Extracellular Protease of Pseudomonas fluorescens CHA0, a Biocontrol Factor with Activity against the Root-Knot Nematode Meloidogyne incognita

In Pseudomonas fluorescens CHA0, mutation of the GacA-controlled aprA gene (encoding the major extracellular protease) or the gacA regulatory gene resulted in reduced biocontrol activity against the root-knot nematode Meloidogyne incognita during tomato and soybean infection. Culture supernatants of strain CHA0 inhibited egg hatching and induced mortality of M. incognita juveniles more strongly than did supernatants of aprA and gacA mutants, suggesting that AprA protease contributes to biocontrol.     

Impact of Biocontrol Pseudomonas fluorescens CHA0 and a Genetically Modified Derivative on the Diversity of Culturable Fungi in the Cucumber Rhizosphere

Little is known about the effects of Pseudomonas biocontrol inoculants on nontarget rhizosphere fungi. This issue was addressed using the biocontrol agent Pseudomonas fluorescens CHA0-Rif, which produces the antimicrobial polyketides 2,4-diacetylphloroglucinol (Phl) and pyoluteorin (Plt) and protects cucumber from several fungal pathogens, including Pythium spp., as well as the genetically modified derivative CHA0-Rif(pME3424). Strain CHA0-Rif(pME3424) overproduces Phl and Plt and displays improved biocontrol efficacy compared with CHA0-Rif. Cucumber was grown repeatedly in the same soil, which was left uninoculated, was inoculated with CHA0-Rif or CHA0-Rif(pME3424), or was treated with the fungicide metalaxyl (Ridomil). Treatments were applied to soil at the start of each 32-day-long cucumber growth cycle, and their effects on the diversity of the rhizosphere populations of culturable fungi were assessed at the end of the first and fifth cycles. Over 11,000 colonies were studied and assigned to 105 fungal species (plus several sterile morphotypes). The most frequently isolated fungal species (mainly belonging to the genera Paecilomyces, Phialocephala, Fusarium, Gliocladium, Penicillium, Mortierella, Verticillium, Trichoderma, Staphylotrichum, Coniothyrium, Cylindrocarpon, Myrothecium, and Monocillium) were common in the four treatments, and no fungal species was totally suppressed or found exclusively following one particular treatment. However, in each of the two growth cycles studied, significant differences were found between treatments (e.g., between the control and the other treatments and/or between the two inoculation treatments) using discriminant analysis. Despite these differences in the composition and/or relative abundance of species in the fungal community, treatments had no effect on species diversity indices, and species abundance distributions fit the truncated lognormal function in most cases. In addition, the impact of treatments at the 32-day mark of either growth cycle was smaller than the effect of growing cucumber repeatedly in the same soil.     

Inactivation of the regulatory gene algU or gacA can affect the ability of biocontrol Pseudomonas fluorescens CHA0 to persist as culturable cells in nonsterile soil

Rifampin-resistant Pseudomonas fluorescens CHA0-Rif and mutants in which the regulatory gene algU (encoding sigma factor sigma(E)) or gacA (encoding a global regulator of secondary metabolism) was inactivated were compared for persistence in three nonsterile soils. Functional algU and (particularly) gacA were needed for CHA0-Rif to maintain cell culturability in soil.     

The Sigma Factor AlgU (AlgT) Controls Exopolysaccharide Production and Tolerance towards Desiccation and Osmotic Stress in the Biocontrol Agent Pseudomonas fluorescens CHA0

A variety of stress situations may affect the activity and survival of plant-beneficial pseudomonads added to soil to control root diseases. This study focused on the roles of the sigma factor AlgU (synonyms, AlgT, RpoE, and ς²²) and the anti-sigma factor MucA in stress adaptation of the biocontrol agent Pseudomonas fluorescens CHA0. The algU-mucA-mucB gene cluster of strain CHA0 was similar to that of the pathogens Pseudomonas aeruginosa and Pseudomonas syringae. Strain CHA0 is naturally nonmucoid, whereas a mucA deletion mutant or algU-overexpressing strains were highly mucoid due to exopolysaccharide overproduction. Mucoidy strictly depended on the global regulator GacA. An algU deletion mutant was significantly more sensitive to osmotic stress than the wild-type CHA0 strain and the mucA mutant were. Expression of an algU′-′lacZ reporter fusion was induced severalfold in the wild type and in the mucA mutant upon exposure to osmotic stress, whereas a lower, noninducible level of expression was observed in the algU mutant. Overexpression of algU did not enhance tolerance towards osmotic stress. AlgU was found to be essential for tolerance of P. fluorescens towards desiccation stress in a sterile vermiculite-sand mixture and in a natural sandy loam soil. The size of the population of the algU mutant declined much more rapidly than the size of the wild-type population at soil water contents below 5%. In contrast to its role in pathogenic pseudomonads, AlgU did not contribute to tolerance of P. fluorescens towards oxidative and heat stress. In conclusion, AlgU is a crucial determinant in the adaptation of P. fluorescens to dry conditions and hyperosmolarity, two major stress factors that limit bacterial survival in the environment.     

Contribution of the Global Regulator Gene gacA to Persistence and Dissemination of Pseudomonas fluorescens Biocontrol Strain CHA0 Introduced into Soil Microcosms

Structural and regulatory genes involved in the synthesis of antimicrobial metabolites are essential for the biocontrol activity of fluorescent pseudomonads and, in principle, amenable to genetic engineering for strain improvement. An eventual large-scale release of such bacteria raises the question of whether such genes also contribute to the persistence and dissemination of the bacteria in soil ecosystems. Pseudomonas fluorescens wild-type strain CHA0 protects plants against a variety of fungal diseases and produces several antimicrobial metabolites. The regulatory gene gacA globally controls antibiotic production and is crucial for disease suppression in CHA0. This gene also regulates the production of extracellular protease and phospholipase. The contribution of gacA to survival and vertical translocation of CHA0 in soil microcosms of increasing complexity was studied in coinoculation experiments with the wild type and a gacA mutant which lacks antibiotics and some exoenzymes. Both strains were marked with spontaneous resistance to rifampin. In a closed system with sterile soil, strain CHA0 and the gacA mutant multiplied for several weeks, whereas these strains declined exponentially in nonsterile soil of different Swiss origins. The gacA mutant was less persistent in nonrhizosphere raw soil than was the wild type, but no competitive disadvantage when colonizing the rhizosphere and roots of wheat was found in the particular soil type and during the period studied. Vertical translocation was assessed after strains had been applied to undisturbed, long (60-cm) or short (20-cm) soil columns, both planted with wheat. A smaller number of cells of the gacA mutant than of the wild type were detected in the percolated water and in different depths of the soil column. Single-strain inoculation gave similar results in all microcosms tested. We conclude that mutation in a single regulatory gene involved in antibiotic and exoenzyme synthesis can affect the survival of P. fluorescens more profoundly in unplanted soil than in the rhizosphere.     

The Viable-but-Nonculturable State Induced by Abiotic Stress in the Biocontrol Agent Pseudomonas fluorescens CHA0 Does Not Promote Strain Persistence in Soil

The effects of oxygen limitation, low redox potential, and high NaCl stress for 7 days in vitro on the rifampin-resistant biocontrol inoculant Pseudomonas fluorescens CHA0-Rif and its subsequent persistence in natural soil for 54 days were investigated. Throughout the experiment, the strain was monitored using total cell counts (immunofluorescence microscopy), Kogure's direct viable counts, and colony counts (on rifampin-containing plates). Under in vitro conditions, viable-but-nonculturable (VBNC) cells of CHA0-Rif were obtained when the strain was exposed to a combination of low redox potential (230 mV) and oxygen limitation. This mimics a situation observed in the field, where VBNC cells of the strain were found in the waterlogged soil layer above the plow pan. Here, VBNC cells were also observed in vitro when CHA0-Rif was subjected to high NaCl levels (i.e., NaCl at 1.5 M but not 0.7 M). In all treatments, cell numbers remained close to the inoculum level for the first 12 days after inoculation of soil, regardless of the cell enumeration method used, but decreased afterwards. At the last two samplings in soil, VBNC cells of CHA0-Rif were found in all treatments except the one in which log-phase cells had been used. In the two treatments that generated high numbers of VBNC cells in vitro, VBNC cells did not display enhanced persistence compared with culturable cells once introduced into soil, which suggests that this VBNC state did not represent a physiological strategy to improve survival under adverse conditions.     

Characterization of spontaneous gacS and gacA regulatory mutants of Pseudomonas fluorescens biocontrol strain CHA0

In Pseudomonas fluorescens strain CHA0, the response regulator gene gacA controls expression of extracellular enzymes and antifungal secondary metabolites, which are important for this strain's biocontrol activity in the plant rhizosphere. Two Tn5 insertion mutants of strain CHA0 that had the same pleiotropic phenotype as gacA mutants were complemented by the gacS sensor kinase gene of P. syringae pv. syringae as well as that of P. fluorescens strain Pf-5, indicating that both transposon insertions had occurred in the gacS gene of strain CHA0. This conclusion was supported by Southern hybridisation using a gacS probe from strain Pf-5. Overexpression of the wild-type gacA gene partially compensated for the gacS mutation, however, the overexpressed gacA gene was not stably maintained, suggesting that this is deleterious to the bacterium. Strain CHA0 grown to stationary phase in nutrient-rich liquid media for several days accumulated spontaneous pleiotropic mutants to levels representing 1.25% of the population; all mutants lacked key antifungal metabolites and extracellular protease. Half of 44 spontaneous mutants tested were complemented by gacS, the other half were restored by gacA. Independent point and deletion mutations arose at different sites in the gacA gene. In competition experiments with mixtures of the wild type and a gacA mutant incubated in nutrient-rich broth, the mutant population temporarily increased as the wild type decreased. In conclusion, loss of gacA function can confer a selective advantage on strain CHA0 under laboratory conditions.     

Persistence and cell culturability of biocontrol strain Pseudomonas fluorescens CHA0 under plough pan conditions in soil and influence of the anaerobic regulator gene anr

Certain fluorescent pseudomonads can protect plants from soil-borne pathogens, and it is important to understand how these biocontrol agents survive in soil. The persistence of the biocontrol strain Pseudomonas fluorescens CHA0-Rif under plough pan conditions was assessed in non-sterile soil microcosms by counting total cells (immunofluorescence microscopy), intact cells (BacLight membrane permeability test), viable cells (Kogure's substrate-responsiveness test) and culturable cells (colony counts on selective plates) of the inoculant. Viable but non-culturable cells of CHA0-Rif (106 cells g-1 soil) were found in flooded microcosms amended with fermentable organic matter, in which the soil redox potential was low (plough pan conditions), in agreement with previous observations of plough pan samples from a field inoculated with CHA0-Rif. However, viable but non-culturable cells were not found in unamended flooded, amended unflooded or unamended unflooded (i.e. control) microcosms, suggesting that such cells resulted from exposure of CHA0-Rif to a combination of low redox potential and oxygen limitation in soil. CHA0-Rif is strictly aerobic. Its anaerobic regulator ANR is activated by low oxygen concentrations and it controls production of the biocontrol metabolite hydrogen cyanide under microaerophilic conditions. Under plough pan conditions, an anr-deficient mutant of CHA0-Rif and its complemented derivative displayed the same persistence pattern as CHA0-Rif, indicating that anr was not implicated in the formation of viable but non-culturable cells of this strain at the plough pan.     

Impact of biocontrol Pseudomonas fluorescens CHA0 and a genetically modified derivative on the diversity of culturable fungi in the cucumber rhizosphere

Little is known about the effects of Pseudomonas biocontrol inoculants on nontarget rhizosphere fungi. This issue was addressed using the biocontrol agent Pseudomonas fluorescens CHA0-Rif, which produces the antimicrobial polyketides 2,4-diacetylphloroglucinol (Phl) and pyoluteorin (Plt) and protects cucumber from several fungal pathogens, including Pythium spp., as well as the genetically modified derivative CHA0-Rif(pME3424). Strain CHA0-Rif(pME3424) overproduces Phl and Plt and displays improved biocontrol efficacy compared with CHA0-Rif. Cucumber was grown repeatedly in the same soil, which was left uninoculated, was inoculated with CHA0-Rif or CHA0-Rif(pME3424), or was treated with the fungicide metalaxyl (Ridomil). Treatments were applied to soil at the start of each 32-day-long cucumber growth cycle, and their effects on the diversity of the rhizosphere populations of culturable fungi were assessed at the end of the first and fifth cycles. Over 11,000 colonies were studied and assigned to 105 fungal species (plus several sterile morphotypes). The most frequently isolated fungal species (mainly belonging to the genera Paecilomyces, Phialocephala, Fusarium, Gliocladium, Penicillium, Mortierella, Verticillium, Trichoderma, Staphylotrichum, Coniothyrium, Cylindrocarpon, Myrothecium, and Monocillium) were common in the four treatments, and no fungal species was totally suppressed or found exclusively following one particular treatment. However, in each of the two growth cycles studied, significant differences were found between treatments (e.g., between the control and the other treatments and/or between the two inoculation treatments) using discriminant analysis. Despite these differences in the composition and/or relative abundance of species in the fungal community, treatments had no effect on species diversity indices, and species abundance distributions fit the truncated lognormal function in most cases. In addition, the impact of treatments at the 32-day mark of either growth cycle was smaller than the effect of growing cucumber repeatedly in the same soil.     

Predominance of Nonculturable Cells of the Biocontrol Strain Pseudomonas fluorescens CHA0 in the Surface Horizon of Large Outdoor Lysimeters

The persistence of the biocontrol agent Pseudomonas fluorescens CHA0 in the surface horizon of 12 large outdoor lysimeters planted with winter wheat, Phacelia tanacetifolia followed by spring wheat, or maize was monitored for 1 year. Soil was inoculated with a spontaneous rifampin-resistant mutant (CHA0-Rif) of CHA0, and the strain was studied by using colony counts, Kogure's direct viable counts, and total counts (immunofluorescence). The number of culturable cells of the inoculant decreased progressively from 8 to 2 log CFU/g of soil or lower. However, culturable cells of CHA0-Rif accounted for less than 1% of the total cells of the inoculant 8 months after release in autumn. Since viable but nonculturable cells represented less than a quarter of the latter, most cells of CHA0-Rif in soil were thus inactive-dormant or dead at that time. Nonculturable cells of the inoculant were predominant also in the surface horizon of the lysimeters inoculated in the spring, and a significant fraction of them were viable. Results suggest that the occurrence of nonculturable cells of CHA0-Rif was influenced by climatic factors (water availability and soil temperature) and the abundance of roots in soil. The fact that the inoculant persisted as mixed populations of cells of different physiological states, in which nonculturable cells were predominant, needs to be taken into account when assessing the autecology of wild-type or genetically modified pseudomonads released into the soil ecosystem.     

Importance of Preferential Flow and Soil Management in Vertical Transport of a Biocontrol Strain of Pseudomonas fluorescens in Structured Field Soil

The large-scale release of wild-type or genetically modified bacteria into the environment for control of plant diseases or for bioremediation entails the potential risk of groundwater contamination by these microorganisms. For a model study on patterns of vertical transport of bacteria under field conditions, the biocontrol strain Pseudomonas fluorescens CHA0, marked with a spontaneous resistance to rifampin (CHA0-Rif), was applied to a grass-clover ley plot (rotation grassland) and a wheat plot. Immediately after bacterial application, heavy precipitation was simulated by sprinkling, over a period of 8 h, 40 mm of water containing the mobile tracer potassium bromide and the dye Brilliant Blue FCF to identify channels of preferential flow. One day later, a 150-cm-deep soil trench was dug and soil profiles were prepared. Soil samples were extracted at different depths of the profiles and analyzed for the number of CHA0-Rif cells and the concentration of bromide and Brilliant Blue FCF. Dye coverage in the soil profiles was estimated by image analysis. CHA0 was present at 10(sup8) CFU/g in the surface soil, and 10(sup6) to 10(sup7) CFU/g of CHA0 was detected along macropores between 10 and 150 cm deep. Similarly, the concentration of the tracer bromide along the macropores remained at the same level below 20 cm deep. Dye coverage in lower soil layers was higher in the ley than in the wheat plot. In nonstained parts of the profiles, the number of CHA0-Rif cells was substantially smaller and the bromide concentration was below the detection limit in most samples. We conclude that after heavy rainfall, released bacteria are rapidly transported in large numbers through the channels of preferential flow to deeper soil layers. Under these conditions, the transport of CHA0-Rif is similar to that of the conservative tracer bromide and is affected by cultural practice.     

The viable-but-nonculturable state induced by abiotic stress in the biocontrol agent Pseudomonas fluorescens CHA0 does not promote strain persistence in soil

The effects of oxygen limitation, low redox potential, and high NaCl stress for 7 days in vitro on the rifampin-resistant biocontrol inoculant Pseudomonas fluorescens CHA0-Rif and its subsequent persistence in natural soil for 54 days were investigated. Throughout the experiment, the strain was monitored using total cell counts (immunofluorescence microscopy), Kogure's direct viable counts, and colony counts (on rifampin-containing plates). Under in vitro conditions, viable-but-nonculturable (VBNC) cells of CHA0-Rif were obtained when the strain was exposed to a combination of low redox potential (230 mV) and oxygen limitation. This mimics a situation observed in the field, where VBNC cells of the strain were found in the water-logged soil layer above the plow pan. Here, VBNC cells were also observed in vitro when CHA0-Rif was subjected to high NaCl levels (i.e., NaCl at 1.5 M but not 0.7 M). In all treatments, cell numbers remained close to the inoculum level for the first 12 days after inoculation of soil, regardless of the cell enumeration method used, but decreased afterwards. At the last two samplings in soil, VBNC cells of CHA0-Rif were found in all treatments except the one in which log-phase cells had been used. In the two treatments that generated high numbers of VBNC cells in vitro, VBNC cells did not display enhanced persistence compared with culturable cells once introduced into soil, which suggests that this VBNC state did not represent a physiological strategy to improve survival under adverse conditions.     

Fusaric Acid-Producing Strains of Fusarium oxysporum Alter 2,4-Diacetylphloroglucinol Biosynthetic Gene Expression in Pseudomonas fluorescens CHA0 In Vitro and in the Rhizosphere of Wheat

The phytotoxic pathogenicity factor fusaric acid (FA) represses the production of 2,4-diacetylphloroglucinol (DAPG), a key factor in the antimicrobial activity of the biocontrol strain Pseudomonas fluorescens CHA0. FA production by 12 Fusarium oxysporum strains varied substantially. We measured the effect of FA production on expression of the phlACBDE biosynthetic operon of strain CHA0 in culture media and in the wheat rhizosphere by using a translational phlA′-′lacZ fusion. Only FA-producing F. oxysporum strains could suppress DAPG production in strain CHA0, and the FA concentration was strongly correlated with the degree of phlA repression. The repressing effect of FA on phlA′-′lacZ expression was abolished in a mutant that lacked the DAPG pathway-specific repressor PhlF. One FA-producing strain (798) and one nonproducing strain (242) of F. oxysporum were tested for their influence on phlA expression in CHA0 in the rhizosphere of wheat in a gnotobiotic system containing a sand and clay mineral-based artificial soil. F. oxysporum strain 798 (FA⁺) repressed phlA expression in CHA0 significantly, whereas strain 242 (FA⁻) did not. In the phlF mutant CHA638, phlA expression was not altered by the presence of either F. oxysporum strain 242 or 798. phlA expression levels were seven to eight times higher in strain CHA638 than in the wild-type CHA0, indicating that PhlF limits phlA expression in the wheat rhizosphere.     

Characterization of the hcnABC Gene Cluster Encoding Hydrogen Cyanide Synthase and Anaerobic Regulation by ANR in the Strictly Aerobic Biocontrol Agent Pseudomonas fluorescens CHA0

The secondary metabolite hydrogen cyanide (HCN) is produced by Pseudomonas fluorescens from glycine, essentially under microaerophilic conditions. The genetic basis of HCN synthesis in P. fluorescens CHA0 was investigated. The contiguous structural genes hcnABC encoding HCN synthase were expressed from the T7 promoter in Escherichia coli, resulting in HCN production in this bacterium. Analysis of the nucleotide sequence of the hcnABC genes showed that each HCN synthase subunit was similar to known enzymes involved in hydrogen transfer, i.e., to formate dehydrogenase (for HcnA) or amino acid oxidases (for HcnB and HcnC). These similarities and the presence of flavin adenine dinucleotide- or NAD(P)-binding motifs in HcnB and HcnC suggest that HCN synthase may act as a dehydrogenase in the reaction leading from glycine to HCN and CO₂. The hcnA promoter was mapped by primer extension; the −40 sequence (TTGGC….ATCAA) resembled the consensus FNR (fumarate and nitrate reductase regulator) binding sequence (TTGAT….ATCAA). The gene encoding the FNR-like protein ANR (anaerobic regulator) was cloned from P. fluorescens CHA0 and sequenced. ANR of strain CHA0 was most similar to ANR of P. aeruginosa and CydR of Azotobacter vinelandii. An anr mutant of P. fluorescens (CHA21) produced little HCN and was unable to express an hcnA-lacZ translational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of the hcnA-lacZ fusion. Mutant CHA21 as well as an hcn deletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused by Thielaviopsis basicola, under gnotobiotic conditions. This effect was most pronounced in water-saturated artificial soil, where the anr mutant had lost about 30% of disease suppression ability, compared with wild-type strain CHA0. These results show that the anaerobic regulator ANR is required for cyanide synthesis in the strictly aerobic strain CHA0 and suggest that ANR-mediated cyanogenesis contributes to the suppression of black root rot.     

Deleterious impact of a virulent bacteriophage on survival and biocontrol activity of Pseudomonas fluorescens strain CHAO in natural soil

Many biotic and abiotic factors affect the persistence and activity of beneficial pseudomonads introduced into soil to suppress plant diseases. One such factor may be the presence of virulent bacteriophages that decimate the population of the introduced bacteria, thereby reducing their beneficial effect. We have isolated a lytic bacteriophage (phi)GP100) that specifically infects the biocontrol bacterium Pseudomonas fluorescens CHA0 and some closely related Pseudomonas strains. phiGP100 was found to be a double-stranded-DNA phage with an icosahedral head, a stubby tail, and a genome size of approximately 50 kb. Replication of phiGP100 was negatively affected at temperatures higher than 25 degrees C. phiGP100 had a negative impact on the population size and the biocontrol activity of P. fluorescens strain CHA0-Rif (a rifampicin-resistant variant of CHA0) in natural soil microcosms. In the presence of phiGP100, the population size of strain CHA0-Rif in soil and on cucumber roots was reduced more than 100-fold. As a consequence, the bacterium's capacity to protect cucumber against a root disease caused by the pathogenic oomycete Pythium ultimum was entirely abolished. In contrast, the phage affected neither root colonization and nor the disease suppressive effect of a phiDGP100-resistant variant of strain CHA0-Rif. To our knowledge, this study is the first to illustrate the potential of phages to impair biocontrol performance of beneficial bacteria released into the natural soil environment.     

Cyanide production by Pseudomonas fluorescens helps suppress black root rot of tobacco under gnotobiotic conditions

Pseudomonas fluorescens CHA0 suppresses black root rot of tobacco, a disease caused by the fungus Thielaviopsis basicola. Strain CHA0 excretes several metabolites with antifungal properties. The importance of one such metabolite, hydrogen cyanide, was tested in a gnotobiotic system containing an artificial, iron-rich soil. A cyanidenegative (hcn) mutant, CHA5, constructed by a gene replacement technique, protected the tobacco plant less effectively than did the wild-type CHA0. Complementation of strain CHA5 by the cloned wild-type hcn⁺ genes restored the strain's ability to suppress disease. An artificial transposon carrying the hcn⁺ genes of strain CHA0 (Tnhcn) was constructed and inserted into the genome of another P.fluorescens strain, P3, which naturally does not produce cyanide and gives poor plant protection. The P3::Tnhcn derivative synthesized cyanide and exhibited an improved ability to suppress disease. All bacterial strains colonized the roots similarly and did not influence significantly the survival of T.basicola in soil. We conclude that bacterial cyanide is an important but not the only factor involved in suppression of black root rot.     

Survival of GacS/GacA Mutants of the Biological Control Bacterium Pseudomonas aureofaciens 30-84 in the Wheat Rhizosphere

GacS/GacA comprises a two-component regulatory system that controls the expression of secondary metabolites required for the control of plant diseases in many pseudomonads. High mutation frequencies of gacS and gacA have been observed in liquid culture. We examined whether gacS/gacA mutants could competitively displace the wild-type populations on roots and thus pose a threat to the efficacy of biological control. The survival of a gac mutant alone and in competition with the wild type on roots was examined in the biological control strain Pseudomonas aureofaciens 30-84. In this bacterium, GacS/GacA controls the expression of phenazine antibiotics that are inhibitory to plant pathogenic fungi and enhance the competitive survival of the bacterium. Wheat seedlings were inoculated with strain 30-84, and bacteria were recovered from roots after 21 days in sterile or nonsterile soil to check for the presence of gacS or gacA mutants. Although no mutants were detected in the inoculum, gacS/gacA mutants were recovered from 29 out of 31 roots and comprised up to 36% of the total bacterial populations. Southern hybridization analysis of the recovered gacA mutants did not indicate a conserved mutational mechanism. Replacement series analysis on roots utilizing strain 30-84 and a gacA mutant (30-84.gacA) or a gacS mutant (30-84.A2) demonstrated that although the mutant population partially displaced the wild type in sterile soil, it did not do so in natural soil. In fact, in natural soil final rhizosphere populations of wild-type strain 30-84 starting from mixtures were at least 1.5 times larger than would be predicted from their inoculation ratio and generally were greater than or equal to the population of wild type alone despite lower inoculation rates. These results indicate that although gacS/gacA mutants survive in natural rhizosphere populations, they do not displace wild-type populations. Better survival of wild-type populations in mixtures with mutants suggests that mutants arising de novo or introduced within the inoculum may be beneficial for the survival of wild-type populations in the rhizosphere.