百合枯萎病的生物防治中具有潜在的应用价值,证实海洋来源的微生物也具有防治陆地植物病原菌的潜力,为植物病害的防治拓宽了思路。     

四株碱性环境真菌铁载体活性筛选

【目的】铁载体(Siderophore)是由微生物产生的一类低分子量金属离子螯合物。在生物医药、环境修复、健康食品等领域均具有广泛应用前景。文献调查显示,针对碱性环境真核微生物铁载体研究尚无相关报道。从中筛选高活性铁载体真菌具有重要意义。【方法】采用铬天青S显色法对分离于云南省碱性湖泊-程海和个旧大屯碱性尾矿土壤的99株真菌进行筛选;利用"分光光度法"对菌株产铁载体能力和铁载体类型进行考察;采用固相萃取(SPE)手段对菌株铁载体富集效果进行考察;根据菌体形态电镜观察和ITS基因系统发育树构建,对菌株进行生物学鉴定。【结果】通过初筛和复筛,确定菌株FEDT-866、FEDT-145、FECH-998和FECH-595均为铁载体高产菌株;所产铁载体类型主要为异羟肟酸型和羧酸型。除菌株FEDT-866外,铁载体活性产物适合采用固相萃取(SPE)方法进行富集。经生物学鉴定,菌株FEDT-866和FECH-998属于曲霉属,分别与Aspergillus tubigensis和A.nomius相似性较高;菌株FECH-595和FEDT-145属于青霉属,分别与Penicillium svalbardense和P.chrysogenum相似性较高。【结论】P.chrysogenum是1种常见的产铁载体真菌。而A.tubigensis、A.nomius及P.svalbardense菌株尚无产铁载体相关报道,可作为铁载体研究良好材料。     

一株产铁载体内生细菌对尖孢镰刀菌的拮抗作用

通过改良蔗糖-天冬氨酸培养基筛选到一株产铁载体的内生细菌HS-4,测定了该菌在不同铁离子浓度下对棉花枯萎病致病菌尖孢镰刀菌(Fusarium oxysporum)的抑菌效果,并结合形态、生理生化、16S rDNA序列同源性和系统发育分析对菌株进行鉴定.结果表明:内生细菌HS-4在MSA培养基中产生荧光型铁载体,其铁载体相对含量为80%.该铁载体在低铁条件下对F.oxysporum具有抑制作用.内生细菌HS-4初步鉴定为萎缩芽孢杆菌(Ba-cillus atrophaeus).     

Ironing out siderophore biosynthesis: a review of non-ribosomal peptide synthetase (NRPS)-independent siderophore synthetases

Iron is required for microbial growth and proliferation. To survive in low-iron environments, some microorganisms secrete ferric iron chelators called siderophores. Siderophore biosynthesis occurs via two pathways: the non-ribosomal peptide synthetase (NRPS) pathway and the NRPS-independent siderophore (NIS) synthetase pathway. NIS enzymes function by adenylating a carboxylic acid substrate, typically citrate, or a derivative, followed by nucleophilic capture of an amine or alcohol and displacement of a citryl intermediate. In this review, we summarize recent advances in NIS biochemistry with a particular focus on structural biology and confirm the classification of NIS enzymes into Types A, A’, B, and C based on substrate specificity. Based on a phylogenetic analysis, we also propose a new subclass of NIS enzymes, Type C’, responsible for dimerization and macrocyclization of complex and substituted amine or amide intermediates. Finally, we describe the role of NIS enzymes in virulence of pathogenic microbes and discuss NIS inhibitors as potential anti-microbial agents.     

A suite of citrate-derived siderophores from a marine Vibrio species isolated following the Deepwater Horizon oil spill

Nearly all microbes require iron for growth. The low concentration of iron found in the ocean makes iron acquisition a particularly difficult task. In response to these low iron conditions, many bacteria produce low-molecular-weight iron-binding molecules called siderophores to aid in iron uptake. We report herein the isolation and structural characterization of a suite of amphiphilic siderophores called the ochrobactins-OH, which are produced by a Vibrio species isolated from the Gulf of Mexico after the 2010 Deepwater Horizon oil spill. The citrate-based ochrobactins-OH are derivatives of aerobactin, replacing the acetyl groups with fatty acid appendages ranging in size from C8 to C12, and are distinctly different from the ochrobactins in that the fatty acid appendages are hydroxylated rather than unsaturated. The discovery of the marine amphiphilic ochrobactin-OH suite of siderophores increases the geographic and phylogenetic diversity of siderophore-producing bacteria.     

The influence of agrobactin on the uptake of ferric iron by plants

Abstract Agrobactin, a linear catechol siderophore, enhances the uptake of ferric iron into young pea and bean plants and therefore the synthesis of chlorophyll.     

微生物嗜铁素介导的铁摄取

嗜铁素是好氧菌和兼性厌氧菌的一种产物,它是微生物在低铁条件下产生的小分子的、特异性的Fe^3＋螯合因子。大多数的好氧和兼性厌氧微生物至少合成一种嗜铁素,由嗜铁素介导的铁摄取可能是细菌最普遍的一种获取铁元素的方式。     

The use of bis(mercaptoacetato-S,O)hydroxoiron(III) complex for the determination of hydroxamates

A rapid, indirect, spectrophometric procedure for the determination of hydroxamates, based on the competition for ferric ions of the bis(mercaptoacetato-S,O)hydroxoiron(III) complex, has been developed. The assay is remarkably free of interferences by common ions, thus rendering it useful in the quantitative determination of hydroxamates in culture fluids and crude preparations.     

Involvement of the siderophore of cowpea Rhizobium in the iron nutrition of the peanut

An iron-inefficient variety of peanut plant, when grown hydroponically with the catechol siderophore of Rhizobium (peanut isolate), showed increased growth and chlorophyll content compared with plants grown with Fe alone. The siderophore, when used in concentrations less than the concentrations of Fe, was still effective in grwoth promotion, indicating that it might function as a shuttle agent, solubilizing and supplying Fe to the plant. Similar results were also obtained with desferrioxamine B.The authors are with the Department of Microbiology and Biotechnology Centre, Faculty of Science, M.S. University of Baroda, Baroda-390 002, Gujarat, India.     

Spectrophotometric ferric ion biosensor from Pseudomonas fluorescens culture

Pseudomonas fluorescens cultures produce fluorescent siderophores. By utilizing optimal conditions for maximizing siderophore production in shake flask cultures of P. fluorescens, we report successful characterization of the culture broth supernatant as a robust ferric ions biosensor. For characterizing the ferric ions biosensor, we tested the effects of pH, buffers, different ferric salts and possible interference by ferrous ions under different solution conditions. We find that the biosensor is very specific to ferric ions only with sensitivity to concentrations as low as 10 microM. Further, the response time of the biosensor is the shortest (approximately 5 min or smaller) for citrate as the accompanying anion with ferric ions. While the response time is longer than that expected of normal biosensors, it is well compensated by the simplicity and economics of the biosensor production. Extremely low standard deviations in several experimental repeats also highlight the robustness of the ferric ions biosensor. Most importantly, the biosensor is extremely easy to use due to its straightforward spectrophotometric applications. We also show the utility of the biosensor with the high resolution technique of fluorescence microscopy. Finally, we report a novel mechanistic finding that siderophores present in the culture broth supernatants have two distinct optically active sites on them, which can be monitored independently in presence or absence of ferric ions.     

In vitro studies of siderophore production by wild type and rifampicin resistant strains of fluorescent Pseudomonads

Wild type (Rif⁻) and rifampicin resistant (Rif⁺) isolates of fluorescent Pseudomonads were grown in King’s B liquid medium in the presence and absence of iron and subcultured every 48 h for 12 days. Growth rates and the proportion of non-fluorescent colonies were measured. All Rif⁺ isolates when grown in the presence of iron produced a significantly higher proportion of non-fluorescent colonies. One Rif⁺ isolate had a reduced growth rate.     

Growth promoting influence of siderophore-producing Pseudomonas strains GRP3A and PRS9 in maize (Zea mays L.) under iron limiting conditions

Maize seeds were bacterized with siderophore-producing pseudomonads with the goal to develop a system suitable for better iron uptake under iron-stressed conditions. Siderophore production was compared in fluorescent Pseudomonas spp. GRP3A, PRS9 and P. chlororaphis ATCC 9446 in standard succinate (SSM) and citrate (SCM) media. Succinate was better suited for siderophore production, however, deferration of media resulted in increased siderophore production in all the strains. Maximum siderophore level (216.23 microg/ml) was observed in strain PRS9 in deferrated SSM after 72 h of incubation. Strains GRP3A and PRS9 were used for plant growth promotion experiments. Strains GRP3A and PRS9 were also antagonistic against the phytopathogens, Colletotrichum dematium, Rhizoctonia solani and Sclerotium rolfsii. Bacterization of maize seeds with strains GRP3A and PRS9 showed significant increase in germination percentage and plant growth. Maximum shoot and root length and dry weight were observed with 10 microM Fe3+ along with bacterial inoculants suggesting application of siderophore producing plant growth promoting rhizobacterial strains in crop productivity in calcareous soil system.