Soil In Situ Enrichment Coupled with RPA-CRISPR/Cas12b for Rapid and Visual On-Site Detection of Fusarium oxysporum in Strawberry

Fusarium oxysporum is a representative soilborne fungal pathogen that causes strawberry wilt, a disease characterized by an extremely high mortality rate that poses a severe threat to the sustainable development of the global strawberry industry. However, traditional detection methods are often time-consuming and dependent on specialized laboratory equipment, while existing soil nucleic acid extraction protocols are highly susceptible to interference from inhibitors in complex matrices, leading to low detection efficiency or high false-negative rates. To address these limitations, this study developed a novel on-site detection platform based on in situ biological enrichment and purification-free nucleic acid release. A specialized enrichment rod targeting F. oxysporum was developed to leverage the tropic growth characteristics of the pathogen, achieving physical separation from the soil matrix and effectively eliminating interference from complex soil inhibitors such as humic acids. The enriched pathogens release nucleic acids via a rapid lysis buffer, which are then neutralized and used directly as templates for recombinase polymerase amplification (RPA)-CRISPR/Cas12b isothermal detection, enabling visual field identification through lateral flow strips. This method requires no specialized instrumentation, achieving a detection limit of 8.5 CFU/g and a sensitivity of 70 copies. After completing 48 h of in situ enrichment, the entire process from rod retrieval to detection completion requires only 40 min (with hands-on operation time <10 min). By effectively circumventing soil inhibitor interference and simplifying complex nucleic acid extraction into a rapid, integrated protocol, this platform provides a critical technical solution for the on-site monitoring and precise control of soilborne pathogens.