Culturing captures members of the soil rare biosphere

Abstract

The ecological significance of rare microorganisms within microbial communities remains an important, unanswered question. Microorganisms of extremely low abundance (the 'rare biosphere') are believed to be largely inaccessible and unknown. To understand the structure of complex environmental microbial communities, including the representation of rare and prevalent community members, we coupled traditional cultivation with pyrosequencing. We compared cultured and uncultured bacterial members of the same agricultural soil, including eight locations within one apple orchard and four time points. Our analysis revealed that soil bacteria captured by culturing were in very low abundance or absent in the culture-independent community, demonstrating unexpected accessibility of the rare biosphere by culturing.

Fig. 1

Community structure varies between culture-based and culture-independent assessments. A. To determine the completeness of the sequencing effort, rarefaction was performed from a minimum of 50 sequences to the median number of sequences observed across all samples (610 sequences, as per QIIME default parameters for the script alpha_rarefaction.py). Error bars are standard error around the mean of 10 subsamples at each level of sequencing effort. B. Omitting singletons has minimal effect on community structure. Communities are ranked by number of OTUs. Closed symbols represent communities including singleton OTUs, and open symbols represent communities without singleton OTUs. Thus, the difference on the y-axis between the closed and open symbols depicts the change in the number of OTUs within a community after omitting singletons. Symbols are scaled in size to the total number of sequences observed within a community before rarefaction.

Fig. 2

Culture-based and culture-independent analyses of soil bacterial communities. A. Number of OTUs shared between culture-based and culture-independent analyses, and number of OTUs unique to each analysis. B. Heat map of 26 most abundant OTUs in the dataset, organized by response patterns (clusters shown by dendrogram). Columns are community samples from the culture-independent method (n = 26, left side of the dashed white line) or the cultured-based method (n = 32, right side of the dashed white line). Each OTU is in a row and colour intensity indicates its relative abundance, with brighter green indicating higher abundance. Asterisk indicates that the OTU could not be identified to the Order level.