L. A. De Weger

Gnotobiotic system for studying rhizosphere colonization by plant growth-promoting Pseudomonas bacteria.

A gnotobiotic system for studying tomato rhizosphere colonization by Pseudomonas bacteria was developed. The system is based on sterile seedlings that are inoculated with one or two strains and subsequently grown in a sterile glass tube containing quartz sand. After 7 days of growth in a climate-controlled growth chamber, the number of bacteria present on the root tip was analyzed. The system was optimized with respect to root morphology, inoculation of the seedling, and isolation of root tip bacteria. With this system, rhizosphere colonization on tomato, radish, wheat, and potato was analyzed. For detailed analysis of tomato rhizosphere colonization by some representative plant growth-promoting rhizo-bacteria, the colonization of known poor, moderate, and good potato root-colonizing Pseudomonas strains and of four Rhizobium strains was determined. All strains colonized the root tips when inoculated as single strains. When inoculated in competition with the efficient root colonizer P. fluorescens strain WCS365, many strains were outcompeted. Mutants of Pseudomonas biocontrol bacteria lacking flagella or the O-antigen of lipopolysaccharide (LPS), which were isolated in previous studies and shown to be impaired in potato rhizosphere colonization in field soil systems, showed a reduced colonization ability in the gnotobiotic system also. The gnotobiotic system was used to screen a collection of 300 random P. fluorescens WCS365::Tn5 mutants for colonization-impaired mutants. Three novel mutants were found that were outcompeted by the wild-type strain in tomato root tip colonization but were not impaired in known colonization traits such as motility, amino acid auxotrophy, and presence of the O-antigenic side chain of LPS. One strain appeared to be a thiamine auxotroph, suggesting that the root does not secrete a sufficient amount of thiamine to support growth of this strain. The other two mutants had a reduced growth rate in laboratory media, suggesting that growth rate is an important colonization factor. As the system is gnotobiotic and devoid of field-soil variables, it can also be used to study the effects of selected biotic and abiotic factors on colonization.

Amino acid synthesis is necessary for tomato root colonization by Pseudomonas fluorescens strain WCS365

In this work the bio-availability of amino acids for the root-colonizing Pseudomonas fluorescens strain WCS365 in the tomato rhizosphere was studied. The amino acid composition of axenically collected tomato root exudate was determined. The results show that aspartic acid, glutamic acid, isoleucine, leucine, and lysine are the major amino acid components. The concentrations of individual amino acids in the rhizosphere of gnotobiotically grown tomato plants were estimated and considered to be too low to support growth of rhizosphere micro-organisms to numbers usually found in the tomato rhizosphere. To test this experimentally, mutants of P. fluorescens WCS365 auxotrophic for the amino acids leucine, arginine, histidine, isoleucine plus valine, and tryptophan were isolated after mutagenesis with Tn5lacZ. Root tip colonization of these mutants was measured after inoculation of germinated tomato seeds and subsequent growth in a gnotobiotic quartz sand system (M. Simons, A. J. van der Bij, I. Brand, L. A. de Weger, C. A. Wijffelman, and B. J. J. Lugtenberg. 1996. Gnotobiotic system for studying rhizosphere colonization by plant growth-promoting Pseudomonas bacteria. Mol. Plant-Microbe Interact. 9:600-607). In contrast to the wild-type strain, none of the five amino acid auxotrophs tested was able to colonize the tomato root tip, neither alone nor after co-inoculation with the wild-type strain. However, addition of the appropriate amino acid to the system restored colonization by the auxotrophic mutants, usually to wild-type levels. Analysis of the root base showed that cells of auxotrophic mutants were still present there. The results show that, although amino acids are present in root exudate, the bio-availability of the tested amino acids is too low to support root tip colonization by auxotrophic mutants of P. fluorescens strain WCS365. The genes that are required for amino acid synthesis are therefore necessary for root colonization. Moreover, these compounds apparently play no major role as nutrients in the tomato rhizosphere.

Quantitative trends in annual totals of five common airborne pollen types (Betula, Quercus, Poaceae, Urtica, and Artemisia), at five pollen-monitoring stations in western Europe

The existence of long-term (20–33 years) trendsin the annual totals of daily airborne pollenconcentrations of five common and/or allergenicwind pollinating taxa was evaluated at fivepollen-monitoring stations in western Europe:Delmenhorst (D), Helmond (NL), Brussels (B),Leiden (NL), and Derby (UK). At all stations,identical or comparable volumetric traps wereused to sample pollen from the air. For grasspollen no increasing or decreasing trends werefound at any station. Trends for birch pollenand oak pollen are increasing, but notsignificant at the stations with the higherannual totals (Delmenhorst and Helmond),probably due to strong year-to-yearfluctuations. At all five stations significantincreasing trends for stinging nettle pollenwere observed. Trends for mugwort pollen aresignificant at all stations, but in differentdirections. Longer periods of observations areneeded to arrive to more definitive conclusionsabout trends in airborne pollenconcentrations.

The ferric-pseudobactin receptor PupA of Pseudomonas putida WCS358: homology to TonB-dependent Escherichia coli receptors and specificity of the protein

The initial step in the uptake of iron via ferric pseudobactin by the plant‐growth‐promoting Pseudomonas putida strain WCS358 is binding to a specific outer‐membrane protein. The nucleotide sequence of the pupA structural gene, which codes for a ferric pseudobactin receptor, was determined. It contains a single open reading frame which potentially encodes a polypeptide of 819 amino acids, including a putative N‐terminal signal sequence of 47 amino acids. Significant homology, concentrated in four boxes, was found with the TonB‐dependent receptor proteins of Escherichia coli. The pupA mutant MH100 showed a residual efficiency of 30% in the uptake of 55HFe3+ complexed to pseudobactin 358, whereas the iron uptake of four other pseudobactins was not reduced at all. Cells of strain WCS374 supplemented with the pupA gene of strain WCS358 could transport ferric pseudobactin 358 but showed no affinity for three other pseudobactins. It is concluded that PupA is a specific receptor for ferric pseudobactin 358, and that strain WCS358 produces at least one other receptor for other pseudobactins.

Characterization of Cell Envelope Protein Patterns of Crop Yield Increasing Root-Colonizing Pseudomonas spp

Yield reduction in high frequency cropping soil is often caused by unknown deleterious micro-organisms. Such yield depressions can be reduced by seed inoculation with fluorescent Pseudomonas spp., which display in vitro antagonism to other micro-organisms (Geels and Schippers, 1983). To explain the beneficial effect of these Pseudomonas spp. on crop yield a mechanism has been suggested in which, at the root-surface a competition for Fe3+ takes place between Pseudomonas spp. and deleterious microorganisms (Kloepper et al., 1980). Under the limiting Fe3+ conditions in soil Pseudomonas spp. are thought to produce a high affinity Fe3+-uptake system, consisting of (i) siderophores, which are Fe3+-chelating agents, and (ii) outer membrane receptor proteins, which have a high affinity and specificity for the matching Fe3+-siderophore complex (Neilands, 1982). These siderophores bind Fe3+, thereby making this essential element unavailable to other micro-organisms, including deleterious micro-organisms. This results in a reduction of the number of deleterious micro-organisms, thus creating a more favourable environment for the plant.

Mutational changes in the O-antigenic side chains of the lipopolysaccharides of Pseudomonas spp. affect colonization of but not adhesion to potato roots

The world wide interest in Pseudomonas spp. as biocontrol agents is mainly based on their in vitro antagonistic potentials combined with their ability to colonize the roots of many plants efficiently. The properties that confer the colonization abilities on these bacteria are largely unknown. This study focuses on the role of the O-antigenic side chain of the LPS (=lipopolysaccharides) of two plant growth-stimulating Pseudomonas strains, WCS358 and WCS374, in the root colonization process.