Evaluation of ready-to-use freezer stocks of a synthetic microbial community for maize root colonization

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 media 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 pre-make and freeze SynComs on a large scale, creating ready-to-use stock 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 standardized de novo construction 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 increases 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 multiple days of laborious growing and combining of multiple microorganisms. We show that ready-to-use SynCom stocks provide comparable results to freshly constructued SynComs and thus allow for large time savings when working with SynComs.