Effect of an Alcaligenes faecalis inoculant strain on bacterial communities in flooded soil microcosms planted with rice seedlings

Abstract

The fate and impact of Alcaligenes faecalis strain A1501R, a rifampicin-resistant derivative of a rice inoculant strain, were studied in flooded silt loam microcosms planted with rice seedlings. Selective plating revealed that strain A1501R survived at high, initially stable and later slowly declining population sizes (10 8–10 6 CFU per gram of dry soil), for 60 days. Inoculant survival in the rice rhizosphere showed a similar trend, and only at one time point (15 days), the inoculant CFU numbers were significantly higher in the rhizosphere than in corresponding bulk soil. A probe for the detection of strain A1501R in soil, based on the 16S rDNA variable region V6, was obtained via PCR amplification with specific primers. Alignment of the sequence of this V6 region with that of a range of different bacterial species indicated that it provided a tool for the detection of strain A1501R in a soil background. Dot blot hybridization of randomly isolated strains and of soil DNA confirmed the usefulness of the probe. Use of the probe in a dilution dot blot hybridization experiment with soil DNA revealed a dynamics of strain A1501R over time similar to that indicated by the CFU counts. However, the presence of background in soil did not allow the detection of inoculant numbers below an estimated 10 5–10 6 cells per gram of soil. At regular times, i.e. 3 h, 15, 30 and 40 days after introduction, bacterial community fingerprints were generated via bacterial 16S rDNA based PCR of soil DNA followed by denaturing gradient gel electrophoresis (DGGE). The introduced strain could be clearly detected by the appearance of a novel, initially strong and progressively weaker, band in the community fingerprints up to roughly 30–40 days of incubation. Indigenous organisms underlying the comigrating bands found after 30–40 days were found to react with the strain A1501R V6 probe. These isolates were identified as Pseudomonas spp. The introduction of strain A1501R did not result in substantial changes in the bacterial community fingerprints. The fingerprints obtained over time in bulk soil were >90–95% similar to each other, and there was no clear trend evidenced via cluster analysis. For the rhizosphere fingerprints, three main clusters (each of >95% similarity), representing the day-0 plus day-15, the day-30, and the day-40 bacterial communities, were found. The communities apparently changed more as a result of rice root growth than due to the presence of A1501R. Community level physiological profiling (CLPP) based on the use of Biolog GN plates was then applied using the soil microbial communities sampled over time. The potential for utilization of substrates of the Biolog system by these microbial communities remained, with a few, varying, exceptions, largely unchanged in spite of the release of strain A1501R. However, significant differences in the utilization of selected substrates were observed between the control and inoculated soils, as evidenced by the application of PCR–DGGE to the bacterial communities inhabiting selected wells of the Biolog plates. In particular, strain A1501R was found to be highly competitive in the presence of lactic acid.