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ABSTRACT Synthetic microbial communities (SynComs) are a valuable tool to study community assembly patterns, host–microbe interactions, and microbe–microbe interactions in a fully controllable setting. Constructing the SynCom inocula for plant–microbe experiments can be time-consuming and difficult because a large number of isolates with different medium requirements and growth rates are grown in parallel and mixed to appropriate titers. A potential workaround to assembling fresh SynCom inocula for every experiment could be to prepare and freeze SynComs on a large scale, creating ready-to-use inocula. The objective of this study was to compare the reproducibility, stability, and colonization ability of freshly prepared versus frozen SynCom inocula. We used a community of seven species known to colonize maize roots. The results from inoculation with the frozen SynCom were as consistent as those of standardizedde novoconstruction of fresh SynCom. Our results indicate that creating frozen SynCom inocula for repeated use in experiments not only saves time but could also improve cross-experiment reproducibility. Although this approach was only validated with one SynCom, it demonstrates a principle that can be tested for improving approaches in constructing other SynComs. IMPORTANCESynthetic communities (SynComs) are an invaluable tool to characterize and model plant–microbe interactions. Multimember SynComs approximate intricate real-world interactions between plants and their microbiome, but the complexity and time required for their construction increase enormously for each additional member added to the SynCom. Therefore, researchers who study a diversity of microbiomes using SynComs are looking for ways to simplify the use of SynComs. In this manuscript, we evaluate the feasibility of creating ready-to-use freezer stocks of a well-studied seven-member SynCom for maize roots. The frozen ready-to-use SynCom stocks work according to the principle of “just add buffer and apply to sterilized seeds or seedlings” and thus can save time applied in multiple days of laborious growing and combining of multiple microorganisms. We show that ready-to-use SynCom stocks provide comparable results to those of freshly constructed SynComs and thus allow for significant time savings when working with SynComs. 

Hybrids account for nearly all commercially planted varieties of maize and many other crop plants because crosses between inbred lines of these species produce first-generation [F₁] offspring that greatly outperform their parents. The mechanisms underlying this phenomenon, called heterosis or hybrid vigor, are not well understood despite over a century of intensive research. The leading hypotheses—which focus on quantitative genetic mechanisms (dominance, overdominance, and epistasis) and molecular mechanisms (gene dosage and transcriptional regulation)—have been able to explain some but not all of the observed patterns of heterosis. Abiotic stressors are known to impact the expression of heterosis; however, the potential role of microbes in heterosis has largely been ignored. Here, we show that heterosis of root biomass and other traits in maize is strongly dependent on the belowground microbial environment. We found that, in some cases, inbred lines perform as well by these criteria as their F₁offspring under sterile conditions but that heterosis can be restored by inoculation with a simple community of seven bacterial strains. We observed the same pattern for seedlings inoculated with autoclaved versus live soil slurries in a growth chamber and for plants grown in steamed or fumigated versus untreated soil in the field. In a different field site, however, soil steaming increased rather than decreased heterosis, indicating that the direction of the effect depends on community composition, environment, or both. Together, our results demonstrate an ecological phenomenon whereby soil microbes differentially impact the early growth of inbred and hybrid maize.