Levels of bacterial community diversity in four arid soils compared by cultivation and 16S rRNA gene cloning

Abstract

Techniques based on amplification of 16S rRNA genes for comparing bacterial communities are now widely used in microbial ecology, but calibration of these techniques with traditional tools, such as cultivation, has been conspicuously absent. In this study, we compared levels of bacterial community diversity in two pinyon rhizosphere soil samples and two between-tree (interspace) soil samples by analyzing 179 cultivated bacterial isolates and 801 16S rRNA genes amplified from extracted soil DNA. Phylotypes were defined by performing a restriction fragment length polymorphism analysis of 16S rRNA gene sequences with the enzymes RsaI and BstUI. The average level of 16S rRNA gene sequence similarity of members of a phylotype was 86.6% based on an analysis of partial sequences. A total of 498 phylotypes were identified among the 16S ribosomal DNA (rDNA) clones, while 34 phylotypes occurred among the cultivated isolates. Analysis of sequences from a subset of the phylotypes showed that at least seven bacterial divisions were represented in the clone libraries, whereas the isolates represented only three. The phylotype richness, frequency distribution (evenness), and composition of the four culture collections and the four clone libraries were investigated by using a variety of diversity indices. Although cultivation and 16S rRNA cloning analyses gave contradictory descriptions of the relative phylotype richness for one of the four environments, the two methods identified qualitatively consistent relationships when levels of evenness were compared. The levels of phylotype similarity between communities were uniformly low (15 to 31%). Both methods consistently indicated that one environment was distinct from the other three. Our data illustrate that while 16S rDNA cloning and cultivation generally describe similar relationships between soil microbial communities, significant discrepancies can occur.

FIG. 1

Division level phylogenetic diversity identified in 16S rDNA clone libraries and culture collections. Division level affiliations were determined by phylogenetic analysis of partial or nearly full-length 16S rDNA sequences from 168 cloned 16S rDNA obtained primarily from the S0 and C0 clone libraries and from 35 bacterial isolates from the four culture collections. Bact.-Cyto.-Flexibact., Bacteroides-Cytophaga-Flexibacter.

FIG. 2

Phylotype richness curves for clone and culture libraries. Sampling curves were calculated by rarefaction (21, 43).

FIG. 3

Relationship between phylotype abundance and detection frequency for 16S rDNA clone libraries and culture collections. The average abundance of each phylotype was calculated across all eight libraries. Phylotypes were then grouped according to the number of libraries in which each phylotype appeared. The mean phylotype abundance for each group was calculated by using the average abundance values previously calculated for individual phylotypes. Only one phylotype appeared in six libraries. The error bars indicate 95% confidence intervals.