Influence of temperature on the motility of Pseudomonas oryzihabitans and control of Globodera rostochiensis

The motility and efficacy of Pseudomonas oryzihabitans as a biocontrol agent against the potato cyst nematode Globodera rostochiensis were studied with respect to temperature. The influence of soil moisture on bacterial movement was also tested. In a closed container trial, P. oryzihabitans significantly reduced invasion of second stage juveniles (J2) of G. rostochiensis in potato roots, its effect being more marked at 25 and 21 degreesC than at 17 degreesC. P. oryzihabitans motility in vitro was optimal at 26 degreesC and inhibited at temperatures below 18 degreesC. In soil, both temperature and matric potential affected bacterial movement. At 16 degreesC its movement and survival were suppressed, but they were unaffected at 25 degreesC. At both temperatures the biocontrol agent moved faster in the wetter (- 0.03 MPa) than in the drier soil (- 0.1 MPa). These results suggest that temperature is a key factor in determining the potential of P. or.yzihabitans as a biocontrol agent. (C) 2003 Elsevier Science Ltd. All rights reserved.

Influence of temperature on the motility of Pseudomonas oryzihabitans and control of Globodera rostochiensis

The motility and efficacy of Pseudomonas oryzihabitans as a biocontrol agent against the potato cyst nematode Globodera rostochiensis were studied with respect to temperature. The influence of soil moisture on bacterial movement was also tested. In a closed container trial, P. oryzihabitans significantly reduced invasion of second stage juveniles (J2) of G. rostochiensis in potato roots, its effect being more marked at 25 and 21 degreesC than at 17 degreesC. P. oryzihabitans motility in vitro was optimal at 26 degreesC and inhibited at temperatures below 18 degreesC. In soil, both temperature and matric potential affected bacterial movement. At 16 degreesC its movement and survival were suppressed, but they were unaffected at 25 degreesC. At both temperatures the biocontrol agent moved faster in the wetter (- 0.03 MPa) than in the drier soil (- 0.1 MPa). These results suggest that temperature is a key factor in determining the potential of P. or.yzihabitans as a biocontrol agent. (C) 2003 Elsevier Science Ltd. All rights reserved.

Involvement of phenazine-1-carboxylic acid, siderophores and hydrogen cyanide in suppression of Rhizoetonia solani and Pythium spp. damping-off by Pseudomonas oryzihabitans and Xenorhabdus nematophila

Entomopathogenic bacterial strains Pseudomonas (Flavimonas) oryzihabitans and Xenorhabdus nematophilus, both bacterial symbionts of the entomopathogenic nematodes Steinernema abbasi and S. carpocapsae have been recently used for suppression of soil-borne pathogens. Bacterial biocontrol agents (P. oryzihabitans and X nematophila) have been tested for production of secondary metabolites in vitro and their fungistatic effect,on mycelium and spore development of soil-borne pathogens. Isolates of Pythium spp. and Rhizoctonia solani, the causal agent of cotton damping-off, varied in sensitivity in vitro to the antibiotics phenazine-I-carboxylic acid (PCA), cyanide (HCN) and siderophores produced by bacterial strains shown previously to have potential for biological control of those pathogens. These findings affirm the role of the antibiotics PCA, HCN and siderophores in the biocontrol activity of these entomopathogenic strains and support earlier evidence that mechanisms of secondary metabolites are responsible for suppression of damping-off diseases. In the present studies colonies of R oryzihabitans showed production of PCA with presence of crystalline deposits after six days development and positive production where found as well in the siderophore's assay when X nematophila strain indicated HCN production in the in vitro assays. In vitro antifungal activity showed that bacteria densities of 101 to 10(6)cells/ml have antifungal activity in different media cultures. The results show further that isolates of Pythium spp. and R. solani insensitive to PCA, HCN and siderophores are present in the pathogen population and provide additional justification for the use of mixtures of entomopathogenic strains that employ different mechanisms of pathogen suppression to manage damping-off.

The control of root-knot nematodes (Meloidogyne spp.) by Pseudomonas oryzihabitans and its immunological detection on tomato roots

Pseudomonas oryzihabitans, a bacterium associated with the entomopathogenic nematode Steinernema abbasi, was evaluated for its potential to colonise roots and thereby control a field population of root-knot nematodes. Immunological techniques were developed to detect root colonisation of P. oryzihabitans on tomato roots using a specific polyclonal antibody raised against vegetative bacterial cells. In vitro, bacterial cell filtrates were also shown significantly to inhibit juveniles hatching. In a glasshouse pot experiment, there were 22 and 82% fewer females in roots of plants treated with suspensions containing 10(3) and 10(6) cells ml(-1) of P oryzihabitans, respectively. In addition, there were significantly fewer egg masses produced; however, the numbers of eggs per egg mass did not differ significantly. The relationship between root colonisation and nematode control is discussed.

Genomic and genetic analyses of diversity and plant interactions of Pseudomonas fluorescens

Background: Pseudomonas fluorescens are common soil bacteria that can improve plant health through nutrient cycling, pathogen antagonism and induction of plant defenses. The genome sequences of strains SBW25 and Pf0-1 were determined and compared to each other and with P. fluorescens Pf-5. A functional genomic in vivo expression technology (IVET) screen provided insight into genes used by P. fluorescens in its natural environment and an improved understanding of the ecological significance of diversity within this species. Results: Comparisons of three P. fluorescens genomes (SBW25, Pf0-1, Pf-5) revealed considerable divergence: 61% of genes are shared, the majority located near the replication origin. Phylogenetic and average amino acid identity analyses showed a low overall relationship. A functional screen of SBW25 defined 125 plant-induced genes including a range of functions specific to the plant environment. Orthologues of 83 of these exist in Pf0-1 and Pf-5, with 73 shared by both strains. The P. fluorescens genomes carry numerous complex repetitive DNA sequences, some resembling Miniature Inverted-repeat Transposable Elements (MITEs). In SBW25, repeat density and distribution revealed 'repeat deserts' lacking repeats, covering approximately 40% of the genome. Conclusions: P. fluorescens genomes are highly diverse. Strain-specific regions around the replication terminus suggest genome compartmentalization. The genomic heterogeneity among the three strains is reminiscent of a species complex rather than a single species. That 42% of plant-inducible genes were not shared by all strains reinforces this conclusion and shows that ecological success requires specialized and core functions. The diversity also indicates the significant size of genetic information within the Pseudomonas pan genome.

Phylogenetic analysis of the pPT23A plasmid family of Pseudomonas syringae

The pPT23A plasmid family of Pseudomonas syringae contains members that contribute to the ecological and pathogenic fitness of their P. syringae hosts. In an effort to understand the evolution of these plasmids and their hosts, we undertook a comparative analysis of the phylogeny of plasmid genes and that of conserved chromosomal genes from P. syringae. In total, comparative sequence and phylogenetic analyses were done utilizing 47 pPT23A family plasmids (PFPs) from 16 pathovars belonging to six genomospecies. Our results showed that the plasmid replication gene (repA), the only gene currently known to be distributed among all the PFPs, had a phylogeny that was distinct from that of the P. syringae hosts of these plasmids and from those of other individual genes on PFPs. The phylogenies of two housekeeping chromosomal genes, those for DNA gyrase B subunit (gyrB) and primary sigma factor (rpoD), however, were strongly associated with genomospecies of P. syringae. Based on the results from this study, we conclude that the pPT23A plasmid family represents a dynamic genome that is mobile among P. syringae pathovars.

Thioquinolobactin, a pseudomonas siderophore with antifungal and anti-pythium activity

Under conditions of iron limitation Pseudomonas fluorescens ATCC 17400 produces two siderophores, pyoverdine, and a second siderophore quinolobactin, which itself results from the hydrolysis of the unstable molecule 8-hydroxy-4-methoxy-2-quinoline thiocarboxylic acid (thioquinolobactin). Pseudomonas fluorescens ATCC 17400 also displays a strong in vitro antagonism against the Oomycete Pythium, which is repressed by iron, suggesting the involvement of a siderophore(s). While a pyoverdine-negative mutant retains most of its antagonism, a thioquinolobactin-negative mutant only slowed-down Pythium growth, and a double pyoverdine-, thioquinolobactin-negative mutant, which does not produce any siderophore, totally lost its antagonism against Pythium. The siderophore thioquinolobactin could be purified and identified from spent medium and showed anti-Pythium activity, but it was quickly hydrolysed to quinolobactin, which we showed has no antimicrobial activity. Analysis of antagonism-affected transposon mutants revealed that genes involved in haem biosynthesis and sulfur assimilation are important for the production of thioquinolobactin and the expression of antagonism.

The use of bioluminescence for monitoring in planta growth dynamics of a Pseudomonas syringae plant pathogen

The use of bioluminescence was evaluated as a tool to study Pseudomonas syringae population dynamics in susceptible and resistant plant environments. Plasmid pGLITE, containing the luxCDABE genes from Photorhabdus luminescens, was introduced into Pseudomonas syringae pv. phaseolicola race 7 strain 1449B, a Gram-negative pathogen of bean (Phaseolus vulgaris). Bacteria recovered from plant tissue over a five-day period were enumerated by counting numbers of colony forming units and by measurement of bioluminescence. Direct measurement of bioluminescence from leaf disc homogenates consistently reflected bacterial growth as determined by viable counting, but also detected subtle effects of the plant resistance response on bacterial viability. This bioluminescence procedure enables real time measurement of bacterial metabolism and population dynamics in planta, obviates the need to carry out labour intensive and time consuming traditional enumeration techniques and provides a sensitive assay for studying plant effects on bacterial cells.

The use of gaseous ozone and gas packaging to control populations of Salmonella infantis and Pseudomonas aeruginosa on the skin of chicken portions

Chilled breasts of chicken were inoculated with Salmonella infantis or Pseudomonas aeruginosa and then given one of the following treatments: (i) exposure to gaseous ozone (>2000 ppm for up to 30 min); (ii) storage under 70% CO2:30% N-2; and (iii) exposure to gaseous ozone (>2000 ppm for 15 min) followed by storage under 70% CO2:30% N-2; all storage at 7degreesC. Gaseous ozone reduced the counts of samnonellae by 97(Y,, and pseudomonads by 95%, but indigenous coliforms were unaffected. Under the modified atmosphere, the cell count of S. infantis was reduced by 72% following initial exposure and then stabilised, coliforms grew, but Ps. aeruginosa behaved like S. infantis-initial reduction (58%) followed by stability. Exposure to gaseous ozone followed by gas packaging allowed survival of S. infantis, Ps. aeruginosa and coliforms over 9 days at 7degreesC, but there was no evidence of any sensory deterioration. It is proposed that the latter treatment could, in a modified form perhaps, be used to reduce the contamination of chicken carcasses with salmonellae and improve their shelf-life. (C) 2004 Elsevier Ltd. All rights reserved.

Testing temperature-induced proteomic changes in the plant-associated bacterium Pseudomonas fluorescens SBW25.

Traits used by bacteria to enhance ecological performance in natural environments are not well understood. Recognizing that the saprophytic plant-colonizing bacterium Pseudomonas fluorescens SBW25 experiences temperatures in its natural environment significantly cooler than the 28°C routinely used in the laboratory, we identified proteins differentially expressed between 28°C and the more environmentally relevant temperature of 14°C. Of 2102 protein isoforms, 32 were temperature responsive and identified by mass spectrometry. Seven of these (OmpR, MucD, GuaD, OsmY and three of unknown function, Tee1, Tee2 and Tee3) were selected for genetic and ecological analyses. In each instance, changes in protein expression with temperature were mirrored by parallel transcriptional changes. The fitness contribution of the genes encoding each of the seven proteins was larger at 14°C than 28°C and included two cases of trade-offs (enhanced fitness at one temperature and reduced fitness at the other – mucD and tee2 deletions). The relationship between the fitness effects of genes in vitro and in vivo was variable, but two temperature-responsive genes – osmY and mucD – contribute substantially to the ability of P. fluorescens to colonize the plant environment.

Comparative genome analysis provides insights into the evolution and adaptation of Pseudomonas syringae pv. aesculi on Aesculus hippocastanum.

A recently emerging bleeding canker disease, caused by Pseudomonas syringae pathovar aesculi (Pae), is threatening European horse chestnut in northwest Europe. Very little is known about the origin and biology of this new disease. We used the nucleotide sequences of seven commonly used marker genes to investigate the phylogeny of three strains isolated recently from bleeding stem cankers on European horse chestnut in Britain (E-Pae). On the basis of these sequences alone, the E-Pae strains were identical to the Pae type-strain (I-Pae), isolated from leaf spots on Indian horse chestnut in India in 1969. The phylogenetic analyses also showed that Pae belongs to a distinct clade of P. syringae pathovars adapted to woody hosts. We generated genome-wide Illumina sequence data from the three E-Pae strains and one strain of I-Pae. Comparative genomic analyses revealed pathovar-specific genomic regions in Pae potentially implicated in virulence on a tree host, including genes for the catabolism of plant-derived aromatic compounds and enterobactin synthesis. Several gene clusters displayed intra-pathovar variation, including those encoding type IV secretion, a novel fatty acid biosynthesis pathway and a sucrose uptake pathway. Rates of single nucleotide polymorphisms in the four Pae genomes indicate that the three E-Pae strains diverged from each other much more recently than they diverged from I-Pae. The very low genetic diversity among the three geographically distinct E-Pae strains suggests that they originate from a single, recent introduction into Britain, thus highlighting the serious environmental risks posed by the spread of an exotic plant pathogenic bacterium to a new geographic location. The genomic regions in Pae that are absent from other P. syringae pathovars that infect herbaceous hosts may represent candidate genetic adaptations to infection of the woody parts of the tree.