Monitoring impact of a pesticide treatment on bacterial soil communities by metabolic and genetic fingerprinting in addition to conventional testing procedures

Abstract

Herbogil (dinoterb), a reference herbicide, the mineral oil Oleo (paraffin oil used as an additive to herbicides), and Goltix (metamitron) were taken as model compounds for the study of impacts on microbial soil communities. After the treatment of soil samples, effects on metabolic sum parameters were determined by monitoring substrate-induced respiration (SIR) and dehydrogenase activity, as well as carbon and nitrogen mineralization. These conventional ecotoxicological testing procedures are used in pesticide registration. Inhibition of biomass-related activities and stimulation of nitrogen mineralization were the most significant effects caused by the application of Herbogil. Even though Goltix and Oleo were used at a higher dosage (10 times higher), the application of Goltix resulted in smaller effects and the additive Oleo was the least-active compound, with minor stimulation of test parameters at later observation times. The results served as a background for investigation of the power of "fingerprinting" methods in microbial ecology. Changes in catabolic activities induced by treatments were analyzed by using the 95 carbon sources provided by the BIOLOG system. Variations in the complex metabolic fingerprints demonstrated inhibition of many catabolic pathways after the application of Herbogil. Again, the effects of the other compounds were expressed at much lower levels and comprised stimulations as well as inhibitions. Testing for significance by a multivariate t test indicated that the sensitivity of this method was similar to the sensitivities of the conventional testing procedures. The variation of sensitive carbon sources, as determined by factor weights at different observation times, indicated the dynamics of the community shift induced by the Herbogil treatment in more detail. DNA extractions from soil resulted in a collection of molecules representing the genetic composition of total bacterial communities. Distinct and highly reproducible community patterns, or genetic fingerprints, resulting from application of the different herbicides were obtained by the sequence-specific separation of partial 16S rDNA amplification products in temperature gradient gel electrophoresis. Significant pattern variations were quantified. For detailed analysis, application-responsive bands from the Herbogil and Oleo treatments were sequenced and their tentative phylogenetic positions were identified. Data interpretation and the potentials and biases of the additional observation windows on microbial communities are discussed.

FIG. 1

Results of the traditional methods. SIR data represent the integration of CO₂ production of soil samples taken at the given sampling times for 6 h after the addition of glucose. For dehydrogenase activity, amounts of (reduced) triphenyl formazan (TPF), as determined with a spectrophotometer at the different sampling times, are shown. Changes in nitrogen mineralization were analyzed by a colorimetric assay at each sampling time. The concentrations observed at the beginning (day 0) were subtracted from actual experimental data. For long-term respiration, the cumulative carbon mineralization, determined by titration in a KOH solution, is shown.

FIG. 2

Pixel pattern representation of BIOLOG community patterns. Readings of BIOLOG GN microplates, 5 weeks after inoculation with bacterial fractions of the experimental variants, are shown. The different pixel patterns represent the means of six replicates each after incubation for 32 h at 28°C, corrected by blank subtraction (OD₀).

FIG. 3

TGGE community patterns 8 weeks after pesticide application. PCR-amplified 16S rDNA fragments (positions 968 to 1401) of control and challenged bacterial communities separated on a TGGE gel. Bands extracted, reamplified, and cloned for sequencing are indicated (clones 3 to 5, 17, and 24). A broad region with single-stranded DNA (ss-DNA) can be identified by its reddish color in the silver-stained gel. Lanes: 1 through 3, control; 4 through 6, Herbogil; 7 through 9, Oleo; 10 through 12, Goltix.

FIG. 4

TGGE pattern comparison: control versus Herbogil treatment. Data of Fig. 3 are represented as a scan of OD readings. The calculated median OD readings from the upper gel sections (approximately 40%) of replicate lanes were used. Data were adjusted by plotting the OD as a fraction of the integrated pattern intensity. The slightly different migration of corresponding bands was corrected according to visual inspection.

FIG. 5

TGGE pattern comparisons: impacts of Oleo and Goltix. The differences between the median OD readings of the Oleo and Goltix patterns and those of the control lanes were calculated by subtraction. The OD readings were adjusted and corrected as described for Fig. 4.

FIG. 6

Phylogenetic positions of the cut-out sequences. Clones correspond to the bands and peaks marked in Fig. 3. Bar, sequence difference of 10%.