拮抗放线菌B-20的种类鉴定及其16S rDNA序列分析

对分离自东北蔬菜保护地土壤的1株拮抗放线菌菌株B-20进行了形态特征、培养特征、生理生化、细胞壁组分分析及16S rDNA序列分析。基内菌丝无横隔、不断裂,气生菌丝多分枝;孢子丝波曲至螺旋形,孢子椭圆形,表面光滑。细胞壁化学组分Ⅰ型。以16s rDNA序列为基础构建了包括13株相关种属细菌在内的系统发育树。根据多相分类鉴定结果表明,放线菌B-20的上述特征与淡紫灰链霉菌(Streptomyces lavendulae)的高度一致。二者的16S rDNA序列的相似性达到了99%。因此,可将链霉菌B-20定名为淡紫灰链霉菌B-20 (S.lavendulae B-20)。     

一株拮抗链霉菌的鉴定及其多相分类特征

通过16S r RNA基因序列分析一株拮抗放线菌JXNU03的系统发育关系,通过生理生化试验分析其生理生化特征,利用菌丝生长率法和杯碟法测定其拮抗活性。结果发现,放线菌JXNU03在高氏一号培养基上培养时,基内菌丝发达,分支多且不断裂,气生菌丝发育良好,孢子丝直形或螺旋状,孢子表面带刺,细胞水解液中检测到葡萄糖,未检测出特征性糖,糖类型属于C型,细胞壁氨基酸中含有L,L-DAP,属于细胞壁Ⅰ型,16S r RNA基因序列与灭癌素链霉菌的同源性高达98%,其发酵液对革兰氏阳性细菌、革兰氏阴性细菌、酵母菌和霉菌等都具有较强的拮抗作用。因此放线菌JXNU03被鉴定为灭癌素链霉菌,记为灭癌素链霉菌JXNU03(Streptomyes gancidicus JXNU03),是一株对细菌、酵母菌和霉菌均有较强拮抗活性的链霉菌,具有潜在开发价值。     

高温链霉菌24＃的初步研究

从土壤中筛选得到一株产广谱、高活性抗真菌物质的链霉菌 2 4 # ,经测定对 2 5种植物病原真菌有显著拮抗作用 ,其次生代谢产物对病菌菌丝有断裂、扭曲、缢缩等致畸效应。 16SrDNA序列分析显示本菌株与模式菌株链霉菌DSM4 4 2 93T的 16SrDNA同源性为 98 93% ,但菌株形态特征、培养特征、细胞壁化学组分、生理生化特性等均不同于模式菌株 ,且DNA杂交率只有 2 2 5 4 % ,建议为链霉菌属的一个新种 ,命名为山东链霉菌 (Streptomycesshandon gensissp .nov .)。     

一株抗万古霉素耐药菌的抗生素产生菌AR1148的鉴定

从土壤中分离得到一株放线菌AR1148,其代谢产物对万古霉素耐药肠球菌有较明显的抑菌活性。该菌株的基内菌丝无横隔,气生菌丝丰茂,分枝较好,孢子椭圆形,表面光滑。菌丝细胞壁工型,含有L-2,6-二氨基庚二酸（LL-DAP）和甘氨酸。菌株AR1148归属于链霉菌属金色类群。根据16SrDNA序列分析,该菌株与现有种差异明显,聚类在不同的分支。定名为Streptomyces sp．AR1148。     

一株生防圆突起链霉菌的分类鉴定

菌株SCY311是从河南省凤凰山土壤样品中分离到的对多种植物病原真菌具有拮抗活性的一株放线菌。为了明确其分类地位, 在形态特征、培养特征、生理生化特征、细胞壁组分测定等传统分类学方法的基础上, 测定和分析了菌株的16S rRNA基因序列。结果表明, 菌株SCY311在高氏一号培养基上生长良好, 基内菌丝呈褐色; 气生菌丝灰色至鼠灰色, 不产生可溶性色素, 无吸水现象; 孢子链卷曲, 末端形成闭合或开放螺旋; 孢子椭圆或圆柱状, 表面形成结节状突起; 生理生化特征和在国际链霉菌计划(ISP)培养基上的培养特     

番茄灰霉菌拮抗放线菌LA-5的筛选及鉴定

利用稀释涂布法从番茄根际土壤中分离放线菌,并以番茄灰霉菌为靶标,利用对峙培养法和牛津杯法筛选拮抗放线菌,得到一株具有较强抑菌活性的放线菌LA-5.通过培养特征、生理生化特性及基于16S rDNA 序列系统进化分析,将菌株LA-5初步鉴定为链霉菌.复筛结果显示,LA-5发酵滤液对番茄灰霉菌孢子萌发及菌丝生长均有明显的抑制作用,其中100倍发酵滤液对孢子萌发抑制率和菌丝生长抑制率均在50%以上;受抑制菌落呈白色,气生菌丝萎缩稀疏,菌丝纤细、分支明显减少.离体防效试验显示,菌株LA-5发酵原液对番茄灰霉病防效可达83.4%.该菌株有望开发为防治番茄灰霉病的生防菌株.     

一株产蓝色素链霉菌18-A-5的鉴定与系统发育分析

从原始热带雨林土壤中,分离到一株产蓝色色素菌株18-A-5,对其进行了系统分类学研究。形态学特征观察表明,在高氏合成一号培养基上初产蓝绿色色素,日久为深蓝色,基内菌丝蓝色,气生菌丝灰白色,产灰色孢子,孢子丝直或柔曲,形成长孢子链,孢子圆柱形。其DNA的G＋C摩尔分数为62.4%,16S rDNA序列分析结果(GenBank登陆号为EU054353),18-A-5与生靛链霉菌Streptomyces indigoferus ATCC23924^T 、草绿色链霉菌Streptomyces herbaricolor ATCC23924^T具有极高的同源性,达100％,聚类分析表明,18-A-5与生靛链霉菌Streptomyces indigoferus、草绿色链霉菌Streptomyces herbaricolor两株菌聚类在一起,分支置信度为74%。结合生理生化特性、细胞壁化学组成分析、脂肪酸分析等将菌株18-A-5定名为草绿色链霉菌Streptomyces herbaricolor。并对该蓝绿色可溶性色素性质进行了耐酸碱性、热稳定性、抗菌谱等初步分析。     

玉米大斑病生防放线菌的筛选鉴定及发酵条件优化

【目的】从土壤中筛选对玉米大斑病菌具有较强拮抗作用的放线菌菌株。【方法】采用稀释涂布法分离;采用平板对峙法、牛津杯法、抑制菌丝生长速率法、抑制孢子萌发法进行拮抗菌的筛选;根据菌株BZ45的形态与培养特征、生理生化特性、16SrDNA序列分析对其进行鉴定。通过单因素试验和正交设计试验优化培养基组分及发酵条件。【结果】通过分离筛选得到一株具有强抑制作用的放线菌菌株BZ45,它对常见的8种病原真菌均有拮抗作用,菌株BZ45的发酵滤液对玉米大斑病菌(Setosphaeria turcica)CC9的菌丝生长和孢子萌发均有较强的抑制作用。菌株BZ45与链霉菌中的壮观链霉菌(Streptomyces spectabilis)的亲缘关系较近,且形态与培养特征、生理生化特性与壮观链霉菌的基本相符。研究表明其最佳发酵配方和培养条件为:果糖1.5%、蛋白胨3.0%、KH2PO40.1%、NaCl 0.04%、CaCO30.1%,起始pH为7.2,装瓶量50 mL/250mL,28℃,200 r/min,种子液接种量为10%,摇瓶培养4 d。【结论】菌株BZ45鉴定为壮观链霉菌(Streptomyces spectabilis),菌株BZ45对玉米大斑病菌(Setosphaeria turcica)CC9显示出较强的拮抗作用。     

一株拮抗番茄叶霉病菌的放线菌筛选、鉴定及发酵条件研究

菌株xjy是一株从新疆棉田土壤中筛选出的对番茄叶霉病菌(Fulviafulva)有较强拮抗作用的放线菌,其对23种常见植物病原真菌和6种细菌有较好的抑制效果,抗菌谱广。其形态特征、培养特性、生理生化特性、细胞壁组分与放线菌中的淡紫灰链霉菌(Streptomyces lavendulae)相同,16SrDNA序列同源性达99.6%。研究发现该菌的摇瓶发酵配方和培养条件为:大豆粉2%、葡萄糖2%、NaCl0.8%、CaCO30.2%、(NH4)2SO40.32%,起始pH7.0、28℃、180r/mim、摇瓶培养6d,这些结果为该菌株今后的应用、抗生素的分离提纯和产业化提供了实验依据。     

苹果树腐烂病菌拮抗放线菌的筛选、鉴定及防效

以苹果树腐烂病菌为靶标菌,通过对峙法和生长速率法对分离自苹果树根际土壤的放线菌进行筛选,对筛选出的拮抗菌株通过形态学和分子生物学特征进行鉴定,并测定了拮抗菌ZZ-9发酵滤液对苹果树腐烂病菌孢子萌发和菌丝生长的影响及离体枝条防效.结果表明: 经对峙初筛,15株放线菌对苹果树腐烂病菌具有抑菌作用,占所分离株数的18.8%,其中抑制率>50%的有8株.复筛结果表明,ZZ-9对腐烂病菌抑制率最高,达96.4%,显著高于其他菌株;通过培养特征、生理生化特性及16S rDNA序列分析将菌株ZZ-9初步鉴定为娄彻氏链霉菌,其在GenBank上的序列登录号为KT986228;不同稀释倍数的ZZ-9发酵滤液对腐烂病孢子萌发及菌丝生长均有明显的抑制作用,其中50倍发酵滤液对孢子萌发抑制率和菌丝生长抑制率均达80%以上,且受抑制菌丝颜色加深,分支增多,末端膨大、畸形,出现原生质浓缩与释放现象;离体枝条防效试验表明,菌株ZZ-9发酵原液对苹果树腐烂病防效可达75%以上,表明该菌株可作为防治苹果树腐烂病的生防菌株.     

抗肿瘤海绵放线菌的分离及菌株HA01184的鉴定

目的：对采自海南、湛江等海域的海绵样品进行放线菌选择性分离,采用其发酵液进行抗肿瘤活性筛选,并对活性较好的菌株进行鉴定。方法：用含50μg／mL重铬酸钾为抑制剂的海水高氏一号合成培养基分离培养海绵放线菌;以MTT法进行菌株的抗肿瘤活性筛选;通过培养特征、形态特征、生理生化特征、16S rDNA序列测定及系统发育分析,对菌株HA01184进行鉴定。结果与结论：海水高氏一号合成培养基用于海绵放线菌分离培养具有很好的选择性,从海绵样品中共分离得到放线菌165株,细胞毒活性达80％以上的阳性菌株有10株,其中菌株HA01184的发酵液细胞毒活性为90％。结合形态观察、生理生化特征和16S rDNA序列比对分析,将HA01184归于链霉菌属,可能是来自海洋环境的一个潜在新种。     

Neoasaia chiangmaiensis gen. nov., sp. nov., a novel osmotolerant acetic acid bacterium in the alpha-Proteobacteria

An acetic acid bacterium, designated as isolate AC28(T), was isolated from a flower of red ginger (khing daeng in Thai; Alpinia purpurata) collected in Chiang Mai, Thailand, at pH 3.5 by use of a glucose/ethanol/acetic acid (0.3%, w/v) medium. A phylogenetic tree based on 16S rRNA gene sequences for 1,376 bases showed that isolate AC28(T) constituted a cluster along with the type strain of Kozakia baliensis. However, the isolate formed an independent cluster in a phylogenetic tree based on 16S-23S rDNA internal transcribed spacer (ITS) region sequences for 586 bases. Pair-wise sequence similarities of the isolate in 16S rRNA gene sequences for 1,457 bases were 93.0-88.3% to the type strains of Asaia, Kozakia, Swaminathania, Acetobacter, Gluconobacter, Gluconacetobacter, Acidomonas, and Saccharibacter species. Restriction analysis of 16S-23S rDNA ITS regions discriminated isolate AC28(T) from the type strains of Asaia and Kozakia species. Cells were non-motile. Colonies were pink, shiny, and smooth. The isolate produced acetic acid from ethanol. Oxidation of acetate and lactate was negative. The isolate grew on glutamate agar and mannitol agar. Growth was positive on 30% D-glucose (w/v) and in the presence of 0.35% acetic acid (w/v), but not in the presence of 1.0% KNO(3) (w/v). Ammoniac nitrogen was hardly assimilated on a glucose medium or a mannitol medium. Production of dihydroxyacetone from glycerol was weakly positive. The isolate did not produce a levan-like polysaccharide on a sucrose medium. Major isoprenoid quinone was Q-10. DNA base composition was 63.1 mol% G+C. On the basis of the results obtained, Neoasaia gen. nov. was proposed with Neoasaia chiangmaiensis sp. nov. The type strain was isolate AC28(T) (=BCC 15763(T) =NBRC 101099(T)).     

Cloning and characterization of a gene involved in aerial mycelium formation in Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is essentially required for aerial mycelium formation and streptomycin production in Streptomyces griseus. A DNA fragment which induced aerial mycelium formation and sporulation in an A-factor-deficient mutant strain, S. griseus HH1, was cloned from this strain on a high-copy-number plasmid. Subcloning and nucleotide sequencing revealed that one open reading frame with 218 amino acids, named AmfC, served as a multicopy suppressor of the aerial mycelium-defective phenotype of the A-factor-deficient strain. The amfC gene did not restore A-factor or streptomycin production, indicating that amfC is involved in aerial mycelium formation independently of secondary metabolic function. Disruption of the chromosomal amfC gene in the wild-type S. griseus strain caused a severe reduction in the abundance of spores but no effect on the shape or size of the spores. The infrequent sporulation of the amfC disruptant was reversed by introduction of amfC on a plasmid. The amfC-defective phenotype was also restored by the orf1590 gene but not by the amfR-amfA-amfB gene cluster. Nucleotide sequences homologous to the amfC gene were distributed in all of 12 Streptomyces species tested, including Streptomyces coelicolor A3(2). The amfC homolog of S. coelicolor A3(2) was cloned and its nucleotide sequence was determined. The AmfC products of S. griseus and S. coelicolor A3(2) showed a 60% identity in their amino acid sequences. Introduction of the amfC gene of S. coelicolor A3(2) into strain HH1 induced aerial mycelium formation and sporulation, which suggests that both play the same functional role in morphogenesis in the strains.     

斑点叉尾鮰一株致病菌的分离鉴定及系统发育分析

从发生急性流行性传染病的斑点叉尾肝、肾分离到一高致病性的菌株(CCF00024),经人工感染实验证实其为该病的病原菌。对该菌的形态、生理生化及16S rDNA序列分析结果表明,其为非发酵型,严格需氧,革兰氏阴性杆菌,极生多鞭毛,对除麦芽糖和甘露糖以外的多种糖类不能利用产酸,氧化酶阴性,DNA酶、蛋白酶、脲酶、赖氨酸脱羧酶阳性,MR阴性。在以该菌16S rDNA序列(GenBank登录号AY970826)和GenBank及RDP数据库内同源性较高的细菌16S rDNA序列构建的系统发育树中,分离菌CCF00024与嗜麦芽寡养单胞菌(Stenotrophomonasmaltophilia)聚在一簇,特别是与S.maltophiliaM5-1的同源性最高,其序列相似性达99.6%,结合形态和生理生化特点将其鉴定为嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia)。     

ε-聚赖氨酸产生菌新菌株的筛选和产物结构鉴定

通过对Nishikawa的方法进行改进,在广东各地土样中筛选到一株产量为0.846 g/L的新ε-聚赖氨酸(ε-PL)产生菌株,命名为Str-8。对Str-8菌株进行形态、生理生化和16S rDNA分析,初步确定为不吸水链霉菌Streptomyces ahygroscopic。纯化的发酵产物通过水解、质谱、紫外光谱等性质确定为ε-PL。     

Phylogenetic characterization and in situ detection of a Cytophaga-Flexibacter-Bacteroides phylogroup bacterium in Tuber borchii vittad. Ectomycorrhizal mycelium

Mycorrhizal ascomycetous fungi are obligate ectosymbionts that colonize the roots of gymnosperms and angiosperms. In this paper we describe a straightforward approach in which a combination of morphological and molecular methods was used to survey the presence of potentially endo- and epiphytic bacteria associated with the ascomycetous ectomycorrhizal fungus Tuber borchii Vittad. Universal eubacterial primers specific for the 5' and 3' ends of the 16S rRNA gene (16S rDNA) were used for PCR amplification, direct sequencing, and phylogenetic analyses. The 16S rDNA was amplified directly from four pure cultures of T. borchii Vittad. mycelium. A nearly full-length sequence of the gene coding for the prokaryotic small-subunit rRNA was obtained from each T. borchii mycelium studied. The 16S rDNA sequences were almost identical (98 to 99% similarity), and phylogenetic analysis placed them in a single unique rRNA branch belonging to the Cytophaga-Flexibacter-Bacteroides (CFB) phylogroup which had not been described previously. In situ detection of the CFB bacterium in the hyphal tissue of the fungus T. borchii was carried out by using 16S rRNA-targeted oligonucleotide probes for the eubacterial domain and the Cytophaga-Flexibacter phylum, as well as a probe specifically designed for the detection of this mycelium-associated bacterium. Fluorescent in situ hybridization showed that all three of the probes used bound to the mycelium tissue. This study provides the first direct visual evidence of a not-yet-cultured CFB bacterium associated with a mycorrhizal fungus of the genus Tuber.     

1株土壤放线菌的分类鉴定及其抗菌活性的研究

对从土壤微生物中筛选到的放线菌菌株1356进行分类学和抗菌活性的研究。采用多相分类法,对菌株的形态特征、培养特征、生理生化特性及16 SrRNA基因序列进行了研究。结果表明:该菌株的形态特征、培养特征、生理生化特性为链霉菌属的特征;16S rDNA序列分析及系统进化树分析表明其序列与灰色产色链霉菌的同源性最高;该菌株的发酵产物对番茄叶霉、白色念珠菌、小麦根腐菌等17种真菌均有不同程度的抑制作用。放线菌1356菌株具有广谱抗真菌活性而对细菌无作用;初步确定其为链霉菌属灰色产色链霉菌的一个亚种。     

Intrageneric structure of the genus Gluconobacter analyzed by the 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences

Forty-nine strains belonging to the genus Gluconobacter were re-examined with respect to their species identification based on the sequences of the 16S rDNA and 16S-23S rDNA internal transcribed spacer regions (ITS). A phylogenetic tree constructed from the 16S rDNA sequences indicated the presence of five clusters corresponding, respectively, to the major five species of the genus Gluconobacter, namely G. albidus, G. cerinus, G. frateurii, G. oxydans (type species), and G. thailandicus. The type strain of G. asaii, NBRC 3276T (T=type strain) was included in the G. cerinus cluster, which is consistent with the report that G. asaii is a junior subjective synonym of G. cerinus. Existence of the G. albidus, G. cerinus, G. frateurii, G. oxydans, and G. thailandicus clusters was also recognized by the ITS sequence analysis. Both sequence analyses revealed that the G. cerinus and G. frateurii clusters were heterogeneous. The G. cerinus cluster comprised three strains of G. cerinus and one strain of G. frateurii, while the G. frateurii cluster included ten strains of G. frateurii, three of G. cerinus, and eleven of G. oxydans. These results suggest that phenotypic differences among Gluconobacter species are ambiguous and the species definition must be re-evaluated. The 16S rDNA and ITS sequences determined in this study are valuable for the identification and phylogenetic analysis of Gluconobacter species.