Culturomics from field-grown crop plants using dilution-to-extinction, two-step library preparation and amplicon sequencing

Culturomics approaches have advanced microbial research by enabling the high-throughput isolation and characterization of a broader range of bacterial taxa, including some previously considered unculturable. Here, we present the testing and optimization of a protocol for isolating and identifying hundreds of cultivable microbes from field-grown plants. This protocol was tested and optimized using the root microbiomes of field-grown corn and pea plants under varying environmental conditions in North Dakota, USA. By employing dilution-to-extinction culturing and a two-step barcoding PCR strategy targeting the V4 region of the 16S rRNA gene, we identified over 200 unique bacterial isolates. The optimized bioinformatic pipeline, built around the DADA2 package, ensured accurate amplicon sequence variant (ASV) detection and taxonomy assignment. The resulting bacterial isolates span diverse phylogenetic groups, including plant-associated taxa known for promoting plant growth and mitigating stress. Our findings highlight the value of culturomics in generating microbial collections for synthetic community design and advancing plant-microbe interaction research. The protocols scalability, cost-effectiveness, and robust performance demonstrate its potential for widespread application in agricultural microbiome studies. Impact statementHigh-throughput isolation and characterization of cultivable microbes from plant microbiomes is crucial for advancing microbiome research. However, efficiently recovering a diverse range of bacterial taxa remains a challenge due to high costs and labor-intensive protocols. Our optimized culturomics protocol integrates dilution-to-extinction culturing with a two-step barcoding PCR strategy, enhancing recovery rates and reducing costs while maintaining high accuracy. By employing next-generation sequencing (NGS) and a streamlined bioinformatic pipeline built around the robust DADA2 workflow for amplicon sequencing, the method enables the scalable recovery of hundreds of unique bacterial isolates. This approach makes significant advancements over traditional culturing methods and other high-throughput cultivation protocols, providing an efficient, cost-effective platform for generating microbial collections essential for synthetic communities, comparative genomics, and agricultural microbiome applications. Data summaryAll protocols, sequence data, and analysis codes have been made publicly available in open-access repositories. The authors confirm all supporting data, code, and protocols have been provided within the article or through supplementary data files.