Isolation and characterization of novel bacterial strains for integrated solar-bioelectrokinetic of soil contaminated with heavy petroleum hydrocarbons.

This study investigated the isolation and characterization of three novel bacterial strains; Acinetobacter calcoaceticus, Sphingobacterium multivorum, and Sinorhizobium, isolated form agriculture land. From three hundred strains of bacteria, the three isolates were identified for their superior diesel degradation ability by a series of bench-scale tests. The isolates were further investigated in bench tests for their ability to grow in different diesel fuel concentrations, temperature and pH; degrade diesel fuel in vitro; and for the identification of functional genes. Semi-pilot bioelectrokinetic tests were conducted in three electrokinetic cells. An innovative electrode configuration was adopted to stabilize the soil pH and water content during the test. The genes expressed in the diesel degradation process including Lipases enzymes Lip A, LipB, Alk-b2, rubA, P450, and 1698/2041 were detected in the three isolates. The results showed that the solar panel voltage output is in agreement with the trapezoid model. The temperatures in the cells were found to be 5-7 °C higher than the ambient temperature. The electrode configuration succeeded in stabilizing the soil pH and water content, preventing the development of a pH gradient, important progress for the survival of bacteria. The diesel degradation in the soil after bioelectrokinetic tests were 20-30%, compared to 10-12% in the controls. The study succeeded in developing environmentally friendly technology employing novel bacterial strains to degrade diesel fuel and utilizing solar panels to produce renewable energy for bioelectrokinetics during the winter season.

Characterization and complete genome analysis of the surfactin-producing, plant-protecting bacterium Bacillus velezensis 9D-6.

BACKGROUND: Bacillus velezensis is an endospore-forming, free-living soil bacterium with potential as a biopesticide against a broad spectrum of microbial pathogens of plants. Its potential for commercial development is enhanced by rapid replication and resistance to adverse environmental conditions, typical of Bacillus species. However, the use of beneficial microbes against phytopathogens has not gained dominance due to limitations that may be overcome with new biopesticidal strains and/or new biological knowledge. RESULTS: Here, we isolated B. velezensis strain 9D-6 and showed that it inhibits the in vitro growth of prokaryotic and eukaryotic pathogens, including the bacteria Bacillus cereus , Clavibacter michiganensis, Pantoea agglomerans, Ralstonia solanacearum, Xanthomonas campestris, and Xanthomonas euvesicatoria; and the fungi Alternaria solani, Cochliobolus carbonum, Fusarium oxysporum, Fusarium solani, Gibberella pulicaris, Gibberella zeae, Monilinia fructicola, Pyrenochaeta terrestris and Rhizoctonia solani. Antimicrobial compounds with activity against Clavibacter michiganensis were isolated from B. velezensis 9D-6 and characterized by high resolution LC-MS/MS, yielding formulae of C52H91N7O13 and C53H93N7O13, which correspond to [Leu7] surfactins C14 and C15 (also called surfactin B and surfactin C), respectively. We further sequenced the B. velezensis 9D-6 genome which consists of a single circular chromosome and revealed 13 gene clusters expected to participate in antimicrobial metabolite production, including surfactin and two metabolites that have not typically been found in this species - ladderane and lantipeptide. Despite being unable to inhibit the growth of Pseudomonas syringae DC3000 in an in vitro plate assay, B. velezensis 9D-6 significantly reduced root colonization by DC3000, suggesting that 9D-6 uses methods other than antimicrobials to control phytopathogens in the environment. Finally, using in silico DNA-DNA hybridization (isDDH), we confirm previous findings that many strains currently classified as B. amyloliquefaciens are actually B. velezensis. CONCLUSIONS: The data presented here suggest B. velezensis 9D-6 as a candidate plant growth promoting bacterium (PGPB) and biopesticide, which uses a unique complement of antimicrobials, as well as other mechanisms, to protect plants against phytopathogens. Our results may contribute to future utilization of this strain, and will contribute to a knowledge base that will help to advance the field of microbial biocontrol.

Clavibacter michiganensis ssp. michiganensis: bacterial canker of tomato, molecular interactions and disease management.

Bacterial canker disease is considered to be one of the most destructive diseases of tomato (Solanum lycopersicum), and is caused by the seed-borne Gram-positive bacterium Clavibacter michiganensis ssp. michiganensis (Cmm). This vascular pathogen generally invades and proliferates in the xylem through natural openings or wounds, causing wilt and canker symptoms. The incidence of symptomless latent infections and the invasion of tomato seeds by Cmm are widespread. Pathogenicity is mediated by virulence factors and transcriptional regulators encoded by the chromosome and two natural plasmids. The virulence factors include serine proteases, cell wall-degrading enzymes (cellulases, xylanases, pectinases) and others. Mutational analyses of these genes and gene expression profiling (via quantitative reverse transcription-polymerase chain reaction, transcriptomics and proteomics) have begun to shed light on their roles in colonization and virulence, whereas the expression of tomato genes in response to Cmm infection suggests plant factors involved in the defence response. These findings may aid in the generation of target-specific bactericides or new resistant varieties of tomato. Meanwhile, various chemical and biological controls have been researched to control Cmm. This review presents a detailed investigation regarding the pathogen Cmm, bacterial canker infection, molecular interactions between Cmm and tomato, and current perspectives on improved disease management.

Complete Genome Sequence of Acinetobacter calcoaceticus CA16, a Bacterium Capable of Degrading Diesel and Lignin.

We report here the complete assembled genome sequence of Acinetobacter calcoaceticus CA16, which is capable of utilizing diesel and lignin as a sole carbon source. CA16 contains a 4,110,074-bp chromosome and a 5,920-bp plasmid. The assembled sequences will help elucidate potential metabolic pathways and mechanisms responsible for CA16's hydrocarbon degradation ability.

Complete Genome Sequence of Burkholderia cenocepacia CR318, a Phosphate-Solubilizing Bacterium Isolated from Corn Root.

Here, we report the complete genome sequence of the phosphate-solubilizing bacterium Burkholderia cenocepacia CR318, consisting of three circular chromosomes of 3,511,146 bp, 3,097,552 bp, and 1,056,069 bp. The data presented will facilitate further insight into the mechanisms of phosphate solubilization and its application for agricultural and ecological sustainability.

Isolation, identification and characterization of Paenibacillus polymyxa CR1 with potentials for biopesticide, biofertilization, biomass degradation and biofuel production.

BACKGROUND: Paenibacillus polymyxa is a plant-growth promoting rhizobacterium that could be exploited as an environmentally friendlier alternative to chemical fertilizers and pesticides. Various strains have been isolated that can benefit agriculture through antimicrobial activity, nitrogen fixation, phosphate solubilization, plant hormone production, or lignocellulose degradation. However, no single strain has yet been identified in which all of these advantageous traits have been confirmed. RESULTS: P. polymyxa CR1 was isolated from degrading corn roots from southern Ontario, Canada. It was shown to possess in vitro antagonistic activities against the common plant pathogens Phytophthora sojae P6497 (oomycete), Rhizoctonia solani 1809 (basidiomycete fungus), Cylindrocarpon destructans 2062 (ascomycete fungus), Pseudomonas syringae DC3000 (bacterium), and Xanthomonas campestris 93-1 (bacterium), as well as Bacillus cereus (bacterium), an agent of food-borne illness. P. polymyxa CR1 enhanced growth of maize, potato, cucumber, Arabidopsis, and tomato plants; utilized atmospheric nitrogen and insoluble phosphorus; produced the phytohormone indole-3-acetic acid (IAA); and degraded and utilized the major components of lignocellulose (lignin, cellulose, and hemicellulose). CONCLUSIONS: P. polymyxa CR1 has multiple beneficial traits that are relevant to sustainable agriculture and the bio-economy. This strain could be developed for field application in order to control pathogens, promote plant growth, and degrade crop residues after harvest.

Complete Genome Sequence of Arthrobacter sp. Strain LS16, Isolated from Agricultural Soils with Potential for Applications in Bioremediation and Bioproducts.

Here we report the complete genomic sequence of the bacterium Arthrobacter sp. strain LS16, consisting of a single circular chromosome of 3.85 Mb with no identified plasmid. Data contained within will facilitate future genetic modification and engineering of the Arthrobacter sp. LS16 metabolic network to enhance traits relevant to bioremediation and bioproducts.

Occurrence of copper-resistant strains and a shift in Xanthomonas spp. causing tomato bacterial spot in Ontario.

Field strains of tomato bacterial spot pathogen (Xanthomonas euvesicatoria, X. vesicatoria, X. perforans, and X. gardneri) were characterized for sensitivity to copper and species composition. A total of 98 strains were isolated from symptomatic leaf and fruit samples collected from 18 tomato fields in Ontario. In greenhouse pathogenicity tests, most of the field strains caused severe (37 strains) to highly severe (23 strains) symptoms on 'Bonny Best' tomato plants, whereas 38 strains caused moderate symptoms. In MGY agar plates amended with various concentrations of copper sulfate, 11 strains were completely sensitive (no growth) and 87 strains were resistant (grew on 1.0 mmol/L or higher copper concentration). PCR analysis of the hrp gene cluster followed by restriction digestion with HaeIII and sequencing identified X. gardneri (35 strains) and X. perforans (26 strains) as predominant species and X. euvesicatoria and X. vesicatoria as less common species in Ontario tomato fields. Separation of field strains into various species was also confirmed with starch hydrolysis activity on agar medium. Moreover, 72 field strains produced shiny greenish-yellow colonies surrounded by a milky zone on xanthomonad differential (Xan-D) medium, and the colonies of 26 strains did not produce a milky zone. Thirty-four strains could not be clustered into any species and 25 of those strains were negative for the hrp gene PCR and also did not produce a milky zone around colonies on Xan-D medium. Our results suggest a widespread existence of copper-resistant strains and an increase in X. perforans strains of bacterial spot pathogen in Ontario. This information on copper resistance and species composition within bacterial spot pathogens in Ontario will be helpful for developing effective disease management strategies, making cultivar selection, and breeding new tomato cultivars.

Complete Genome Sequence of Paenibacillus polymyxa CR1, a Plant Growth-Promoting Bacterium Isolated from the Corn Rhizosphere Exhibiting Potential for Biocontrol, Biomass Degradation, and Biofuel Production.

Here we report the complete genome sequence of the bacterium Paenibacillus polymyxa CR1 (accession no. CP006941), which consists of one circular chromosome of 6,024,666 bp with 5,283 coding sequences (CDS), 87 tRNAs, and 12 rRNA operons. Data presented will allow for further insights into the mechanisms underpinning agriculturally and industrially relevant processes.

Isolation, characterization, and effect of fluorescent pseudomonads on micropropagated sugarcane.

In this study, we report on the isolation, identification, and characterization of seven fluorescent pseudomonads isolated from the roots, shoots, and rhizosphere soil of sugarcane and their impacts on the growth of sugarcane plantlets. 16S rRNA gene sequence of five isolates showed close homology with Pseudomonas putida, one with Pseudomonas graminis, and one with Pseudomonas fluorescens. Physiological and biochemical characterizations were determined using API50CH and QTS24 identification kits. The isolates were also subjected to tests for various known growth promoting properties including production of indole acetic acid, the ability to fix nitrogen via the presence of the nifH gene, and ability to solubilize phosphate. Biological control potential was determined from agar diffusion assays of HCN production and production of antifungal compounds against local isolates of Colletotrichum falcatum (that induces red-rot disease of sugarcane). Direct plant growth promoting effects were tested on sugarcane plantlets in tissue culture under gnotobiotic conditions. All seven isolates provided significant increases in fresh and dry masses but only five strains increased shoot height.

Characterization of a phenazine and hexanoyl homoserine lactone producing Pseudomonas aurantiaca strain PB-St2, isolated from sugarcane stem.

A novel strain of fluorescent pseudomonad (PB-St2) was isolated from surface-sterilized stems of sugarcane grown in Pakistan. The bacterium was identified as Pseudomonas aurantiaca on the basis of 16S rRNA gene sequence analysis and results from physiological and biochemical characteristics carried out with API50 CH and QTS 24 bacterial identification kits. Assays using substrate specific media for enzymes revealed lipase and protease activities but cellulase, chitinase, or pectinase were not detected. The bacterium was unable to solubilize phosphate or produce indole acetic acid. However, it did produce HCN, siderophores, and homoserine lactones. In dual culture assays on agar, the bacterium showed antifungal activity against an important pathogen of sugarcane in Pakistan, namely Colletotrichum falcatum, as well as for pathogenic isolates of Fusarium oxysporium, F. lateritium but not against F. solani. The antifungal metabolites were identified using thin-layer chromatography, UV-spectra, and MALDI-TOFF spectra and shown to be phenazine-1-carboxylic acid (PCA), 2-hydroxyphenazine (2-OH-PHZ), and N-hexanoyl homoserine lactone (HHL) (assessed using only TLC data). The capacity of this bacterium to produce HCN and 2-OH-PHZ, as well as to inhibit the growth of C. falcatum, has not been previously reported.

Azospirillum zeae sp. nov., a diazotrophic bacterium isolated from rhizosphere soil of Zea mays.

Two free-living nitrogen-fixing bacterial strains, N6 and N7(T), were isolated from corn rhizosphere. A polyphasic taxonomic approach, including morphological characterization, Biolog analysis, DNA-DNA hybridization, and 16S rRNA, cpn60 and nifH gene sequence analysis, was taken to analyse the two strains. 16S rRNA gene sequence analysis indicated that strains N6 and N7(T) both belonged to the genus Azospirillum and were closely related to Azospirillum oryzae (98.7 and 98.8 % similarity, respectively) and Azospirillum lipoferum (97.5 and 97.6 % similarity, respectively). DNA-DNA hybridization of strains N6 and N7(T) showed reassociation values of 48 and 37 %, respectively, with A. oryzae and 43 % with A. lipoferum. Sequences of the nifH and cpn60 genes of both strains showed 99 and approximately 95 % similarity, respectively, with those of A. oryzae. Chemotaxonomic characteristics (Q-10 as quinone system, 18 : 1omega7c as major fatty acid) and G+C content of the DNA (67.6 mol%) were also similar to those of members of the genus Azospirillum. Gene sequences and Biolog and fatty acid analysis showed that strains N6 and N7(T) differed from the closely related species A. lipoferum and A. oryzae. On the basis of these results, it is proposed that these nitrogen-fixing strains represent a novel species. The name Azospirillum zeae sp. nov. is suggested, with N7(T) (=NCCB 100147(T)=LMG 23989(T)) as the type strain.

Sphingobacterium canadense sp. nov., an isolate from corn roots.

A free-living Gram-negative bacterial strain CR11(T) was isolated from corn roots. Polyphasic taxonomy was performed, including API20 NE and API50 CH bacterial identification kits, Biolog analysis, lipids and fatty acid analysis, DNA-DNA hybridization, 16S rRNA and cpn60 gene sequence analyses. 16S rRNA gene sequence analysis indicated that strain CR11(T) belonged to the genus Sphingobacterium and was closely related to Sphingobacterium multivorum IFO 14947(T) (98% similarity) and Sphingobacterium. thalpophilum ATCC 43320(T) (97% similarity). DNA-DNA hybridization showed 11% and 13% DNA re-association with S. multivorum LMG 8342(T) and S. thalpophilum LMG 11520(T), respectively. Major fatty acids (16:0, 15:0 iso and 17:0 iso 3-OH) and the G+C content of the DNA (40.5 mol%), were also similar to those of the genus Sphingobacterium. The predominant respiratory quinone was MK-7. In all analyses, including phenotypic characterization, this isolate was found to be different from the closely related species, S. multivorum and S. thalpophilum. On the basis of these results, this strain represents a new species within the genus Sphingobacterium. The name Sphingobacterium canadense sp. nov. is suggested and the type strain is CR11(T) (=NCCB 100125(T)=LMG 23727(T)).

Azospirillum canadense sp. nov., a nitrogen-fixing bacterium isolated from corn rhizosphere.

A free-living diazotrophic strain, DS2(T), was isolated from corn rhizosphere. Polyphasic taxonomy was performed including morphological characterization, Biolog analysis, and 16S rRNA, cpn60 and nifH gene sequence analyses. 16S rRNA gene sequence analysis indicated that strain DS2(T) was closely related to the genus Azospirillum (96 % similarity). Chemotaxonomic characteristics (DNA G+C content 67.9 mol%; Q-10 quinone system; major fatty acid 18 : 1omega7c) were also similar to those of the genus Azospirillum. In all the analyses, including phenotypic characterization using Biolog analysis and comparison of cellular fatty acids, this isolate was found to be different from the closely related species Azospirillum lipoferum, Azospirillum oryzae and Azospirillum brasilense. On the basis of these results, a novel species is proposed for this nitrogen-fixing strain. The name Azospirillum canadense sp. nov. is suggested with the type strain DS2(T) (=NCCB 100108(T)=LMG 23617(T)).

Isolation and identification of Gluconacetobacter azotocaptans from corn rhizosphere.

Six acetic acid producing, diazotrophic bacteria were isolated from soil adhering to corn roots. These isolates were shown to be Gluconacetobacter azotocaptans and they shared some features with G. johannae and G. diazotrophicus but differed on the basis of colony morphology on different media, use of carbon sources and use of l-amino acids as a nitrogen source. The species identity was confirmed using 16S rDNA sequence analysis, PCR amplification of 16S rRNA gene with species-specific primers and amplified rDNA restriction analysis. This is the first report of the presence of this bacteria on corn plants. Scope of the paper: This is the first report of the occurrence and association of Gluconacetobacter azotocaptans with corn.