Dynamics of the linuron hydrolase libA gene pool size in response to linuron application and environmental perturbations in agricultural soil and on-farm biopurification systems

Abstract

libA, a gene encoding a novel type of linuron hydrolase, was recently identified in the linuron-mineralizing Variovorax sp. strain SRS16. In order to assess the contribution of libA to linuron degradation in environmental settings, libA abundance was monitored in response to the application of linuron and to environmental perturbations in agricultural soil microcosms and microcosms simulating the matrix of on-farm biopurification systems. libA numbers were measured by real-time PCR and linked to reported data of Variovorax community composition and linuron mineralization capacity. In the soil microcosms and one biopurification system setup, libA numbers responded to the application of linuron and environmental changes in congruency with the modulation of linuron mineralization capacity and the occurrence of a particular Variovorax phylotype (phylotype A). However, in another biopurification system setup, no such correlations were found. Our data suggest that in the simulated environmental settings, the occurrence of libA can be linked to the linuron mineralization capacity and that libA is primarily hosted by Variovorax phylotype A strains. However, the results also suggest that, apart from libA, other, as-yet-unknown isofunctional genes play an important role in linuron mineralization in the environment.

Fig 1

Dynamics of libA number in water- or linuron-fed SMs as determined by qPCR. The results for water-fed SMs are presented in dark gray, the results for SMs with a single linuron treatment are presented in white, and the results for discontinuously linuron-treated SMs are presented in pale gray. The smaller bars within the bars showing the libA gene number represent the previously reported linuron mineralization lag times (indicative for the linuron mineralization capacity) for the same samples (5). The x axis shows the sampling times. The numbers on the left y axis (log scale) show the number of copies of libA per gram of dry soil. The numbers on the right y axis show the linuron mineralization lag time in days. The qPCR data are averages from duplicate measurements performed on the three replicates of each setup. The standard deviations (SD) are presented on the bars. Two-way repeated measures ANOVA was used to trace significant differences between treatments and time points (P < 0.05). Average libA numbers and average linuron mineralization lag times that differ significantly are indicated by different letters. Below the graph, the Variovorax community composition as recorded by Variovorax-specific PCR-DGGE (5) for the corresponding setups and time points is presented. Bands representing the different Variovorax phylotypes as reported by Bers et al. (5) are indicated in the DGGE profiles.

Fig 2

Correlation between the number of libA copies per g (dry weight) of soil (x) and the lag time (y) of linuron mineralization (as reported previously [5, 20]) in SMs. libA numbers are presented in log scale on the x axis. The data from different setups are indicated with different symbols (squares, water-fed SMs; triangles, single linuron-fed SMs; diamonds, discontinuously linuron-fed SMs). Equations of the logarithmic (full line) and power (dashed line) correlation curves are presented in boxes with corresponding patterns. All data points were used for the power correlation curve. The data points used for the logarithmic correlation (presented by the full line) are indicated by filled symbols.

Fig 3

Dynamics of libA gene number in BMs inoculated with a linuron-primed soil (L⁺ and L⁻) (top) and a non-linuron-primed soil (C⁺ and C⁻) (bottom) as determined by qPCR. (Top) Light gray and dark gray bars represent the libA numbers for setups L⁻ and L⁺, respectively. (Bottom) Black bars represent the libA numbers for setup C⁺. libA numbers determined for setup C⁻ were always below the detection limit. The smaller bars within the black bars represent the previously reported linuron mineralization lag times (indicative of the linuron mineralization capacity) for setup C⁺, while the white bars show those reported for setup C⁻ (21, 22). The numbers on the x axis show the weeks of incubation after which the samples were taken. The numbers on the left y axis (log scale) show the number of copies of libA per gram of dry soil. The numbers on the right y axis show the linuron mineralization lag time in days. The qPCR data are averages from duplicate measurements performed on three replicates of each setup. “NL” marks each time point in the treatments where the libA abundance was below the detection limit. Since libA numbers were only above the detection limit for replicate BM3 in BMs of setup C⁺, bars are based on values for BM3 only, except for week 42 when libA was detected in all three replicates. “NM” marks each time point in the treatments where no mineralization was observed. Linuron mineralization lag times for setup C⁺ at weeks 2 and 17 are of BM3 only. In all cases, the SD values are presented on the bars. Two-way repeated-measures ANOVA was used to trace significant differences between treatments and time points (P < 0.05). Average libA numbers and average linuron mineralization lag times that differ significantly are indicated by different letters. Above the graphs a time scheme of the sequential environmental perturbations applied to the different BM setups is shown. Sampling times are marked with arrows, with the numbers indicating the week of sampling after startup. Below the graphs, the Variovorax community composition as recorded by Variovorax-specific PCR-DGGE and reported by Sniegowski et al. (20, 21) for the corresponding setups and time points is presented. Bands representing the different Variovorax phylotypes as reported by Bers et al. (5) are indicated in the DGGE profiles.

Fig 4

Correlation between the number of libA copies per g (dry weight) of soil (x) and the lag time (y) of linuron mineralization (as reported previously [5, 20]) in BMs. The libA numbers are presented in log scale on the x axis. The data from different setups are indicated with different symbols (+, BMs of setup L⁺; ●, BMs of setup L⁻). The equation of the logarithmic (full line) correlation curve is presented in the box.