Effect of Two Plant Species, Flax (Linum usitatissinum L.) and Tomato (Lycopersicon esculentum Mill.), on the Diversity of Soilborne Populations of Fluorescent Pseudomonads

Suppression of soilborne disease by fluorescent pseudomonads may be inconsistent. Inefficient root colonization by the introduced bacteria is often responsible for this inconsistency. To better understand the bacterial traits involved in root colonization, the effect of two plant species, flax (Linum usitatissinum L.) and tomato (Lycopersicon esculentum Mill.), on the diversity of soilborne populations was assessed. Fluorescent pseudomonads were isolated from an uncultivated soil and from rhizosphere, rhizoplane, and root tissue of flax and tomato cultivated in the same soil. Species and biovars were identified by classical biochemical and physiological tests. The ability of bacterial isolates to assimilate 147 different organic compounds and to show three different enzyme activities was assessed to determine their intraspecific phenotypic diversity. Numerical analysis of these characteristics allowed the clustering of isolates showing a high level (87.8%) of similarity. On the whole, the populations isolated from soil were different from those isolated from plants with respect to their phenotypic characteristics. The difference in bacteria isolated from uncultivated soil and from root tissue of flax was particularly marked. The intensity of plant selection was more strongly expressed with flax than with tomato plants. The selection was, at least partly, plant specific. The use of 10 different substrates allowed us to discriminate between flax and tomato isolates. Pseudomonas fluorescens biovars II, III, and V and Pseudomonas putida biovar A and intermediate type were well distributed among the isolates from soil, rhizosphere, and rhizoplane. Most isolates from root tissue of flax and tomato belonged to P. putida bv. A and to P. fluorescens bv. II, respectively. Phenotypic characterization of bacterial isolates was well correlated with genotypic characterization based on repetitive extragenic palindromic PCR fingerprinting.     

Influence of Two Plant Species (Flax and Tomato) on the Distribution of Nitrogen Dissimilative Abilities within Fluorescent Pseudomonas spp

The distribution of nitrogen-dissimilative abilities among 317 isolates of fluorescent pseudomonads was studied. These strains were isolated from an uncultivated soil and from the rhizosphere, rhizoplane, and root tissue of two plant species (flax and tomato) cultivated on this same soil. The isolates were distributed into two species, Pseudomonas fluorescens (45.1%) and Pseudomonas putida (40.4%), plus an intermediate type (14.5%). P. fluorescens was the species with the greatest proportion of isolates in the root compartments and the greatest proportion of dissimilatory and denitrifying strains. According to their ability to dissimilate nitrogen, the isolates have been distributed into nondissimilatory and dissimilatory strains, nitrate reducers and true denitrifiers with or without N(inf2)O reductase. The proportion of dissimilatory isolates was significantly enhanced in the compartments affected by flax and tomato roots (55% in uncultivated soil and 90 and 82% in the root tissue of flax and tomato, respectively). Among these strains, the proportion of denitrifiers gradually and significantly increased in the root vicinity of tomato (44, 68, 75, and 94% in uncultivated soil, rhizosphere, rhizoplane, and root tissue, respectively) and was higher in the flax rhizoplane (66%) than in the uncultivated soil. A higher proportion of N(inf2)O reducers was also found in the root compartments. This result was particularly clear for tomato. It is hypothesized that denitrification could be a selective advantage for the denitrifiers in the root environment and that this process could contribute to modify the specific composition of the bacterial communities in the rhizosphere.     

OprF polymorphism as a marker of ecological niche in Pseudomonas

OprF is the major outer-membrane protein of Pseudomonas sensu stricto (rRNA group I). In addition to playing a role as porin, membrane structural protein and root adhesion, this pleiotropic protein shows a length polymorphism corresponding to two types of OprF, termed OprF type 1 and OprF type 2. In a previous work, all the P. fluorescens isolated from bulk soil (non-rhizospheric) were shown to possess oprF type 1, while all the clinical P. fluorescens isolates and most rhizospheric strains corresponded to type 2. In this study, we further investigated the relation between the OprF polymorphism and the ecological niche by developing a culture-independent approach (a ratio polymerase chain reaction) to measure the percentage of each oprF type in environmental DNA samples, including two different soils and three different cultured plants (flax, wheat and grassland). Although the proportions of oprF type 2 between rhizospheric samples were quite variable, they were always very significantly higher (P<0.001) than the proportions of oprF type 2 of the adjacent bulk soil where the vast majority of oprF (>95%) corresponded to type 1. We discuss the potential applications of this ecological fingerprint in an agronomic and taxonomic point of view.     

Phenylacetic acid-producing Rhizoctonia solani represses the biosynthesis of nematicidal compounds in vitro and influences biocontrol of Meloidogyne incognita in tomato by Pseudomonas fluorescens strain CHA0 and its GM derivatives

AIMS: The aim of the present investigation was to determine the influence of Rhizoctonia solani and its pathogenicity factor on the production of nematicidal agent(s) by Pseudomonas fluorescens strain CHA0 and its GM derivatives in vitro and nematode biocontrol potential by bacterial inoculants in tomato. METHODS AND RESULTS: One (Rs7) of the nine R. solani isolates from infected tomato roots inhibited seedling emergence and caused root rot in tomato. Thin layer chromatography revealed that culture filtrates of two isolates (Rs3 and Rs7) produced brown spots at Rf-values closely similar to synthetic phenylacetic acid (PAA), a phytotoxic factor. Filtrates from isolate Rs7, amended with the growth medium of P. fluorescens, markedly repressed nematicidal activity and PhlA'-'LacZ reporter gene expression of the bacteria in vitro. On the contrary, isolate Rs4 enhanced nematicidal potential of a 2,4-diacetylphloroglucinol overproducing mutant, CHA0/pME3424, of P. fluorescens strain CHA0 in vitro. Therefore, R. solani isolates Rs4 and Rs7 were tested more rigorously for their potential to influence biocontrol effectiveness of the bacterial agents. Methanol extract of the culture filtrates of PAA-producing isolate Rs7 resulting from medium amended with phenylalanine enhanced fungal repression of the production of nematicidal agents by bacteria, while amendments with zinc or molybdenum eliminated such fungal repression, thereby restoring bacterial potential to cause nematode mortality in vitro. A pot experiment was carried out, 3-week-old tomato seedlings were infested with R. solani isolates Rs4 or Rs7 and/or inoculated with Meloidogyne incognita, the root-knot nematode. The infested soil was treated with aqueous cell suspensions (10(8) CFU) of P. fluorescens strain CHA0 or its GM derivatives or left untreated (as a control). Observations taken 45 days after nematode inoculation revealed that, irrespective of the bacterial treatments, galling intensity per gram of fresh tomato roots was markedly higher in soil amended with isolate Rs4 than in Rs7-amended soils. Soil amendments with R. solani and the bacterial antagonists resulted in substantial reductions of the number of galls per gram of root. These results are contradictory to those obtained under in vitro conditions where culture filtrates of PAA-positive Rs7 repressed the production of nematicidal compounds. Plants grown in Rs7-amended soils, with or without bacterial inoculants, had lesser shoot and root weights than plants grown in nonamended or Rs4-amended soils. Moreover, amendments with Rs7 substantially retarded root growth and produced necrotic lesions that reduced the number of entry sites for invasion and subsequent infection by nematodes. Populations of P. fluorescens in the tomato rhizosphere were markedly higher in Rs7-amended soils. CONCLUSIONS: PAA-producing virulent R. solani drastically affects the potential of P. fluorescens to cause death of M. incognita juveniles in vitro and influences bacterial effectiveness to suppress nematodes in tomato roots. SIGNIFICANCE AND IMPACT OF THE STUDY: As most agricultural soils are infested with root-infecting fungi, including R. solani, it is likely that some PAA-producing isolates of the fungus may also be isolated from such soils. The inhibitory effect of PAA-producing R. solani on the biosynthesis of nematicidal agent(s) critical in biocontrol may reduce or even eliminate the effectiveness of fluorescent pseudomonads against root-knot nematodes, both in nursery beds and in field conditions. Introduction of bacterial inoculants, for the control of any plant pathogen, should be avoided in soils infested with PAA-producing R. solani. Alternatively, the agents could be applied together with an appropriate quantity of fungicide or chemicals such as zinc to create an environment more favourable for bacterial biocontrol action.     

Species variation and plasmid incidence among fluorescent Pseudomonas strains isolated from agricultural and industrial soils

Abstract: A total of 132 different fluorescent Pseudomonas strains were isolated from several agricultural and industrial soils. The bacteria from the two different soil environments were compared for species and biotype variation, antibiotic and heavy metal resistance profiles, ability to degrade polyaromatic hydrocarbons, and plasmid incidence. Irrespective of the soil type, the isolates belonged to Pseudomonas fluorescens biotypes I–VI and Pseudomonas putida biotype B. Except for a streptomycin resistant isolate from one of the industrial soils, all the strains had the same antibiotic resistance profile. However, there was a higher incidence of heavy metal resistance and polyaromatic hydrocarbon degradation phenotypes in the isolates from industrial soils than from the agricultural soils. Only 2 out of 68 strains from agricultural soil were found to carry plasmids, while 28 out of 64 strains from industrial soil had plasmids. A majority of the plasmids (56%) were estimated to be larger than 50 kb, indicating that they could encode transfer functions. However, transferability as indicated by the ability to mobilize an IncQ plasmid (tra⁻, mob⁺), was observed with only one plasmid. None of the plasmid(s) containing isolates hybridized to a ³²P-labelled repP probe suggesting that none of the indigenous plasmids in the soil fluorescent Pseudomonas strains was related to the IncP group of conjugative plasmids commonly associated with resistance and catabolic genes.     

Fluorescent pseudomonads in the rhizosphere of plants and their relation to root exudates

Fluorescent pseudomonads were present in chernozem soil not influenced by plant roots (10(3)-10(4) per g dry soil) in the rhizosphere soil of various plants (10(4)-10(5) per g soil) and on roots (10(3) to 10(7) per g fresh roots), depending on the species and age of the plant. Relative species representation of fluorescent pseudomonads changed on the roots and in the plant rhizosphere as compared with free soil. Pseudomonas fluorescens, representing 60-93% of the population of fluorescent pseudomonads predominated on the roots of all plants investigated. Somewhat different results were obtained in rhizosphere soil. Relatively higher numbers of P. fluorescens were detected in the rhizosphere soil of cucumber and maize, numbers in the rhizosphere soil of wheat were practically the same as in free soil and higher numbers of P. putida were found in the rhizosphere soil of barley. Almost all components contained in the root exudates of the plants studied, including beta-pyrazolylalanine from the root exudates of cucumbers were utilized as carbon and energy sources. Root exudates of wheat and maize were utilized by the strain P. putida K2 with an efficiency of 73-91%, depending on species and age of the plant.     

The Composition of Fluorescent Pseudomonad Populations Associated with Roots Is Influenced by Plant and Soil Type

Populations of fluorescent pseudomonads isolated from an uncultivated soil and from the roots of two plant species were previously shown to differ (P. Lemanceau, T. Corberand, L. Gardan, X. Latour, G. Laguerre, J.-M. Boeufgras, and C. Alabouvette, Appl. Environ. Microbiol. 61:1004-1012, 1995). The diversities of fluorescent pseudomonads, from two uncultivated soils and from the roots of two plant species cultivated in these two soils, were compared. The phenotypic diversity of the bacterial isolates was characterized on the basis of biochemical and physiological tests and on the basis of their ability to utilize 147 different organic compounds. The genotypic diversity of the isolates was characterized on the basis of the types of 16S genes coding for rRNA (rDNA), their repetitive extragenic palindromic patterns by PCR, and plasmid profiles. Taxonomic identification of the isolates was achieved with both biochemical and physiological tests and by comparing their 16S rDNA types to those of reference and type strains of fluorescent Pseudomonas spp. Numerical analysis of phenotypic characteristics allowed the clustering of isolates that showed high levels of similarity. This analysis indicated that both soil type and host plant had an effect on the diversity of fluorescent pseudomonads. However, of the two factors studied, the soil was clearly the dominating one. Indeed, the populations associated with the roots of each plant species varied from one soil to the other. This variation could possibly be ascribed to the differences recorded between the phenotypically diverse populations of fluorescent pseudomonads from the two uncultivated soils. The plant selection was, at least partly, plant specific. It was not related to bacterial species and biovars or to the presence of plasmid DNA. The phenotypic clustering of isolates was well correlated with genotypic characterization by repetitive extragenic palindrome-PCR fingerprinting.     

Rapid identification of environmental isolates of Pseudomonas aeruginosa, P. fluorescens and P. putida by SDS-PAGE analysis of whole-cell protein patterns

Abstract Environmental isolates of fluorescent pseudomonads grown to early stationary phase in glucose-enriched Luria broth were treated with proteinase K in sodium dodecylsulphate (SDS) lysis buffer and subsequently analyzed by polyacrylamide gel electrophoresis (PAGE). Four silver-staining protein-fragment bands could be used for rapid identification at the species level. Pseudomonas aeruginosa isolates were easily recognized by a unique banding pattern. Isolates considered to be P. fluorescen from biochemical and physiological tests (classical biotypes I, II, III, IV and V) also had a characteristic banding pattern, which in turn was different from that of P. putida isolates (classical biotype A). A residual group representing intermediate isolates of P. fluorescens (new biotype VI of Barrett et al., J. Gen. Microbiol. 132, 1986) or P. putida (biotype B) had a banding pattern similar to that of classical P. fluorescens biotypes. On the other hand, a group representing other intermediate isolates of P. putida (new biotype C of Barrett et al., J. Gen. Microbiol. 132, 1986) had a unique banding pattern resembling that of classical P. putida biotype A. A small number of protein fragment bands appearing in SDS-PAGE analysis of whole-cell lysates seems adequate for a rapid identification at the species level of P. aeruginosa, P. fluorescens and P. putida isolated from natural environments.     

Sequence diversity of the OprD protein of environmental Pseudomonas strains

OprD has been widely described for Pseudomonas aeruginosa at both structural and functional levels. Here, we describe the sequence diversity of the OprD proteins from other fluorescent Pseudomonads. We analysed the sequence of the oprD gene in each of the 49 Pseudomonas isolates, mostly putida and fluorescens species, obtained from various environmental sources, including soil, rhizosphere and hospitals. Phylogeny based on OprD sequences distinguished three well-separated clusters in the P. fluorescens species whereas P. putida isolates formed only one cluster. The OprD sequences were generally well conserved within each cluster whereas on the opposite, they were highly variable from one cluster to another and particularly with regards to the cluster of P. aeruginosa. Predicted secondary structures, based on the topological model elaborated for P. aeruginosa, suggest signatures in the large extracellular loops of OprD, which are linked to the OprD-based clusters. Correlations between these OprD-based clusters and ecological niches, growth on various carbon sources and antibiotic sensitivity were investigated.     

Large Pseudomonas phages isolated from barley rhizosphere

Abstract: Five bacteriophages infecting common fluorescent pseudomonads ( Pseudomonas fluorescens and Pseudomonas putida ) were isolated from barley rhizosphere soil. Morphological and molecular characteristics of the phages are described together with selected phage-host interactions. All phages belonged to the Myoviridae family with isometrical heads on contractile tails; 4 of them were unusually large and had complex protein and DNA profiles. The large phages had estimated genome sizes of 200 kb or more. Restriction enzyme analyses and DNA-DNA hybridizations showed that all isolates represented different phage species. None of the isolates were observed to establish lysogeny with the main host strain, P. putida MM1. The large phages multiplied slowly on their hosts, producing very small plaques; one-step growth experiments with one of the large phages (Psp 4) hence demonstrated a long latent period (2.5 h) and a very small burst size (10 particles). One of the large phages (Psp 3) was abundant in the rhizosphere (approx. 10⁴ pfu g⁻¹ soil) and had a particularly broad host range which extended to both fluorescent ( Pseudomonas aeruginosa, P. fluorescens, P. putida and Pseudomonas chlororaphis ) and non-fluorescent (Pseudomonas stutzeri) Pseudomonas spp. occurring in soil. The ecological importance of the large Pseudomonas phages must be further studied, but their slow multiplication rates suggested a possible mechanism of balanced phage-host co-existence in the rhizosphere.     

Genetic diversity and biological control activity of novel species of closely related pseudomonads isolated from wheat field soils in South Australia

Rhizobacteria closely related to two recently described species of pseudomonads, Pseudomonas brassicacearum and Pseudomonas thivervalensis, were isolated from two geographically distinct wheat field soils in South Australia. Isolation was undertaken by either selective plating or immunotrapping utilizing a polyclonal antibody raised against P. brassicacearum. A subset of 42 isolates were characterized by amplified 16S ribosomal DNA restriction analysis (ARDRA), BIOLOG analysis, and gas chromatography-fatty acid methyl ester (GC-FAME) analysis and separated into closely related phenetic groups. More than 75% of isolates tested by ARDRA were found to have >95% similarity to either Pseudomonas corrugata or P. brassicacearum-P. thivervalensis type strains, and all isolates had >90% similarity to either type strain. BIOLOG and GC-FAME clustering showed a >70% match to ARDRA profiles. Strains representing different ARDRA groups were tested in two soil types for biological control activity against the soilborne plant pathogen Gaeumannomyces graminis var. tritici, the causative agent of take-all of wheat and barley. Three isolates out of 11 significantly reduced take-all-induced root lesions on wheat plants grown in a red-brown earth soil. Only one strain, K208, was consistent in reducing disease symptoms in both the acidic red-brown earth and a calcareous sandy loam. Results from this study indicate that P. brassicacearum and P. thivervalensis are present in Australian soils and that a level of genetic diversity exists within these two novel species but that this diversity does not appear to be related to geographic distribution. The result of the glasshouse pot trial suggests that some isolates of these species may have potential as biological control agents for plant disease.     

The role of soil microbes in plant sulphur nutrition

Chemical and spectroscopic studies have shown that in agricultural soils most of the soil sulphur (>95%) is present as sulphate esters or as carbon-bonded sulphur (sulphonates or amino acid sulphur), rather than inorganic sulphate. Plant sulphur nutrition depends primarily on the uptake of inorganic sulphate. However, recent research has demonstrated that the sulphate ester and sulphonate-pools of soil sulphur are also plant-bioavailable, probably due to interconversion of carbon-bonded sulphur and sulphate ester-sulphur to inorganic sulphate by soil microbes. In addition to this mineralization of bound forms of sulphur, soil microbes are also responsible for the rapid immobilization of sulphate, first to sulphate esters and subsequently to carbon-bound sulphur. The rate of sulphur cycling depends on the microbial community present, and on its metabolic activity, though it is not yet known if specific microbial species or genera control this process. The genes involved in the mobilization of sulphonate- and sulphate ester-sulphur by one common rhizosphere bacterium, Pseudomonas putida, have been investigated. Mutants of this species that are unable to transform sulphate esters show reduced survival in the soil, indicating that sulphate esters are important for bacterial S-nutrition in this environment. P. putida S-313 mutants that cannot metabolize sulphonate-sulphur do not promote the growth of tomato plants as the wild-type strain does, suggesting that the ability to mobilize bound sulphur for plant nutrition is an important role of this species.     

Survival of, and induced stress resistance in, carbon-starved Pseudomonas fluorescens cells residing in soil.

We investigated the survival, cell length, and development of general stress resistance in populations of Pseudomonas fluorescens R2f and its rifampin-resistant mutant, R2f Rpr, following exposure to carbon starvation conditions in liquid cultures and residence in two different soils, Flevo silt loam (FSL) and Ede loamy sand (ELS). In much the same way as was recently shown for P. putida KT2442, carbon-starved P. fluorescens R2f populations revealed enhanced resistance to otherwise lethal treatments, such as exposure to ethanol, high temperature, osmotic tension, and oxidative stress. A large population of nonculturable P. fluorescens R2f Rpr cells arose shortly after their introduction into ELS soil, whereas the formation of nonculturable cells was not observed in FSL soil. Also, the inoculant cell (based on immunofluorescence) and CFU counts decreased faster in ELS soil than in FSL soil. Introduction of carbon-starved instead of exponential-growth-phase R2f Rpr cells into ELS soil did not affect bacterial survival. The inoculant cell length decreased in soil, and no large differences in cell length in the two soil types were observed. Addition of glucose to ELS soil resulted in a stable cell length of R2f Rpr cells, whereas carbon-starved cells introduced into ELS soil remained small. Exponentially growing R2f Rpr cells developed enhanced resistance to ethanol, high temperature, osmotic tension, and oxidative stress within 1 day in both soils, whereas cells introduced into ELS soil amended with glucose showed decreased resistance. Cells that were carbon starved prior to introduction into ELS soil showed unchanged stress resistance levels upon residence in soil.     

A comparative evaluation of five typing techniques for determining the diversity of fluorescent pseudomonads

Five typing methods were evaluated, utilising 63 strains of fluorescent pseudomonads, to assess their usefulness as tools to study the bacterial diversity within this complex group. The methods used were Biolog metabolic profiling, restriction fragment length polymorphism ribotyping, PCR ribotyping, and repetitive element sequence-based PCR (rep-PCR) utilising BOX and enterobacterial repetitive intergenic consensus (ERIC) primers. Cluster analysis of the results clearly demonstrated the considerable homogeneity of Pseudomonas aeruginosa isolates and, conversely, the heterogeneity within the other species, in particular P. putida and P. fluorescens, which need further taxonomic investigation. Biolog metabolic profiling enabled the best differentiation among the species. Rep-PCR proved to be highly discriminatory, more so than the other DNA fingerprinting techniques, demonstrating its suitability for the analysis of highly clonal isolates. RFLP ribotyping, PCR ribotyping, and rep-PCR produced specific clusters of P. aeruginosa isolates, which corresponded to their origins of isolation, hence we recommend these methods for intraspecific typing of bacteria.     

Survival of rifampin-resistant mutants of Pseudomonas fluorescens and Pseudomonas putida in soil systems.

The fate of spontaneous chromosomal rifampin-resistant (Rifr) mutants of Pseudomonas putida and Pseudomonas fluorescens in sterile and live organic soil from which they were isolated was studied. In sterile native-soil assays, a Rifr mutant of P. putida showed no decrease in competitive fitness when compared with the wild-type parent. However, mutants of P. fluorescens were of two general categories. Group 1 showed no difference from the wild type in terms of growth rate, competitive fitness, and membrane protein composition. Group 2 showed a slower growth rate in both minimal and enriched media and an altered membrane protein profile. These mutants also demonstrated decreased competitive fitness compared with the wild-type strain. In live soil, the Rifr P. putida strain persisted throughout the 38-day test period with a decay rate of 0.7 log10 CFU/g of soil per 10 days. A group 1 Rifr P. fluorescens mutant maintained its inoculated titer for 7 to 10 days and then decayed at a rate of 0.2 to 0.4 log10 CFU/g of soil per 10 days. A group 2 Rifr P. fluorescens mutant remained at its titer for 1 to 5 days before decaying at a two- to threefold-faster rate. These findings indicate that rifampin resistance may not be an innocuous mutation in some pseudomonads and that marked strains should be compared with wild-type parents before being used as monitors of parental strain survival. Colonization of sterile soil with either the wild-type or mutant strain precluded normal colonization of the second added strain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Survival of rifampin-resistant mutants of Pseudomonas fluorescens and Pseudomonas putida in soil systems

The fate of spontaneous chromosomal rifampin-resistant (Rifr) mutants of Pseudomonas putida and Pseudomonas fluorescens in sterile and live organic soil from which they were isolated was studied. In sterile native-soil assays, a Rifr mutant of P. putida showed no decrease in competitive fitness when compared with the wild-type parent. However, mutants of P. fluorescens were of two general categories. Group 1 showed no difference from the wild type in terms of growth rate, competitive fitness, and membrane protein composition. Group 2 showed a slower growth rate in both minimal and enriched media and an altered membrane protein profile. These mutants also demonstrated decreased competitive fitness compared with the wild-type strain. In live soil, the Rifr P. putida strain persisted throughout the 38-day test period with a decay rate of 0.7 log10 CFU/g of soil per 10 days. A group 1 Rifr P. fluorescens mutant maintained its inoculated titer for 7 to 10 days and then decayed at a rate of 0.2 to 0.4 log10 CFU/g of soil per 10 days. A group 2 Rifr P. fluorescens mutant remained at its titer for 1 to 5 days before decaying at a two- to threefold-faster rate. These findings indicate that rifampin resistance may not be an innocuous mutation in some pseudomonads and that marked strains should be compared with wild-type parents before being used as monitors of parental strain survival. Colonization of sterile soil with either the wild-type or mutant strain precluded normal colonization of the second added strain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular and phenotypic characterization of Pseudomonas spp. isolated from milk

Putative Pseudomonas spp. isolated predominantly from raw and processed milk were characterized by automated ribotyping and by biochemical reactions. Isolates were biochemically profiled using the Biolog system and API 20 NE and by determining the production of proteases, lipases, and lecithinases for each isolate. Isolates grouped into five coherent clusters, predominated by the species P. putida (cluster A), P. fluorescens (cluster B), P. fragi (as identified by Biolog) or P. fluorescens (as identified by API 20 NE) (cluster C), P. fragi (as identified by Biolog) or P. putida (as identified by API 20 NE) (cluster D), and P. fluorescens (cluster E). Isolates within each cluster also displayed similar enzyme activities. Isolates in clusters A, C, and D were generally negative for all three enzyme activities; isolates in cluster B were predominantly positive for all three enzyme activities; and isolates in cluster E were negative for lecithinase but predominantly positive for protease and lipase activities. Thus, only isolates from clusters B and E produced enzyme activities associated with dairy product flavor defects. Thirty-eight ribogroups were differentiated among the 70 isolates. Ribotyping was highly discriminatory for dairy Pseudomonas isolates, with a Simpson's index of discrimination of 0.955. Isolates of the same ribotype were never classified into different clusters, and ribotypes within a given cluster generally showed similar ribotype patterns; thus, specific ribotype fragments may be useful markers for tracking the sources of pseudomonads in dairy production systems. Our results suggest that ribogroups are generally homogeneous with respect to nomenspecies and biovars, confirming the identification potential of ribotyping for Pseudomonas spp.     

Pseudomonas fluorescens biovar V: its resolution into distinct component groups and the relationship of these groups to other P. fluorescens biovars, to P. putida, and to psychrotrophic pseudomonads associated with food spoilage

A numerical taxonomic analysis was performed to evaluate the appropriateness of a single biovar designation (biovar V) for all Pseudomonas fluorescens isolates negative for denitrification, levan production and phenazine pigmentation and to determine the relationship of biovar V strains to other taxa within the same Pseudomonas RNA homology group. Seventy-two strains assigned to P. fluorescens biovar V and four strains of P. fragi were characterized and the data subjected to a numerical taxonomic analysis along with comparable data for 17 previously characterized strains of this biovar and 89 P. putida strains. Seven distinct biovar V clusters containing three or more strains were revealed, and the carbon sources useful for their differentiation were identified. Cluster 1 (38 strains) closely resembled two atypical P. fluorescens I strains. It was also related to P. fluorescens biovar IV and to P. fragi. Cluster 2 (5 strains) was related to cluster 1. Cluster 3 (7 strains) was identical to a major group of meat spoilage psychrotrophic pseudomonads (P. lundensis). Cluster 4 (3 strains) was not related to any other group examined. Cluster 5 consisted of six isolates initially designated P. putida A along with four P. fluorescens biovar V strains all of which resembled P. putida more than they resembled the other P. fluorescens groups. Cluster 6 (16 strains) was distinct from the other biovar V clusters, but was closely related to P. fluorescens biovars I and II. Cluster 7 (3 strains) shared many characteristics with cluster 5. Separate P. fluorescens biovar designations are proposed for cluster 6 and for the combined clusters 1 and 2. A new P. putida biovar is proposed for the combined clusters 5 and 7.     

Genetic Diversity and Biological Control Activity of Novel Species of Closely Related Pseudomonads Isolated from Wheat Field Soils in South Australia

Rhizobacteria closely related to two recently described species of pseudomonads, Pseudomonas brassicacearum and Pseudomonas thivervalensis, were isolated from two geographically distinct wheat field soils in South Australia. Isolation was undertaken by either selective plating or immunotrapping utilizing a polyclonal antibody raised against P. brassicacearum. A subset of 42 isolates were characterized by amplified 16S ribosomal DNA restriction analysis (ARDRA), BIOLOG analysis, and gas chromatography-fatty acid methyl ester (GC-FAME) analysis and separated into closely related phenetic groups. More than 75% of isolates tested by ARDRA were found to have >95% similarity to either Pseudomonas corrugata or P. brassicacearum-P. thivervalensis type strains, and all isolates had >90% similarity to either type strain. BIOLOG and GC-FAME clustering showed a >70% match to ARDRA profiles. Strains representing different ARDRA groups were tested in two soil types for biological control activity against the soilborne plant pathogen Gaeumannomyces graminis var. tritici, the causative agent of take-all of wheat and barley. Three isolates out of 11 significantly reduced take-all-induced root lesions on wheat plants grown in a red-brown earth soil. Only one strain, K208, was consistent in reducing disease symptoms in both the acidic red-brown earth and a calcareous sandy loam. Results from this study indicate that P. brassicacearum and P. thivervalensis are present in Australian soils and that a level of genetic diversity exists within these two novel species but that this diversity does not appear to be related to geographic distribution. The result of the glasshouse pot trial suggests that some isolates of these species may have potential as biological control agents for plant disease.     

Isolation and characterization of ACC deaminase-producing fluorescent pseudomonads, to alleviate salinity stress on canola (Brassica napus L.) growth

Salinity stress is of great importance in arid and semi-arid areas of the world due to its impact in reducing crop yield. Under salinity stress, the amount of 1-aminocyclopropane-1-carboxylate (ACC), a precursor for ethylene production in plants, increases. Here, we conducted research under the hypothesis that isolated ACC deaminase-producing Pseudomonas fluorescens and Pseudomonas putida can alleviate the stressful effects of salinity on canola (Brassica napus L.) growth. The experiments were conducted in the Soil and Water Research Institute, Tehran, Iran. Seven experimental stages were conducted to isolate and characterize ACC deaminase-producing Pseudomonas fluorescens strains and to determine factors enhancing their growth and, consequently, their effects on the germination of canola seeds. Under salinity stress, in 14% of the isolates, ACC deaminase activity was observed, indicating that they were able to utilize ACC as the sole N-source. Bacterial strains differed in their ability to synthesize auxin and hydrogen cyanide compounds, as well as in their ACC deaminase activity. Under salinity stress, the rate of germinating seeds inoculated with the strains of ACC deaminase-producing Pseudomonas fluorescens and Pseudomonas putida, and seedling growth was significantly higher. These results indicate the significance of soil biological activities, including the activities of plant growth-promoting bacteria, in the alleviation of soil stresses such as salinity on plant growth.