Molecular analysis of bacterial community structure and diversity in unimproved and improved upland grass pastures

Abstract

Bacterial community structure and diversity in rhizospheres in two types of grassland, distinguished by both plant species and fertilization regimen, were assessed by performing a 16S ribosomal DNA (rDNA) sequence analysis of DNAs extracted from triplicate soil plots. PCR products were cloned, and 45 to 48 clones from each of the six libraries were partially sequenced. Phylogenetic analysis of the resultant 275 clone sequences indicated that there was considerable variation in abundance in replicate unfertilized, unimproved soil samples and fertilized, improved soil samples but that there were no significant differences in the abundance of any phylogenetic group. Several clone sequences were identical in the 16S rDNA region analyzed, and the clones comprised eight pairs of duplicate clones and two sets of triplicate clones. Many clones were found to be most closely related to environmental clones obtained in other studies, although three clones were found to be identical to culturable species in databases. The clones were clustered into operational taxonomic units at a level of sequence similarity of >97% in order to quantify diversity. In all, 34 clusters containing two or more sequences were identified, and the largest group contained nine clones. A number of diversity, dominance, and evenness indices were calculated, and they all indicated that diversity was high, reflecting the low coverage of rDNA libraries achieved. Differences in diversity between sample types were not observed. Collector's curves, however, indicated that there were differences in the underlying community structures; in particular, there was reduced diversity of organisms of the alpha subdivision of the class Proteobacteria (alpha-proteobacteria) in improved soils.

FIG. 1

Neighbor-joining tree showing the relationship of grassland rhizosphere clones to reference members of the α-proteobacteria based on analysis of 327 bases of aligned 16S rDNA sequences. Clones exhibiting >97% sequence similarity are included in numbered clusters. The horizontal scale represents the extent of variation within each cluster, and brackets indicate clones belonging to the same group. Bootstrap values are shown for nodes that had >50% support in a bootstrap analysis of 100 replicates. Sequences obtained from unimproved and improved grassland soils are designated by the prefixes SAF and SL, respectively, followed by replicate numbers (SAF1, SAF2, etc.). The clones obtained during other direct analyses of environmental samples are MHP17 (21), MC74 (36), MC77 (36), TM69 (30), and TM28 (30). Sequences whose designations begin with the prefix S represent bacterial isolates (23). For convenience, the tree was pruned from a larger tree containing additional sequences from reference bacteria. The scale bar indicates an estimated change of 10%.

FIG. 2

Neighbor-joining tree showing the relationship of grassland rhizosphere clones to reference members of the β- and γ-proteobacteria based on analysis of 373 bases of aligned 16S rDNA sequences. The sequence designated PVB_3 is a clone obtained during another direct analysis of an environmental sample (24). Sequences whose designations begin with the prefix S represent bacterial isolates (23). For other sequence nomenclature and branch labeling, see the legend to Fig. 1.

FIG. 3

Neighbor-joining tree showing the relationship of grassland rhizosphere clones to reference actinomycetes based on analysis of 379 bases of aligned 16S rDNA sequences. Sequences whose designations begin with the prefix S represent bacterial isolates (23). For other sequence nomenclature and branch labeling see the legend to Fig. 1.

FIG. 4

Neighbor-joining tree showing the relationship of grassland rhizosphere clones to reference bacteria based on analysis of 276 bases of aligned 16S rDNA sequences. The positions of all other clones, which are associated with the α-, β-, and γ-proteobacteria and the actinomycetes, are shown in Fig. 1 to 3. The clones obtained during other direct analyses of environmental samples are MC18 (17), all other sequences whose designations begin with the prefix MC (36), and sequences whose designations begin with the prefixes TM (30) DA (10), and SBR (2). Sequences whose designations begin with the prefix S represent bacterial isolates (23). For other sequence nomenclature and branch labeling, see the legend to Fig. 1.

FIG. 5

Collector’s curves for α-proteobacterial clones from triplicate unimproved and improved soil libraries. Clones were grouped into OTUs at a level of sequence similarity of >97%.