High-Throughput Cultivation and Dilution-to-Extinction Protocol for Field-Grown Crops v1

This protocol describes a high-throughput cultivation method for isolating and identifying bacteria from field-grown crop plants using dilution-to-extinction, two-step library preparation, and amplicon sequencing. The approach enables the systematic recovery of diverse bacterial strains from root-associated microbiomes while minimizing competition from fast-growing species. The protocol integrates automated liquid handling, culture-based microbial isolation, and high-throughput sequencing to streamline bacterial cultivation and identification. The expected results include the isolation of a diverse set of cultivable root-associated bacteria, comprehensive taxonomic identification using 16S rRNA amplicon sequencing, and the generation of a curated culture collection for downstream applications, including functional screening and synthetic community design. This method improves efficiency, reproducibility, and scalability in microbial cultivation studies.