Treatment drives bacterial community composition
We used 16S rRNA gene amplicon sequencing classified into ASVs to
characterize both the native microbial communities and communities
generated by experimental inoculations as a representation for the
overall biotic differences. For parental ecotypes growing under natural
habitats, PCoA of bacterial ASV counts revealed strong location/ecotype
and compartment effects (i.e. soil, rhizosphere, and root) across axes
one and two, respectively (Fig. 1a). Permanova mirrored these results
with location/ecotype explaining the most variance (R2= 0.21, P < 0.001) and compartment explaining the
second most (R2 = 0.15, P < 0.001;
Table 1). Microbiota varied significantly in alpha diversity between
compartments, but not between location (Fig. 1b). Phylum level
distributions were overall consistent between microbiota of plants
growing at the two natural locations with Proteobacteria ,Actinobacteria , and Acidobacteria being dominant members
(Fig. 1c, Fig. S1a), which is congruent with results from previous
root-associated microbiome studies (Edwards et al. 2014; Lundberg et al.
2012; Singer et al. 2019; Wagner et al. 2016). Only three relatively low
abundance phyla displayed significant differences between
location-ecotype: WPS-2 and Entotheonellaeota in the rhizosphere
and Rokubacteria in the root (Fig. S2). Conversely, microbiota
from the two locations were more divergent at the ASV level and we
identified 735 unique ASVs which were differentially abundant by
compartment (440 in soil, 251 in rhizosphere, and 401 in roots; Fig.
1c).
We next analyzed microbiota acquired under experimental conditions in
the greenhouse by sampling roots and rhizosphere from the RIL parents,
along with soil from unplanted pots. PCoA revealed that inoculum and
compartment significantly impacted microbiota composition (Fig. 1d).
Alpha diversity was also impacted by compartment and inoculum: in
general, plants inoculated with native soil slurries hosted microbiota
with greater Shannon diversity compared to plants with heat-killed, mock
microbiota (Fig. 1e). As expected, when comparing the effect of inoculum
source within heat killed or native conditions, we found that microbial
communities of plants and soil with heat-killed inocula were
significantly more similar than if the inoculum was unsterilized and
this effect was consistent independent of compartment (Fig. S3). Similar
trends were observed at the phylum level where there were many more
differentially abundant phyla by soil source with intact inoculum vs.
heat treated (Fig. S1b, c). When identifying ASVs whose abundance was
impacted by soil inoculation source, many more ASVs were differentially
abundant in comparisons between native inoculum compared to heat killed
(Fig. 1f). When analyzed together, we found that greenhouse and field
microbiomes formed distinct communities, yet were still identifiable by
soil source (Fig. S4a, more in Appendix S1). These results indicate that
heat sterilization of inoculum dampens the effect of soil source on
compositions of the resulting microbiome and that plants inoculated with
native microbiota host significantly different communities in the
rhizosphere and roots.