Microbial Inoculum Collection and Treatment Soil Preparation
RIL and parental replicates were grown in the presence and absence of native microbiome inoculations obtained from the locations where parents were collected (Lady Bird Johnson Wildflower Center (WFC) in Austin, TX for var. hallii and the Corpus Christi Botanical Garden (CCBG), in Corpus Christi, TX for var. filipes ; native soils properties in Appendix S1 and Table S1).
To create treatments, we mixed 1% by volume of native soil inoculum (to minimize the effect of nutritive and textural soil properties from the inoculum) with a twice-autoclaved horticultural soil mix of compost, decomposed granite and vermiculite (Thunder Dirt, Geo Growers, Austin, TX) and incubated it for two weeks in closed 400-liter plastic containers (as in Edwards et al. 2019). We selected this particular commercial soil mix because it homogenizes well and facilitates root extraction and cleaning. For soils utilized in the control treatments, the 1% soil inoculum was twice autoclaved over a 24-hour period before mixing and incubation. Given the nature of this large-scale greenhouse experiment under an open-air environment, true sterility of the control treatments is not possible and thus we refer to the treatments by their inoculum source: microbiome treatments as Austin Inoculated (AI) and Corpus Inoculated (CI), and control treatments as Mock Austin Inoculated (MAI) and Mock Corpus Inoculated (MCI). Nevertheless, we feel this system allows us to assess the holistic impact of soil microbes, above and beyond what could be obtained from studies of individual microbes or experiments under more artificial conditions.