Effect of Genetically Modified Pseudomonas putida WCS358r on the Fungal Rhizosphere Microflora of Field-Grown Wheat

We released genetically modified Pseudomonas putida WCS358r into the rhizospheres of wheat plants. The two genetically modified derivatives, genetically modified microorganism (GMM) 2 and GMM 8, carried the phz biosynthetic gene locus of strain P. fluorescens 2-79 and constitutively produced the antifungal compound phenazine-1-carboxylic acid (PCA). In the springs of 1997 and 1998 we sowed wheat seeds treated with either GMM 2, GMM 8, or WCS358r (approximately 10⁷ CFU per seed), and measured the numbers, composition, and activities of the rhizosphere microbial populations. During both growing seasons, all three bacterial strains decreased from 10⁷ CFU per g of rhizosphere sample to below the limit of detection (10² CFU per g) 1 month after harvest of the wheat plants. The phz genes were stably maintained, and PCA was detected in rhizosphere extracts of GMM-treated plants. In 1997, but not in 1998, fungal numbers in the rhizosphere, quantified on 2% malt extract agar (total filamentous fungi) and on Komada's medium (mainly Fusarium spp.), were transiently suppressed in GMM 8-treated plants. We also analyzed the effects of the GMMs on the rhizosphere fungi by using amplified ribosomal DNA restriction analysis. Introduction of any of the three bacterial strains transiently changed the composition of the rhizosphere fungal microflora. However, in both 1997 and 1998, GMM-induced effects were distinct from those of WCS358r and lasted for 40 days in 1997 and for 89 days after sowing in 1998, whereas effects induced by WCS358r were detectable for 12 (1997) or 40 (1998) days. None of the strains affected the metabolic activity of the soil microbial population (substrate-induced respiration), soil nitrification potential, cellulose decomposition, plant height, or plant yield. The results indicate that application of GMMs engineered to have improved antifungal activity can exert nontarget effects on the natural fungal microflora.     

Repeated introduction of genetically modified Pseudomonas putida WCS358r without intensified effects on the indigenous microflora of field-grown wheat

To investigate the impact of genetically modified, antibiotic-producing rhizobacteria on the indigenous microbial community, Pseudomonas putida WCS358r and two transgenic derivatives were introduced as a seed coating into the rhizosphere of wheat in two consecutive years (1999 and 2000) in the same field plots. The two genetically modified microorganisms (GMMs), WCS358r::phz and WCS358r::phl, constitutively produced phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (DAPG), respectively. The level of introduced bacteria in all treatments decreased from 10(7) CFU per g of roots soon after sowing to less than 10(2) CFU per g after harvest 132 days after sowing. The phz and phl genes remained stable in the chromosome of WCS358r. The amount of PCA produced in the wheat rhizosphere by WCS358r::phz was about 40 ng/g of roots after the first application in 1999. The DAPG-producing GMMs caused a transient shift in the indigenous bacterial and fungal microflora in 1999, as determined by amplified ribosomal DNA restriction analysis. However, after the second application of the GMMs in 2000, no shifts in the bacterial or fungal microflora were detected. To evaluate the importance of the effects induced by the GMMs, these effects were compared with those induced by crop rotation by planting wheat in 1999 followed by potatoes in 2000. No effect of rotation on the microbial community structure was detected. In 2000 all bacteria had a positive effect on plant growth, supposedly due to suppression of deleterious microorganisms. Our research suggests that the natural variability of microbial communities can surpass the effects of GMMs.     

Effect of genetically modified Pseudomonas putida WCS358r on the fungal rhizosphere microflora of field-grown wheat

We released genetically modified Pseudomonas putida WCS358r into the rhizospheres of wheat plants. The two genetically modified derivatives, genetically modified microorganism (GMM) 2 and GMM 8, carried the phz biosynthetic gene locus of strain P. fluorescens 2-79 and constitutively produced the antifungal compound phenazine-1-carboxylic acid (PCA). In the springs of 1997 and 1998 we sowed wheat seeds treated with either GMM 2, GMM 8, or WCS358r (approximately 10(7) CFU per seed), and measured the numbers, composition, and activities of the rhizosphere microbial populations. During both growing seasons, all three bacterial strains decreased from 10(7) CFU per g of rhizosphere sample to below the limit of detection (10(2) CFU per g) 1 month after harvest of the wheat plants. The phz genes were stably maintained, and PCA was detected in rhizosphere extracts of GMM-treated plants. In 1997, but not in 1998, fungal numbers in the rhizosphere, quantified on 2% malt extract agar (total filamentous fungi) and on Komada's medium (mainly Fusarium spp.), were transiently suppressed in GMM 8-treated plants. We also analyzed the effects of the GMMs on the rhizosphere fungi by using amplified ribosomal DNA restriction analysis. Introduction of any of the three bacterial strains transiently changed the composition of the rhizosphere fungal microflora. However, in both 1997 and 1998, GMM-induced effects were distinct from those of WCS358r and lasted for 40 days in 1997 and for 89 days after sowing in 1998, whereas effects induced by WCS358r were detectable for 12 (1997) or 40 (1998) days. None of the strains affected the metabolic activity of the soil microbial population (substrate-induced respiration), soil nitrification potential, cellulose decomposition, plant height, or plant yield. The results indicate that application of GMMs engineered to have improved antifungal activity can exert nontarget effects on the natural fungal microflora.     

Ascomycete communities in the rhizosphere of field-grown wheat are not affected by introductions of genetically modified Pseudomonas putida WCS358r

A long-term field experiment (1999-2002) was conducted to monitor effects on the indigenous microflora of Pseudomonas putida WCS358r and two transgenic derivatives constitutively producing phenazine-1-carboxylic acid (PCA) or 2,4-diacetylphloroglucinol (DAPG). The strains were introduced as seed coating on wheat into the same field plots each year. Rhizosphere populations of ascomycetes were analysed using denaturing gradient gel electrophoresis (DGGE). To evaluate the significance of changes caused by the genetically modified microorganisms (GMMs), they were compared with effects caused by a crop rotation from wheat to potato. In the first year, only the combination of both GMMs caused a significant shift in the ascomycete community. After the repeated introductions this effect was no longer evident. However, cropping potato significantly affected the ascomycete community. This effect persisted into the next year when wheat was grown. Clone libraries were constructed from samples taken in 1999 and 2000, and sequence analysis indicated ascomycetes of common genera to be present. Most species occurred in low frequencies, distributed almost evenly in all treatments. However, in 1999 Microdochium occurred in relatively high frequencies, whereas in the following year no Microdochium species were detected. On the other hand, Fusarium-like organisms were low in 1999, and increased in 2000. Both the DGGE and the sequence analysis revealed that repeated introduction of P. putida WCS358r had no major effects on the ascomycete community in the wheat rhizosphere, but demonstrated a persistent difference between the rhizospheres of potato and wheat.     

Characterization of type IV pilus genes in plant growth-promoting Pseudomonas putida WCS358.

In a search for factors that could contribute to the ability of the plant growth-stimulating Pseudomonas putida WCS358 to colonize plant roots, the organism was analyzed for the presence of genes required for pilus biosynthesis. The pilD gene of Pseudomonas aeruginosa, which has also been designated xcpA, is involved in protein secretion and in the biogenesis of type IV pili. It encodes a peptidase that processes the precursors of the pilin subunits and of several components of the secretion apparatus. Prepilin processing activity could be demonstrated in P. putida WCS358, suggesting that this nonpathogenic strain may contain type IV pili as well. A DNA fragment containing the pilD (xcpA) gene of P. putida was cloned and found to complement a pilD (xcpA) mutation in P. aeruginosa. Nucleotide sequencing revealed, next to the pilD (xcpA) gene, the presence of two additional genes, pilA and pilC, that are highly homologous to genes involved in the biogenesis of type IV pili. The pilA gene encodes the pilin subunit, and pilC is an accessory gene, required for the assembly of the subunits into pili. In comparison with the pil gene cluster in P. aeruginosa, a gene homologous to pilB is lacking in the P. putida gene cluster. Pili were not detected on the cell surface of P. putida itself, not even when pilA was expressed from the tac promoter on a plasmid, indicating that not all the genes required for pilus biogenesis were expressed under the conditions tested. Expression of pilA of P. putida in P. aeruginosa resulted in the production of pili containing P. putida PilA subunits.     

Isolation and analysis of genes involved in siderophore biosynthesis in plant-growth-stimulating Pseudomonas putida WCS358

The plant-growth-stimulating Pseudomonas putida WCS358 was mutagenized with transposon Tn5. The resulting mutant colony bank was screened for mutants defective in the biosynthesis of the fluorescent siderophore. A total of 28 mutants, divided into six different classes, were isolated that were nonfluorescent or defective in iron acquisition or both. These different types of mutants together with the probable overall structure of the siderophore, i.e., a small peptide chain attached to a fluorescing group, suggest a biosynthetic pathway in which the synthesis of the fluorescing group is preceded by the synthesis of the peptide part. A gene colony bank of P. putida WCS358 was constructed with the broad-host-range cosmid vector pLAFR1. This genomic library, established in Escherichia coli, was mobilized into the 28 individual mutants, screening for transconjugants restored in fluorescence or growth under iron-limiting conditions or both. A total of 13 cosmids were found to complement 13 distinct mutants. The complementation analysis revealed that at least five gene clusters, with a minimum of seven genes, are needed for siderophore biosynthesis. Some of these genes seem to be arranged in an operon-like structure.     

Impact of Field Release of Genetically Modified Pseudomonas fluorescens on Indigenous Microbial Populations of Wheat

In a field release experiment, an isolate of Pseudomonas fluorescens, which was chromosomally modified with two reporter gene cassettes (lacZY and Kan(supr)-xylE), was applied to spring wheat as a seed coating and subsequently as a foliar spray. The wild-type strain was isolated from the phylloplane of sugar beet but was found to be a common colonizer of both the rizosphere and phylloplane of wheat as well. The impact on the indigenous microbial populations resulting from release of this genetically modified microorganism (GMM) was compared with the impact of the unmodified, wild-type strain and a nontreated control until 1 month after harvest of the crop. The release of the P. fluorescens GMM and the unmodified, wild-type strain resulted in significant but transient perturbations of some of the culturable components of the indigenous microbial communities that inhabited the rhizosphere and phylloplane of wheat, but no significant perturbations of the indigenous culturable microbial populations in nonrhizosphere soil were found. Fast-growing organisms that did not produce resting structures (for example, fluorescent pseudomonads and yeasts) seemed to be most sensitive to perturbation. In terms of hazard and risk to the environment, the observed microbial perturbations that resulted from this GMM release may be considered minor for several reasons. First, the recombinant P. fluorescens strain caused changes that were, in general, not significantly different from those caused by the unmodified wild-type strain; second, perturbations resulting from bacterial inoculations were mainly small; and third, the release of bacteria had no obvious effects on plant growth and plant health.     

Effect of pseudobactin 358 production by Pseudomonas putida WCS358 on suppression of fusarium wilt of carnations by nonpathogenic Fusarium oxysporum Fo47.

Nonpathogenic Fusarium oxysporum Fo47b10 combined with Pseudomonas putida WCS358 efficiently suppressed fusarium wilt of carnations grown in soilless culture. This suppression was significantly higher than that obtained by inoculation of either antagonistic microorganism alone. The increased suppression obtained by Fo47b10 combined with WCS358 only occurred when Fo47b10 was introduced at a density high enough (at least 10 times higher than that of the pathogen) to be efficient on its own. P. putida WCS358 had no effect on disease severity when inoculated on its own but significantly improved the control achieved with nonpathogenic F. oxysporum Fo47b10. In contrast, a siderophore-negative mutant of WCS358 had no effect on disease severity even in the presence of Fo47b10. Since the densities of both bacterial strains at the root level were similar, the difference between the wild-type WCS358 and the siderophore-negative mutant with regard to the control of fusarium wilt was related to the production of pseudobactin 358. The production of pseudobactin 358 appeared to be responsible for the increased suppression by Fo47b10 combined with WCS358 relative to that with Fo47b10 alone.     

Effect of pseudobactin 358 production by Pseudomonas putida WCS358 on suppression of fusarium wilt of carnations by nonpathogenic Fusarium oxysporum Fo47.

Nonpathogenic Fusarium oxysporum Fo47b10 combined with Pseudomonas putida WCS358 efficiently suppressed fusarium wilt of carnations grown in soilless culture. This suppression was significantly higher than that obtained by inoculation of either antagonistic microorganism alone. The increased suppression obtained by Fo47b10 combined with WCS358 only occurred when Fo47b10 was introduced at a density high enough (at least 10 times higher than that of the pathogen) to be efficient on its own. P. putida WCS358 had no effect on disease severity when inoculated on its own but significantly improved the control achieved with nonpathogenic F. oxysporum Fo47b10. In contrast, a siderophore-negative mutant of WCS358 had no effect on disease severity even in the presence of Fo47b10. Since the densities of both bacterial strains at the root level were similar, the difference between the wild-type WCS358 and the siderophore-negative mutant with regard to the control of fusarium wilt was related to the production of pseudobactin 358. The production of pseudobactin 358 appeared to be responsible for the increased suppression by Fo47b10 combined with WCS358 relative to that with Fo47b10 alone.     

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 55Fe3+ 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.     

Siderophore-mediated uptake of Fe3+ by the plant growth-stimulating Pseudomonas putida strain WCS358 and by other rhizosphere microorganisms.

Under iron-limited conditions, Pseudomonas putida WCS358 produces a siderophore, pseudobactin 358, which is essential for the plant growth-stimulating ability of this strain. Cells of strain WCS358, provided that they have been grown under Fe3+ limitation, take up 55Fe3+ from the 55Fe3+-labeled pseudobactin 358 complex with Km and Vmax values of 0.23 microM and 0.14 nmol/mg of cell dry weight per min, respectively. Uptake experiments with cells treated with various metabolic inhibitors showed that this Fe3+ uptake process was dependent on the proton motive force. Furthermore, strain WCS358 was shown to be able to take up Fe3+ complexed to the siderophore of another plant-beneficial P. fluorescens strain, WCS374. The tested pathogenic rhizobacteria and rhizofungi were neither able to grow on Fe3+-deficient medium in the presence of pseudobactin 358 nor able to take up 55Fe3+ from 55Fe3+-pseudobactin 358. The same applies for three cyanide-producing Pseudomonas strains which are supposed to be representatives of the minor pathogens. These results indicate that the extraordinary ability of strain WCS358 to compete efficiently for Fe3+ is based on the fact that the pathogenic and deleterious rhizosphere microorganisms, in contrast to strain WCS358 itself, are not able to take up Fe3+ from Fe3+-pseudobactin 358 complexes.     

Polymerase chain reaction for verification of fluorescent colonies of Erwinia chrysanthemi and Pseudomonas putida WCS358 in immunofluorescence colony staining

The potential of polymerase chain reaction (PCR) for verifying the identity of colonies stained by the immunofluorescence colony-staining (IFC) procedure was investigated. Using primers directed against conserved sequences of the pectate lyase-genes coding for isozymes PLa, PLd and PLe of Erwinia chrysanthemi , the authors confirmed the identity of 96% of 20 fluorescent target colonies, punched from IFC-stained samples with pure cultures. In pour plates with mixtures of Erw. chrysanthemi and non-target colonies from potato peel extracts, the identity of 90% of 113 target colonies was confirmed.
Using primers directed against sequences of the ferric-pseudobactin receptor gene pupA of Pseudomonas putida WCS358, the identity of 96% of 22 target colonies was confirmed in IFC-stained samples with pure cultures. In pour plates with mixtures of Ps. putida WCS358 and non-target bacteria from compost extracts, the identity of 59% of 108 fluorescent colonies was confirmed by PCR. It was shown that components from non-target bacteria lowered the threshold level of PCR for Ps. putida WCS358     

Suppression of fusarium wilt of carnation by Pseudomonas putida WCS358 at different levels of disease incidence and iron availability

Treatment with Pseudomonas putida WCS358r, a rifampicin‐resistant derivative of strain WCS358, significantly reduced fusarium wilt of carnation grown in rockwool if disease incidence was moderate, but not if disease incidence was high. Differences in disease incidence could intentionally be established by varying the inoculum density of the pathogen Fusarium oxysporum f. sp. dianthi (Fod). The effectiveness of disease suppression by WCS358r increased with decrease of inoculum density and consequently decrease of disease incidence. WCS358r and a Tn5 marked derivative of WCS358 (B243) reduced fusarium wilt of carnation most effectively if a low iron availability for the pathogen was established by adding unferrated or only partially ferrated ethylenediamine [di(o‐hydroxyphenylacetic) acid]. A Tn5 mutant of WCS358 defective in siderophore biosynthesis (JM218) did not reduce disease incidence. Siderophore production and inhibition of Fod by WCS358r in vitro decreased with increasing iron availability, supporting the more effective disease suppression by strains WCS358r and B243 at low iron availability. Siderophore‐mediated competition for iron was shown to be the mechanism of suppression of fusarium wilt of carnation by P. putida WCS358. Its effectivity was highest at a low iron availability and at a moderate disease incidence.     

Identification and characterization of the exbB, exbD and tonB genes of Pseudomonas putida WCS358: their involvement in ferric-pseudobactin transport

Catechol-cephalosporins are siderophore-like antibiotics which are taken up by cells of Pseudomonas putida WCS358 via the ferric-siderophore transport pathway. Mutants of strain WCS358 were isolated that are resistant to high concentrations of these antibiotics. These mutants failed to grow under iron-limiting conditions, and could not utilize different ferric-siderophores. The mutants fall in three complementation groups. The nucleotide sequence determination identified three contiguous open reading frames, which were homologous to the exbB, exbD and tonB genes of Escherichia coli respectively. The deduced amino acid sequence of P. putida ExbB showed 58.6% homology with its E. coli homologue, but, unlike the E. coli protein, it has a N-terminal extension of 91 amino acids. The ExbD proteins are 64.8% homologous, whereas the TonB proteins only show 27.7% homology. The P. putida exbB gene could complement an E. coli exbB mutation, but the TonB proteins were not interchangeable between the species. It is concluded that P. putida WCS358 contains an energy-coupling system between the membranes for active transport across the outer membrane, which is comprised of a TonB-like energy-transducing protein and two accessory proteins. This system is similar to, but not completely compatible with, the E. coli system.