INTRODUCTION

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The effective use of microbial agents, such as the pseudomonads, to remedy plant diseases, decontaminate soil, improve texture, and enhance plant nutrition requires that such agents be ecologically adapted to the location where they are released. However, inoculation of selected microbes into soil or on seeds often results in rapid loss of their population within a short time (2). Such phenomena and the factors influencing this decline have been reviewed (23).

DeFlaun et al. (9) isolated two Tn5 insertion mutants of Pseudomonas fluorescens strain Pf0-1 that were deficient in attachment to soil particles and seeds and were nonmotile. The locus affected by these Tn5 insertions was identified as adnA. In complementation experiments, cloned wild-type adnA restored the wild-type phenotype described for the Tn5 insertion mutants (7a). Sequencing and expression experiments revealed that AdnA is a homologue of FleQ (7a), a transcriptional activator of the NtrC/NifA family in Pseudomonas aeruginosa which, in association with sigma 54, mediates transcription of structural genes for flagellar synthesis (4).

Other regulation factors have been linked to fitness in natural environments, e.g., RosR in Rhizobium etli and GacS in Pseudomonas syringae (6, 12, 13). RosR is likely to regulate exopolysaccharide production, whereas GacS has been implicated in regulation of several activities, such as swarming, antibiotic, toxin, and siderophore production in the genus Pseudomonas (11, 14, 18, 19, 24). The rosR mutant showed a reduced ability to grow in the rhizosphere and compete for nodule occupancy under laboratory conditions (3, 6). In field experiments, the gacS mutant attained significantly lower populations than those of the parent strain in bean foliage, but there was no effect on the populations in soil surrounding germinating seeds (12). These experiments were conducted in either laboratory conditions or for the short term and studied the effects of regulation factors on fitness of soil influenced by roots or bean foliage.

In this study, we examined the role of adnA on long-term survival, spread, and competence in soil under natural field conditions. We present evidence that adnA provides a fitness advantage in P. fluorescens in soil and discuss future research.

	MATERIALS AND METHODS

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Bacterial strains and semiselective media. To determine how adnA affects soil persistence and spread of P. fluorescens under natural field conditions, we used rifampin-resistant (Rif^r) strains Pf0-2 and Pf0-5a, derivatives of Pf0-1 and Pf0-5, respectively. Pf0-1 was first isolated by Compeau et al. (8) and was later used by DeFlaun et al. (9) in sand and seed attachment assays. Pf0-5 was generated by Tn5 mutagenesis and described as a nonflagellated, nonmotile, attachment-deficient mutant of Pf0-1 (9).

7

Inoculation site and procedure. The experiment was conducted in an agricultural field situated at the University of Massachusetts Agricultural Experiment Station in Waltham. The experimental site, a fallow area which had not been cultivated for at least 10 years, was prepared with conventional tillage implements. An area of 20 by 10 m was fenced in chicken wire, and a plot of 1 m² was demarked by wooden stakes. Prior to inoculation, soil samples were taken to determine soil characteristics (Table 1) and CFU of indigenous bacteria on Mac agar resistant to rifampin and kanamycin. Plating assays indicated that indigenous bacteria with these characteristics were below the limit of detection (12 CFU/g of fresh soil). Soil cores were extracted using 0.8- by 13-cm brass soil samplers (model GC-1; La Motte Co., Chesterton, Md.), which were washed, individually wrapped in aluminum foil, and autoclaved between samplings.

Assay for soil populations of Pf0-2 and Pf0-5a and sampling strategy. Figure 1 illustrates the soil plot and sampling plan used in this study. We slightly modified the design described by Krimsky et al. (17) to assess risk of microbial release. To measure spread of the strains, values for log CFU per gram of soil were determined at the inoculation plug (0 to 2 cm) and 1.5, 4.5, 13.5, and 40 cm from the inoculation plug (Fig. 1A). Soil fractions corresponding to depths of 0 to 6.5, 6.5 to 13, 13 to 15.5, 15.5 to 18, 18 to 21.5, and 21.5 to 25 cm below (Fig. 1B) were sampled. For measuring soil populations 1.5, 4.5, 13.5, and 40 cm from the inoculation plug, we established the four cardinal points in the outer ring of the soil plots and used a compass clockwise to determine angles randomly for sampling sites (Fig. 1A). Measurements at and below the inoculation plug were taken inside the circle demarked by the inoculation plug at different days after inoculation (dai). The inoculation plug was 5.75 cm in diameter, while the soil core samplers were 0.8 cm in diameter, which allowed ample room for several samplings. To measure persistence, the plot was sampled periodically from 0 to 231 dai and finally 365 dai. Soil corers were inserted to the desired depth and were then wrapped, labeled, and transported for analysis on the same day. For sampling at depths of 18 and 25 cm, two corers were bound end to end with duct tape. To maintain the integrity of the soil plot after sampling, the holes left by plug removal were filled with soil from a nearby uninoculated site. Because the bacterial reservoir was depleted any time that soil samples were taken, a new set of sites were generated for subsequent sampling times. A second independent plot with the same sampling strategy but with fewer time points and depths sampled was examined in parallel.

View larger version (26K): [in this window] [in a new window]   FIG. 1.   Diagram of sampling strategy and field plot inoculated with P. fluorescens strains Pf0-2 and Pf0-5a. (A) Top view of plot. The inoculation plug (shaded area) consisted of a cone with a diameter of 5.75 cm and a volume of 41 ml. The four cardinal points were established just beyond the outer ring of the plots. Sampling sites at test distances (1.5 and 4.5 cm) were determined by using a compass clockwise and randomly picking an angle between 315 and 44°, 45 and 134°, 135 and 224°, and 225 and 314° for north, east, south, and west, respectively. Rings indicating distances away from the site of inoculation were demarked with toothpicks. Distances of 13.5 and 40 cm from the point of inoculation were also tested, but no test organisms were detected. Plug is drawn to scale. (B) Side view of field plot. Movement of the strains below the site of inoculation was measured in soil fractions of depths (in centimeters) of 0 to 2, 2 to 6.5, 6.5 to 13, 13 to 15.5, 15.5 to 18, 18 to 21.5, and 21.5 to 25. Movement of the strains from the site of inoculation was measured at distances of 1.5 and 4.5 cm from the site and at depths of 0 to 6.5, 6.5 to 13, 13 to 15.5, 15.5 to 18, 18 to 21.5, and 21.5 to 25 cm below the inoculation site. Side view is not drawn to scale.

Statistical analysis. Plate counts were adjusted to CFU/gram of dry soil and log₁₀ transformed. For measurements at and below the inoculation plug, we used a Student's t test of paired observations to determine whether the proportion of the strains at each depth remained similar to the nominal proportions in the inoculum (1.5:1, Pf0-2:Pf0-5a). At each timepoint tested, we paired values for log CFU/gram of soil from Pf0-2 with values for log CFU/gram of soil from Pf0-5a and calculated the difference in the pair. The number of pairs (replicates) corresponded to the number of times sampled. For example, for the 0 to 2-cm depth we measured populations at 7, 17, 31, 45, 73, and 231 dai. Then we determined the mean difference over six samplings and tested against the difference between the populations of the strains in the inoculum.

	RESULTS

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Precipitation and soil water content. Figure 2A and B show precipitation in millimeters and soil water content of the plots up to 70 dai, respectively. Precipitation was registered after any appreciable rain occurred. Total rainfall on the plots was 287.6 mm with an average of 4.1 mm/day. Soil water content was determined by removing 2 to 10 g of soil at various depths and taking soil weight before and after drying the samples for 24 h at 60°C. Soil water content was more variable at the surface and ranged from 7 to 35%, compared to 22 to 27% at 13 cm, but the averages were 23% for both depths. The values for water content at 18 and 25 cm were very similar to those from 13 cm (data not shown). During the winter season (between 70 and 231 dai), precipitation and soil water content were not registered.

View larger version (29K): [in this window] [in a new window]   FIG. 2.   Precipitation in millimeters (A) and soil water content (B) for plots inoculated with P. fluorescens for 70 days at depths of 0 to 1.5 cm and 13 cm. Precipitation is reported as the average per day from 60 to 70 dai.

Effect of adnA on vertical movement in soil. Mean representations and culturable populations of Pf0-2 and Pf0-5a (adnA mutant) in soil at the surface (depth 0 to 2 cm) and from fractions at depths of 2 to 6.5, 6.5 to 13, 13 to 15.5, 15.5 to 18, 18 to 21.5, and 21.5 to 25 cm below the inoculation plug were determined (Table 2). Pf0-2 significantly increased its representation in soil from 1.5:1 (Pf0-2:Pf0-5a) at the time of inoculation, to 6:1, 5:1, 49:1, 99:1, and 6:1 in soil fractions corresponding to depths of 2 to 6.5, 6.5 to 13, 13 to 15.5, 15.5 to 18, and 18 to 21.5 cm through 231 days, respectively. There were no significant differences between the representation of both strains at the surface (depth, 0 to 2 cm) and at the depth of 21.5 to 25 cm. Populations were highest at the surface and declined with depth.

Effect of adnA on movement from the inoculation site. Populations for Pf0-2 and Pf0-5a in soil fractions below the 1.5-cm-depth test point were assayed (Fig. 3). At the total population level, values for log CFU/gram of soil reached 3.5 in soil fractions spanning from 0 to 18 cm (Fig. 3A to D), whereas numbers in soil fractions obtained from 18- to 25-cm depths did not exceed 0.8 log units (Fig. 3E and F). Values for log CFU/gram of soil appeared more variable in the 0- to 6.5-cm fraction (Fig. 3A). Pf0-2 titers were significantly higher than those of Pf0-5a for most of the dai sampled for soil fractions of 0 to 6.5, 6.5 to 13, and 13 to 15.5 cm (Fig. 3A to C) and for all dai sampled for the 15.5- to 18-cm fraction (Fig. 3D). Of note, in the soil fractions corresponding to 18 to 25 cm, only Pf0-2 was detected (Fig. 3E and F).

View larger version (62K): [in this window] [in a new window]   FIG. 3.   Soil populations of P. fluorescens strains Pf0-2 (wild type) and Pf0-5a (adnA mutant) at different depths 1.5 cm from the point of inoculation: 0 to 6.5 cm (A), 6.5 to 13 cm (B), 13 to 15.5 cm (C), 15.5 to 18 cm (D), 18 to 21.5 cm (E), and 21.5 to 25 cm (F). On day 0, strains were mixed and inoculated to 7.84 total log CFU/g of soil. At different times, 0.5- to 1.8-g samples were taken and strains were enumerated by plating serial 10-fold dilutions on Mac agar plates containing rifampin alone or rifampin and kanamycin. Means were determined from four replicates. Significant differences were determined by ANOVA. *, **, statistically significant differences between soil populations of the two strains at P  0.1 and 0.05, respectively; +, no Pf0-5a detected. Results are representative of two experiments.

View larger version (36K): [in this window] [in a new window]   FIG. 4.   Soil populations of P. fluorescens strains Pf0-2 (wild type) and Pf0-5a (adnA mutant) at different depths 4.5 cm from the point of inoculation: 0 to 6.5 cm (A), 6.5 to 13 cm (B), 13 to 15.5 cm (C), and 15.5 to 18 cm (D). Strains were inoculated and enumerated as described for Fig. 3. Means were determined from four replicates. Significant differences were determined by ANOVA. *, **, statistically significant differences between soil populations of the two strains at P  0.1 and 0.05, respectively; +, no Pf0-5a detected; @, both strains not detected. Results are representative of two experiments.

Distribution of total CFU of test strains from the point of inoculation. There were no significant differences in soil total populations (sum of Pf0-2 and Pf0-5a) in terms of log CFU/gram of soil at four cardinal points (north, east, south, and west) at 1.5 cm from and 6.5 to 13 cm below the inoculation site. These data suggest that bacterial movement of P. fluorescens in the soil conditions tested proceeded in a radial fashion at similar rates (data not shown).

	DISCUSSION

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This study provides evidence that the transcriptional activator AdnA confers a long-term fitness advantage in P. fluorescens under field conditions. The mutant strain Pf0-5a has a single Tn5 insertion which is in the adnA gene (9). Complementation experiments with cloned adnA confirmed that its inactivation led to the adhesion-deficient and nonflagellated phenotypes (7a). In two separate, yearlong experiments, Pf0-5a showed a significant decrease in its survival and persistence in soil compared to the parent strain, Pf0-2 (Table 2). Moreover, Pf0-5a was not detected in the deeper soil fractions or as frequently or as numerously as Pf0-2 at 1.5 and 4.5 cm from the inoculation site (Fig. 3 and 4).

We determined soil populations by a standard plating technique, which has been shown to provide information for a small fraction of total and metabolically active cells (1, 7, 21). However, Johnsen et al. (16) recently showed that quantitative PCR correlated well with culturable counts of Pseudomonas in soil obtained on selective media.

As proposed previously (7a), the product of adnA has high similarity to FleQ, a transcriptional factor of the NtrC/NifA family of regulators. Such activators generally work as part of a two-component regulatory system which, under the right environmental conditions and in association with sigma 54, mediates nitrogen fixation in Rhizobium and formation of flagella in P. aeruginosa (4, 20). Although the adnA mutation may have pleiotropic effects, we speculate that its primary function is in flagellum synthesis because of its location in a region of flagellar genes and homology to FleQ, a transcription factor for flagellum synthesis. We favor the notion that the effects in soil are principally related to motility. DeFlaun et al. (9) showed that under water-saturated conditions the mutant strain moves down faster than the parent strain in soil columns; however, the adnA mutant strain was not detected in the deepest soil fractions (Fig. 3E and F). A major means of bacterial spread and transport in soil is by percolating water (22). In our case, the average soil water content was 23%, well below the threshold for percolating water. Perhaps motility is important for spread under non-water-saturated soil conditions. Similar results were reported for nonmotile variants of Rhizobium leguminosarum biovar phaseoli, which did not spread as well as the wild type from the point of inoculation in soils that were moderately wet (15).

Despite this evidence, we cannot rule out the possibility that adnA is influencing spread and persistence in soil of P. fluorescens by activating transcription of other systems. To address this, we have undertaken experiments that will identify genes regulated by adnA. As is the case with identifying genes affected by transcriptional factors (5), we foresee three functions regulated by adnA: genes important for soil fitness, other genes involved in regulation, and genes affecting other cellular functions. As adnA is predicted to be a transcriptional activator, this gene provides a handle to study environment-bacterium interactions and adaptation to soil environments.