Isolation and characterization of soybean-associated bacteria and their potential for plant growth promotion

Endophytic and epiphytic bacteria were isolated from two soybean cultivars (Foscarin and Cristalina). Significant differences were observed in bacterial population densities in relation to season of isolation, soybean growth phase and the tissues from which the isolates were obtained. The isolates were identified by partial 16S rDNA sequence analysis, with most of the isolates belonging to the Pseudomonaceae, Burkholderiacea and Enterobacteriaceae groups. The potential of the isolates for plant growth promotion was evaluated by screening for indoleacetic acid (IAA) production and mineral phosphate solubilization; 34% of endophytic bacteria produced IAA and 49% were able to solubilize mineral phosphate whereas only 21% of epiphytic bacteria produced IAA although 52% were able to solubilize mineral phosphate. A high frequency of IAA producing isolates occurred in the early ripening Foscarin cultivar whereas a high percentage of phosphate solubilizing isolates were obtained from plants in the initial development stage (V6). We also found that 60% of endophytic and 69% of epiphytic isolates that produced IAA and solubilized mineral phosphate were also able to fix nitrogen in vitro. The soybean-associated bacteria showing characteristics related to plant growth promotion were identified as belonging to the genera Pseudomonas, Ralstonia, Enterobacter, Pantoea and Acinetobacter.     

大豆根际土壤中氢氧化细菌的分离、筛选和基本特征

土壤中的氢氧化细菌能够利用土壤中的H2为能源并同化CO2,增加其种群数量并促进作物生长.采用气体循环培养体系(持续通氢气装置),通过电解水的方式产生H2,与通入的空气混合,形成流速为280ml.min-1、含H2量为41.6～125μmol.L-1的混合气体.采用矿质盐固体培养基,在适当的H2、O2和CO2下以H2作为唯一能源分离氢氧化细菌.采用此方法从大豆根际土壤样品中分离出40余株细菌,对其进行耗氢能力测定表明,有20株菌具有氧化氢功能和自养生长能力,初步判断这20株菌为氢氧化细菌,并测定了菌落形态及生理生化特征.     

Elemental sulfur production during mixotrophic growth on hydrogen and thiosulfate of thermophilic hydrogen-oxidizing bacteria

In this study we measured the exogenous production and the intracellular content of elemental sulfur (S⁰) in the thermophilic sulfur-oxidizing bacteriaHydrogenobacter spp. andBacillus schlegelii during mixolithoautotrophic growth on hydrogen and thiosulfate. Under these conditions, all strains produced and released white-yellow hydrophilic S⁰ particles into the growth medium. Hydrophilic S⁰ was separated from cells by a differential low-speed centrifugation procedure. The S⁰ pellets were dried, and the S⁰ was purified by column chromatography and by thin-layer chromatography (TLC). The S⁰ TLC-band could be stained with triphenyltetrazolium chloride and piperidine procedure. Determination of intracellular S⁰ content was performed from fresh cells absolutely free of exogenous S⁰ particles. quantitation of S⁰ was performed by high-performance liquid chromatography, colorimetric thiocyanate procedure, and by UV-spectra analyses. All the strains studied, in particularB. schlegelii strains, released significant quantities of S⁰ into the growth media. In contrast, the intracellular S⁰ content was very low. Significant rhodanese activity in the presence of thiosulfate was measured with toluenepermeabilized cells and cell-free extracts ofB. schlegelii (type strain) andHydrogenobacter spp. (strain T3).     

Isolation and characterization of different plant associated bacteria and their potential to degrade polychlorinated biphenyls

In our experiments the effect of different plants on microbial activities resulting in degradation and PCB removal from long-term contaminated soil was evaluated. Total bacteria and bacteria representing the dominating microflora within rhizosphere of individual plant species – tobacco (Nicotiana tabacum), black nightshade (Solanum nigrum), horseradish (Armoracia rusticana) and goat willow (Salix caprea) planted in PCB contaminated soil as well as from the same, but non-vegetated PCBs soil, were isolated and biochemically characterized. PCB bacterial degraders, stimulated by root exudates of individual plants, were detected after isolation from rhizosphere soil and precultivation on minimal medium with biphenyl as the sole carbon source. Detection of BphA1 gene (first gene of bacterial aerobic PCB degradative pathway) in genomes of rhizosphere microorganisms was performed by nested PCR technique using previously designed specific primers. Rhizosphere of individual plants contained different bacterial species, mostly gram-negative non-fermenting bacteria of Pseudomonas, Agrobacterium, Ochrobactrum and other species. Gene BphA1 was identified in genome of only several of them. From tested species, S. caprea and A. rusticana have shown to be promising candidates for rhizoremediation purposes.     

Isolation and characterization of 2,4-dichlorophenoxyacetic acid-catabolizing bacteria and their biodegradation efficiency in soil

Bacterial isolates (NJ 10 and NJ 15) capable of degrading the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were isolated from agricultural soil by enrichment culture technique. The isolates exhibited substantial growth in mineral salt medium supplemented with 0.1–0.5% of 2,4-D as a sole source of carbon and energy. Based on their morphological, cultural and biochemical characteristics, the isolates NJ 10 and NJ 15 have been identified as Pseudomonas species and Pseudomonas aeruginosa, respectively. Biodegradation studies in a soil microcosm enriched with pure cultures of the isolates demonstrated a time-dependent disappearance of 2,4-D from the 100 mg/kg herbicide-amended soil. The HPLC data analysis revealed 96.6 and 99.8% degradation in the soil inoculated with the pure cultures of isolates NJ 10 and NJ 15, respectively with in 20 days of incubation at 30 °C. Both the isolates showed significant solubilization of inorganic phosphate [Ca₃(PO₄)₂] on the specific Pikovskaya's medium.     

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.     

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.     

Effects of ammonia on growth and morphology of thermophilic hydrogen-oxidizing methanogenic bacteria

Abstract: Seven bacteriophage (Φ1R, Φ10W, Φ3R, Φ30W, Φ1261 M, Φ1261 V and Φgor3V), specific for the Rhizobium galegae species and representing three morphotypes, were isolated from different locations in Finland and in New Zealand. DNA was isolated from these phage and from phage Φ1R-3 and Φ1R', which were derived from Φ1R in the laboratory, and analyzed by restriction endonuclease digestion and dot blot DNA hybridization. The sizes of the phage DNAs were estimated to range from 45.1–114.6 kb. The restriction patterns revealed four different phage genotypes, which correlated with the isolation hosts. DNA hybridization showed that the four genotypes were distantly related. The genotypes were distinguished when purified phage protein was analyzed in SDS-PAGE gels.     

Isolation and characterization of phenol-catabolizing bacteria from a coking plant

New phenol degrading bacteria with high biodegradation activity and high tolerance were isolated as Burkholderia cepacia PW3 and Pseudomonas aeruginosa AT2. Both isolates could grow aerobically on phenol as a sole carbon source even at 3 g/l. The whole-cell kinetic properties for phenol degradation by strains PW3 and AT2 showed a Vmax of 0.321 and 0.253 mg/l/min/(mg protein), respectively. The metabolic pathways for phenol biodegradation in both strains were assigned to the meta-cleavage activity of catechol 2,3-dioxygenase.     

Isolation, characterization, and use for plant growth promotion under salt stress, of ACC deaminase-producing halotolerant bacteria derived from coastal soil

In total, 140 halotolerant bacterial strains were isolated from both the soil of barren fields and the rhizosphere of six naturally growing halophytic plants in the vicinity of the Yellow Sea, near the city of Incheon in the Republic of Korea. All of these strains were characterized for multiple plant growth promoting traits, such as the production of indole acetic acid (IAA), nitrogen fixation, phosphorus (P) and zinc (Zn) solubilization, thiosulfate (S2O3) oxidation, the production of ammonia (NH3), and the production of extracellular hydrolytic enzymes such as protease, chitinase, pectinase, cellulase, and lipase under in vitro conditions. From the original 140 strains tested, on the basis of the latter tests for plant growth promotional activity, 36 were selected for further examination. These 36 halotolerant bacterial strains were then tested for 1- aminocyclopropane-1-carboxylic acid (ACC) deaminase activity. Twenty-five of these were found to be positive, and to be exhibiting significantly varying levels of activity. 16S rRNA gene sequencing analyses of the 36 halotolerant strains showed that they belong to 10 different bacterial genera: Bacillus, Brevibacterium, Planococcus, Zhihengliuella, Halomonas, Exiguobacterium, Oceanimonas, Corynebacterium, Arthrobacter, and Micrococcus. Inoculation of the 14 halotolerant bacterial strains to ameliorate salt stress (150 mM NaCl) in canola plants produced an increase in root length of between 5.2% and 47.8%, and dry weight of between 16.2% and 43%, in comparison with the uninoculated positive controls. In particular, three of the bacteria, Brevibacterium epidermidis RS15, Micrococcus yunnanensis RS222, and Bacillus aryabhattai RS341, all showed more than 40% increase in root elongation and dry weight when compared with uninoculated saltstressed canola seedlings. These results indicate that certain halotolerant bacteria, isolated from coastal soils, have a real potential to enhance plant growth under saline stress, through the reduction of ethylene production via ACC deaminase activity.     

Isolation and characterization endophytic bacteria from hyperaccumulator Sedum alfredii Hance and their potential to promote phytoextraction of zinc polluted soil

The aim of this study was to isolate and characterize endophytic bacteria from roots, stems and leaves of Zn/Cd hyperaccumulator Sedum alfredii. Endophytic bacteria were observed in roots, stems and leave of S. alfredii, with a significantly higher density in roots, followed by leave and stems. A total of fourteen bacterial endophytes were isolated and are closely related phylogenetically to Pseudomonas, Bacillus, Stenotrophomonas, Acinetobacte by 16S rRNA sequence analysis. Most of the endophytic bacteria were found to exhibit high Zn and Cd resistance characteristics, but difference existed among this isolates. The fourteen endophytic bacteria all had the capacity to produce IAA. Moreover, strains VI₈L₁, VI₈L₂, VI₈L₄, VI₈R₂, VI₈R₃ and II₂R₃ could solubilize Ca₃(PO₄)₂, strains VI₈L₂, II₈L₄ and VI₈R₂ could produce siderophore, and strains VI₈L₂ and VI₈R₃ had the capacity of nitrogen fixation. Both plate and broth assay proved that strain VI₈L₁, VI₈L₂, II₈L₄ and VI₈R₂ were able to effectively solubilize ZnCO₃ and Zn₃(PO₄)₂. The filtrate liquid media after growth of strains VI₈L₁, VI₈L₂, II₈L₄ and VI₈R₂ extracted much higher Zn from artificially ZnCO₃ and Zn₃(PO₄)₂ contaminated soils than those extracted by axenic SMS broth, and the filtrates of the culture media supporting growth of strains VI₈L₂, II₈L₄ and VI₈R₂ also extracted significantly greater quantities of Zn from the Dabaoshan contaminated soils. This Zn mobilizing, plant growth promoting and metal resistant endophytic bacteria may offer promise as inoculants to increase soil Zn bioavailability and improve growth and Zn accumulation by S. alfredii.     

The use of disposable gaas generating kits for the growth of hydrogen-oxidizing bacteria and the determination of hydrogen autotrophy

A simplified procedure for the determination of autotropic growth of hydrogen-oxidizing bacteria has been developed. The method uses commercially available disposable hydrogen and carbon dioxide kits, commonly used in anaerobic bacteriology, to produce a gaseous atmosphre containing by volume approximately 41% hydrogen, 6% carbon dioxide, 11% oxygen and 42% nitrogen. The atmosphere was suitable for the growth of strains assigned to the species Alcaligenes eutrophus, Alcaligenes paradoxus, Paracoccus denitrificans, Pseudomonas facilis, Pseudomonas flava, Pseudomonas palleronii, Pseudomonas saccahrophilia and Rhodococcus sp. (‘Nocardia opaca’). The method can also be used for the screening of hydrogen-oxidizing ability in bacterial isolates, thus eliminating the need for complex gas mixing devices or expensive gas mixtures.     

Isolation and characterization of polyphenols-degrading bacteria from olive-mill wastewaters polluted soil

In this study 28 bacterial strains, isolated from greenwaters-polluted-soil, were investigated for their ability to grow in presence of phenols added to Mineral Basal Medium (MBM) in aerobic conditions. In particular, three of them were found to be able to use as sole carbon source phenol, cathecol, caffeic acid and ferulic acid with efficiency ranging from 76% (phenol in 5 days, millimolar concentration from 3.7 10^?2 to 9 10^?3) to 95% (ferulic acid in 2 days millimolar concentration from 6.8 10^?1 to 3 10^?2). For these strains the taxonomic position was studied by amplification and sequencing of 16S rRNA genes. The isolated strains were classified belonging to Arthrobacter sulfureus, Pseudomonas synxantha and Pseudomonas oryzihabitans. Noteworthy, for the first time such Pseudomonas strains have been shown to be able to use polyphenols as the only carbon source in vitro. In fact, to the best of our knowledge, this kind of study were not done on Ps. Synxantha, while it was recently shown the ability of P. oryzihabitans to degrade catechol. These findings may open to new biotechnological applications for the degradation of polyphenols.     

Isolation,characterization and growth kinetics of bacteria metabolizing pentachlorophenol

Summary A soil bacterium capable of utilizing pentachlorophenol (PCP) as a sole source of carbon and energy was examined for its morphological, biochemical, cultural, and physiological characteristics. The organism was a member of the coryneform group of bacteria, probably in the genus Arthrobacter. The isolate exhibited a doubling time of 4–5 h while growing on either glucose or PCP as the sole carbon source. The growth rate on PCP was essentially constant between 10–135 mg/l. At higher concentrations of PCP the growth rate was inhibited. The organism was found to be an excellent scavenger of PCP; a Monod saturation constant of 1.12 mg/l was obtained from chemostat measurements.     

Endophytic bacteria isolated from orchid and their potential to promote plant growth

Twelve endophytic bacteria were isolated from the meristem of in vitro Cymbidium eburneum orchid, and screened according to indole yield quantified by colorimetric assay, in vitro phosphate solubilization, and potential for plant growth promotion under greenhouse conditions. Eight strains with positive results were classified into the genus Paenibacillus by FAME profile, and evaluated for their ability to increase survival and promote the growth of in vitro germinated Cattleya loddigesii seedlings during the acclimatization process. The obtained results showed that all strains produced detectable indole levels and did not exhibit potential for solubilizing inorganic phosphate. Particularly, an increase of the total biomass and number of leaves was observed. Two strains of Paenibacillus macerans promoted plant growth under greenhouse conditions. None of the treatments had a deleterious effect on growth of inoculated plants. These results suggest that these bacterial effects could be potentially useful to promote plant growth during seedling acclimatization in orchid species other than the species of origin.     

Isolation and partial characterization of normal and defective bacteriophages of gram-negative hydrogen bacteria

Widespread defective lysogeny was detected in Alcaligenes eutrophus by electron microscopic analysis of cultures. Mitomycin C treatment of the cultures resulted in the production of defective (inco-) particles. Polysheaths were produced both with and without induction.With the simultaneous isolation technique six phages were isolated for hydrogen-oxidizing strains of the new species Pseudomonas pseudoflava. The phages were able to replicate under autotrophic conditions and were found to have a very restricted host range. Electron microscopic analysis allowed classification into two structural groups. Group I contained phages with contractile tails; group II contained phages with flexible, noncontractile tails. All but one (gb) of the new phages were shown to be temperate by isolation of lysogens and induction with mitomycin C.Non-Standard Abbreviations OD optical density - SM-medium standard mineral medium - KS-medium Kaserer medium - NB nutrient broth - p.f.u. plaque forming unit - c.f.u. colony forming unit - e.o.p. efficiency of plating - Do-phage buffer Dopatka-phage buffer - PTA phosphotungstic acid     

Isolation of endophytic bacteria from embryogenic suspension culture of banana and assessment of their plant growth promoting properties

Endophytic microorganisms have been reported from various plants. In the current study, somatic embryogenic cultures of banana (Musa accuminata AAA cv. Grand Naine) were found to have association with endophytic bacteria which were present initially in a covert state. The presence of bacteria was detected only in suspension cultures derived from the somatic embryogenic cultures. The bacteria isolated from embryogenic cell suspension culture were identified as Ralstonia sp. and Bacillus sp. The Ralstonia sp. interestingly showed the presence of various plant growth promoting properties including indole acetic acid and siderophore production. Also the strain was found to have the potential to solubilize phosphate and produce ammonia. Growth enhancement effect of Ralstonia sp. on Vigna radiata seedlings showed promising results and the growth parameters were found to be statistically significant when compared to control. Identification and confirmation of the plant growth promoting properties of Ralstonia sp. makes the study significant with promising applications.