Leveraging microbiomes to promote soil health and plant growth requires understanding underlying molecular mechanisms through reproducible experimental systems. In a global collaborative effort involving five laboratories, we aimed to advance reproducibility in microbiome studies by leveraging recently developed fabricated ecosystems (EcoFAB 2.0). Our inter-laboratory ring trial utilized synthetic bacterial communities (SynComs) to study root microbiome recruitment (analyzed by 16S amplicon sequencing) and exudate composition (analyzed by LC-MS/MS) of the model grass Brachypodium distachyon in EcoFAB 2.0. All participating laboratories replicated permutation-dependent changes in plant phenotype, root exudates, and bacterial community structure. Additionally, we identified factors determining bacterial colonization patterns through comparative genomics of SynCom members, fluorescent microscopy, motility assays, and exudate utilization. The study provides benchmark datasets and detailed protocols to achieve reproducibility and best practices, demonstrating how standardized SynComs and EcoFAB can promote scientific collaboration, improve comparability, and accelerate the application of findings, thus enhancing our understanding of microbiomes in plant and ecosystem science.  The 16S rRNA amplicon sequencing data are available via NCBI (https://www.ncbi.nlm.nih.gov/) as BioProject PRJNA1151037. All raw data, including plant phenotypes, sterility tests, metabolite identifications, and in vitro assays are available via Figshare at https://doi.org/10.6084/m9.figshare.c.7373842. The untargeted metabolomics outputs (HILIC-pos) with features annotations and .mzml files are available via GNPS2 at https://gnps2.org/status?task=2ccbf82840724c99a2acc2c9e512a302. Raw LC-MS/MS files are available in .raw format at MassIVE (https://massive.ucsd.edu/) under ID number MSV000095476 or via https://doi.org/10.25345/C5Q23RB6B. The protocol is available at protocols.io via https://dx.doi.org/10.17504/protocols.io.kxygxyydkl8j/v1. The annotated bacterial genomes can be accessed via IMG/M (https://img.jgi.doe.gov/) by searching for either the isolate name, taxon ID, GOLD Project ID, or by using the links in Table S2.
The U.S. Department of Energy Office of Science, Office of Biological and Environmental Research Trial Ecosystem Advancement for Microbiome Science (TEAMS) project under contract DE-AC02-05CH11231 to Lawrence Berkeley National Laboratory.