Field-deployable LAMP assay for rapid and specific detection of Fusarium oxysporum causing corm rot in saffron.

A field-deployable and highly specific loop-mediated isothermal amplification (LAMP) assay was developed and validated for the rapid detection of Fusarium oxysporum, the causal agent of corm rot in saffron. Corm rot poses a serious threat to saffron (Crocus sativus L.) cultivation, especially during the flowering stage, resulting in significant yield losses. This study focused on the molecular identification and rapid detection of F. oxysporum in saffron-growing regions of Kishtwar, Jammu and Kashmir. Cultures of F. oxysporum and eleven other microbial isolates were obtained, and molecular identification was conducted via Sanger sequencing using universal ITS1-F and ITS4-R primers for fungi and 27F/1492R primers for bacteria. Complete genome sequences of all isolates were retrieved from NCBI for comparative analysis. A unique 500 bp sequence specific to F. oxysporum, located between positions 5,500-6,000 on chromosome XII, was identified for designing specific PCR and LAMP primers, which were validated both in silico and experimentally (gel electrophoresis and SYBR Green dye). The LAMP detection method proved simple, sensitive, and suitable for early, on-site detection, as it yields positive results exclusively for F. oxysporum. Sensitivity analysis showed detection as low as 2 ng/μL of DNA, suitable for early on-site diagnosis. The operational simplicity requiring only a basic heat source and straightforward result interpretation makes it suitable for field use in resource-limited settings. Early diagnosis enables timely interventions to limit disease spread and crop losses, while the adaptability of the assay supports broader use in plant health monitoring and crop disease management.