Bacterial seed endophytes in developing seedlings of Spathoglottis plicata

Background: Bacterial endophytes equip plants with the ability to navigate biotic and abiotic challenges. Introducing a consortium of these beneficial microbes into our crop plants would address the many challenges of sustainable agriculture, particularly if these endophytes were passed generationally. However, our knowledge of seedling colonization by seed endophytes is limited and has remained a data-bottleneck critical to resolving mechanisms that drive successful placement of seed endophytes within the plant body. In this work, we aimed to visualize these early stages of colonization and tie bacterial endophytes species to key events in migration from the seedcoat to the seedling leaf.Results: The requisite system for this study was an orchid, Spathoglottis plicata, since whole-seeding imaging was necessary. Microbiome analysis revealed the core genera as Mesorhizobium, Bradyrhizobium, Ralstonia, Kocuria and Acinetobacter, and demonstrated that as the germinating orchid protocorm formed a first leaf, and later, a root, the microbiome community structure shifted. All flagellated seed endophytes were visualized using confocal LSM with Alexa Fluor 488 during germination. The endophytes aggregated into bacterial mats, located to the shoot meristem and moved into the first leaf, where endophytes dispersed into smaller aggregates and targeted the stomata for access to the leaf interior. Many of the core genera were abundant during meristem targeting, while Hydrotalea, Brevibacillus, Rothia, and Bacillus peaked in OTUs during stomatal targeting. Immunolocalization studies with Arabidopsis during germination revealed a similar pattern of colonization.Conclusions: The whole-seeding approach in this study unveiled a more detailed story of the dynamic changes that seed endophytes undergo during germination. Bacterial endophytes appear to work in concert to make their way to the plant interior, which is a plausible explanation for the shifts in community structure as seed endophytes aggregate, target the meristem and localize to stomata during plant organ formation. Identification of genera and species associated with key colonization events provide a starting point for future co-culture experiments to understand the essential microbial interactions during colonization.