The role of motility in the colonization of potato roots by Pseudomonas bacteria was studied. Four TnS-induced flagella-less mutants of the plant-growth-stimulating P. fluorescens WCS374 appeared to be impaired in their ability to colonize growing potato roots.Certain fluorescent Pseudomonas isolates are able to increase plant growth (3,10,16,28) or to protect plants against microbial pathogens (17,25). Our research is focused on strains that can be used to eliminate potato yield reductions as observed in Dutch fields in which potatoes are frequently grown (28). These yield reductions can be abolished by treatment of the seed potatoes with particular Pseudomonas strains (3, 16, 27, 28,). Essential for this beneficial effect of the bacteria on plants is the production of fluorescent siderophores, 22) which supposedly enable the Pseudomonas cells to scavenge most of the Fe3`ions from the Fe3+-poor soil, thereby depriving deleterious microorganisms of this essential element. A second factor which is supposed to be essential for efficient protection of the roots against deleterious microorganisms is the delivery of siderophores along the whole root system of the plant, which requires efficient colonization of the potato roots. Although root colonization is very important in nature, virtually nothing is known about it at the molecular level. We studied the role of motility of Pseudomonas bacteria in the colonization of potato roots. MATERIALS ANID METHODSStrains and growth conditions. The potato root isolate Pseudomonas fluorescens WCS374 (7, 9, 10) is resistant to nalidixic acid (25 ,ug/ml). Strain JM3741 is a TnS-marked derivative of WCS374 which was isolated by J. Marugg. It does not differ significantly from the parental strain in its root-colonizing ability, its siderophore production, its growth rate in the complex King B medium or in minimal medium, and its motility (P. A. H. M. Bakker, unpublished data). Escherichia coli CSH52 harboring the mobilizable plasmid pSUP202 (Apr Cmr Kmr Tcr) and strain S17-1 harboring pSUP2021 (=pSUP202 with Tn5 inserted into the gene for tetracycline resistance) were obtained from R. Simon (29). Pseudomonas strains were grown at 28°C and E. ccli strains were grown at 37°C in King B medium (15) under vigorous aeration. For the root colonization assay TnSlabeled strains were cultivated at 28°C for 48 h on King B medium solidified with 1.6% agar and supplemented with kanamycin (50 ,ug/ml), The TnS-labeled strains were resistant to kanamycin (200 ,ug/ml) and streptomycin (200 ,ug/ml) * Corresponding author.(TnS encodes both kanamycin and streptomycin resistance in Pseudomonas species).Isolation of Tn5-induced nonmotile mutants. TnS-induced mutants were obtained by the method of Simon et al. (29) with slight modifications as described by Marugg et al. (22). Briefly, E. coli S17-1 harboring pSUP2021 was mated with P. fluorescens WCS374 for 3 to 4 h at 28°C. Transconjugants were selected on King B agar plates containing 25 ,ug of kanamycin per ml and 20 ,g of nalidixic acid per ml. Colon...

To visualize simultaneously different populations of pseudomonads in the rhizosphere at the single cell level in a noninvasive way, a set of four rhizosphere-stable plasmids was constructed expressing three different derivatives of the green fluorescent protein (GFP), namely enhanced cyan (ECFP), enhanced green (EGFP), enhanced yellow (EYFP), and the recently published red fluorescent protein (RFP; DsRed). Upon tomato seedling inoculation with Pseudomonas fluorescens WCS365 populations, each expressing a different autofluorescent protein followed by plant growth for 5 days, the rhizosphere was inspected using confocal laser scanning microscopy. We were able to visualize simultaneously and clearly distinguish from each other up to three different bacterial populations. Microcolonies consisting of mixed populations were frequently observed at the base of the root system, whereas microcolonies further toward the root tip predominantly consisted of a single population, suggesting a dynamic behavior of microcolonies over time. Since the cloning vector pME6010 has a broad host range for gram-negative bacteria, the constructed plasmids can be used for many purposes. In particular, they will be of great value for the analysis of microbial communities, for example in processes such as biocontrol, biofertilization, biostimulation, competition for niches, colonization, and biofilm formation.

The fungus Fusarium oxysporum f. sp. radicis-lycopersici causes foot and root rot of tomato plants, which can be controlled by the bacteria Pseudomonas fluorescens WCS365 and P. chlororaphis PCL1391. Induced systemic resistance is thought to be involved in biocontrol by P. fluorescens WCS365. The antifungal metabolite phenazine-1-carboxamide (PCN), as well as efficient root colonization, are essential in the mechanism of biocontrol by P. chlororaphis PCL1391. To understand the effects of bacterial strains WCS365 and PCL1391 on the fungus in the tomato rhizosphere, microscopic analyses were performed using different autofluorescent proteins as markers. Tomato seedlings were inoculated with biocontrol bacteria and planted in an F. oxysporum f. sp. radicis-lycopersici-infested gnotobiotic sand system. Confocal laser scanning microscope analyses of the interactions in the tomato rhizosphere revealed that i) the microbes effectively compete for the same niche, and presumably also for root exudate nutrients; ii) the presence of either of the two bacteria negatively affects infection of the tomato root by the fungus; iii) both biocontrol bacteria colonize the hyphae extensively, which may represent a new mechanism in biocontrol by these pseudomonads; and iv) the production of PCN by P. chlororaphis PCL1391 negatively affects hyphal growth and branching, which presumably affects the colonization and infecting ability of the fungus.

Transcript profiling is crucial to study biological systems, and various platforms have been implemented to survey mRNAs at the genome scale. We have assessed the performance of the CATMA microarray designed for Arabidopsis (Arabidopsis thaliana) transcriptome analysis and compared it with the Agilent and Affymetrix commercial platforms. The CATMA array consists of gene-specific sequence tags of 150 to 500 bp, the Agilent (Arabidopsis 2) array of 60mer oligonucleotides, and the Affymetrix gene chip (ATH1) of 25mer oligonucleotide sets. We have matched each probe repertoire with the Arabidopsis genome annotation (The Institute for Genomic Research release 5.0) and determined the correspondence between them. Array performance was analyzed by hybridization with labeled targets derived from eight RNA samples made of shoot total RNA spiked with a calibrated series of 14 control transcripts. CATMA arrays showed the largest dynamic range extending over three to four logs. Agilent and Affymetrix arrays displayed a narrower range, presumably because signal saturation occurred for transcripts at concentrations beyond 1,000 copies per cell. Sensitivity was comparable for all three platforms. For Affymetrix GeneChip data, the RMA software package outperformed Microarray Suite 5.0 for all investigated criteria, confirming that the information provided by the mismatch oligonucleotides has no added value. In addition, taking advantage of replicates in our dataset, we conducted a robust statistical analysis of the platform propensity to yield false positive and false negative differentially expressed genes, and all gave satisfactory results. The results establish the CATMA array as a mature alternative to the Affymetrix and Agilent platforms.