Studying plant–microbe–soil interactions is challenging due to their high complexity and variability in natural ecosystems. While fabricated ecosystems provide opportunities to recapitulate aspects of these systems in reduced complexity and controlled environments, inoculation can be a significant source of variation. To tackle this, we evaluated how different bacterial inoculation practices and plant harvesting time points affect the reproducibility of a synthetic microbial community (SynCom) in association with the model grass Brachypodium distachyon. We tested three microbial inoculation practices, seed inoculation, transplant inoculation, and seedling inoculation, and two harvesting points, early (14-day-old plants) and late (21 days postinoculation). We grew our plants and bacterial strains in sterile devices (EcoFABs) and characterized the microbial community from root, rhizosphere, and sand using 16S ribosomal RNA gene sequencing. The results showed that inoculation practices significantly affected the rhizosphere microbial community when harvesting at an early time point but not at the late time point. As the SynCom showed a persistent association with B. distachyon at 21 days postinoculation regardless of inoculation practices, we assessed the reproducibility of each inoculation method and found that transplant inoculation showed the highest reproducibility. Moreover, plant biomass was not adversely affected by transplant inoculation treatment. We concluded that bacterial inoculation while transplanting coupled with a later harvesting time point gives the most reproducible microbial community in the EcoFAB−B. distachyon−SynCom fabricated ecosystem. We recommend this method as a standardized protocol for use with fabricated ecosystem experimental systems.

Harnessing beneficial microorganisms is seen as a promising approach to enhance sustainable agriculture production. Synthetic communities (SynComs) are increasingly being used to study relevant microbial activities and interactions with the plant host. Yet, the lack of community standards limits the efficiency and progress in this important area of research. To address this gap, we recommend three actions: (1) defining reference SynComs; (2) establishing community standards, protocols and benchmark data for constructing and using SynComs; and (3) creating an infrastructure for sharing strains and data. We also outline opportunities to develop SynCom research through technical advances, linking to field studies, and filling taxonomic blind spots to move towards fully representative SynComs.