A Monitoring System for Green Fluorescence Protein Gene-transformed Fusarium oxysporum in Melon Seedlings

Abstract

Using melon seedlings at the cotyledon stage and genetically marked fungi, a system for monitoring pathogenic and nonpathogenic Fusarium oxysporum was devised in the present study. Protoplasts were prepared from three formae speciales (melonis, radicis-lycopersici and fragariae )of F. oxysporum and transformed with a synthetic gene for green fluorescence protein. Transformants were primarily isolated in the presence of hygromycin B and then screened by the emission of bright green fluorescence. Roots of melon seedlings were inoculated with fluorescing microconidia of these fungi, and fungal infection behavior was traced. Using fluorescence microscopy, we directly observed not only the fungus at the root surface, but also the mycelia elongating in the trachea of roots. Both pathogenic and nonpathogenic fungi germinated and hyphae elongated superficially on the surface of root. Only pathogenic fungi caused root necrosis at the inoculation site. Hyphae grew within the stem to induce constriction or cracking of lower hypocotyls, then causing wilting of the seedlings. Infection behavior of genetically marked pathogenic and nonpathogenic F. oxysporum could be successfully monitored after inoculation of cotyledons of seedlings.