Experiment 1: Window of Opportunity

To test for the age at which plants are most susceptible to root colonization, we introduced soil microbes to plant roots of different developmental stages. Plants were planted at time intervals to create cohorts of different ages in order to be able to apply the soil inoculum at one point in time, avoiding storage effects on the inoculum. Therefore, the only difference among the groups was the age at which they received microbial inoculum (for a more detailed description of the planting and harvesting setup, see Figure 1). At the time of inoculation, plants represented different life stages (seeds, seedlings, adult plants, budding plants, and flowering plants) and ages (0, 1, 7, 8, and 9 weeks old).

Schematic overview of Experiment 1: Window of opportunity. At each harvest, root samples of Setaria viridis were collected. Harvest 1 controls for community composition prior to inoculation and includes plants of representing different developmental stages (flowering, budding, non-reproducing mature plant, seedling, and seed). Harvest 2 was collected 2 weeks after inoculation to control for the length of exposure to the inoculum, and Harvest 3 was collected when the plant was 12 weeks old to control for the age of the plant. The age of the plants at the time of harvest is listed in the diagram.

Before inoculation, one-third of the plants from all age categories were harvested to determine root microbial communities before inoculation (Harvest 1; for a detailed summary of how many samples were acquired and retained from each harvest, see Supplementary Table S1). The remaining plants were inoculated with a soil-slurry consisting of soil collected from the top 20 cm of rhizosphere soil of a single mature wild specimen of Setaria viridis (growing at University of British Columbia – Okanagan campus; 49.939975N, -119.399264W) and autoclaved de-ionized water. Briefly, collected soil was mixed with 250 ml water and sieved through a 2-mm mesh before 1 ml was applied to the rock wool plugs of the plants.

Around 2 weeks after inoculation, half of the remaining plants were harvested to control for the length of exposure to soil (Harvest 2). Roots were collected for DNA extraction (described below). The remaining plants were grown until they reached a total of 12 weeks of age (i.e., 3, 4, 5, 11, or 12 weeks after inoculation; Harvest 3) after which they were harvested and sampled for root communities. By 12 weeks, all plants were mature plants that had not yet started to senesce.