The spermosphere is the transient, immediate zone of soil around imbibing and germinating seeds, rich in microbial activity. It represents a habitat where there is contact between seed-associated microbes and soil microbes, but is studied less compared to other plant habitats. Previous studies on spermosphere microbiology were primarily culture-based or did not sample the spermosphere soil as initially defined in space and time. Thus, the objectives of this study were to develop an efficient strategy to collect spermosphere soils around imbibing soybean and cotton in non-sterile soil and investigate factors contributing to changes in microbial communities. The method employed sufficiently collected spermosphere soil as initially defined in space (3-10 mm soil around a seed) by constraining the soil sampled with an cork borer and confining the soil to a 12-well microtiter plate. Spermosphere prokaryote composition changed over time and depended on the crop within six hours after seeds were sown. By 12 to 18 hours, crops had unique microbial communities in spermosphere soils. Prokaryote evenness dropped following seed imbibition and the proliferation of copiotroph soil bacteria in the phyla Proteobacteria and Firmicutes. Due to their long history of plant growth promotion, prokaryote OTUs in Bacillus, Paenibacillus, Burkholderia, Massilia, Azospirillum, and Pseudomonas were notable genera enriched in the cotton and soybean spermosphere. There was no consistent evidence that fungal communities changed like prokaryotes. However, fungi and prokaryotes were hub taxa in cotton and soybean spermosphere networks. Additionally, the enriched taxa were not hubs in networks, suggesting other taxa besides the copiotrophic may be important for spermosphere communities. Overall, this study advances knowledge in the assembly of the plant microbiome early in a plant's life, which may have plant health implications in more mature plant growth stages.

The central hypothesis of this research was that plant species and seed exudate release would alter the assembly of microbes in the spermosphere soil. Our research investigated the response of microbes to the initial burst of nutrients into the spermosphere soil, filling knowledge gaps from previous studies that pregerminated seeds under sterile conditions.