Effect of phenylurea herbicides on soil microbial communities estimated by analysis of 16S rRNA gene fingerprints and community-level physiological profiles

Abstract

The effect of three phenyl urea herbicides (diuron, linuron, and chlorotoluron) on soil microbial communities was studied by using soil samples with a 10-year history of treatment. Denaturing gradient gel electrophoresis (DGGE) was used for the analysis of 16S rRNA genes (16S rDNA). The degree of similarity between the 16S rDNA profiles of the communities was quantified by numerically analysing the DGGE band patterns. Similarity dendrograms showed that the microbial community structures of the herbicide-treated and nontreated soils were significantly different. Moreover, the bacterial diversity seemed to decrease in soils treated with urea herbicides, and sequence determination of several DGGE fragments showed that the most affected species in the soils treated with diuron and linuron belonged to an uncultivated bacterial group. As well as the 16S rDNA fingerprints, the substrate utilization patterns of the microbial communities were compared. Principal-component analysis performed on BIOLOG data showed that the functional abilities of the soil microbial communities were altered by the application of the herbicides. In addition, enrichment cultures of the different soils in medium with the urea herbicides as the sole carbon and nitrogen source showed that there was no difference between treated and nontreated soil in the rate of transformation of diuron and chlorotoluron but that there was a strong difference in the case of linuron. In the enrichment cultures with linuron-treated soil, linuron disappeared completely after 1 week whereas no significant transformation was observed in cultures inoculated with nontreated soil even after 4 weeks. In conclusion, this study showed that both the structure and metabolic potential of soil microbial communities were clearly affected by a long-term application of urea herbicides.

FIG. 1

Viable heterotrophic bacterial counts from different soil samples treated or not treated with urea herbicides. Error bars represent standard deviations. Values with a different letter are significantly different from each other (P < 0.05).

FIG. 2

PCA (A) and CA (B) performed on the BIOLOG GN fingerprints of the extracts of the soils treated with diuron (■), chlorotoluron (●), diuron+linuron (▾), and the control soil (⧫).

FIG. 2

PCA (A) and CA (B) performed on the BIOLOG GN fingerprints of the extracts of the soils treated with diuron (■), chlorotoluron (●), diuron+linuron (▾), and the control soil (⧫).

FIG. 3

DGGE analysis of 16S rDNA fragments of different soil samples treated or not treated with urea herbicides. The 16S rDNA genes were amplified with the primer set P338f plus P518r. For the dendrograms of community relatedness, the percent similarity was calculated on the basis of two band-based coefficients, the Jaccard and area-sensitive coefficients.

FIG. 4

(A) DGGE analysis of 16S rDNA fragments of pooled soil samples collected in March 1998, treated or not treated with urea herbicides. Lanes: 1, nontreated soil; 2, diuron; 3, diuron+linuron; 4, chlorotoluron. (B) DGGE analysis of individual soil samples collected in August 1998 from two different locations per herbicide-treated plot. Lanes: 1 and 2, nontreated; 3 and 4, diuron; 5 and 6, diuron+linuron; 7 and 8, chlorotoluron. The 16S rDNA genes were amplified with the primer set P63f plus P518r. Gels A and B were not run under exactly the same conditions, which explains the difference in distances between bands. C4 indicates the fragment that was excised and sequenced.

FIG. 5

(a to c) Transformation of urea herbicides in enrichment cultures. For each herbicide, nontreated soil (○) and soil with a 10-year history of treatment (■) were used as the inoculum. (d to f) For each herbicide enrichment, the DGGE patterns from duplicate cultures were obtained after 10 days of incubation: T, enrichments inoculated with treated soil; NT, enrichments inoculated with nontreated soil. The 16S rDNA genes were amplified with primer set P63f and P518r. The arrow in panel f indicates a strong difference in the profile between treated and nontreated soil. a and d, diuron; b and e, chlorotoluron; c and f, linuron.

FIG. 6

Comparison of DGGE fingerprints between linuron treated and nontreated soil. Lanes: 1, nontreated soil before enrichment; 2 and 3, microbial communities enriched from nontreated and linuron-treated soil, respectively; 4, linuron-treated soil before enrichment. The enrichments were carried out in minimal medium containing 25 mg of linuron per liter. The 16S rDNA genes were amplified with primer set P63f plus P518r.