Impact of protists on the activity and structure of the bacterial community in a rice field soil

Abstract

Flooded rice fields have become a model system for the study of soil microbial ecology. In Italian rice fields, in particular, aspects from biogeochemistry to molecular ecology have been studied, but the impact of protistan grazing on the structure and function of the prokaryotic community has not been examined yet. We compared an untreated control soil with a gamma-radiation-sterilized soil that had been reinoculated with a natural bacterial assemblage. In order to verify that the observed effects were due to protistan grazing and did not result from sterilization, we set up a third set of microcosms containing sterilized soil that had been reinoculated with natural assemblage bacteria plus protists. The spatial and temporal changes in the protistan and prokaryotic communities were examined by denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analysis, respectively, both based on the small-subunit gene. Sequences retrieved from DGGE bands were preferentially affiliated with Cercozoa and other bacteriovorous flagellates. Without protists, the level of total DNA increased with incubation time, indicating that the level of the microbial biomass was elevated. Betaproteobacteria were preferentially preyed upon, while low-G + C-content gram-positive bacteria became more dominant under grazing pressure. The bacterial diversity detectable by T-RFLP analysis was greater in the presence of protists. The level of extractable NH4+ was lower and the level of extractable SO4(2-) was higher without protists, indicating that nitrogen mineralization and SO4(2-) reduction were stimulated by protists. Most of these effects were more obvious in the partially oxic surface layer (0 to 3 mm), but they could also be detected in the anoxic subsurface layer (10 to 13 mm). Our observations fit well into the overall framework developed for protistan grazing, but with some modifications pertinent to the wetland situation: O2 was a major control, and O2 availability may have limited directly and indirectly the development of protists. Although detectable in the lower anoxic layer, grazing effects were much more obvious in the partially oxic surface layer.

FIG. 1.

Effects of different inocula on O₂ gradients measured after 10 h (A), 7 days (B), 14 days (C), and 21 days (D). The values are means ± standard errors (n = 3).

FIG. 2.

Effects of different microbial inocula on O₂ consumption (A) and CO₂ emission (B). Open bars, sterilized soil inoculated with protists and bacteria (treatment P+B); solid bars, sterilized soil inoculated with bacteria (treatment B); cross-hatched bars, control. The values are means ± standard errors (n = 3). Columns marked with different letters are significantly different (P < 0.05, as determined by Tukey's honestly significant difference test). dw, dry weight.

FIG. 3.

Effects of different microbial inocula on extractable NH₄⁺ levels in the upper layer (0 to 3 mm) (A) and the lower layer (10 to 13 mm) (B). Open bars, sterilized soil inoculated with protists and bacteria (treatment P+B); solid bars, sterilized soil inoculated with bacteria (treatment B); cross-hatched bars, control. The values are means ± standard errors (n = 3). Columns marked with different letters are significantly different (P < 0.05, as determined by Tukey's honestly significant difference test). dw, dry weight.

FIG. 4.

Effects of different microbial inocula on extractable SO₄²⁻ levels in the upper layer (0 to 3 mm) (A) and the lower layer (10 to 13 mm) (B). Open bars, sterilized soil inoculated with protists and bacteria (treatment P+B); solid bars, sterilized soil inoculated with bacteria (treatment B); cross-hatched bars, control. The values are means ± standard errors (n = 3). Columns marked with different letters are significantly different (P < 0.05, as determined by Tukey's honestly significant difference test). dw, dry weight.

FIG. 5.

Effects of different microbial inocula on the amount of DNA extracted from the upper layer (0 to 3 mm) (A) and the lower layer (10 to 13 mm) (B). Open bars, sterilized soil inoculated with protists and bacteria (treatment P+B); solid bars, sterilized soil inoculated with bacteria (treatment B); cross-hatched bars, control. The values are means ± standard errors (n = 3). Columns marked with different letters are significantly different (P < 0.05, as determined by Tukey's honestly significant difference test). dw, dry weight.

FIG. 6.

DGGE banding patterns of 18S rRNA gene partial sequences. (A) Comparison of all treatments and layers at day 0. (B and C) Comparison of days 7 to 21 for the upper layer (0 to 3 mm) (B) and the lower layer (10 to 13 mm) (C). Three replicate microcosms were analyzed per day and treatment. P+B, sterilized soil inoculated with protists and bacteria; B, sterilized soil inoculated with bacteria; Cont., control; S, γ-irradiation-sterilized soil; C, intact soil before sterilization. Bands with different mobilities are indicated by different designations. For the phylogenetic affiliation see Table 2. Note that the same designation may indicate different bands in the different gels. Only bands that appeared after day 0 are indicated in panels B and C.

FIG. 7.

Correspondence analysis of bacterial communities based on T-RFLP patterns. Circles, sterilized soil inoculated with protists and bacteria (treatment P+B); triangles, sterilized soil inoculated with bacteria (treatment B); squares, control. Solid symbols, upper layer; open symbols, lower layer. Day 0 is indicated by arrows, and data from the same treatments are connected with lines in the order of time series. The values are means ± standard errors (n = 3).