Using a Control to Better Understand Phyllosphere Microbiota

<div><p>An important data gap in our understanding of the phyllosphere surrounds the <i>origin</i> of the many microbes described as phyllosphere communities. Most sampling in phyllosphere research has focused on the collection of microbiota without the use of a control, so the opportunity to determine which taxa are actually driven by the biology and physiology of plants as opposed to introduced by environmental forces has yet to be fully realized. To address this data gap, we used plastic plants as inanimate controls adjacent to live tomato plants (phyllosphere) in the field with the hope of distinguishing between bacterial microbiota that may be endemic to plants as opposed to introduced by environmental forces. Using 16S rRNA gene amplicons to study bacterial membership at four time points, we found that the vast majority of all species-level operational taxonomic units were shared at all time-points. Very few taxa were unique to phyllosphere samples. A higher taxonomic diversity was consistently observed in the control samples. The high level of shared taxonomy suggests that environmental forces likely play a very important role in the introduction of microbes to plant surfaces. The observation that very few taxa were unique to the plants compared to the number that were unique to controls was surprising and further suggests that a subset of environmentally introduced taxa thrive on plants. This finding has important implications for improving our approach to the description of core phytobiomes as well as potentially helping us better understand how foodborne pathogens may become associated with plant surfaces.</p></div

Unique OTUs in Control and Phyllosphere Environments.

<p>Unique OTUs in Control and Phyllosphere Environments.</p

Network relationships in Control and Phyllosphere.

<p>Computing Spearman’s correlation coefficients with associated P values, (P< 0.05) after correction using FDR for all pairwise relationships of genera in control and phyllosphere samples (using Cytoscape v3x for visualization <a href="http://www.Cytoscape.org" target="_blank">www.Cytoscape.org</a>), 21 pairwise correlations were shared (C) between control and phyllosphere samples. A total of 37 correlations were unique to phyllosphere (B) and 23 correlations were unique to controls (A). Correlations unique to the phyllosphere appear increased among members of Bacilli, and Betaproteobacteria, including genera such as <i>Ralstonia</i>, <i>Staphylococcus</i>, and <i>Arthrobacter</i>. For example, <i>Ralstonia</i> has many significant relationships in phyllosphere samples but none in controls.</p

NDMS of 16S Communities from Store, Control and Phyllosphere Environments at 4 time-points.

<p><b>3a</b> Bray-Curtis ordination colored by environment (control (plastic), phyllosphere, and store bacterial communities). A very clear separation of store microbiota (green) is evident, however no significant separation between communities associated with control (red) and phyllosphere (purple) is evident. <b>3b</b> Bray Curtis ordination colored by time-point. Time-point 0 (T0) and Time-point 1 (T1) appear to separate from each other and also from time-points 2 (T2) and 3 (T3), however T2 and T3 are more similar. For both 1a and 1b, the ellipse defines the upper 95^th percentile limit of the assumed distribution.</p

Shared and Unique OTUs for Phyllosphere and Control Environments.

<p>Shared and Unique OTUs for Phyllosphere and Control Environments.</p

Bacterial Genera Unique to Phyllosphere and Control Samples.

<p>Using the total depth of sequences generated for Time-point 0 (16,074 sequences per independent replicate) (N = 9) for phyllosphere (live tomato plants) and control (plastic plants), we were able to identify 63 OTUs that were unique to the phyllosphere environment and 3, 249 OTUs that were unique to the control (plastic plant) environment.</p