Lopez-Echartea Eglantina, Dusek Nicholas, Misialek Mallory, Mahmud-Un-Nabi Mohammad Al, Williamson Riley, Marathe Komal, Geddes Barney A
Department of Microbiological Sciences, North Dakota State University, Fargo, ND, USA.
Center for Computationally Assisted Science and Technology, North Dakota State University, Fargo, ND, USA.
Microbiology (Reading). 2025 Jun;171(6). doi: 10.1099/mic.0.001571.
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 ND, 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 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 protocol's scalability, cost-effectiveness and robust performance demonstrate its potential for widespread application in agricultural microbiome studies.
文化组学方法通过实现更广泛细菌类群的高通量分离和表征，推动了微生物研究的发展，其中包括一些以前被认为不可培养的细菌。在此，我们展示了一种从田间种植的植物中分离和鉴定数百种可培养微生物的方案的测试和优化。该方案在美国北达科他州不同环境条件下，使用田间种植的玉米和豌豆植物的根微生物群进行了测试和优化。通过采用稀释至灭绝培养法和针对16S rRNA基因V4区域的两步条形码PCR策略，我们鉴定出了200多种独特的细菌分离株。围绕DADA2软件包构建的优化生物信息学流程，确保了准确的扩增子序列变异检测和分类学归属。所得细菌分离株涵盖了不同的系统发育群体，包括以促进植物生长和缓解胁迫而闻名的与植物相关的类群。我们的研究结果凸显了文化组学在为合成群落设计生成微生物集合以及推进植物 - 微生物相互作用研究方面的价值。该方案的可扩展性、成本效益和强大性能证明了其在农业微生物组研究中广泛应用的潜力。
