Biolog GN Microplates Research Articles

New and dangerous bacterial disease of oleander Pseudomonas savastanoi pv. Nerii in greenhouses of ukraine

Object: Study of symptoms and etiology of oleander tuberculosis causative agents and determination of causes of their appearance in conditions of the closed ground of the greenhouses of Ukraine. Methods: Samples for research were selected in greenhouses of Botanical Garden named after acad. A. V. Fomin from different kinds and forms of oleander (Nerium oleander L.). The microbiological and phytopathological analysis was carried out after K. I. Beltyukova. Biolog GN MicroPlate system was used to identify isolated bacterial strains. Calculations for bacteria identification in relation to genus, species and other taxonomic units are based on the similarity index. Results: On oleander plants, the greatest disease progress shows itself on the branches of the second year of development. Up to 5 bacterial nodes of different sizes can be formed on one branch. Galls can be formed on the leaves both on the upper (62.1%) and lower (37.9%) sides. Bacteria actively spread with water from branches and leaves, especially when spraying plants from a hose. The infection degree of different varieties of oleander was not the same. Such varieties and forms as of Nerium oleander Album, Nerium oleander Album Plenum suffer from bacteriosis the most. Conclusions: For the first time in Ukraine tuberculosis Pseudomonas savastanoi pv. nerii was detected on oleander (Nerium oleander L.). The infection degree of different varieties of oleander was not the same. Nerium oleander Album, Nerium oleander Album Plenum, are the most severely leased, Nerium oleander Flavescens, Nerium oleander Lacteum are leased to the lesser extent. The spread of the disease is favored by some methods of this group of plants growing: pruning plants, branches, and leaves bruise.

Newly isolated alkalophilic Advenella species bioaugmented in activated sludge for high p-cresol removal

In this work, an alkalophilic bacterium (LVX-4) capable of using p-cresol as sole source of carbon and energy was screened and isolated from soil polluted by used oil. Phylogenetic (16S rRNA) and phenotypic characterization using Biolog GN microplates and API 20NE strips indicated that LVX-4 strain is a new Advenella species. It showed both the capability to degrade of p-cresol at high concentrations (750 mg/L) and to use p-cresol for growth in a pH from 7 to 10, although the optimum pH was 9. Moreover bioaugmentation of activated sludge with this strain lead to the complete removal of p-cresol in less than 100 h. This is the first study that shows the potential of Advenella sp. to be bioaugmented in activated sludge system for p-cresol biodegradation.

First Report of Dickeya solani Causing Soft Rot in Imported Bulbs of Hyacinthus orientalis in China

A bacterial pathogen, Dickeya solani, emerged as a major threat to potato (Solanum tuberosum) production in Europe in 2004 and has spread to many potato-growing regions via international trade. In December 2013, soft rot symptoms were observed in hyacinth (Hyacinthus orientalis) bulbs imported from the Netherlands into China at Ningbo Port. Diseased bulbs gave off an offensive odor. The base and internal parts of diseased bulbs rotted, and the margins of diseased tissues showed brown discoloration. Isolation on nutrient agar glucose (NAG) medium resulted in dominating colonies of characteristic "fried egg" morphology (1). One colony was chosen for further investigation and tentatively named "isolate 6165-3." Under microscopic visualization after gram stain, the cells of isolate 6165-3 were gram-negative, motile, and rod shaped. The isolate was then identified as a member of genus Dickeya using the Biolog GN microplate. The 16S rRNA, recA, and dnaX sequences of isolate 6165-3 were subsequently determined and deposited in GenBank with accession numbers KM405240, KM405241, and KM405242, sharing 99% (16S rRNA), 100% (recA), and 100% (dnaX) nucleotide identity with those of known D. solani isolates, respectively. By this means, the isolate 6165-3 was identified as D. solani (1,2). To confirm the pathogenicity of the isolate, four plants each of 30-day-old hyacinth, 14-day-old potato, and 60-day-old moth orchid (Phalaenopsis amabilis) were inoculated with suspensions of the isolate with a concentration of 108 CFU/ml in sterile water by stabbing. Plants were incubated in a climate chamber at 28°C during the day and 24°C during the night with a relative humidity of 93% and a photoperiod of 12/12 h. Plants inoculated with sterile water were included as negative controls. After 2 or 3 days, typical symptoms such as water-soaked lesions and soft rot developed around the inoculation point, while the negative controls remained symptomless. Koch's postulates were fulfilled by re-isolating bacteria from lesions, which had identical sequence and morphology characters with the inoculated isolate. This is the first report of intercepted D. solani on hyacinth bulbs imported from the Netherlands into China, indicating that D. solani can spread via hyacinth. Further spread of the pathogen into potato production might lead to immeasurable economic consequences for China.

Optimization of Cu, Hg and Cd removal by Enterobacter cloacae by ferric ammonium citrate precipitation

Iron precipitating organisms play a significant role in the formation of ferric hydroxide precipitate, which acts as strong adsorbent for toxic metal. In this respect four different iron precipitating cultures were isolated from Hutti gold mine surface winze water sample on citrate agar medium. The best isolate was screened out for metal removal study on the basis of fast visual iron precipitation. The selected isolate was identified as Enterobacter sp. based on routine biochemical tests and Biolog GN microplate results and as Enterobacter cloacae subsp. dissolvens by 16S rRNA gene sequence analysis (GenBank accession number EU429448). Influence of medium composition, medium initial pH, the influence of inoculum size, effect of various media and ferric ammonium citrate concentration were studied on metal removal in shake flask experiments. Under the optimized conditions studied, E. cloacae showed <TEX>$94{\pm}2$</TEX>, <TEX>$95{\pm}2$</TEX> and <TEX>$70{\pm}2%$</TEX> of cadmium, copper and mercury removal from a simulated waste in shake flask studies. In lab scale column reactor more than 85% of copper and mercury removal was achieved.

First Report of Pectobacterium carotovorum subsp. carotovorum Causing Soft Rot of Orostachys japonica in Korea.

Orostachys japonica (Maxim) A. Berger is an important traditional medicine in Korea. The extract of this plant has antioxidant activity and suppresses cancer cell proliferation (1). From summer through fall of 2012 and 2013, a high incidence (~10% to 30%) of disease outbreaks of all plants characterized by water-soaked lesions and soft rot with a stinky odor was observed in cultivated O. japonica around Uljin (36°59'35.04″N, 126°24'1.51″E), Korea. Water-soaked lesions were first observed on the stem base of plants. Subsequently, the plants collapsed, although the upper portion remained asymptomatic. Thereafter, the lesions expanded rapidly over the entire plant. To isolate potential pathogens from infected leaves, small sections (5 to 10 mm2) were excised from the margins of lesions. Ten bacteria were isolated from ten symptomatic plants. Three representative isolates from different symptomatic plants were used for identification and pathogenicity tests. Isolated bacteria were gram negative, pectolytic on crystal violet pectate agar, nonfluorescent on King's medium B, and elicited a hypersensitive response in tobacco plants. All isolates caused soft rot of potato tubers. These isolates also differed from isolates of Erwinia chrysanthemi (Ech) that they were insensitive to erythromycin and did not produce phosphatase. These isolates differed from known strains of E. carotovora subsp. atroseptica in that they did not produce reducing substances from sucrose (2). Use of the Biolog GN microplate and the Release 4.0 system identified the isolate as Pectobacterium carotovorum subsp. carotovorum with 81.2% similarity. The 16S rRNA of the isolated bacteria was amplified by PCR and sequenced as described by Weisburg et al. (3). A BLAST analysis for sequence similarity of the 16S rRNA region revealed 99% similarity with nucleotide sequences for P. carotovorum subsp. carotovorum isolates (KC790305, KC790280, JF926758, JX196705, and AB680074). The pathogenicity of three bacterial isolates was examined on three 2-year-old O. japonica plants by adding 50 μl of a bacterial suspension containing 108 CFU/ml when wounding the leaves with sterile needles. Ten control plants were inoculated with sterilized water. After inoculation, plants were maintained in a growth chamber at 25°C with relative humidity ranging from 80 to 90%. After 2 to 3 days, tissue discoloration, water-soaked lesions, and soft rot developed around the inoculation point. Severe symptoms of soft rot and darkening developed on leaves of inoculated plants within 3 to 5 days after inoculation. All controls remained healthy during these experiments. The bacterial strains re-isolated from the parts of the leaf showing the symptoms and identified as P. carotovorum subsp. carotovorum on the basis of the biochemical and physiological tests, as well as Biolog system. The results obtained for pathogenicity, Biolog analysis, and molecular data corresponded with those for P. carotovorum subsp. carotovorum. To our knowledge, this is the first report of the presence of P. carotovorum on O. japonica in Korea.

Isolation and Identification of Bacteria Micro Flora of White Shrimp, Litopenaeus vannamei, with Antagonistic Properties Against Vibrio Species

The emergence of antibiotic-resistant bacterial pathogen has led aquaculture attentions to the use of probiotics as an alternative to antibiotics. This study was conducted to isolate bacterial micro flora from digestive tract of healthy juvenile white shrimp, Litopenaeus vannamei based on antagonistic activity against shrimp pathogen, Vibrio parahaemolyticus. In this study, potential probiotic strains were isolated using replica plating method to screen the bacteria with antagonism properties. Six isolates were identified as Pseudomonas sp. using conventional biochemical tests and Biolog GN microplates. In addition three Vibrio species including V. parahaemolyticus, Vibrio alginolyticus and Vibrio cholerae were isolated from moribund shrimp and used as pathogen in this study. The pathogenicity of three Vibrio species was tested on shrimp L. vannamei by injecting 0.1 mL of 107 CFU mL-1 of each pathogen into the third abdominal segment. Among three Vibrio species only V. parahaemolyticus found to be highly virulent to shrimp with 43% cumulated mortality after 10 days. Subsequently, all Pseudomonas sp. isolates were tested for antibacterial activity against three Vibrio species using cross streak assay. Strong antibacterial activity was recorded for Pseudomonas sp. isolates number 5, 7, 15 and 30 against three pathogens. In addition a reasonable antibacterial activity was observed for isolates number 9 and 12. On the basis of great antibacterial activity of Pseudomonas sp. isolates these species may be considered for future challenge experiments in shrimp as a very promising alternative to the use of antibiotics.

Assessment of growth condition for a candidate probiotic, Shewanella algae, isolated from digestive system of a healthy juvenile Penaeus monodon

To conquer disease problem in shrimp industries, probiotic biocontrol is a well-known remedy now. The antagonistic ability of separated isolates from different parts of juvenile P. monodon was screened against shrimp Vibrio pathogens, V. parahaemolyticus and V. alginolyticus. The most antagonistic effect was observed for an isolate that primarily identified as Shewanella algae using conventional method followed by Biolog GN and GP microplates. Since adaptability to the host optimum cultural condition of the target organism is of the great importance, response surface methodology, with central composite design, was applied to assess log cell count response of S. algae in different incubation conditions. Therefore, four independent variables were assumed as: temperature (10–50°C), pH (6–10), NaCl concentration (0–50‰) and time (12–60 h). The coefficients of multiple determinations (R 2) for the responses log cell count of S. algae being 0.827. Temperature was the merely significant independent variable that affected the log cell count of the candidate probiotic. The candidate probiotic was revealed a reasonable growth response in quite wide range of temperature, pH and NaCl concentration in which the maximum levels were in same range of optimum shrimp culture.

First Report of Pectobacterium carotovorum Causing Soft Rot of Opium Poppy in Spain.

Opium poppy (Papaver somniferum L.) is an economically important pharmaceutical crop in Spain. Approximately 8,000 ha are cultivated annually in southern and central Spain. To improve yields, opium poppy cultivation is expanding to more humid or irrigated areas of Spain. In the springs of 2005 and 2007, we observed poppy plants with wilt and stem rot symptoms in irrigated, commercial opium poppy (cv. Nigrum) at Carmona and Écija, which are in Seville Province in southern Spain. Closer observations of affected plants revealed darkening and water soaking of the leaves and stem at the soil level, wilting of the lower leaves or the entire plant, and dark brown discoloration of stem vascular tissues and pith of the plant. Severely affected plants became completely rotten and collapsed. Isolations from symptomatic tissues on nutrient agar consistently yielded bacterial colonies. Pure cultures of four representative bacterial strains (two per each of affected field and year of isolation) were used in triplicate for a comparative analysis of biochemical and physiological traits in the 'carotovora' group of Erwinia (1) with known isolates of Pectobacterium carotovorum subsp. carotovorum, P. carotovorum subsp. atrosepticum, and Dickeya chrysanthemi. The isolates from opium poppy were gram negative, facultatively anaerobic, oxidase negative, catalase positive, grew at 37°C, and did not produce gas from D-glucose. Acid was produced from D(+)-arabinose, lactose, and D(+)-trehalose, but not from α-D-methylglucoside. In addition, the opium poppy bacterial isolates caused soft rot on potato slices within 24 h at 25°C and did not induce a hypersensitive reaction on tobacco leaves. Use of the Biolog GN microplates and the OmniLog ID 1.2 system identified the four poppy isolates as P. carotovorum (showing a 66.7% similarity with the subsp. carotovorum). Pathogenicity of poppy isolates was tested on three 6-week-old opium poppy plants (cv. Nigrum) by injecting 100 μl of a bacterial suspension containing 108 CFU/ml in the basal stem. Plants that served as controls were injected with sterile water. Plants were incubated in a growth chamber adjusted to 28°C, 90% relative humidity, and a 14-h photoperiod of fluorescent light of 360 μE·m-2·s-1. Severe symptoms of soft rot and darkening developed on stems of inoculated plants within 3 to 5 days after inoculation. No symptoms developed on control plants. Bacterial strains reisolated from inoculated plants were identified as P. carotovorum on the basis of the Biolog system, as well as biochemical and physiological characters. To our knowledge, this is the first report of P. carotovorum causing soft rot of commercial opium poppy crops in Spain and elsewhere. The presence of this bacterial pathogen to irrigated crops and humid areas may pose an important constraint on the yield of opium poppy crops in Spain.

Cultivable bacterial composition and BIOLOG catabolic diversity of biofilm communities developed on Phragmites australis

Biofilm samples formed on submerged young and old stems of reed, Phragmites australis (Cav.) Trin ex Steudel were taken during summer at different sites of Lake Velencei, Hungary. BIOLOG GN microplates were used to analyze the patterns of sole carbon source utilizations by microbial communities. From the carbon sources, carbohydrates and amino acids were preferred by all microbial communities. In the case of the old reed stem samples, higher number of carbohydrates, carboxylic acids and polymers were used than in young samples. Biofilm bacterial communities from the old reed samples of the nature conservation area of the lake used the highest number of (≥50% of the available) substrates. In principal component analysis (PCA), the metabolic potential of the microbial communities from the middle open water region of the lake showed the smallest variability. The variability within metabolic potential of the reed stem microbial communities from a given sampling site was the largest in the case of samples originating from the western, reed-covered nature conservation area. A total of 251 bacterial isolates obtained after serial dilutions and plating onto different media were characterized by traditional phenotypic tests. The strains showed high activities mainly in the hydrolysis of certain biopolymers (gelatine and casein). PCA was used to evaluate the phenotypic variability of strain groups of different sampling sites. The two open water regions were similar to each other, and separated from the western reed covered part of the lake. Similarly to the BIOLOG community-level physiological profiles, strain groups of the young and old reed stem samples originating from the nature conservation area had the largest metabolic potential. On the basis of 16S rDNA sequence analysis, 23 representative strains with different ARDRA patterns were identified. The cultivation-based investigations of bacterial diversity showed characteristic differences in the number of identified taxa in connection with the sampling sites. No characteristic differences could be observed according to medium or sample type (young, first year and more than 1-year old stems) among the identified species. 16S rDNA sequence comparisons resulted in the identification of the genera Aureobacterium, Arthrobacter, Kocuria, Microbacterium, Micrococcus, Rhodococcus, Bacillus, Marinibacillus, Rhodobacter, Defluvibacter, Pseudomonas, Klebsiella, Serratia and Aeromonas. The results of the cultivation-based and BIOLOG investigations revealed characteristic differences in the bacterial community composition and activities of the open water region and the reed covered nature conservation part of the lake.

PAH biodegradation in surfactant‐water systems based on the theory of cohesive energy density (CED)

AbstractThe influence of two non‐ionic surfactant additives (Triton X‐100 and Brij 35) on polycyclic aromatic hydrocarbon (PAH) biodegradation was evaluated using the chemical molecular interaction method, which is based on the theory of cohesive energy density (CED). The results indicated that PAHs have relatively higher CED values because aromatic compounds with labile π are more polarized and this encourages molecular attractions involving induced dipole force. Under different PAH‐surfactant compositions, similar CED values, which facilitated their intermolecular attractions through π–π electron interactions, gave rise to a similar biodegradation pattern. For example, when induced enzymes were able to target the same molecular bonding on the PAH and Triton X‐100, rapid degradation rates were observed in both systems. The distribution of the PAH in the monomer or micelle surfactant bulk affected the rate of PAH biodegradation. Quantification of the bacterial activity by applying specific oxygen uptake requirements (SOUR) identified an effect involving chemical molecular interaction. Changes in the physiological characteristics as measured by BioLog GN microplate measurements were identifiable and also could be related to the chemical structure of the dual substrates. Community‐level physiological profiling (CLPP) changed when PAH biodegradation took place in the presence of the different surfactants. Cluster analysis using matching coefficient and carbon degradation potential values also varied with the different PAH‐surfactant compositions. Copyright © 2007 Society of Chemical Industry

Bacterial diversity in soils around a lead and zinc mine

Five samples of soil collected from a lead and zinc mine were used to assess the effect of combined contamination of heavy metals on soil bacterial communities using a polyphasic approach including characterization of isolates by culture method, community level catabolic profiling in BIOLOG GN microplates, and genetic community fingerprinting by denaturing gradient gel electrophoresis of 16S rDNA fragments amplified by PCR from community DNA (PCR-DGGE). The structure of the bacterial community was affected to a certain extent by heavy metals. The PCR-DGGE analysis of 16S rRNA genes showed that there were significant differences in the structure of the microbial community among the soil samples, which were related to the contamination levels. The number of bacteria and the number of denaturing gradient gel electrophoresis (DGGE) bands in the soils increased with increasing distance from the lead and zinc mine tailing, whereas the concentration of lead (Pb) and cadmium (Cd) was decreased. Heavily polluted soils could be characterized by a community that differs from those of lightly polluted soils in richness and structure of dominating bacterial populations. The clustering analysis of the DGGE profiles showed that the bacteria in all the five samples of soil belonged to three clusters. The data from the BIOLOG analysis also showed the same result. This study showed that heavy metal contamination decreased both the biomass and diversity of the bacterial community in soil.

Genetic Characterization and Real-Time PCR Detection of Burkholderia glumae, a Newly Emerging Bacterial Pathogen of Rice in the United States.

Panicle blight of rice (Oryza sativa), caused by the bacterium Burkholderia glumae, is one of the most important new diseases in rice production areas of the southern United States. In this study, pathogenic strains were isolated from diseased panicles in Arkansas rice fields and examined using the Biolog GN microplate system, whole cell fatty acid methyl ester analysis (FAME), rep-polymerase chain reaction (PCR) genomic DNA fingerprinting, and 16S-23S ribosomal DNA (rDNA) intergenic transcribed spacer (ITS) sequence analysis. The B. glumae isolates from Arkansas can be divided into two major groups, but their genetic diversity was relatively low as revealed by 16S-23S rDNA ITS sequence analysis. Since no practical method existed, up to now, for testing the presence of B. glumae in rice seeds, we have developed in this study a real-time PCR method that is effective in detecting and identifying the pathogen in seed lots and in whole plants. The specific PCR primers were designed based on the 16S-23S rDNA ITS sequence of several representative isolates from Arkansas and Japan. This method is highly sensitive, rapid, and reliable, and has great potential for analyzing large numbers of samples without the need for DNA extraction or agarose gel electrophoresis. Although vertical resistance has not been observed among tested rice cultivars, LM-1 and Drew exhibited considerable resistance to B. glumae infection based on disease lesion size and the bacterial growth in planta.

Rapid effects of plant species diversity and identity on soil microbial communities in experimental grassland ecosystems

Changes in plant community structure, including the loss of plant diversity may affect soil microbial communities. To test this hypothesis, plant diversity and composition were experimentally varied in grassland plots cultivated with monocultures or mixtures of 2, 3 or 4 species. We tested the effects of monocultures versus mixtures and of plant species composition on culturable soil bacterial activity, number of substrates used and catabolic diversity, microbial biomass N, microbial respiration, and root biomass. These properties were all measured 10 months after seeding the experiment. Soil bacterial activity, number of substrates used and catabolic diversity were measured in the different plant communities using BIOLOG GN and GP microplates, which are redox-based tests measuring capacity of soil culturable bacteria to use a variety of organic substrates. Microbial biomass N, microbial respiration, and root biomass were insensitive to plant diversity. Culturable soil microbial activity, substrates used and diversity declined with declining plant diversity. Their activity, number of substrates used and diversity were significantly higher in plots with 3 and 4 plant species than in monocultures and in plots with 2 species. There was also an effect of plant species composition. Culturable soil microbial activity and diversity was higher in the four-species plant community than in any of the plant monocultures suggesting that the effect of plant diversity could not be explained by the presence of a particular plant species. Our results showed that changes in plant diversity and composition in grassland ecosystems lead to a rapid response of bacterial activity and diversity.

First report of Pseudomonas savastanoi pv. nerii on oleander in the Czech Republic

The bacterium Pseudomonas savastanoi pv. nerii was identified as the causal agent of parenchymatous galls on leaves of potted oleander plants grown at Brno, Moravia, in 2004. The plants had originated from cuttings made from firm shoots of a supposedly asymptomatic plant grown in and introduced from the Mediterranean region. The Biolog GN microplate system was used to identify the isolated bacterial strains. Successful inoculation of Nerium oleander seedlings proved the pathogenicity of the isolates. This is the first record of P. savastanoi pv. nerii in the Czech Republic. :

Characterization of soil health in an Italian polluted site by using microorganisms as bioindicators

The soil status inside and outside the chemical ACNA factory (Cengio, Italy) has been investigated by assessing chemical and biological parameters. Three sites, two (A and B) located inside the so-called high-risk zone and one (C) outside the factory were considered. Chemical analysis performed in the first metre of depth indicated the presence of inorganic pollution (copper and arsenic) in site A and organic (1,2,3,4-tetrachlorobenzene and 2,4-dichloroaniline) and inorganic (arsenic and mercury) contaminations in site B. Site C did not show any significant contamination and was considered as control. Biological analyses were performed by evaluating functional (substrate utilization pattern and enzymatic activities of the soil extracts) and structural (bacterial population density, composition and structure) diversity of the soil bacterial community as indicator of soil health. Moreover, 20 bacterial strains per site, able to grow on minimal medium added with 2,4-dichloroaniline as sole carbon source, were selected and identified. The metabolic profile of soil extracts, characterized by using Biolog GN MicroPlates, coupled with hierarchical clustering, showed a high similarity between the microbial communities isolated from sites A and B. The highest amounts of enzymatic activities were recorded in site C. Bacterial density in all sites did not differ significantly and a prevalence of r-strategist (fast growing and copiotrophic) bacteria was observed. The composition of the bacterial community showed a higher similarity between sites A and B than with site C. Only few bacterial species were common to the three sites: as a consequence, a prevalence of bacterial species typically associated to each site was observed. The fraction of bacteria able to grow on 2,4-dichloroaniline as sole carbon source on the total culturable bacteria in each site was lower than 0.1%. Bacterial identifications were performed by using Biolog MicroPlates. By considering the bacterial strains able to grow on minimal medium added with 2,4-dichloroaniline, a higher similarity between sites A and C than with B site was recorded. In conclusion, the presence of different chemical pollutants did not affect the bacterial density but rather the community structure. The characterization of soil chemical properties, metabolic fingerprinting, enzymatic activities and bacterial community structure could be useful tools for the evaluation of the soil health.

Proline-based modulation of 2,4-diacetylphloroglucinol and viable cell yields in cultures of Pseudomonas fluorescens wild-type and over-producing strains

The antifungal compound 2,4-diacetylphloroglucinol (DAPG) is produced in the rhizosphere of wheat by pseudomonad populations responsible for the natural biological control phenomenon known as "take-all decline." Studies were conducted to elucidate the impact of DAPG and its co-product 2,4,6-trihydroxyacetophenone (THA) on the production of Pseudomonas fluorescens for biological control. Increasing DAPG from 0.1 g/l to 0.5 g/l and THA from 0.05 g/l to 0.5 g/l significantly inhibited the growth and lowered the yield of viable bacteria in liquid cultures. On further examination of these metabolites applied in seed coatings, levels of DAPG and THA exceeding 0.05 mg/g seed significantly reduced wheat germination percentages. The three-way interaction of DAPG, THA, and culture medium ingredients was significant, and greatest seed germination loss (40-50%) was observed when 0.5 mg DAPG and 0.25 mg THA were combined in a coating of 0.5 ml culture medium per gram of seed. Based on the results of Biolog GN microplate, flask, and fermentor screens of C sources, proline was found to optimize the viable cell yields of the P. fluorescens strains tested. The combination of proline with glucose and urea as C and N sources in growth media could be optimized to minimize DAPG production and maximize the vitality of P. fluorescens Q8R1-96 and Q69c-80:miniTn5:phl20 (DAPG over-producer). In production cultures, the proline supply rate offers a potentially useful means to optimize the biological control agent yield and quality.

Characterization of cultivable heterotrophic bacterial communities in Cr-polluted and unpolluted soils using Biolog and ARDRA approaches

The bacterial communities of two soils with different chromium levels were characterized by Biolog carbon substrate utilization patterns and amplified 16S ribosomal DNA restriction analysis (ARDRA). For each bacterial community sample, cell suspensions containing 10,000 or 100 colony-forming units (CFU) were inoculated in each well of Biolog-GN microplates. The number of carbon compounds utilized by the bacterial community consisting of 100 CFU from unpolluted soil was significantly lower than that detected for the bacterial community consisting of 10,000 CFU. The size of inoculum did not substantially influence the percentage of carbon sources utilized by the Cr-polluted soil bacterial community. ARDRA approach was applied to about 100 bacterial isolates for each soil sample. A similar number of clusters for Gram-negative bacteria were found in both soils, but there were differences in percentages of isolates belonging to each group and specific genomic groups were found in each soil. Pseudomonas was the dominant taxon in both soils. Comparing the ARDRA clusters obtained from Gram-positive isolates it was evident that the culturable bacterial communities of Cr-polluted and unpolluted soils were dominated by the genus Arthrobacter and the genus Bacillus, respectively.

Heterotrophic Community‐Level Physiological Profiles of Domestic Wastewater Following Treatment by Small Constructed Subsurface Flow Wetlands

Seasonal treatment performance of small-surface flow wetlands was evaluated during their second operational year and compared with community-level physiological profiles (CLPP) of the heterotrophic bacterial community obtained from sole-carbon source utilization patterns in BIOLOG GN (Haywood, California) microplates. The CLPP patterns varied significantly by season, indicating reduced functional diversity in the heterotrophic community during warmer months of active plant growth (April through October). Principal component analysis (PCA) suggested that the functional community differed in planted versus unplanted wetlands during the growing season. Wetlands generally improved wastewater quality; however, treatment performance was reduced in the second year. Despite differences in the heterotrophic community suggested by CLPP, treatment efficiency with respect to removal of five-day biochemical oxygen demand or reduction in fecal indicator organisms generally was not significantly changed as a function of growing season or plant treatment.

Incidence of Antibiotic Resistance in Coliforms from Drinking Water and Their Identification Using the Biolog and the API Identification Systems

Antibiotic-resistant bacteria were common in samples collected from an intermittent water distribution system in Lebanon. Multiply-resistant isolates were also present and most commonly to amoxycillin, cephalexin and sulfamethoxazole/trimethoprim. The aminoglycosides (amikacin, gentamicin and kanamycin) were the most effective, with almost all tested strains showing susceptibility to these antimicrobial agents. Both the Biolog GN MicroPlates and the API 20E strips can be used for the identification of coliform bacteria isolated from potable water, but the outcome of the identification should be viewed with caution. 51% of isolates were assigned similar identities by both the Biolog MicroPlates and the API 20E strips. The similarity at the species level was lower (33%) compared to that at the genus level (67%). The identification of Escherichia coli strains, which represented 30% of all tested organisms, showed 95% similarity in the assigned genus and species using both identification schemes.

DNA Based Classification of Food Associated Enterobacteriaceae Previously Identified by Biolog GN Microplates

Enterobacteriaceae are frequently isolated from food products and it is essential to have methods for correct identification for both food hygiene and epidemiology reasons. Phenotypic methods are not always sufficient and have to be supplemented by DNA based methods. In the present study, 70 strains of Enterobacteriaceae derived from milk, fish and meat that had previously been identified by Biolog GN Microplates were genomically classified together with 15 representative type strains of species of Enterobacteriaceae. The field strains were dominated by Hafnia alvei, Serratia liquefaciens and Rahnella aquatilis. All strains were subjected to temporal temperature gel electrophoresis (TTGE) analysis using amplicons encompassing the V3, V4 and V9 variable regions of the 16S rRNA gene. Selected strains were analysed by ribotyping and partial 16S rDNA sequencing. The type strains were differentiated into 10 different TTGE groups. Two of the groups contained two type strains. Enterobacter aerogenes and Klebsiella planticola were not distinguished due to their identical sequences and Yersinia ruckeri and Citrobacter freundii showed the same migration pattern. The 70 food strains could be differentiated into 14 TTGE groups where 33 strains (47.1%) could be assigned to TTGE groups including type or reference strains. Rahnella strains were dispersed into three TTGE groups of which one group corresponded to Rahnella genomospecies 1 and one to genomospecies 3. The grouping of Rahnella strains was supported by ribotyping and phylogenetic analysis. TTGE can be a useful additional tool for identification on the species level of food related Enterobacteriaceae.