两株具有芘降解功能的植物内生细菌的分离筛选及其特性

从植物体内筛选具有多环芳烃(PAHs)降解功能的内生细菌并定殖于植物体,有望有效地去除植物体内PAHs,从而减低植物污染风险。采用富集培养法,从长期受PAHs污染的植物体内分离筛选出2株能以芘为唯一碳源和能源生长的内生细菌BJ03和BJ05,经形态观察、生理生化特性及16S rDNA序列同源性分析,将2株菌分别鉴定为不动杆菌属(Acinetobacter sp.)和库克氏菌属(Kocuria sp.)。并研究了2株内生细菌对芘的降解能力及环境条件对其降解芘的影响。结果表明,菌株BJ03和BJ05在以浓度为50 mg/L的芘为唯一碳源生长时,于30℃、150 r/min摇床培养15 d后,对芘的降解率分别为65.0%和53.3%。2株菌在pH值(6.0—9.0)、温度(25—40℃)和盐浓度(NaCl含量为0—15 g/L)条件下生长良好,且皆为好氧生长,通气量越大,菌株生长越旺盛,对芘的降解能力越强。添加C、N源可有效促进菌株BJ03和BJ05的生长,加速其对芘的降解速率。当外加C源为蔗糖、N源为酵母膏时,2株菌在30℃摇床培养4 d后,对芘的降解率分别高达71.1%和55.3%。2株菌的细胞表面疏水率最大分别为93.7%和43.9%,对四环素和利福平敏感,而对其它多种抗生素具有较强的抗性。     

Inoculating wheat (Triticum aestivum L.) with the endophytic bacterium Serratia sp. PW7 to reduce pyrene contamination

This research was conducted to find an optimal inoculation way for a pyrene-degrading endophytic Serratia sp. PW7 to colonize wheat for reducing pyrene contamination. Three inoculation ways, which are soaking seeds in inocula (TS), dipping roots of seedlings in inocula (TR), and spraying inocula on leaves of seedlings (TL), were used in this study. Inoculated seedlings and noninoculated seedlings (CK) were, respectively, cultivated in Hoagland solutions supplemented with pyrene in a growth chamber. The results showed that strain PW7 successfully colonized the inoculated seedlings in high numbers, and significantly promoted the growth of seedlings (TS and TR). More importantly, strain PW7 reduced pyrene levels in the seedlings and the Hoagland solutions. Compared to the noninoculated seedlings, the pyrene contents of the inoculated seedlings were decreased by 35.7-86.3% in the shoots and by 26.8–60.1% in the roots after 8-day cultivation. By comparing the efficiencies of decreasing pyrene residues, it can be concluded that TR was an optimal inoculation way for endophytic strains to colonize the inoculated plants and to reduce the pyrene contamination. Our findings provide an optimized inoculation way to reduce organic contamination in crops by inoculating plants with functional endophytic bacteria.     

Analysis of the abilities of endophytic bacteria associated with banana tree roots to promote plant growth

A total of 40 endophytic bacterial isolates obtained from banana tree roots were characterized for their biotechnological potential for promoting banana tree growth. All isolates had at least one positive feature. Twenty isolates were likely diazotrophs and formed pellicles in nitrogen-free culture medium, and 67% of these isolates belonged to the genus Bacillus sp. The isolates EB-04, EB-169, EB-64, and EB-144 had N fixation abilities as measured by the Kjeldahl method and by an acetylene reduction activity assay. Among the 40 isolates, 37.5% were capable of solubilizing inorganic phosphate and the isolates EB-47 and EB-64 showed the highest solubilization capacity. The isolate EB-53 (Lysinibacillus sp.) had a high solubilization index, whereas 73% of the isolates had low solubilization indices. The synthesis of indole-3-acetic acid (IAA) in the presence of L-tryptophan was detected in 40% of the isolates. The isolate EB-40 (Bacillus sp.) produced the highest amount of IAA (47.88 μg/ml) in medium supplemented with L-tryptophan and was able to synthesize IAA in the absence of L-tryptophan. The isolates EB-126 (Bacillus subtilis) and EB-47 (Bacillus sp.) were able to simultaneously fix nitrogen, solubilize phosphate and produce IAA in vitro. The results of this study demonstrated that the isolates analyzed here had diverse abilities and all have the potential to be used as growth-promoting microbial inoculants for banana trees.     

In-situ enumeration and probing of pyrene-degrading soil bacteria

Inferences about which microorganisms degrade polycyclic aromatic hydrocarbons in contaminated soils have largely been obtained using culture-based techniques, despite the low percentage of microorganisms in soil that are believed to be culturable. We used a substrate-responsive direct viable count method to identify and quantify potential polycyclic aromatic hydrocarbon-degrading bacteria in a soil containing petroleum wastes. Bacteria were extracted and their response to substrates determined in the presence of DNA gyrase inhibitors, which cause viable and active cells to elongate. When yeast extract, a widely used carbon source, was added as a growth substrate, together with nalidixic acid, piromidic acid and ciprofloxacin, a significant increase in elongated cells to 47%, 37% and 22%, respectively, was observed within 24 h. With pyrene as the main substrate, 10 mg L(-1) of nalidixic acid or piromidic acid caused 18-22% and 8-12%, respectively, of the cells to elongate within 24 h; whereas the effect of 0.5 mg L(-1) ciprofloxacin was not significant until 53 h later. Enlarged cells were identified and enumerated by fluorescent in situ hybridization, using Alpha-, Beta- and Gammaproteobacteria, and domain Bacteria-specific probes. The Bacteria-specific probe detected 35-71% of the total microorganisms detected by the DNA-binding dye 4,6-diamidino-2-phenylindole. Initially, 44%, 13% and 5% of the total bacteria in the soil extract were Alpha-, Beta- and Gammaproteobacteria, respectively. Without pyrene or a gyrase inhibitor, these subgroups decreased to 30% of the total population but were predominant with piromidic acid or unchanged with ciprofloxacin when pyrene was the main substrate. The proportion of elongated Alpha- and Betaproteobacteria (potential pyrene degraders) increased significantly (P<0.05). This approach links phylogenetic information with physiological function in situ without the conventional cultivation of bacteria and can be used to probe and enumerate degradative groups at even a finer level of discrimination.     

具溶磷能力的植物内生促生细菌的分离筛选及其生物多样性

【目的】具溶磷能力的植物内生促生细菌的分离筛选及生物多样性的研究将有助于扩大溶磷微生物来源、丰富功能内生细菌资源库及开发新的改善土壤磷素营养途径。【方法】结合内生细菌分离方法从油菜和玉米体内分离筛选具溶磷能力的内生细菌,测定菌株摇瓶条件下的溶磷能力,并研究其产生吲哚乙酸(IAA)、铁载体、1-氨基环丙烷-1-羧酸(ACC)脱氨酶等的特性,采用16SrDNA限制性酶切片段长度多态性分析(RFLP)研究了具溶磷能力的植物内生促生细菌的遗传多样性,并挑选典型菌株进行了鉴定。【结果】分离筛选到32株具稳定溶磷能力的植物内生细菌,所有菌株都能从磷酸钙中释放出有效磷并使培养液pH值降低,释放的有效磷浓度最高达到537.6mg/L。分离自油菜的供试细菌都能产生吲哚乙酸和铁载体,分离自玉米的供试细菌中有68.4%的菌株产生吲哚乙酸,63.2%的菌株产生铁载体,63.2%的菌株具ACC脱氨酶活性。分离菌株在76%相似性水平上可聚类分为8个群。11株典型菌株归属于泛菌属(Pantoea)、假单胞菌属(Pseudomonas)、伯克霍尔德氏菌属(Burkholderia)、不动杆菌属(Acinetobacter)及罗尔斯顿菌属(Ralstonia)等5个属。【结论】油菜和玉米体内溶磷细菌具有丰富多样的生物学特性和遗传多样性。     

枸骨益生菌的筛选及其抑菌作用

采用涂布平板法分离药用植物枸骨可培养内生细菌,采用对峙法和发酵滤液培养法筛选对植物病原菌具高抑制作用的菌株,通过镜检和显微摄影观察处理组菌丝的变化,对16SrDNA PCR产物进行测序和同源性分析菌株的系统发育关系,通过形态、生理生化特征和16SrDNA序列比对确定菌株的分类地位.从枸骨健康植株的根、茎、叶及果实中分离得到85株内生细菌,筛选出对烟草赤星病菌、稻瘟病菌、棉花枯萎病菌等有较强抑菌作用的10株菌,初步鉴定为4属7种.其中,抑菌效果最强的GG78(60.3％)、GG31 (48.1％)、GG3(61.0％)分别属于阴沟肠杆菌、路德维希肠杆菌和蜡样芽孢杆菌.受抑制病原菌菌丝均发生畸形、扭曲,局部膨大形成原生质浓缩球、菌丝基部出现毛发状细分支等现象,可能与内生菌分泌到胞外具有一定抑菌或杀菌作用的代谢物质如抗生素、水解酶类、生物碱等有关.     

Procedures to reduce contamination of cell cultures

Summary The most frequent causes of cell culture contamination are poor techniques and housekeeping and inadequate sterility testing of supplies and culture media. The most common contaminants of cell cultures areMycoplasma, Torula sp., andPseudomonas sp. Routine procedures used in this laboratory to control or prevent accidental contamination include the use of filtered laminar air flow in transfer rooms, elimination of antibiotics wherever possible, careful execution of aseptic procedures, periodic review and discussion of techniques, sterility testing of all media components, use of approved clothing, and effective cleaning and disinfection procedures. These studies were supported by a grant from the John A. Hartford Foundation General Research Support Grant FR-5582 from the National Institutes of Health, Grant in Aid Contract M-43 from the State of New Jersey, and by Grant CA-04953-11 from the National Cancer Institute.     

Diversity of endophytic bacteria in ginseng and their potential for plant growth promotion

Endophytic bacteria have been found in virtually every plant studied, where they colonize the internal tissues of their host plant and can form a range of different beneficial relationships. The diversity of bacterial endophytes associated with ginseng plants of varying age levels in Korea was investigated. Fifty-one colonies were isolated from the interior of ginseng stems. Although a mixed composition of endophyte communities was recovered from ginseng based on the results of 16S rDNA analysis, bacteria of the genus Bacillus and Staphylococcus dominated in 1-year-old and 4-year-old plants, respectively. Phylogenetic analysis revealed four clusters: Firmicutes, Actinobacteria, α-Proteobacteria, and γ-Proteobacteria, with Firmicutes being predominant. To evaluate the plant growth promoting activities, 18 representative isolates were selected. Amplification of nifH gene confirmed the presence of diazotrophy in only two isolates. Half of the isolates solubilized mineral phosphate. Except four, all the other endophytic isolates produced significant amounts of indole acetic acid in nutrient broth. Iron sequestering siderophore production was detected in seven isolates. Isolates E-I-3 (Bacillus megaterium), E-I-4 (Micrococcus luteus), E-I-8 (B. cereus), and E-I-20 (Lysinibacillus fusiformis) were positive for most of the plant growth promoting traits, indicating their role in growth promotion of ginseng.     

Metabolic potential of endophytic bacteria

1. Download : Download high-res image (234KB)
2. Download : Download full-size image

     

Fluorescence in situ hybridization for phytoplasma and endophytic bacteria localization in plant tissues

In the present study, we developed a rapid and efficient fluorescence in situ hybridization assay (FISH) in non-embedded tissues of the model plant Catharanthus roseus for co-localizing phytoplasmas and endophytic bacteria, opening new perspectives for studying the interaction between these microorganisms.     

玉米品种遗传多态性与根系内生细菌种群的相互关系

对辽宁省14个玉米主栽品种根系内主要细菌种群的分析结果表明,芽孢杆菌属（Bucillus ssp．）为玉米内生细菌的主要种群,其它种属包括肠杆菌属、沙雷氏杆菌属、假单孢菌属、黄单孢菌属和棍状杆菌属。内生细菌在不同玉米品种和不同生育期之间存在程度不同的差异。从90条随机引物中筛选出条带清晰、重复性好的13个引物,对14个玉米主栽品种进行遗传多态性的RAPD分析,共扩增出条带139条,其中多态性条带101条,多态性比率72．7％。利用SPSS统计分析软件对14个玉米品种在DNA水平和内生细菌种群水平上进行聚类分析,结果表明,品种亲缘关系在这两个水平上表现基本一致,即品种内生细菌的种类及其数量在很大程度上受品种的遗传背景控制。     

Increased plant growth and copper uptake of host and non-host plants by metal-resistant and plant growth-promoting endophytic bacteria

The effects of inoculation with two metal-resistant and plant growth-promoting endophytic bacteria (Burkholderia sp. GL12 and Bacillus megaterium JL35) were evaluated on the plant growth and Cu uptake in their host Elsholtzia splendens and non-host Brassica napus plants grown in natural Cu-contaminated soil. The two strains showed a high level of ACC deaminase activities. In pot experiments, inoculation with strain GL12 significantly increased root and above-ground tissue dry weights of both plants, consequently increasing the total Cu uptake of E. splendens and Brassica napus by 132% and 48.2% respectively. Inoculation with strain JL35 was found to significantly increase not only the biomass of B. napus, consequently increasing the total Cu uptake of B. napus by 31.3%, but Cu concentration of E. splendens for above-ground tissues by 318% and roots by 69.7%, consequently increasing the total Cu uptake of E. splendens by 223%. The two strains could colonize the rhizosphere soils and root interiors of both plants. Notably, strain JL35 could colonize the shoot tissues and significantly increase the translocation factors and bioaccumulation factors of E. splendens. These results suggested that Burkholderia sp. GL12 and B. megaterium JL35 were valuable bacterial resource which had the potential in improving the efficiency of Cu phytoextraction by E. splendens and B. napus in a natural Cu-contaminated soil.     

Isolation and characterization of plant growth promoting endophytic diazotrophic bacteria from Korean rice cultivars

We have isolated 576 endophytic bacteria from the leaves, stems, and roots of 10 rice cultivars and identified 12 of them as diazotrophic bacteria using a specific primer set of nif gene. Through 16S rDNA sequence analysis, nifH genes were confirmed in the two species of Penibacillus, three species of Microbacterium, three Bacillus species, and four species of Klebsiella. Rice seeds treated with these plant growth-promoting bacteria (PGPB) showed improved plant growth, increased height and dry weight and antagonistic effects against fungal pathogens. In addition, auxin and siderophore producing ability, and phosphate solubilizing activity were studied for the possible mechanisms of plant growth promotion. Among 12 isolates tested, 10 strains have shown higher auxin producing activity, 6 isolates were confirmed as strains with high siderophore producing activity while 4 isolates turned out to have high phosphate-solubilizing activity. These results strongly suggest that the endophytic diazotrophic bacteria characterized in this study could be successfully used to promote plant growth and inducing fungal resistance in plants.     

Utilizing genetically engineered bacteria to produce plant-specific glucosides

Plant-derived glucosides have attracted much attention due to their widespread applications. This class of products is difficult to isolate or to synthesize in pure form because of the resulting low yields. Thus, simple approaches for the generation of such glucosides would be highly beneficial. We purified and characterized a novel glucosyltransferase from plant cell suspension cultures of Rauvolfia serpentina, which showed rather low substrate specificity. We obtained its cDNA and expressed the active recombinant protein in bacteria (Escherichia coli) with excellent plant-specific glucosylation efficiencies. Compared with the plant system, the bacteria delivered the new enzyme, which was in the form of a soluble or matrix-bound enzyme, approximately 1800 times more efficiently for the synthesis of a wide range of glucosides. More importantly, the engineered E. coli strain allowed for in vivo glucosylation and release of the product into the culture medium, as shown by the formation of arbutin, which is a potent inhibitor of human melanin biosynthesis with commercial value.     

银沙槐内生放线菌抗菌活性及其与内生细菌的拮抗关系

从荒漠濒危植物银沙槐根部分离到内生放线菌12株和内生细菌31株.以大肠杆菌、金黄色葡萄球菌和枯草芽孢杆菌为指示菌对内生放线菌进行了抗菌活性筛选.结果表明：3株放线菌对3种指示菌均有抑制作用,2株完全无抑菌作用.采用菌块抑制法分析内生放线菌对来自同一植物内生细菌的抑制效果,44.9%的组合出现抑制现象,最大抑菌圈直径为28.5mm,最小者仅8.25 mm,主要抑制对象是革兰氏阴性菌和芽孢菌;55.1%的组合无抑制现象.说明内生菌的相互作用具有多样性.     

Isolation of micropropagated strawberry endophytic bacteria and assessment of their potential for plant growth promotion

Twenty endophytic bacteria were isolated from the meristematic tissues of three varieties of strawberry cultivated in vitro, and further identified, by FAME profile, into the genera Bacillus and Sphingopyxis. The strains were also characterized according to indole acetic acid production, phosphate solubilization and potential for plant growth promotion. Results showed that 15 strains produced high levels of IAA and all 20 showed potential for solubilizing inorganic phosphate. Plant growth promotion evaluated under greenhouse conditions revealed the ability of the strains to enhance the root number, length and dry weight and also the leaf number, petiole length and dry weight of the aerial portion. Seven Bacillus spp. strains promoted root development and one strain of Sphingopyxis sp. promoted the development of plant shoots. The plant growth promotion showed to be correlated to IAA production and phosphate solubilization. The data also suggested that bacterial effects could potentially be harnessed to promote plant growth during seedling acclimatization in strawberry.