The adnA transcriptional factor affects persistence and spread of Pseudomonas fluorescens under natural field conditions

Abstract

A soil plot was inoculated with a mixture of Pseudomonas fluorescens Pf0-2, the wild type, and Pf0-5a, a Tn5 insertion mutant in adnA, at 7.84 log CFU/g of soil. Over a period of 231 days, culturable populations of both strains were measured at selected times below and away from the point of inoculation. Pf0-5a did not spread as fast and attained significantly lower populations than Pf0-2. At sample depths below the inoculation site, the adnA mutant showed a significant decrease in CFU/g of soil as compared to Pf0-2. Pf0-2 was first detected at the 1.5-cm annular site at 3 days after inoculation, whereas Pf0-5a required 7 days to travel the same distance. At this distance, the wild-type strain could be detected at a 21.5- to 25-cm depth, whereas Pf0-5a could be detected only as deep as 15.5 to 18 cm. At 4.5 cm from the site of inoculation and in soil fractions corresponding to 13 to 18 cm, Pf0-2 was the only strain detected. These results suggest that the transcription factor AdnA provides a fitness advantage in P. fluorescens, allowing it to spread and survive in soil under field conditions.

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.

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.

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.

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.