Agriculture Reference
In-Depth Information
complexity to more manageable levels is through fractionation of extracted DNA
according to its buoyant density, which is determined by the percentage of base
pairs that are guanine:cytosine (G + C content). This strategy increases the capac-
ity to detect bacterial species (defined as >97% identity of the 16S rRNA-encoding
gene) in clone libraries (Morales et al. 2009). Using clone libraries from G +
C-enrichment, Morales et al. (2009) subsequently designed and tested a collection
of primers for PCR. They discovered significant variability in nontarget detection
and demonstrated that rigorous empirical validation is necessary before new prim-
ers can be used to analyze complex communities using either regular (saturation) or
quantitative PCR (Morales and Holben 2009).
While the construction of clone libraries has been a primary tool for assessing
genetic landscapes in soil, the recent development of next-generation sequencing
techniques offers the opportunity to explore the composition of complex micro-
bial communities in much greater detail. These techniques can document not only
the dominant members but also the newly revealed “rare biosphere” (Sogin et al.
2006)—those species in very low numbers that would otherwise go undetected.
They can also identify other microbes such as Archaea and Fungi that are not
detected in surveys of bacterial 16S genes. Application of next-generation sequenc-
ing to DNA extracted from KBS LTER soils has provided one of the first in-depth
views of how terrestrial microbial communities differ functionally from complex
communities in other ecosystems (Fig. 6.2).
Taxonomic and Functional Diversity
Greater taxonomic diversity within soil microbial communities has been revealed
as the resolving power of analytical methods has improved—from culture collec-
tions to biochemical profiling (fatty acid methyl esters) to DNA sequences (clone
libraries, then metagenomes). The remarkable diversity in soil results in large part
from a wide variety of microbes present in low numbers. For example, analysis of
a 5000-member clone library from the MCSE Conventional system (Table 6.1) led
to an estimate of 3500 bacterial species (defined by 97% sequence identity of 16S
rRNA genes) (Morales et al. 2009). Yet 80% of the 1700 species actually identified
were encountered three times or less, so the projection that the soil contained about
3500 species is almost certainly an underestimate. Similarly, a study of 409 clones
of 18S rDNA from basidiomycete fungi yielded a surprising variety of 241 spe-
cies of basidiomycetes (Lynch and Thorn 2006). While there is not yet a complete
description of the microbial diversity in any soil environment, statistically rigorous
comparisons of the more abundant members of the community are now possible.
An in-depth assessment of the effect of row-crop agriculture on taxonomic
diversity in microbial communities is currently under way across the KBS LTER
landscape. Initial results, based on approximately 15,000 sequences of 16S rRNA
encoding genes for replicated plots in the MCSE Deciduous Forest and Conventional
systems of the MCSE (Table 6.1), revealed approximately 10,000 species in each
treatment (Schmidt et al. unpublished). Surprisingly, despite the dramatic differences
in these ecosystems, there is not an obvious difference in the phylum level compo-
sition of communities. As with other studies of bacterial diversity in soil (Janssen
