Response of a soil bacterial community to grassland succession as monitored by 16S rRNA levels of the predominant ribotypes

Abstract

The composition of predominant soil bacteria during grassland succession was investigated in the Dutch Drentse A area. Five meadows, taken out of agricultural production at different time points, and one currently fertilized plot represented different stages of grassland succession. Since fertilization and agricultural production were stopped, the six plots showed a constant decline in the levels of nutrients and vegetation changes. The activity of the predominant bacteria was monitored by direct ribosome isolation from soil and temperature gradient gel electrophoresis of reverse transcription (RT)-PCR products generated from bacterial 16S rRNA. The amounts of 16S rRNA of 20 predominant ribosome types per gram of soil were monitored via multiple competitive RT-PCR in six plots at different succession stages. These ribosome types mainly represented Bacillus and members of the Acidobacterium cluster and the alpha subclass of the class Proteobacteria. The 20 16S rRNA molecules monitored represented approximately half of all bacterial soil rRNA which was estimated by dot blot hybridizations of soil rRNA with the Bacteria probe EUB338. The grasslands showed highly reproducible and specific shifts of bacterial ribosome type composition. The total bacterial ribosome level increased during the first years after agricultural production and fertilization stopped. This correlated with the collapse of the dominant Lolium perenne population and an increased rate of mineralization of organic matter. The results indicate that there is a true correlation between the total activity of the bacterial community in soil and the amount of bacterial ribosomes.

FIG. 1

Map of the Drentse A area. The plots are located near the Anlooër Diepje Brook and are separated by channels and hedges.

FIG. 2

Comparison of rRNA quantification by dot blot hybridization and multiple competitive (mc) RT-PCR. The upper graph shows the total bacterial rRNA yield per gram (dry weight) of soil as estimated by dot blot hybridization with the EUB338 probe. The lower graph represents the sums of the single values for the 20 ribosome types as calculated by multiple competitive RT-PCR. The vertical bars indicate the standard deviations (n = 4).

FIG. 3

Normalized multiple competitive RT-PCR results for the 20 ribosome types in the six plots. All 1997 values were defined as 1, and all other data points were calculated by using these baseline values. The dotted graphs represent the average rRNA levels during grassland succession based on the sum of the values for all 20 ribosome types (Fig. 2). The 16S rRNA clusters are indicated in parentheses: Bac, Bacillus; HGC, high-G+C-content gram-positive bacteria; Acb, Acidobacterium cluster; Ver, Verrucomicrobium cluster; α-P, α-Proteobacteria. (a) Ribosome types with increasing relative rRNA amounts at the later stages of grassland succession; (b) ribosome types with increased relative rRNA amounts at the intermediate stage of grassland succession; (c) rRNA levels with minute deviations from the average; (d) ribosome types with inconstantly decreasing rRNA levels; (e) dramatically declining rRNA levels during grassland succession. The vertical bars indicate the standard deviations (n = 4).

FIG. 4

Representative TGGE fingerprints of RT-PCR products obtained with primers U968-GC and L1401 for the six plots in successional order from the initial situation in the 1997 plot to the advanced succession stage of the 1967 plot. The ribosome types whose levels decreased during succession are indicated on the left side, and the ribosome types whose levels increased are indicated on the right side. Three ribosome types remained stable without obvious relative changes.