Endophytic microbiome variation among single plant seeds

Like other plant compartments, the seed harbors a microbiome. Seed microbiome members are the first to colonize a germinating seedling, and they may initiate the trajectory of microbiome assembly for the next plant generation. Therefore, the members of the seed microbiome are important for the dynamics of plant microbiome assembly and the vertical transmission of potentially beneficial symbionts. However, it remains challenging to assess the microbiome at the individual seed level (and, therefore, for the future individual plants) due to low endophytic microbial biomass, seed exudates that can select for particular members, and high plant and plastid contamination of resulting reads. Here, we report a protocol for extracting microbial DNA from an individual seed (common bean, Phaseolus vulgaris L.) with minimal disruption of host tissue, which we expect to be generalizable to other medium- and large-seed plant species. We applied this protocol to determine the 16S rRNA V4 and rRNA ITS2 amplicon composition and examine the variability of individual seeds harvested from replicate common bean plants grown under standard, controlled conditions to maintain health. Using DNA extractions from individual seeds, we compared seed-to-seed, pod-to-pod, and plant-to-plant microbiomes, and found highest microbiome variability at the plant level. This suggests that several seeds from the same plant could be pooled for microbiome assessment, given experimental designs that apply treatments at the parent plant level. This study adds protocols and insights to the growing toolkit of approaches to understand the plant-microbiome engagements that support the health of agricultural and environmental ecosystems.