Abstract:

Rootstalk rot of Hibiscus mutabilis is primarily caused by fungi of the genus Fusarium，with F. oxysporum，F. solani，and F. equiseti identified as the dominant pathogenic species. In this study，a loop-mediated isothermal amplification（LAMP）assay targeting the EF-1α gene was developed for the detection of these three Fusarium species. The specificity of the primers designed for each species was validated，and the reagents and reaction conditions for the LAMP assay were optimized. Results demonstrated that the LAMP primers exhibited high specificity，allowing for accurate detection of the target Fusarium species. The assay was capable of distinguishing each species，even in mixed infections，through the use of a species-specific primer. In a 25 μL reaction volume，optimal concentrations of Mg²⁺， dNTPs，and the colorimetric indicator hydroxy naphthol blue（HNB）were determined to be 10 mmol · L^-1，1.4 mmol · L^-1，and 100 μmol · L^-1，respectively. Under these conditions，the assay performed at 63 ℃ for 60 min achieved a detection sensitivity of 10^-3 ng · μL^-1. In simulated environmental samples，the LAMP assay successfully detected the inoculated Fusarium species in the rhizosphere soil of Hibiscus. mutabilis within 12 hours post-inoculation，regardless of single or mixed species presence. Additionally，the pathogens were detectable within plant tissues as early as 24 hours after inoculation.

Key words: Hibiscus mutabilis, rootstalk rot, Fusarium, LAMP