Detection and Incidence of Specific Species of Spoilage Bacteria on Fish: II. Relative Incidence of Pseudomonas putrefaciens and Fluorescent Pseudomonads on Haddock Fillets

Pseudomonas putrefaciens has been found to constitute one of the major species of spoilage bacteria on haddock fillets. The initial population of this organism on fillets of high bacterial quality is uniformly below 4% and most frequently no greater than 1%. During refrigerated storage, the organism increases at a more rapid rate than the total psychrophilic population, comprising 50 to 90% of the total population when the total count exceeds 10(6)/g of tissue. Fluorescent pseudomonads were shown to constitute a second group of predominant pseudomonads constituting up to 19.3% of the total population after 8 days of refrigerated storage. Of a total of 45 fluorescent pseudomonads isolated from haddock fillets, 14 (31.1%) were found to be potent fish spoilers. The use of a soft-agar-gelatin plating technique showed a parallel increase of proteolytic organisms with total count indicating that proteolytic organisms other than P. putrefaciens and fluorescent pseudomonads increase at a slower rate than these two groups.     

Extracellular Deoxyribonuclease Production by Anaerobic Bacteria

The production of extracellular deoxyribonuclease was examined with anaerobic organisms isolated from clinical specimens. Nuclease activity was extraordinarily common. All strains of Fusobacterium, including eight species, as well as Bacteroides fragilis and B. melaninogenicus, displayed enzyme activity. Whereas the gram-positive bacteria were generally less productive, all strains of Clostridium perfringens, Peptostreptococcus intermedius, and P. anaerobius specifically produced deoxyribonuclease. The test is taxonomically valuable, particularly in the characterization of gram-positive cocci, since a deoxyribonuclease-producing coccus indicates P. intermedius or P. anaerobius. Additionally, possession of the enzyme may prove to be a useful correlate of the potential pathogenicity of anaerobes.     

Characterization of Exopolysaccharides Produced by Plant-Associated Fluorescent Pseudomonads

A total of 214 strains of plant-associated fluorescent pseudomonads were screened for the ability to produce the acidic exopolysaccharide (EPS) alginate on various solid media. The fluorescent pseudomonads studied were saprophytic, saprophytic with known biocontrol potential, or plant pathogenic. Approximately 10% of these strains exhibited mucoid growth under the conditions used. The EPSs produced by 20 strains were isolated, purified, and characterized. Of the 20 strains examined, 6 produced acetylated alginate as an acidic EPS. These strains included a Pseudomonas aeruginosa strain reported to cause a dry rot of onion, a strain of P. viridiflava with soft-rotting ability, and four strains of P. fluorescens. However, 12 strains of P. fluorescens produced a novel acidic EPS (marginalan) composed of glucose and galactose (1:1 molar ratio) substituted with pyruvate and succinate. Three of these strains were soft-rotting agents. Two additional soft-rotting strains of P. fluorescens produced a third acidic novel EPS composed of rhamnose, mannose, and glucose (1:1:1 molar ratio) substituted with pyruvate and acetate. When sucrose was present as the primary carbon source, certain strains produced the neutral polymer levan (a fructan) rather than an acidic EPS. Levan was produced by most strains capable of synthesizing alginate or the novel acidic EPS containing rhamnose, mannose, and glucose but not by strains capable of marginalan production. It is now evident that the group of bacteria belonging to the fluorescent pseudomonads is capable of elaborating a diverse array of acidic EPSs rather than solely alginate.     

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.     

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.     

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.     

Identification of exopolysaccharides produced by fluorescent pseudomonads associated with commercial mushroom (Agaricus bisporus) production.

The acidic exopolysaccharides (EPSs) from 63 strains of mushroom production-associated fluorescent pseudomonads which were mucoid on Pseudomonas agar F medium (PAF) were isolated, partially purified, and characterized. The strains were originally isolated from discolored lesion which developed postharvest on mushroom (Agaricus bisporus) caps or from commercial lots of mushroom casing medium. An acidic galactoglucan, previously named marginalan, was produced by mucoid strains of the saprophyte Pseudomonas putida and the majority of mucoid strains of saprophytic P. fluorescens (biovars III and V) isolated from casing medium. One biovar II strain (J1) of P. fluorescens produced alginate, a copolymer of mannuronic and guluronic acids, and one strain (H13) produced an apparently unique EPS containing neutral and amino sugars. Of 10 strains of the pathogen "P. gingeri," the causal agent of mushroom ginger blotch, 8 gave mucoid growth on PAF. The "P. gingeri" EPS also was unique in containing both neutral sugar and glucuronic acid. Mucoid, weakly virulent strains of "P. reactans" produced either alginate or marginalan. All 10 strains of the pathogen P. tolaasii, the causal agent of brown blotch of mushrooms were nonnmucoid on PAF. Production of EPS by these 10 strains plus the 2 nonmucoid strains of "P. gingeri" also was negative on several additional solid media as well as in two broth media tested. The results support our previous studies indicating that fluorescent pseudomonads are a rich source of novel EPSs.     

Biodiversity of rice (Oryza sativa L.) and sugarcane (Saccharum officinarum L.) rhizosphere pseudomonads

Fluorescent pseudomonads were isolated from the rhizosphere of rice and sugarcane and examined for their biodiversity. All fifty strains of the fluorescent pseudomonads produced indole acetic acid. Among these pseudomonads, halves of sugarcane rhizosphere isolates and one isolate from the rice rhizosphere exhibited phosphate solubilization activity. On the contrary, majority of the rice rhizosphere pseudomonads, and one isolate from sugarcane rhizosphere exhibited antifungal activity. These fluorescent pseudomonads were further classified based on their growth and biochemical characteristics. Those isolates that had same biochemical characteristics were distinguished by random amplification of polymorphic DNA (RAPD). These biochemical and molecular biological methods clearly differentiated fluorescent Pseudomonads of rice and sugarcane rhizosphere.     

Detection of phlD gene in some fluorescent pseudomonads isolated from Iran and its relative with antifungal activities

Fluorescent pseudomonads that produce antibiotic 2,4-diacetylphloroglocinol (2,4-DAPG) are important group of PGRP that inhibit a broad spectrum of plant pathogenic fungi. Studying on genetic diversity of 2,4-diacetylphloroglucinol-producing fluorescent pseudomonads has been shown with special importance. The first step to investigate the genetic diversity of these bacteria is detecting of the genes required for the biosynthesis of this antibiotic. The objectives of the current study were detection of phlD gene within fluorescent pseudomonads by a PCR-based assay, and comparison of phenotypic and genotypic characteristics of fluorescent pseudomonads with proven biocontrol potential against some soil-borne phytopathogenic fungi. We used a collection of 47 fluorescent Pseudomonas spp. some with known biological control activity against Macrophomina phaseolina, Rhizoctonia solani, Phytophthora nicotianae var. parasitica, Pythium sp. and Fusarium sp. in vitro and the potential to produce known secondary metabolites such as, siderophore, HCN and protease. The results indicated that 66, 40.42, 63.82,48.94 and 27.65% of strains revealed antagonistic activity against R. solani, M. phaseolina, Pythium sp., P. nicotianae and Fusarium sp., respectively. Rhizoctonia solani recognized as the most vulnerable fungus. Among 47 strains, 76.59, 97.87 and 17% of strains produced protease, siderophore and HCN, respectively. We could detect phlD gene in strains P-5, P-32, P-47. Strain CHA0 was used as positive control for the detection this gene. Overall, there was no obvious link between the existence of phlD gene and inhibition of fungal growth or production of the antifungal metabolites in vitro. But in some strains such as CHA0 and P-5, we saw a link between the existence of phlD and antifungal activities. Studying on detection and diversity of phlD provides a fundamental knowledge for developing a rapid genetic screening system to identify a potential biocontrol strains.     

Characterization of Pseudomonas Species Isolated from Clinical Specimens

More than 90 morphological and physiological characters of 227 strains of pseudomonads isolated from clinical specimens and 16 reference strains are described. The clinical isolates included P. aeruginosa (apyocyanogenic), P. fluorescens, P. putida, P. pseudomallei, P. cepacia, P. acidovorans, P. alcaligenes, P. pseudoalcaligenes, P. stutzeri, P. putrefaciens, P. maltophilia, and P. diminuta.     

A Numerical Taxonomic Study of Pseudomonas-like Bacteria Isolated from Fish in Southeastern Queensland and their Association with Spoilage

Pseudomonas -like bacteria isolated from fresh and spoiling fish in southeastern Queensland were subjected to a wide range of physiological and nutritional tests. The results of these tests, together with those of 20 named strains, were analysed numerically, resulting in the formation of 11 groups. Most of the isolates clustered into group 1 and group 2 which also contained the bulk of the strains able to produce spoilage odours when grown in a tryptic digest of fish muscle at 2°C. Almost all of the group 1 organisms produced sulphydryl type odours, had only 50 mol % G + C and were identified as strains of Alteromonas putrefaciens which were deficient in the ability to produce H₂S detectable in Peptone Iron Agar. Certain of the group 2 strains produced fruity and sulphydryl type odours, but these organisms were not distinguishable from other strains in this group not producing odours. Group 2 strains were highly related to Pseudomonas fragi and were intermediate in properties between Ps. fluorescens and Ps. putida. The remaining nine minor groups contained few organisms able to produce spoilage odours.     

Volatile Compounds Produced in Sterile Fish Muscle (Sebastes melanops) by Pseudomonas putrefaciens, Pseudomonas fluorescens, and an Achromobacter Species

Volatile compounds produced by Pseudomonas putrefaciens, P. fluorescens, and an Achromobacter species in sterile fish muscle (Sebastes melanops) were identified by combined gas-liquid chromatography and mass spectrometry. Compounds produced by P. putrefaciens included methyl mercaptan, dimethyl disulfide, dimethyl trisulfide, 3-methyl-1-butanol, and trimethylamine. With the exception of dimethyl trisulfide, the same compounds were produced by an Achromobacter species. Methyl mercaptan and dimethyl disulfide were the major sulfur-containing compounds produced by P. fluorescens.     

Similarities of genome deoxyribonucleic acids ofPseudomonas strains isolated from meat

A total of 29Pseudomonas strains from meat and 14 reference strains of differentPseudomonas species were studied by DNA-DNA hybridization. Of the field strains, 15 were phenotypically similar toP. fragi; the others represented a new group described by Molin and Ternström [4], phenotypically related toP. fragi and the fluorescent pseudomonads; 12 strains of this phenotype formed one major DNA-relatedness group; the type strain ofP. fragi together with nine field strains formed another group. The remaining eight meat isolates could not be assigned to either of the two groups. The intergroup relatedness and the relatedness of both groups to the fluorescent pseudomonads was about 50%. Hybridization data indicate that the phenotype of Molin and Ternström deserves species rank and that this tentative species andP. fragi belong to the group of fluorescent pseudomonads.     

Alginate Production by Plant-Pathogenic Pseudomonads

Eighteen plant-pathogenic and three non-plant-pathogenic pseudomonads were tested for the ability to produce alginic acid as an exopolysaccharide in vitro. Alginate production was demonstrated for 10 of 13 fluorescent plant-pathogenic pseudomonads tested with glucose or gluconate as the carbon source, but not for all 5 nonfluorescent plant pathogens and all 3 non-plant pathogens tested. With sucrose as the carbon source, some strains produced alginate while others produced both polyfructan (levan) and alginate. Alginates ranged from <1 to 28% guluronic acid, were acetylated, and had number-average molecular weights of 11.3 × 10³ to 47.1 × 10³. Polyfructans and alginates were not elicitors of the soybean phytoalexin glyceollin when applied to wounded cotyledon surfaces and did not induce prolonged water soaking of soybean leaf tissues. All or most pseudomonads in rRNA-DNA homology group I may be capable of synthesizing alginate as an exopolysaccharide.     

New Gas Chromatographic Characterization Procedure: Preliminary Studies on Some Pseudomonas Species

Strains of saccharolytic and nonsaccharolytic Pseudomonas species were examined by a new single-step gas chromatographic characterization procedure. Cells were digested in a methanolic solution of tetramethylammonium hydroxide pentahydrate, and the digestates were subjected to gas-liquid chromatographic analysis. The chromatograms were examined for similarities and differences in their overall patterns. A single component was defined for use as an internal qualitative and quantitative standardizing component in order to develop relative retention time-relative peak height profiles of each organism. Comparison of these profiles enabled the characterization of strains of Pseudomonas aeruginosa, P. putida, P. cepacia, P. pseudomallei, P. stutzeri, P. pseudoalcaligenes, P. alcaligenes, P. diminuta, P. denitrificans, and P. acidovorans. The P. maltophilia and P. putrefaciens digestates showed chromatograms which were superficially similar yet easily distinguished as belonging to different species. The chromatograms of these two organisms were very different from those of other pseudomonads.     

Genetic and Functional Diversity among Fluorescent Pseudomonads Isolated from the Rhizosphere of Banana

Fluorescent pseudomonads from banana rhizospheric soil were isolated and screened for the production of enzymes and hormones such as phosphatase, indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylate (ACC) deaminase, protease, and antifungal metabolites. Of 95 isolates, 50 (52%) isolates solubilized tri-calcium phosphate (TCP), 63 (66%) isolates produced plant growth hormone IAA, 10 (11%) isolates exhibited ACC deaminase, and 23 (24%) isolates produced protease. Isolates were screened for antifungal activity toward phytopathogenic fungi. Gene-specific primers have identified the putative antibiotic producing isolates. These putative isolates were grown in the production media and production of antibiotics was confirmed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Genotypic analysis by BOX (bacterial repetitive BOX element)-polymerase chain reaction (PCR) resulted into three distinct genomic clusters at a 50% similarity level and 62 distinct BOX profiles. Based on the sequence similarity of 16S rRNA and construction of subsequent phylogenetic tree analysis, isolates were designated as Pseudomonas monteilii, P. plecoglossicida, P. fluorescens, P. fulva, P. mosselii, P. aeruginosa, P. alcaligenes, and P. pseudoalcaligenes. Present study revealed the genetic and functional diversity among isolates of fluorescent pseudomonads associated with rhizospheric soil of banana and also identified P. monteilii as dominant species. The knowledge on genetic and functional diversity of fluorescent pseudomonads associated with banana rhizosphere is useful to understand their ecological role and for their utilization in sustainable agriculture.     

Immunological properties of protocatechuate 3, 4-dioxygenase isofunctional enzymes.

Antiserum prepared against protocatechuate 3, 4-dioxygenase from Pseudomonas aeruginosa forms precipitin bands without spurs with isofunctional enzymes from different strains of the fluorescent pseudomonads on immunodiffusion plates. Catalytic activity of the isofunctional enzymes was inhibited by an immunoglobulin fraction prepared against the enzyme from organisms of the same genus and not from different genera.     

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.     

Traits of fluorescent Pseudomonas spp. involved in suppression of plant root pathogens.

Certain members of the fluorescent pseudomonad group have been shown to be potential agents for the biocontrol of plant root diseases. The major problems with the commercialization of these beneficial strains are that few wild-type strains contain all the desired characteristics for this process and the performance of strains in different soil and climatic conditions is not reproducible. Consequently, prior to selection and/or improvement of suitable strains for biocontrol purposes, it is necessary to understand the important traits required for this purpose. The production of fluorescent siderophores (iron-binding compounds) and antibiotic compounds has been recognized as important for the inhibition of plant root pathogens. Efficient root colonization is also a prerequisite for successful biocontrol strains. This review discusses some of the characteristics of fluorescent pseudomonads that have been suggested to be important for biocontrol. The genetic organization and regulation of these processes is also examined. This information is necessary for attempts aimed at the improvement of strains based on deregulating pathways or introducing traits from one strain to another. The release of genetically engineered organisms into the environment is governed by regulations, and this aspect is summarized. The commercialization of fluorescent pseudomonads for the biological control of plant root diseases remains an exciting possibility. The understanding of the relevant characteristics will facilitate this process by enabling the direct selection and/or construction of strains which will perform under a variety of environmental conditions.