柑橘间座壳菌绿色荧光蛋白标记菌株的构建及致病性分析OA

Diaporthe citri,an important pathogen of citrus,poses a threat to citrus growth and fruit storage.In this study,a genetic transformation system for Diaporthe citri was developed using polyethylene glycol(PEG)-mediated transformation.A filamentous fungal expression vector,pBARGPE1-Hygro,carrying green fluorescent protein(GFP)and hygromycin resistance genes,was successfully introduced into Diaporthe citri strain ZJUQ7.Building on previous fungal transformation techniques,we established an efficient genetic transformation system for Diaporthe citri.Following hygromycin and green fluorescence screening,biological characterization,fluorescence stability verification,and pathogenicity assays of the transformants,34 stable fluorescent strains were obtained.These strains exhibited hyphal morphology,growth rate,and pathogenicity consistent with the wild-type strain.The hyphae of the ZJUQ7-2 transformant were inoculated onto leaves of Newhall Navel orange,and visible lesions appeared after 3 d.These lesions gradually darkened and expanded,eventually leading to the decay of the entire leaf.Fluorescence microscopy of the inoculation site clearly revealed the infection process of Diaporthe citri.This study successfully constructed a protoplast-mediated genetic transformation system for Diaporthe citri and generated stable GFP-labeled strains,providing key tools and technical support for elucidating the pathogen's life cycle,disease progression,and pathogenic mechanism.