Serial Knockout Isolation Technique (SKIT): targeted isolation of multi-antagonist bacteria.

Traditional approaches to isolating antagonistic bacteria are non-targeted, yield lower recovery of potential multi-antagonists, and require substantial time, human resources, and lab resources. We advocate isolating siderophore-producing bacteria (SPB) or hydrolytic enzyme-producing bacteria (cellulase, protease, or chitinase) using the Serial Enrichment Incubation Technique (SEIT). Further, these recovered bacteria may be tested using the Triple Sugar Iron (TSI) test, which can easily identify Enterobacteriaceae and exclude probable human pathogens. Then, the Hydrocyanic (HCN) production assay may be performed, and prioritize both siderophore- and HCN-positive bacterial isolates in a dual assay against the predominant target phytopathogens first, then others. Here, non-siderophore and enterobacteriaceae members are serially eliminated or knocked out using the Serial Knockout Isolation Technique (SKIT). SKIT is proposed to reduce workflow time for isolating multi-antagonist bacteria, thereby saving time, labor, and lab resources.