Partial 16S rDNA Sequence Analysis Research Articles

Isolation and characterization of Priestia megaterium KD7 for the biological control of pear fire blight.

Erwinia amylovora is a plant pathogen that causes fire blight disease in Rosaceous plants, such as pear and apple. To develop an effective biocontrol method to suppress E. amylovora, a total of 16 bacteria were isolated from pear orchard soil in China and screened for antagonistic activity in vitro. Among them, 9 isolates that exhibited antagonistic activity against E. amylovora were identified, including Bacillus atrophaeus, Priestia megaterium (previously known as Bacillus megaterium) and Serratia marcescens based on the partial 16S rDNA sequence analysis and similarity search. The plate confrontation experiments showed that strain 8 (P. megaterium strain KD7) had strong antagonistic activity against E. amylovora. The methanolic extract from cell-free supernatant of strain KD7 displayed high antibacterial activities against E. amylovora. Furthermore, the active compounds of strain KD7 were separated by thin layer chromatography (TLC) and the amino acids were detected by the presence of a spot with retention factor (Rf) of 0.71. Next, three lipopeptides were identified with high-resolution mass spectrometry (HRMS), including C13-surfactin [M+H]+ at m/z 1008.14, C15-surfactin [M+H]+ at m/z 1036.50, and C14-iturin A [M+H]+ at m/z 1043.17. Strain KD7 showed multiple antibiotic resistance, such as ampicillin, erythromycin, penicillin and tetracycline. The detached pear leaves, twigs and fruits assay showed that both protective and curative action with strain KD7 had the ability to decrease the development of fire blight. Taken together, P. megaterium strain KD7 is a potential effective biocontrol agent against fire blight.

Antagonistic potential of an Egyptian entomopathogenic nematode, compost and two native endophytic bacteria isolates against the root-knot nematode (Meloidogyne incognita) infecting potato under field conditions

BackgroundThe root-knot nematode, Meloidogyne spp., are one of the most dominant and dangerous group of pests. The deformations and discolorations make tubers unmarketable and/or of less quality. Therefore, management of Meloidogyne spp. becomes an obligatory challenge that warrants intervention. Biological control agents are the best alternative tools for controlling plant-parasitic nematodes that comply with the requirements of the development of the green agriculture and that reduce the reliance on these harmful chemicals. Therefore, this study aimed to evaluate the effectiveness of compost singly, and in combinations with the bio-agents Heterorhabditis bacteriophora, and two bacterial isolates Nem 212 and Nem 213 against the root-knot nematode Meloidogyne incognita infecting potato plants under field conditions.ResultsAmong 15 bacterial isolates (Nem205-Nem219) obtained from the rhizosphere of tomato and eggplant from Giza, Egypt, the two isolates (Nem 212 and Nem 213) were molecularly characterized based on the partial 16S rDNA sequencing analysis. These two bacterial isolates were deposited in the GenBank as Bacillus cereus Nem 212 and B. cereus Nem 213 and were tested against M. incognita J2s in vitro. Results showed that the cell filtrates of B. cereus Nem 212 and B. cereus Nem 213 gave the highest percentage of M. incognita J2s mortality (100%), after 48 h of the in vitro application. Moreover, all the applied treatments significantly suppressed the reproductive of M. incognita on potato plants and enhanced the potato crop yield under the field conditions. Compost enriched with B. cereus Nem 212 cell suspension was the most effective treatment. The combination between the bacterial cell suspension and the compost offered an increase in the disease curing and the potato plant growth and yield production, compared to the treatment with compost alone. The entomopathogenic nematode, Heterorhabditis bacteriophora, was relatively less effective in controlling M. incognita on potato, compared to B. cereus Nem 212 and/or B. cereus Nem 213 treatments. However, when compost was enriched with H. bacteriophora, it increased its capability to control the nematodes.ConclusionsThis study provides insights into the practical usage of EPNs H. bacteriophora, and the endophytic bacteria (B. cereus Nem 212 or B. cereus Nem 213) as biocontrol agents against M. incognita on potato plants. The application of compost enriched with the bacterial cell suspensions of either B. cereus Nem 212 or B. cereus Nem 213 and H. bacteriophora within Galleria mellonella cadaver proved efficient control of M. incognita infecting potato plants and improved the growth and yield of potato plants under field conditions.

Production Optimization and Biochemical Characterization of Cellulase from Geobacillus sp. KP43 Isolated from Hot Spring Water of Nepal.

This study is aimed at isolating and identifying a thermophilic cellulolytic bacterium from hot spring water and characterizing thermostable cellulase produced by the isolate. Enrichment and culture of water sample was used for isolation of bacterial strains and an isolate with highest cellulase activity was chosen for the production, partial purification, and biochemical characterization of the enzyme. Different staining techniques, enzymatic tests, and 16s ribosomal DNA (16s rDNA) gene sequencing were used for the identification of the isolate. The cellulase producing isolate was Gram positive, motile, and sporulated rod-shaped bacterium growing optimally between 55°C and 65°C. Based on partial 16s rDNA sequence analysis, the isolate was identified as Geobacillus sp. and was designated as Geobacillus sp. KP43. The cellulase enzyme production condition was optimized, and the product was partially purified and biochemically characterized. Optimum cellulase production was observed in 1% carboxymethyl cellulose (CMC) at 55°C. The molecular weight of the enzyme was found to be approximately 66 kDa on 12% SDS-PAGE analysis. Biochemical characterization of partially purified enzyme revealed the temperature optimum of 70°C and activity over a wide pH range. Further, cellulase activity was markedly stimulated by metal ion Fe2+. Apart from cellulases, the isolate also depicted good xylanase, cellobiase, and amylase activities. Thermophilic growth with a variety of extracellular enzyme activities at elevated temperature as well as in a wide pH range showed that the isolated bacteria, Geobacillus sp. KP43, can withstand the harsh environmental condition, which makes this organism suitable for enzyme production for various biotechnological and industrial applications.

Potential anti-biofilm producing marine actinomycetes isolated from sea sediments in Thailand

Marine actinomycetes are well known as novel resources of bioactive compounds including anti-biofilm agents with various therapeutic applications. In this study, one hundred and one marine actinomycetes were isolated from sea sediments collected from Andaman sea and the Gulf of Thailand. Morphological study and partial 16S rDNA sequence analysis revealed that most of isolates belong to genus Streptomyces (n = 90, 89.11%) and the rest are rare actinomycetes (n = 11, 10.89%) including Actinopolymorpha, Actinomycetospora, Dietzia Nocardiopsis, Micromonospora and Mycobacterium. Among them, ten and thirteen strains significantly reduced more than 60% of biofilm formation of Escherichia coli and Staphylococcus aureus, respectively. But no such activity was found against Pseudomonas aeruginosa. Further analysis of their supernatants indicated that most of marine actinomycetes secreted non-toxic anti-biofilm agents and some accompanied with protease activity. These are preferred characteristics of anti-biofilm substances that potentially restricted biofilm forming of E. coli and S. aureus and simultaneously prevented bacteria to develop resistance.

Bacterial Endophytes Isolated from Plants in Natural Oil Seep Soils with Chronic Hydrocarbon Contamination.

The bacterial endophytic communities of four plants growing abundantly in soils highly contaminated by hydrocarbons were analyzed through culturable and culture-independent means. Given their tolerance to the high levels of petroleum contamination at our study site, we sought evidence that Achillea millefolium, Solidago canadensis, Trifolium aureum, and Dactylis glomerata support high levels of hydrocarbon degrading endophytes. A total of 190 isolates were isolated from four plant species. The isolates were identified by partial 16S rDNA sequence analysis, with class Actinobacteria as the dominant group in all species except S. canadensis, which was dominated by Gammaproteobacteria. Microbacterium foliorum and Plantibacter flavus were present in all the plants, with M. foliorum showing predominance in D. glomerata and both endophytic bacterial species dominated T. aureum. More than 50% of the isolates demonstrated degradative capabilities for octanol, toluene, naphthalene, kerosene, or motor oil based on sole carbon source growth screens involving the reduction of tetrazolium dye. P. flavus isolates from all the sampled plants showed growth on all the petroleum hydrocarbons (PHCs) substrates tested. Mineralization of toluene and naphthalene was confirmed using gas-chromatography. 16S based terminal restriction fragment length polymorphism analysis revealed significant differences between the endophytic bacterial communities showing them to be plant host specific at this site. To our knowledge, this is the first account of the degradation potential of bacterial endophytes in these commonly occurring pioneer plants that were not previously known as phytoremediating plants.

Bioaugmentation with Endophytic Bacterium E6S Homologous to Achromobacter piechaudii Enhances Metal Rhizoaccumulation in Host Sedum plumbizincicola.

Application of hyperaccumulator-endophyte symbiotic systems is a potential approach to improve phytoremediation efficiency, since some beneficial endophytic bacteria are able to detoxify heavy metals, alter metal solubility in soil, and facilitate plant growth. The objective of this study was to isolate multi-metal resistant and plant beneficial endophytic bacteria and to evaluate their role in enhancing plant growth and metal accumulation/translocation. The metal resistant endophytic bacterial strain E6S was isolated from stems of the Zn/Cd hyperaccumulator plant Sedum plumbizincicola growing in metalliferous mine soils using Dworkin and Foster salts minimal agar medium with 1-aminocyclopropane-1-carboxylate (ACC) as the sole nitrogen source, and identified as homologous to Achromobacter piechaudii based on morphological and biochemical characteristics, partial 16S rDNA sequence and phylogenetic analysis. Strain E6S showed high level of resistance to various metals (Cd, Zn, and Pb). Besides utilizing ACC, strain E6S exhibited plant beneficial traits, such as solubilization of phosphate and production of indole-3-acetic acid. Inoculation with E6S significantly increased the bioavailability of Cd, Zn, and Pb in soil. In addition, bacterial cells bound considerable amounts of metal ions in the following order: Zn > Cd >Pb. Inoculation of E6S significantly stimulated plant biomass, uptake and bioaccumulation of Cd, Zn, and Pb. However, E6S greatly reduced the root to shoot translocation of Cd and Zn, indicating that bacterial inoculation assisted the host plant to uptake and store heavy metals in its root system. Inoculation with the endophytic bacterium E6S homologous to A. piechaudii can improve phytostabilization of metalliferous soils due to its effective ability to enhance in situ metal rhizoaccumulation in plants.

<i>Ex Situ</i> Biodegradation of Crude Oil Using Bacterial Isolates From Palm Oil Mill Effluent

Bioremediation of crude oil is quite unique, complex and relatively a slow process. They study area for this research work was a cottage industry located in Orumba North local government area of Anambra state, Nigeria. Evaluating the degradation potential of bacterial isolates from palm oil mill effluent on crude oil was conducted using standard methods, which includes screening, inoculums development and degradation studies to assess the most promising hydrocarbon degraders. Pooled samples collected from different points on the palm oil mill effluent disposal channels were inoculated mineral salt medium containing crude oil to assess degradation abilities of different bacterial species. Data indicated that these bacterial isolates were capable of thriving in the mineral salt medium between the pH range of 6.0-7.0 and bacterial proliferation were quantified by assessing the total viable count which ranged between 2 x 105-9 x 105 cfu/ml based on individual abilities to utilize crude oil as a sole carbon source. Cultural, morphological and biochemical characterizations were conducted on these isolates and Bergey’s manual of determinative bacteriology was used to suggest possible isolates, while molecular identification was done Centre for Agriculture and biosciences International (CABI), UK using partial 16S rDNA sequencing analysis and FASTA algorithm with prokaryote database from EBI. Escherichia fergosonii, Klebsiella variicola and Microccocus luteus were identified. Gas chromatographic analysis was applied to quantify the extent of degradation of crude oil by the isolates. All the isolated bacterial species displayed varying degree of crude hydrocarbon biodegradation.

Sulfate-reducing bacteria mediate thionation of diphenylarsinic acid under anaerobic conditions.

Diphenylarsinic acid (DPAA) is often found as a toxic intermediate metabolite of diphenylchloroarsine or diphenylcyanoarsine that were produced as chemical warfare agents and were buried in soil after the World Wars. In our previous study Guan et al. (J Hazard Mater 241-242:355-362, 2012), after application of sulfate and carbon sources, anaerobic transformation of DPAA in soil was enhanced with the production of diphenylthioarsinic acid (DPTAA) as a main metabolite. This study aimed to isolate and characterize anaerobic soil microorganisms responsible for the metabolism of DPAA. First, we obtained four microbial consortia capable of transforming DPAA to DPTAA at a high transformation rate of more than 80% after 4 weeks of incubation. Sequencing for the bacterial 16S rRNA gene clone libraries constructed from the consortia revealed that all the positive consortia contained Desulfotomaculum acetoxidans species. In contrast, the absence of dissimilatory sulfite reductase gene (dsrAB) which is unique to sulfate-reducing bacteria was confirmed in the negative consortia showing no DPAA reduction. Finally, strain DEA14 showing transformation of DPAA to DPTAA was isolated from one of the positive consortia. The isolate was assigned to D. acetoxidans based on the partial 16S rDNA sequence analysis. Thionation of DPAA was also carried out in a pure culture of a known sulfate-reducing bacterial strain, Desulfovibrio aerotolerans JCM 12613(T). These facts indicate that sulfate-reducing bacteria are microorganisms responsible for the transformation of DPAA to DPTAA under anaerobic conditions.

Biocompatible polyhydroxybutyrate (PHB) production by marine Vibrio azureus BTKB33 under submerged fermentation

Polyhydroxybutyrate (PHB) is known to have applications as medical implants and drug delivery carriers and is consequently in high demand. In the present study the possibilities of harnessing potential PHB-producing vibrios from marine sediments as a new source of PHB was investigated since marine environments are underexplored. Screening of polyhydroxyalkanoate (PHA)-producing vibrios from marine sediments was performed using a fluorescent plate assay followed by spectrophotometric analysis of liquid cultures. Out of 828 isolates, Vibrio sp. BTKB33 showed maximum PHA production of 0.21 g/L and PHA content of 193.33 mg/g of CDW. The strain was identified as Vibrio azureus based on phenotypic characterization and partial 16S rDNA sequence analysis. The strain also produced several industrial enzymes: amylase, caseinase, lipase, gelatinase, and DNase. The FTIR analysis of extracted PHA and its comparison with standard PHB indicated that the accumulated PHA is PHB. Bioprocess development studies for enhancing PHA production were carried out under submerged fermentation conditions. Optimal submerged fermentation conditions for enhanced intracellular accumulation of PHA production were found to be 35 °C, pH −7, 1.5 % NaCl concentration, agitation at 120 rpm, 12 h of inoculum age, 2.5 % initial inoculum concentration, and 36 h incubation along with supplementation of magnesium sulphate, glucose, and ammonium chloride. The PHA production after optimization was found to be increased to 0.48 g/L and PHA content to 426.88 mg/g of CDW, indicating a 2.28-fold increase in production. Results indicated that V. azureus BTKB33 has potential for industrial production of PHB.

Cellulolytic Bacteria Associated with the Gut of Dendroctonus armandi Larvae (Coleoptera: Curculionidae: Scolytinae)

The object of this study was to investigate the cellulolytic bacterial community in the intestine of the Chinese white pine beetle (Dendroctonus armandi) larvae. A total of 91 cellulolytic bacteria were isolated and assigned to 11 genotypes using amplified ribosomal DNA restriction analysis (ARDRA). Partial 16S rDNA sequence analysis and morphological tests were used to assign the 11 representative isolates. The results showed that the isolates belonged to α-Proteobacteria, γ-Proteobacteria and Firmicutes. Members of γ-Proteobacteria were the most frequently represented species and accounted for 73.6% of all the cellulolytic bacteria. The majority of cellulolytic bacteria in D. armandi larva gut were identified as Serratia and accounted for 49.5%, followed by Pseudomonas, which accounted for 22%. In addition, members of Bacillus, Brevundimonas, Paenibacillus, Pseudoxanthomonas, Methylobacterium and Sphingomonas were found in the D. armandi larva gut. Brevundimonas kwangchunensis, Brevundimonas vesicularis, Methylobacterium populi and Pseudoxanthomonas mexicana were reported to be cellulolytic for the first time in this study. Information generated from the present study might contribute towards understanding the relationship between bark beetle and its gut flora.

Improvement of Ni phytostabilization by inoculation of Ni resistant Bacillus megaterium SR28C

The use of metal tolerant plants for the phytostabilization of metal contaminated soil is an area of extensive research and development. In this study the effects of inoculation of Ni-resistant bacterial strains on phytostabilization potential of various plants, including Brassica juncea, Luffa cylindrica and Sorghum halepense, were studied. A Ni-resistant bacterial strain SR28C was isolated from a nickel rich serpentine soil and identified as Bacillus megaterium based on the morphological features, biochemical characteristics and partial 16S rDNA sequence analysis. The strain SR28C tolerated concentrations up to 1200 mg Ni L−1 on a Luria–Bertani (LB) agar medium. Besides, it showed high degree of resistance to various metals (Cu, Zn, Cd, Pb and Cr) and antibiotics (ampicillin, tetracycline, streptomycin, chloramphenicol, penicillin and kanamycin) tested. In addition, the strain bound considerable amounts of Ni in their resting cells. Besides, the strain exhibited the plant growth promoting traits, such as solubilization of phosphate and production of indole-3-acetic acid (IAA) in modified Pikovskayas medium and LB medium, respectively in the absence and presence of Ni. Considering such potential, the effects of SR28C on the growth and Ni accumulation of B. juncea, L. cylindrica and S. halepense, were assessed with different concentrations of Ni in soil. Inoculation of SR28C stimulated the biomass of the test plants grown in both Ni contaminated and non-contaminated soils. Further, SR28C alleviated the detrimental effects of Ni by reducing its uptake and translocation to the plants. This study suggested that the PGPB inoculant due to its intrinsic abilities of growth promotion and attenuation of the toxic effects of Ni could be exploited for phytostabilization of Ni contaminated site.

First Report of a Leaf Spot Disease of Golden Dewdrop (Duranta erecta) Caused by Pseudomonas cichorii and a Xanthomonas Species in Florida.

Duranta erecta (Verbenaceae) is used extensively in southern states as an ornamental shrub and has replaced boxwood as the most common short hedge accenting flower beds. Over the past 2 years, during warm wet periods, dark necrotic leaf spots have been observed on golden dewdrop plants in Florida. Isolations from these spots on Difco nutrient agar (NA) consistently yielded two types of bacterial colonies that were not always simultaneously present: 1) round butyrous, bright yellow and 2) flat cream-colored. Both were 2 mm in size after 48 h, gram-negative, and produced a hypersensitivity reaction (HR) on tobacco cv Hicks. Yellow colony bacteria were oxidase negative and non-fluorescent on King's medium B (KMB) (1). Cream-colored colony bacteria were oxidase positive and fluorescent on KMB. Three isolates of both types were selected for further study. Partial 16S rDNA sequencing and fatty acid analysis (FAME) MIDI Microbial Identification System (Microbial ID, Inc., Newark, DE) were used for identification of strains. The 16S rDNA primers used were; forward primer AMB14 5'-TCCAGCAATGCCGCGTGTGT-3' and reverse primer AMB13 5'-CATCCACCGCTTGTGCGGGT-3'. The PCR program consisted of an initial denaturing cycle of 95°C for 2 min followed by 30 cycles of denaturing at 95°C for 30 s, annealing at 60°C for 40 s and extension at 72°C for 1 min and one final extension at 72°C for 10 min. Using FAME analysis, the three strains of the cream-colored colony type were identified as Pseudomonas cichorii with high similarity values (0.907, 0.961, 0.819) and this corresponded well with the 16S rDNA sequences where 99% sequence identity was observed with P. cichorii strain JBC1 16S ribosomal RNA gene, partial sequence GenBank Accession No. JF951725. Two of the three yellow colony strains were identified by MIDI FAME profiles as Xanthomonas axonopodis pv. manihotis with similarity coefficients of 0.767 and 0.826. The third strain had a low similarity match to X. a. pv. carotae (0.541). The 16S rDNA sequencing of these strains showed 98% sequence identity to X. citri subsp. citri strain SA1 16S ribosomal RNA gene only, partial sequence identity JQ890091.1, thus indicating a possible undescribed X. axonopodis pathovar. To satisfy Koch's postulates, three golden dewdrop 'Golden Mound' plants were sprayed with a suspension of 108 CFU/ml of a 2-day NA culture of each strain, bagged for 24 h to raise humidity, and placed in a greenhouse. A strain of P. cichorii (P409) isolated from chrysanthemum was used as a positive control when comparing cream-colored strains. A saline buffered control was used as a negative control. Within 3 weeks, leaf spots developed on plants sprayed with each of the six strains, including positive control strain of P. cichorii. Reisolations yielded the same type of colony as the originally inoculated strain. Inoculation experiments were repeated three times with a minimum of three plants per isolate with similar results. To our knowledge, this is the first report in the United States of bacterial leaf spot caused by P. cichorii and X. axonopodis on golden dewdrop. An earlier morphological and physiological description of a Xanthomonas sp. was done on Duranta in India in 1962 (2). Due to the difficulty in controlling bacterial diseases and the popularity of Duranta spp. in the landscape, these diseases may present a problem in ornamental trade.

Isolation and Identification of Cellulolytic Bacteria from the Gut of Holotrichia parallela Larvae (Coleoptera: Scarabaeidae)

In this study, 207 strains of aerobic and facultatively anaerobic cellulolytic bacteria were isolated from the gut of Holotrichia parallela larvae. These bacterial isolates were assigned to 21 genotypes by amplified ribosomal DNA restriction analysis (ARDRA). A partial 16S rDNA sequence analysis and standard biochemical and physiological tests were used for the assignment of the 21 representative isolates. Our results show that the cellulolytic bacterial community is dominated by the Proteobacteria (70.05%), followed by the Actinobacteria (24.15%), the Firmicutes (4.35%), and the Bacteroidetes (1.45%). At the genus level, Gram-negative bacteria including Pseudomonas, Ochrobactrum, Rhizobium, Cellulosimicrobium, and Microbacterium were the predominant groups, but members of Bacillus, Dyadobacter, Siphonobacter, Paracoccus, Kaistia, Devosia, Labrys, Ensifer, Variovorax, Shinella, Citrobacter, and Stenotrophomonas were also found. Furthermore, our results suggest that a significant amount of bacterial diversity exists among the cellulolytic bacteria, and that Siphonobacter aquaeclarae, Cellulosimicrobium funkei, Paracoccus sulfuroxidans, Ochrobactrum cytisi, Ochrobactrum haematophilum, Kaistia adipata, Devosia riboflavina, Labrys neptuniae, Ensifer adhaerens, Shinella zoogloeoides, Citrobacter freundii, and Pseudomonas nitroreducens are reported to be cellulolytic for the first time in this study. Our results indicate that the scarab gut is an attractive source for the study of novel cellulolytic microorganisms and enzymes useful for cellulose degradation.

Characterization of methomyl and carbofuran degrading-bacteria from soils of horticultural farms in Rift Valley and Central Kenya

The use of pesticides is very critical in protecting the farmers’ investment in seeds, fertilizer and labour since they provide a sure cover from damage by pests. The use of pesticides is therefore inevitable and the environmental pollution due to pesticides and their residues will continue to be a challenge. In this study, bacterial strains capable of degrading methomyl (S-methyl-N-[(methylcarbamoyl) oxy]-thioacetimidate) and carbofuran (2, 3-dihydro-2, 2-dimethyl-7-benzofuranyl methylcarbamate) were isolated from soils sampled from horticultural farms with a history of pesticide usage. High pressure liquid chromatography was used to monitor biodegradation of both methomyl and carbofuran using reference standards and acetonitrile and water as mobile phases. Partial 16S rDNA sequence analysis indicated that the carbofuran-degrading strains were closely related to members of the genus Pseudomonas and Alcaligenes while the methomyl degrading strains were closely related to members of the genus Flavobacterium and Alcaligenes . The morphological and biochemical characteristics of the isolates also confirmed the phylogenetic signature. The study established that the activities of the esterase and phosphatase enzymes correlated well with biodegradative capability and recommends possible application of the isolates in the in vivo bioremediation of pesticide contaminated soils. Key words : Pesticides, carbofuran, methomyl, biodegradation, bacteria.

Production and Biochemestry-Molecular Analysis of Microbial Community Fermenting Whey as a Potential Probiotic for Use Animals

The aims of this work were: To achieve a simple and low cost propagation of potential probiotic agents using plain whey as a culture medium, study the diversity of the members of the bacterial community (MC) in plain whey and to evaluate the probiotic capacity of this MC. After a systematic selection of agents according to their growing capacity in whey, the constituted MC was considered as a unit. Biochemical characterization of the lactic acid bacteria were performed using the API system. Molecular characterization of the lactic acid bacteria was realized using AFLPTM DNA-fingerprinting, partial 16S rDNA sequence analysis and PCR-denaturing gradient gel electrophoresis (PCR-DGGE). The physiological characterization of yeast was determined with the automated microplate method Allev/Biolomics and using yeast characterization system based on standard taxonomic criteria. The identification molecular was realized by PCR-fingerprinting. The resistance of MC to pH and bile salts were evaluated. The MC was composed of agents from different separated Dominium like Bacteria (Lactobacillum) and Eukaria (yeast). They are multispecies and also multistrain assuring high biodiversity. The MC grew at low pH and different concentrations bile salts.

Antifungal activity and genetic diversity of selected Pseudomonas spp. from maize rhizosphere in Vojvodina

Antibiotic production by plant-associated microorganisms represents an environmentally compatible method of disease control in agriculture. However, a vide application of bacterial strains needs careful selection and genetic characterization. In this investigation, selected Pseudomonas strains were characterized by rep-PCR methods using ERIC and (GTG)5 primers, and partial 16S rDNA sequence analysis. None of strains produced homoserine lactones (C4, C6, C8) as quorum sensing signal molecules. Very poor production of phenazines and no significant fungal inhibition was observed for PS4 and PS6 strains. High amount of phenazines were produced by Pseudomonas sp. strain PS2, which inhibited mycelial growth of 10 phytopatogenic fungi in percent of 25 (Verticillium sp.) to 65 (Fusarium equiseti). Genetic characterization of the Pseudomonas sp. PS2 and evaluation of phenazines production, as the main trait for growth inhibition of phytopathogenic fungi, will allow its application as a biosafe PGPR for field experiments of plant disease control.

Biological characterization of two marine Bdellovibrio-and-like organisms isolated from Daya bay of Shenzhen, China and their application in the elimination of Vibrio parahaemolyticus in oyster

Bdellovibrio-and-like organisms (BALOs) are a group of highly motile delta-proteobacteria that prey on other gram-negative bacteria. However, nothing is known of the application potential of marine BALOs in safeguarding seafood safety. Here, biological characterization of two marine BALOs strains and their application in the elimination of Vibrio parahaemolyticus in oyster (Crassostrea ariakensis) at the laboratory scale were investigated. BALOs strains BDH12 and BDHSH06 were isolated from sediment of Daya bay in Shenzhen of China, with Shewanella putrefaciens strain 12 and V. parahaemolyticus strain SH06 as preys, respectively, when using double layer agar technique. They were identified as BALOs morphologically by transmission electron microscopy, while partial 16S rDNA sequencing analysis revealed that they showed no close relationships with members of the known genera Bdellovibrio, Bacteriolyticum, Bacteriovorax, or Peredibacter. Biological characterizations revealed that both strains had the optimal pH, salinity and temperature at 7.2, 3% and 30 °C, correspondingly. They could not utilize autoclaved, dead cells as hosts. Prey range analysis revealed that individually, BDH12 and BDHSH06 lysed 82.5% (47 strains) and 84.2% (48 strains) of the total 57 preys tested respectively. In combination, they lysed 98.2% (56 of 57) strains. All strains of V. parahaemolyticus, Vibrio cholerae and Vibrio alginolyticus tested could be lysed by both strains. A 7-day laboratory-scale V. parahaemolyticus elimination experiment in oyster showed that in the control, the cell counts of total vibrios and V. parahaemolyticus strain Vp plus in water and in oyster intestines were on the rise, whereas in the BALOs treated groups, their numbers were down from 8.09±0.05 log CFU/ml and 8.02±0.04 log CFU/ml to 2.39±0.01 log CFU/ml and 2.33±0.01 log CFU/ml, respectively. The same patterns could also be observed in oyster intestines. Results of this study indicate the feasibility of using BALOs to biologically control or even eliminate V. parahaemolyticus in seafood oyster.

Halomonas sp. nov., an EPA-Producing Mesophilic Marine Isolate from the Indian Ocean

Marine samples from the Indian Ocean were used to isolate and characterize the organisms with respect to their fatty acid profiles. Six mesophilic isolates (MBRI 6, MBRI 8, MBRI 9, MBRI 10, MBRI 12 and MBRI 13) were obtained from three different water samples. They were i) Gram-negative, ii) catalase positive, iii) produced acid from glucose and maltose, iv) tolerated 5 to 15% NaCI v) except MBRI 9, showed pH tolerance in the range of 5.0 to 9.0 with optimum pH 7.0 to 8.0 v) grew well at 30 degrees C and were able to grow in the range of 15 to 45 degrees C. EPA, an essential omega-3 fatty acid, was produced by these isolates in the range of 12 to 60% at 30 degrees C. MBRI 12 was found to be a potential source as it produced 60% EPA. This isolate was further identified by partial 16S rDNA sequencing and phylogenetic analysis revealed that the strain belonged to Gammaproteobacteria and was closely related to Halomonas bolviensis (96% sequence similarity, 570 bp). Thus a new genus of Halomonas may be included in earlier reported EPA- producing prokaryotic genera affiliated to the Gammaproteobacteria.

The protective effect of Bdellovibrio-and-like organisms (BALO) on tilapia fish fillets against Salmonella enterica ssp. enterica serovar Typhimurium

To characterize freshwater Bdellovibrio-and-like organisms (BALO) isolated in China and examine their potential in controlling growth of Salmonella enterica ssp. enterica serovar Typhimurium on tilapia fillets. Four BALO isolates were recovered from a pond in Yanzhou of Shandong province, China, with Salm. Typhimurium as prey using double-layer agar method. Partial 16S rDNA sequencing analysis identified BD2GL, BD5GL and BDXGL as Bdellovibrio bacteriovorus and BD2GS as a Peredibacter sp. Lysis experiments on 32 potentially pathogenic strains revealed that BALO lysis rates are in the range of 56.3-65.6%. On the five Salmonella strains tested, only BD2GS achieved 100% lysis rate. When applied on tilapia fillets against Salm. Typhimurium, BD2GS showed its growth control potential. Cell increments of Salm. Typhimurium were significantly lower (P < 0.05) in two BD2GS-treated groups compared to control and low-dose group (BD2GS to prey ratio, 1 : 1) was more effective than high-dose group (BD2GS to prey ratio, 10 : 1) in controlling Salm. Typhimurium growth. Results of this study indicated that BD2GS could control Salm. Typhimurium growth on tilapia fillets. BALO could be used as a live protective culture in controlling bacterial growth and ensure food safety.

Microbial community dynamics in anaerobic bioreactors and algal tanks treating piggery wastewater

Integrated biosystem is becoming a major aspect of wastewater management practice. Microbial communities in piggery wastewater sampled from anaerobic (thermophilic and mesophilic) and aerobic digesters (algal tanks) during waste remediation were analyzed by culture-independent techniques based on polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The use of Muyzer's 314F-GC, 518R bacterial primers, and archaeal A934F, 1309R primers followed by partial 16s rDNA sequence analysis of the main bands from DGGE revealed the presence of unknown and as yet uncultured microorganisms but also showed functional and ecologically significant denitrifying, acetogenic bacteria along with autotrophic, hydrogenotrophic, and acetoclastic methanogen archaea. Thermophilic digesters were dominated by gamma-Proteobacteria, Methanothermobacter sp., while mesophilic digesters showed dominance by Firmicutes, uncultured bacteria, Methanosarcina, and Methanoculleus genera. Under aerobic conditions within algal tanks, pH rose from 7.17 to 9.32, with a significant decrease in total ammonia nitrogen, chemical oxygen demand, and soluble phosphorus levels. PCR-DGGE proved a useful tool for investigating the dynamics of microbial community in the bio-processing of piggery wastewater. Knowledge of the microbial communities involved in digestion of piggery wastewater will allow optimization of integrated biosystem by removing the main pollutants like inorganic ammonium-nitrogen, phosphorus, and pathogens from intensive farming system.