Taxonomic heterogeneity of the collection strains of fluorescent pseudomonads

Typing of 13 strains of fluorescent pseudomonads from the Belarusian collection of nonpathogenic microorganisms (BIM) by ERIC-PCR and BOX-PCR revealed high level of genetic heterogeneity in bacteria, most of which have been previously identified as Pseudomonas fluorescens according to the classical scheme. Evaluation of the similarities of the 16S rRNA gene sequences and their phylogenetic analysis excluded affiliation of the bacteria under study within the same species and allowed them to be distributed within three relatively distant clusters of the genus Pseudomonas phylogenetic tree. While eight strains fell into the phylogenetic group of P. fluorescens, only one of them could be identified as P. fluorescens. Four strains clustered within the P. vancouverensis phylogenetic group, formed by new species, which have been described mainly according to the evaluation of genome relationships. One bacterium was related to a stable branch that did not contain any type strains of the known Pseudomonas spp. These results indicate taxonomic heterogeneity of collection strains of the fluorescent pseudomonads and demonstrate the necessity of identification of them considering the requirements of phylogenetic bacterial taxonomy.     

A phenotypic and genetic study of sucrose nonfermenting strains ofVibrio mimicus andVibrio cholerae

Sixty-six strains unable to ferment sucrose and resemblingVibrio mimicus andV. cholerae were submitted to an extensive phenotypic characterization. DNA-DNA homology among selected strains and the type strain ofV. cholerae was studied by the S1 endonuclease method. Seven sucrose-negative strains were shown to have the phenotypic properties of and a high percentage DNA relatedness toV. cholerae and a low level of homology withV. mimicus. Eight luminescent strains phenotypically most closely resembledV. mimicus; however, two of these were shown to have a high level of DNA homology withV. cholerae and a low level of relatedness toV. mimicus. A single strain was found to be phenotypically and genetically unrelated to eitherV. cholerae orV. mimicus and may represent a new species. The remaining strains were phenotypically shown to beV. mimicus, and selected strains were shown to have a high percentage DNA homology withV. mimicus and a low level of homology withV. cholerae. Problems associated with the identification of these strains and differential traits are discussed.     

Dehydrogenase activity of pseudomonas species

Single-strain cultures of Pseudomonas fragi, P. fluorescens, P. putrefaciens, and strains of two marine species, Pseudomonas type I and Pseudomonas type II, were found to be capable of reducing added acetaldehyde, proprionaldehyde, and butyraldehyde to the corresponding alcohols at 21 C. All species studied reduced propionaldehyde at 6 C. P. fragi, Pseudomonas type I, and Pseudomonas type II reduced butanone at 6 and 21 C. P. fragi and Pseudomonas type II reduced acetone at 21 C. Dehydrogenase activity was found in some cultures in which growth was not evident. Under aerobic conditions, a strain of P. fragi reduced added propionaldehyde to n-propanol quantitatively within 36 hr at 21 C.     

Preliminary Studies of Fluorescent Pseudomonads Capable of Growth at 41 C in Swimming Pool Waters

During the summer of 1973 cultures of Pseudomonas aeruginosa and other fluorescent pseudomonads capable of growth at 41 C were obtained from swimming pool waters at a training center for the mentally retarded. Isolates were subjected to selected physiological tests, pyocine typing, and immunotyping. High counts of P. aeruginosa or other fluorescent pseudomonads consisted mainly of single predominant types. Both P. aeruginosa strains and unidentified fluorescent Pseudomonas strains predominated in pool waters at different times.     

Fluorescence Spectroscopy as a Promising Tool for a Polyphasic Approach to Pseudomonad Taxonomy

Fluorescence spectroscopy is an emerging tool for the analysis of biomolecules from complex matrices. We explored the potentialities of the method for the pseudomonad taxonomic purpose at the genus and species level. Emission spectra of three intrinsic fluorophores (namely, NADH, tryptophan, and the complex of aromatic amino acids and nucleic acid) were collected from whole bacterial cells. Their comparisons were performed through principal component analysis and factorial discriminant analysis. Reference strains from the Xanthomonas, Stenotrophomonas, Burkholderia, and Pseudomonas genera were well separated, with sensitivity and selectivity higher than 90%. At the species level, P. lundensis, P. taetrolens, P. fragi, P. chlororaphis, and P. stutzeri were also well separated, in a distant group, from P. putida, P. pseudoalcaligenes, and P. fluorescens. These results are in agreement with the generally admitted rRNA and DNA bacterial homology grouping but they also provide additional information about strain relatedness. In the case of environmental isolates, the method allows good discrimination, even for strains for which ambiguity still remained after PCR and API 20NE identification. Rapid, easy to perform, and low cost, fluorescence spectroscopy provides substantial information on cell components. Statistical analysis of collected data allows in-depth comparison of strains. Our results strongly support the view that fluorescence spectroscopy fingerprinting can be used as a powerful tool in a polyphasic approach to pseudomonad taxonomy.     

Characterization of Pseudomonas spp. Associated with Spoilage of Gilt-Head Sea Bream Stored under Various Conditions

The population dynamics of pseudomonads in gilt-head sea bream Mediterranean fish (Sparus aurata) stored under different conditions were studied. Phenotypic analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins were performed to identify a total of 106 Pseudomonas strains isolated from S. aurata stored under different temperatures (at 0, 10, and 20°C) and packaging conditions (air and a modified atmosphere of 40% CO₂-30% N₂-30% O₂). Pseudomonas lundensis was the predominant species, followed by Pseudomonas fluorescens, while Pseudomonas fragi and Pseudomonas putida were detected less frequently. Fluorescent Pseudomonas strains dominated under air conditions, while proteolytic and less lipolytic strains dominated under modified-atmosphere packaging. Different storage conditions appear to govern the selection of pseudomonads in gilt-head sea bream fish.     

Potential for improving pea production by co-inoculation with fluorescent Pseudomonas and Rhizobium

Seed bacterization with five plant growth promoting fluorescent Pseudomonas strains isolated from Indian and Swedish soils and three Rhizobium leguminosarumbiovar viceae strains isolated from Swedish soils were shown to promote plant growth in Pisum sativum L. cv. Capella. Co-inoculation of the fluorescent pseudomonads and Rhizobium improved plant growth in terms of shoot height, root length and dry weight. Both the fluorescent pseudomonads and Rhizobium were shown to exhibit a wide range of antifungal activity against pathogens specific to pea. Seed bacterization with plant growth promoting strains alone and together with a rhizobial isolate, R 361-27 reduced the number of infected peas grown in Fusarium oxysporum infested soils. We found that the introduced organisms were able to colonize the roots, which was confirmed using immunofluorescence staining and drug resistant mutant strains. In a synthetic culture medium, all the plant growth promoting fluorescent pseudomonads strains produced siderophores, which shown to express antifungal and antibacterial activity. Our results suggest the potential use of these bacteria to induce plant growth and disease suppression in sustainable agriculture production systems.     

Characterization of esculin-positive<Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> strains isolated from an underground brook

A group of sixteen esculin-positive fluorescent pseudomonads isolated from an underground brook flowing through a cave complex was characterized by biotyping, multiple enzyme restriction fragment length polymorphism analysis of 16S rDNA (MERFLP), ribotyping and whole-cell fatty-acid methyl-esters analysis (FAME). All strains were phenotypically close to Pseudomonas fluorescens, but they revealed high biochemical variability as well as some reactions atypical for P. fluorescens species. Because identification of pseudomonads by of biochemical testing is often unclear, further techniques were employed. Fingerprints obtained by MERFLP clearly showed that all strains represent P. fluorescens species. Ribotyping separated the strains analyzed into four groups corresponding almost completely (with the exception of one strain) to the clustering based on biochemical profiles. FAME analysis grouped all the strains into one cluster together with the P. putida (biotype A, B), P. chlororaphis and P. fluorescens biotype F representatives, but differentiated them from other FAME profiles of all pseudomonads included in the standard library TSBA 40 provided by MIDI, Inc.     

Determination of the sensitivity of bacteria to barium ions,a taxonomic marker of the genus <Emphasis Type="Italic">Pseudomonas</Emphasis>

Comparative efficacy of the determination of the sensitivity of bacterial cells to barium ions was evaluated on a synthetic nutrient medium, FMH agar, Mueller-Hinton agar, and AGV agar. The synthetic nutrient medium developed for this study contained L-proline and L-glutamine as the sole nitrogen and carbon source, which promoted growth of all Pseudomonas strains and ensured the minimal level of barium binding. The sensitivity of 80 strains belonging to 11 Pseudomonas species, including the type strains, as well as of 80 strains of 22 other bacterial species, was studied. The sensitivity of bacteria to barium ions was determined by using serial dilutions of barium chloride in the nutrient medium. The highest level of analytical sensitivity of pseudomonads to barium ions was determined on the synthetic nutrient medium: the minimal inhibitory concentration (MIC) values of barium chloride ranged from 0.5 to 6 g/L, the MIC₉₀ value was 2 g/L. At the same time, 86.1% of all strains of fluorescent Pseudomonas species produced fluorescein on the control BaCl₂-free synthetic nutrient medium. For representatives of other genera grown on all the studied nutrient media, the MIC values of barium chloride ranged from 20 to 50 g/L. The proposed method for determination of the sensitivity of bacteria to barium ions using the synthetic nutrient medium with 6 g/L of barium chloride as a criterion for the classification of barium-sensitive strains to the genus Pseudomonas is suitable for standardization.     

The phylogeny of fluorescent pseudomonads in an unflooded rice paddy soil

The purpose of this research was to determine the diversity and distribution of fluorescent pseudomonads in an unflooded rice paddy soil. A region of the 16S ribosomal RNA gene from isolates was amplified using PCR and subsequently analysed by sequence analysis for bacterial identification and phylogenetic classification. A total of 117 fluorescent pseudomonads, representing between 10 and 21 species, were isolated from two sampling sites within the same paddy (designated as soils C and S). The isolates were found to be ≥96% homologous with known sequences, and were most closely related to the followingPseudomonas species:P. antarctica, P. costantini, P. extremorientalis, P. frederiksbergensis, P. kilonensis, P. koreensis, P. lini, P. mandelii, P. poae, P. rhodesiae, andP. veronii. Of these matches, the bulk of the isolates (49%) were affiliated withP. mandelii. In soils C and S, phylogenetic analysis revealed that 35 and 82 isolates co-clustered with 39 and 59% of 66 fluorescent pseudomonad type strains, respectively.     

Characterization of Pseudomonas fuscovaginae and Differentiation from Other Fluorescent Pseudomonads Occurring on Rice in Burundi

In various pathogenicity, serological, physiological and biochemical tests, performed in Belgium and Japan, the Pseudomonas fuscovaginae strains associated above 1,350 m elevation in Burundi with sheath brown rot of rice, rusty seed and black, stripes on seedlings, were found to be similar to reference strains of this pathogen from Japan. The same bacteria was detected on rice seeds imported from Asia to Burundi. Beside the serological characteristics, P. fuscovaginae can be differentiated from other oxidase and arginine dihydrolase positive non-pathogenic fluorescent pseudomonads, also isolated from lesions on rice seedlings, by the simultaneous occurrence of no production of 2-ketogluconate, acid production from trehalose, but not from inositol. Occasionally, other symptoms inducing, oxidase positive, fluorescent pseudomonads, different from any described species, were isolated from rice seedlings and sheath rot in Burundi.     

Lack of genetic relatedness betweenYersinia philomiragia (the “philomiragia” bacterium) andYersinia species

Four strains ofYersinia philomiragia were studied biochemically and genetically. They showed a high degree of phenotypic similarity and formed a homogenous DNA relatedness group. We could not demonstrate any significant DNA relatedness between this group and otherYersinia species, other Enterobacteriaceae, orPasteurella multocida. Y. philomiragia is phenotypically quite different from other yersiniae and Enterobacteriaceae. These DNA and phenotypic data indicate thatY. philomiragia is not a member of the genusYersinia or a member of the family Enterobacteriaceae. It is suggested that, pending further study, this organism be referred to as the “Philomiragia” bacterium.     

Phylogenetically closely related pseudomonads isolated from arthropods exhibit differential insect-killing abilities and genetic variations in insecticidal factors

Strains belonging to the Pseudomonas protegens and Pseudomonas chlororaphis species are able to control soilborne plant pathogens and to kill pest insects by producing virulence factors such as toxins, chitinases, antimicrobials or two-partner secretion systems. Most insecticidal Pseudomonas described so far were isolated from roots or soil. It is unknown whether these bacteria naturally occur in arthropods and how they interact with them. Therefore, we isolated P. protegens and P. chlororaphis from various healthy insects and myriapods, roots and soil collected in an agricultural field and a neighbouring grassland. The isolates were compared for insect killing, pathogen suppression and host colonization abilities. Our results indicate that neither the origin of isolation nor the phylogenetic position mirror the degree of insecticidal activity. Pseudomonas protegens strains appeared homogeneous regarding phylogeny, biocontrol and insecticidal capabilities, whereas P. chlororaphis strains were phylogenetically and phenotypically more heterogenous. A phenotypic and genomic analysis of five closely related P. chlororaphis isolates displaying varying levels of insecticidal activity revealed variations in genes encoding insecticidal factors that may account for the reduced insecticidal activity of certain isolates. Our findings point towards an adaption to insects within closely related pseudomonads and contribute to understand the ecology of insecticidal Pseudomonas.     

Effect of osmolarity and dehydration on alginate production by fluorescent pseudomonads

Alginate is produced as an exopolysaccharide by many fluorescent pseudomonads. However, pseudomonads often have a nonmucoid phenotype in standard laboratory media. Growth in the presence of 0.3M sodium chloride or 3–5% ethanol reportedly can lead to the generation of mucoid variants of nonmucoid strains ofPseudomonas aeruginosa. We wished to determine whether alginate production by other fluorescent pseudomonads is affected by sodium chloride and ethanol. Eight alginate-producing strains of saprophytic and phytopathogenic pseudomonads were grown as broth cultures containing 0–0.7M sodium chloride or 0–5% ethanol for 24–30 h at 28° or 35°C. Culture supernatant fluids were subjected to ethanol precipitation, and the amount of alginate present was estimated by measuring the uronic acid content. The presence of sodium chloride and ethanol caused significant stimulation of alginate production by all strains tested exceptP. viridiflava ATCC 13223 andP. fluorescens W4F1080. The optimal concentration of sodium chloride ranged from 0.2 to 0.5M; that for ethanol ranged from 1 to 3%. Moreover, inclusion of the nonmetabolizable, nonionic solute sorbitol showed a similar stimulation of alginate production. The stimulation of alginate production by high medium osmolarity and dehydration appears to be a trait shared by fluorescent pseudomonads.Reference to brand or firm name does not constitute endorsement by the U.S. Department of Agriculture overothers of a similar nature not mentioned.     

Fluorescent Pseudomonas Isolates Pathogenic on Witloof Chicory Leaves

Nineteen phytopathogenic fluorescent Pseudomonas isolates were isolated from diseased witloof chicory (Cichorium intybus L. var. foliosum Hegi). They were compared with six Ps. fluorescens strains from culture collections, using SDS-PAGE of total cell proteins. The fluorescent pseudomonads examined showed seven different fingerprint types. The major group of phytopathogenic fluorescent pseudomonas revealed a fingerprint type which was frequently found on healthy chicory roots and leaves too. Some, but not all, of the isolates from healthy plants produced typical disease symptoms upon artificial inoculation of etiolated chicory leaves. Infectivity titrations showed a reduced efficiency of the latent pathogen PGSB-7228 to cause disease symptoms on the chicory leaves. The virulence of the latent pathogen is induced in the presence of wounded chicory tissue.     

Production of biosurfactants and antibiotics by fluorescent pseudomonads isolated from a closed hydroponic system equipped with a slow filter

The presence of antibiotic- and biosurfactant-producing strains of fluorescent pseudomonads in a closed hydroponic system equipped with a slow filter was investigated. A total of 271 strains of pseudomonads were isolated before the filter, from the filter skin and from the effluent. Production of biosurfactants was determined using the drop-collapse method. The ability of the strains to inhibit the growth of the plant pathogens Pythium ultimum, Phytophthora cryptogea and Fusarium oxysporum was determined using dual culture plating. The influence of carbon sources on production was determined for selected strains, which also were identified to species level. Production of antibiotics or biosurfactants was observed to be a common trait among the fluorescent pseudomonads within the closed hydroponic system and it was affected by the filter. Pythium ultimum was the pathogen that was most sensitive to antibiotics produced by the fluorescent pseudomonads. The results indicated a strong influence of nutritional resources on antibiotic and biosurfactant production.     

Agglutination, Adherence, and Root Colonization by Fluorescent Pseudomonads

Two fractions of agglutination activity towards fluorescent pseudomonads were detected in root washes of potato, tomato, wheat, and bean. High-molecular-mass (>10⁶ Da) components in crude root washes agglutinated only particular saprophytic, fluorescent Pseudomonas isolates. Ion-exchange treatment of the crude root washes resulted in preparations of lower-molecular-mass (10⁵ to 10⁶ Da) fractions which agglutinated almost all Pseudomonas isolates examined. Also, components able to suppress agglutination reactions of pseudomonads with the lower-molecular-mass root components were detected in crude root washes of all crops studied. Pseudomonas isolates were differentially agglutinated by both types of root components. The involvement of these two types of root components in short-term adherence and in colonization was studied in potato, tomato, and grass, using Pseudomonas isolates from these crops. Short-term adherence of isolates to roots was independent of their agglutination with either type of root components. With agglutination-negative mutants, the high-molecular-mass components seemed to be involved in adherence of Pseudomonas putida Corvallis to roots of all crops studied. Short-term adherence to roots of four Pseudomonas isolates could be influenced by addition of both crude and ion-exchange-treated root washes, depending on their agglutination phenotype with these root wash preparations. Potato root colonization by 10 different isolates from this crop, over a period of 7 days, was not correlated with their agglutination phenotype. Agg^- mutants of P. putida Corvallis were not impaired in root colonization. It is concluded that the root agglutinins studied can be involved in short-term adherence of pseudomonads to roots but do not play a decisive role in their root colonization.     

Simultaneous Detection of Pseudomonas fragi, P. lundensis, and P. putida from Meat by Use of a Multiplex PCR Assay Targeting the carA Gene

Species-specific primers and a multiplex PCR assay were developed for the simultaneous identification and differentiation of Pseudomonas fragi, P. lundensis, and P. putida based on the coamplification of different portions of the small subunit of the carbamoyl phosphate synthase gene (carA). The carA multiplex PCR was used to detect the presence of the three Pseudomonas species from beef, chicken, and pork samples and proved to be effective in showing their evolution during the storage of meat.     

Numerical taxonomy of Pseudomonas based on published records of substrate utilization

Data published by R. Y. Stanier, N. J. Palleroni, M. Doudoroff and their colleagues on Pseudomonas have been analysed by numerical taxonomy. Records on 401 strains were used, representing 155 characters, mostly utilization of substrates as carbon-energy sources. Twenty-nine phenons were recognized, which included 394 strains: the remaining 7 remained unclustered. The results were in very good accord with the conclusions of these authors. Almost all phenons were well separated with very little overlap. Many of them corresponded to distinct species, and others corresponded to recognized biotypes. Some small groups may represent unnamed new species.Analyses by Gower's Coefficient showed five major groupings: A) the fluorescent pseudomonads; B) biochemically active species (Pseudomonas cepacia, P. pseudomallei and allies); D) P. solanacearum and allies; and E) P. mallei. P. diminuta does not appear to be clearly distinct from P. vesicularis, nor does P. alcaligenes appear clearly distinct from P. pseudoalcaligenes. There may, however, be some difference between P. multivorans and P. cepacia.Analyses using the Pattern Coefficient differed mainly in the relationships shown by a few of the metabolically active species. Of the two coefficients, the Pattern Coefficient gave results that were in somewhat better agreement with evidence from nucleic acids, but it showed an unexpectedly close relationship between P. solanacearum and P. cepacia.     

Growth promotion and inhibition by antibiotic-producing fluorescent pseudomonads on citrus roots

Summary Forty-three strains of feeder root colonizing fluorescent pseudomonads from rough lemon (Citrus jambhiri Lush.) roots were examined for effects on rough lemon and sweet orange (Citrus sinensis Osbeck) seedlings. Plants inoculated with a single bacterial soil-drench had, after 10 months, a range of stimulatory (to 116%) and inhibitory effects (to 52%). Stimulatory bacteria particularly increased growth of root systems. Cultivar-specific inhibition and stimulation was evident in inoculations of rough lemon and sweet orange seedlings. Populations of fluorescent rhizobacteria on inoculated and noninoculated, as well as on stimulated and nonstimulated seedlings, did not differ significantly (10.8×10⁶ to 30.3×10⁶ CFU/g root). Population of fluorescent rhizobacteria on seedlings were higher than populations on feeder roots from grove trees (2.8 to 5.7×10⁶ CFU/g). Ninety-four and 81% of 251 fluorescent strains produced antibiotics against the fungusGeotrichum candidum and the bacteriumErwinia stewartii, respectively. Antibiotic activities of 90% of the antibiotic producing strains were repressed by Fe³⁺, indicating siderophore production. In comparison, only 9.6 and 15% of 94 randomly selected nonfluorescentPseudomonas strains were antibiotic producers. Differences between stimulatory and inhibitory or neutral bacteria were not apparent from antibiosis tests. On the basis of physiological tests,Pseudomonas putida was the most abundant (>62%) pseudomonad species on rough lemon roots. Growth stimulating strains appeared to be in bothP. putida andP. fluorescens groups. FewP. aeruginosa strains were identified on citrus roots.Florida Agricultural Experiment Stations Journal Series No.