Artificial Microbiome-Selection to Engineer Microbiomes That Confer Salt-Tolerance to Plants

We develop a method to artificially select for rhizosphere microbiomes that confer salt-tolerance to the model grass Brachypodium distachyon. We differentially propagate microbiomes within the background of a non-evolving, highly-inbred plant population, and therefore only microbiomes evolve in our experiment, but not the plants. To optimize methods, we conceptualize artificial microbiome-selection as a special case of indirect selection: We do not measure microbiome properties directly, but we use host performance (e.g., biomass; seed set) as an indicator to infer association with rhizosphere microbiomes that confer salt-tolerance to a plant. We previously called this indirect-selection scheme host-mediated indirect selection on microbiomes (Mueller & Sachs 2015). Our methods aim to maximize evolutionary changes due to differential microbiome-propagation, while minimizing some (but not all) ecological processes affecting microbiome composition. Specifically, our methods aim to maximize microbiome perpetuation between selection-cycles and maximize response to artificial microbiome-selection by (a) controlling microbiome assembly when inoculating seeds at the beginning of each selection cycle; (b) using low-carbon soil to enhance host-control mediated by carbon secretions of plants during initial microbiome assembly and subsequent microbiome persistence; (c) fractionating microbiomes before transfer between plants to perpetuate and select only on bacterial and viral (but not fungal) microbiome components; and (d) ramping of salt-stress between selection-cycles to minimize the chance of over-stressing plants. Our selection protocol generates microbiomes that enhance plant fitness after only 1-3 rounds of artificial selection on rhizosphere microbiomes. Relative to fallow-soil control treatments, artificially-selected microbiomes increase plant fitness by 75% under sodium-sulfate stress, and by 38% under aluminum-sulfate stress. Relative to null control treatments, artificially-selected microbiomes increase plant fitness by 13% under sodium-sulfate stress, and by 12% under aluminum-sulfate stress. When testing microbiomes after nine rounds of differential microbiome propagation, the effect of bacterial microbiomes selected to confer tolerance to sodium-sulfate stress appears specific (these microbiomes do not confer tolerance to aluminum-sulfate stress), but the effect of microbiomes selected to confer tolerance to aluminum-sulfate stress appears non-specific (selected microbiomes ameliorate both sodium- and aluminum-sulfate stresses). Complementary metagenomic analyses of the artificially selected microbiomes will help elucidate metabolic properties of microbiomes that confer specific versus non-specific salt-tolerance to plants.