Molecular and culture-based analyses of prokaryotic communities from an agricultural soil and the burrows and casts of the earthworm Lumbricus rubellus - PubMed (original) (raw)

Molecular and culture-based analyses of prokaryotic communities from an agricultural soil and the burrows and casts of the earthworm Lumbricus rubellus

Michelle A Furlong et al. Appl Environ Microbiol. 2002 Mar.

Abstract

The microbial populations in no-till agricultural soil and casts of the earthworm Lumbricus rubellus were examined by culturing and molecular methods. Clone libraries of the 16S rRNA genes were prepared from DNA isolated directly from the soil and earthworm casts. Although no single phylum dominated the soil library of 95 clones, the largest numbers of clones were from Acidobacteria (14%), Cytophagales (13%), Chloroflexi (8%), and gamma-Proteobacteria (8%). While the cast clone library of 102 clones was similar to the soil library, the abundances of several taxa were different. Representatives of the Pseudomonas genus as well as the Actinobacteria and Firmicutes increased in number, and one group of unclassified organisms found in the soil library was absent in the cast library. Likewise, soil and cast archaeal 16S rRNA gene libraries were similar, although the abundances of some groups were different. Two hundred and thirty aerobic bacteria were also isolated on general heterotrophic media from casts, burrows, and soil. The cast isolates were both phenotypically and genotypically different from the soil isolates. The cast isolates were more likely to reduce nitrate, grow on acetate and Casamino Acids, and utilize fewer sugars than the soil isolates. On the basis of their ribotypes, the cast isolates were dominated by Aeromonas spp. (28%), which were not found in the soil isolates, and other gamma-Proteobacteria (49%). In contrast, the soil isolates were mostly Actinobacteria (53%), Firmicutes (16%), and gamma-Proteobacteria (19%). Isolates obtained from the sides of earthworm burrows were not different from the soil isolates. Diversity indices for the collections of isolates as well as rRNA gene libraries indicated that the species richness and evenness were decreased in the casts from their levels in the soil. These results were consistent with a model where a large portion of the microbial population in soil passes through the gastrointestinal tract of the earthworm unchanged while representatives of some phyla increase in abundance.

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Figures

FIG. 1.

FIG. 1.

Phylogenetic tree of the γ-Proteobacteria clones and isolates from Horseshoe Bend. Sequence names are coded. For the clones, the names are in boldface where the letter C or S before the number indicates cast or soil, respectively, e.g., C102. For the isolates from 1996 and 1997, the names include the letter C, S, or B, followed by a unique number; the letter M or D; and the number 1 or 2. The first letter indicates the habitat: cast, soil, or burrow, respectively. The first number is the sample number. The second letter indicates the isolation medium: MMSA or DNB, respectively. The last number indicates whether the isolate was the first or second isolate from the particular sample, e.g., S84D1. For the isolates from 1999, the names include the letters NC or NS for cast or soil isolates, respectively, and a unique number, e.g., NC8. Brackets after sequence names denote the total number of sequences in the OTU when that number was greater than 1. Accession numbers appear in parentheses for all reference sequences. Open circles (○) denote bootstrap support of ≥95%, and closed circles (•) denote bootstrap support of ≥50%. The scale bar represents the Jukes-Cantor evolutionary distance.

FIG. 2.

FIG. 2.

Phylogenetic tree of the Horseshoe Bend clones and isolates associated with the Firmicutes phylum. Notation is as described for Fig. 1.

FIG. 3.

FIG. 3.

Phylogenetic tree of the Horseshoe Bend clones and isolates associated with the Actinobacteria phylum except Rubrobacter. Notation is as described for Fig. 1.

FIG. 4.

FIG. 4.

Phylogenetic tree of the Horseshoe Bend clones and isolates associated with the Rubrobacteria. Groups denoted on the right side of the tree follow the nomenclature of Holmes et al. (33). Other notation is as described for Fig. 1.

FIG. 5.

FIG. 5.

LIBSHUFF comparison of HSB soil and cast clones. Homologous coverage curves are shown with open circles (○) and heterologous coverage curves with closed circles (•). Solid lines indicate the difference between the homologous and heterologous coverage curves at each value of D as determined by the Cramér-von Mises test statistic, and broken lines denote the 95% value of the random shufflings. (A) Comparison of the HSB cast clones (X) to the HSB soil clones (Y); (B) comparison of the HSB soil clones (X) to the HSB cast clones (Y).

FIG. 6.

FIG. 6.

(A) Phylogenetic tree of the soil archaeal clones from Horseshoe Bend. RFLP patterns are in parentheses after clone names. Other notation is as described for Fig. 1. (B) Distribution of RFLP patterns of archaeal clones in soil and cast clone libraries.

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