Cultivating native bacteria from roots of plants grown in a given environment is essential for dissecting the functions of the root microbiota for plant growth and health with strain-specific resolution. In this study, we established a straightforward protocol for high-throughput bacterial isolation from fresh root samples using limiting dilution to ensure that most cultured bacteria originated from only one microorganism. This is followed by strain characterization using a two-sided barcode polymerase chain reaction system to identify pure and heterogeneous bacterial cultures. Our approach overcomes multiple difficulties of traditional bacterial isolation and identification methods, such as obtaining bacteria with diverse growth rates while greatly increasing throughput. To facilitate data processing, we developed an easy-to-use bioinformatic pipeline called ‘Culturome’ (https://github.com/YongxinLiu/Culturome) and a graphical user interface web server (http://bailab.genetics.ac.cn/culturome/). This protocol allows any research group (two or three lab members without expertise in bioinformatics) to systematically cultivate root-associated bacteria within 8–9 weeks.

从特定环境中生长的植物根部培养原生细菌对于剖析根部微生物群对植物生长和健康的功能以及特定菌株的分辨率至关重要。在这项研究中，我们建立了一个简单的协议，使用有限稀释从新鲜根样品中分离高通量细菌，以确保大多数培养的细菌仅来自一种微生物。随后使用双侧条形码聚合酶链反应系统进行菌株表征，以识别纯和异质细菌培养物。我们的方法克服了传统细菌分离和鉴定方法的多重困难，例如在大大提高通量的同时获得具有不同生长速率的细菌。为了便于数据处理，我们开发了一个名为“Culturome”（https://github.com/YongxinLiu/Culturome）的易于使用的生物信息学管道和一个图形用户界面网络服务器（http://bailab.genetics.ac.cn/culturome/） . 该协议允许任何研究小组（两个或三个没有生物信息学专业知识的实验室成员）在 8-9 周内系统地培养根相关细菌。