Microcosms were used to examine whether pesticide-primed soils could be preferentially used over nonprimed soils for bioaugmentation of on-farm biopurification systems (BPS) to improve pesticide mineralization. Microcosms containing a mixture of peat, straw and either linuron-primed soil or nonprimed soil were irrigated with clean or linuron-contaminated water. The lag time of linuron mineralization, recorded for microcosm samples, was indicative of the dynamics of the linuron-mineralizing biomass in the system. Bioaugmentation with linuron-primed soil immediately resulted in the establishment of a linuron-mineralizing capacity, which increased in size when fed with the pesticide. Also, microcosms containing nonprimed soil developed a linuron-mineralizing population, but after extended linuron feeding. Additional experiments showed that linuron-mineralization only developed with some nonprimed soils. Concomitant with the increase in linuron degradation capacity, targeted PCR-denaturing gradient gel electrophoresis showed the proliferation of a Variovorax phylotype related to the linuron-degrading Variovorax sp. SRS16 in microcosms containing linuron-primed soil, suggesting the involvement of Variovorax in linuron degradation. The correlation between the appearance of specific Variovorax phylotypes and linuron mineralization capacity was less clear in microcosms containing nonprimed soil. The data indicate that supplementation of pesticide-primed soil results in the establishment of pesticide-mineralizing populations in a BPS matrix with more certainty and more rapidly than the addition of nonprimed soil.

Real-time PCR and PCR-denaturing gradient gel electrophoresis (DGGE) approaches that specifically target the Variovorax 16S rRNA gene were developed to estimate the number and diversity of Variovorax in environmental ecosystems. PCR primers suitable for both methods were selected as such that the enclosed sequence showed maximum polymorphism. PCR specificity was maximized by combining PCR with a targeted endonuclease treatment of template DNA to eliminate 16S rRNA genes of the closely related Acidovorax. DGGE allowed the grouping of PCR amplicons according to the phylogenetic grouping within the genus Variovorax. The toolbox was used to assess the Variovorax community dynamics in agricultural soil microcosms (SMs) exposed to the phenylurea herbicide linuron. Exposure to linuron resulted in an increased abundance within the Variovorax community of a subgroup previously linked to linuron degradation through cultivation-dependent isolation. SMs that were treated only once with linuron reverted to the initial community composition 70 days after linuron exposure. In contrast, SMs irrigated with linuron on a long-term base showed a significant increase in Variovorax number after 70 days. Our data support the hypothesis that the genus Variovorax is involved in linuron degradation in linuron-treated agricultural soils.

Mobile genetic elements (MGEs) are considered as key players in the adaptation of bacteria to degrade organic xenobiotic recalcitrant compounds such as pesticides. We examined the prevalence and abundance of IncP-1 plasmids and IS1071, two MGEs that are frequently linked with organic xenobiotic degradation, in laboratory and field ecosystems with and without pesticide pollution history. The ecosystems included on-farm biopurification systems (BPS) processing pesticide-contaminated wastewater and soil. Comparison of IncP-1/IS1071 prevalence between pesticide-treated and nontreated soil and BPS microcosms suggested that both IncP-1 and IS1071 proliferated as a response to pesticide treatment. The increased prevalence of IncP-1 plasmids and IS1071-specific sequences in treated systems was accompanied by an increase in the capacity to mineralize the applied pesticides. Both elements were also encountered in high abundance in field BPS ecosystems that were in operation at farmyards and that showed the capacity to degrade/mineralize a wide range of chlorinated aromatics and pesticides. In contrast, IS1071 and especially IncP-1, MGE were less abundant in field ecosystems without pesticide history although some of them still showed a high IS1071 abundance. Our data suggest that MGE-containing organisms were enriched in pesticide-contaminated environments like BPS where they might contribute to spreading of catabolic genes and to pathway assembly.

On-farm biopurification systems (BPS) treat pesticide-contaminated wastewater of farms through biodegradation. Adding pesticide-primed soil has been shown to be beneficial for the establishment of pesticidedegrading populations in BPS. However, no data exist on the response of pesticide-degrading microbiota, either endogenous or introduced with pesticide-primed soil, when BPS are exposed to expected less favorable environmental conditions like cold periods, drought periods, and periods without a pesticide supply. Therefore, the response of microbiota mineralizing the herbicide linuron in BPS microcosm setups inoculated either with a linuron-primed soil or a nonprimed soil to a sequence of such less favorable conditions was examined. A period without linuron supply or a drought period reduced the size of the linuron-mineralizing community in both setups. The most severe effect was recorded for the setup containing nonprimed soil, in which stopping the linuron supply decreased the linuron degradation capacity to nondetectable levels. In both systems, linuron mineralization rapidly reestablished after conventional operation conditions were restored. A cold period and feeding with a pesticide mixture did not affect linuron mineralization. The changes in the linuron-mineralizing capacity in microcosms containing primed soil were associated with the dynamics of a particular Variovorax phylotype that previously had been associated with linuron mineralization. This study suggests that the pesticide-mineralizing community in BPS is robust in stress situations imposed by changes in environmental conditions expected to occur on farms. Moreover, it suggests that, in cases where effects do occur, recovery is rapid after restoring conventional operation conditions.The treatment of pesticide-contaminated wastewater in onfarm biopurification systems (BPS) is a low-cost and effortless solution for farmers to minimize the direct losses of pesticides to surface water (6). On-farm BPS operate as biofilters in which the pesticides are removed from the wastewater by biodegradation and sorption processes occurring in the biofilter matrix. The matrix in a BPS, designated a biomix, is composed of a mixture of soil, peat, and straw or other organic waste materials (6, 9). The addition of pesticide-primed soil to BPS has been proposed as an alternative for bioaugmentation with axenic cultures of specialized pesticide-degrading bacteria to accelerate pesticide degradation and avoid the production of toxic metabolites (7, 18). Sniegowski et al. (18) showed that bioaugmentation with a linuron-primed soil containing linuron-mineralizing microorganisms immediately resulted in the establishment of a linuron mineralization capacity in BPS microcosms. The size of the linuron-mineralizing populations in the system further increased when the microcosms were fed linuron. BPS microcosms inoculated with nonprimed soils also developed a linuron mineralization capacity but only after a much longer period of linuron supply. In the BPS microcosms contain...

ABSTRACTlibA, a gene encoding a novel type of linuron hydrolase, was recently identified in the linuron-mineralizingVariovoraxsp. strain SRS16. In order to assess the contribution oflibAto linuron degradation in environmental settings,libAabundance 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.libAnumbers were measured by real-time PCR and linked to reported data ofVariovoraxcommunity composition and linuron mineralization capacity. In the soil microcosms and one biopurification system setup,libAnumbers responded to the application of linuron and environmental changes in congruency with the modulation of linuron mineralization capacity and the occurrence of a particularVariovoraxphylotype (phylotype A). However, in another biopurification system setup, no such correlations were found. Our data suggest that in the simulated environmental settings, the occurrence oflibAcan be linked to the linuron mineralization capacity and thatlibAis primarily hosted byVariovoraxphylotype A strains. However, the results also suggest that, apart fromlibA, other, as-yet-unknown isofunctional genes play an important role in linuron mineralization in the environment.