Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel electrophoresis: plant-dependent enrichment and seasonal shifts revealed

Abstract

The bacterial rhizosphere communities of three host plants of the pathogenic fungus Verticillium dahliae, field-grown strawberry (Fragaria ananassa Duch.), oilseed rape (Brassica napus L.), and potato (Solanum tuberosum L.), were analyzed. We aimed to determine the degree to which the rhizosphere effect is plant dependent and whether this effect would be increased by growing the same crops in two consecutive years. Rhizosphere or soil samples were taken five times over the vegetation periods. To allow a cultivation-independent analysis, total community DNA was extracted from the microbial pellet recovered from root or soil samples. 16S rDNA fragments amplified by PCR from soil or rhizosphere bacterium DNA were analyzed by denaturing gradient gel electrophoresis (DGGE). The DGGE fingerprints showed plant-dependent shifts in the relative abundance of bacterial populations in the rhizosphere which became more pronounced in the second year. DGGE patterns of oilseed rape and potato rhizosphere communities were more similar to each other than to the strawberry patterns. In both years seasonal shifts in the abundance and composition of the bacterial rhizosphere populations were observed. Independent of the plant species, the patterns of the first sampling times for both years were characterized by the absence of some of the bands which became dominant at the following sampling times. Bacillus megaterium and Arthrobacter sp. were found as predominant populations in bulk soils. Sequencing of dominant bands excised from the rhizosphere patterns revealed that 6 out of 10 bands resembled gram-positive bacteria. Nocardia populations were identified as strawberry-specific bands.

FIG. 1

Experimental field design. See Materials and Methods for details.

FIG. 2

Comparison of DGGE patterns of 16S rDNA fragments amplified from bulk soil DNA (lanes 2 to 5) or from rhizosphere DNA of strawberry (lanes 6 to 9), potato (lanes 11 to 14), or oilseed rape (lanes 15 to 18) at sampling time 2.3; lanes 1, 10, and 19, standard.

FIG. 3

Rhizosphere effect. Median similarity (Pearson correlation coefficient [r]) between soil and rhizosphere communities (left bar) compared with the similarity within a treatment (rhizosphere or soil) (right bar) at different sampling times. If the median r values of two treatments (soil and rhizosphere samples at a certain time) differed more than expected from natural variability (as estimated by quartiles), then a relevant effect was assumed. Sampling event numbers indicate the year (number to left of decimal) and months after planting (number to right of decimal).

FIG. 4

Plant-dependent diversity. Median similarity (Pearson correlation [r]) between strawberry and oilseed rape (a), strawberry and potato (b), and between potato and oilseed rape (c) rhizosphere communities (left bar) compared with the similarity within a treatment (right bar) at different sampling times. If the median r values of two treatments differed more than expected from natural variability (as estimated by quartiles), then a relevant effect was assumed. See the legend to Fig. 3 for an explanation of sampling event numbers.

FIG. 5

Seasonal shifts of rhizosphere bacterial communities. DGGE analysis of 16S rDNA fragments amplified from rhizosphere DNA taken at different sampling times in the second year. Analyses of strawberry (a), potato (b), and oilseed rape (c) are shown. Lanes 1 and 22, standard; lanes 2 to 5, sampling time 2.1; lanes 6 to 9, sampling time 2.2; lanes 10 to 13, sampling time 2.3; lanes 14 to 17, sampling time 2.4; lanes 18 to 21, sampling time 2.5. Arrows indicate bands showing seasonal shifts in the relative abundance of the bacterial population.