Use Of The Green Fluorescent Protein Variant GFPuv For Analysis Of Pseudomonas Syringae PV. Actinidiae Infection, Movement And Colonization In Kiwifruit

Pseudomonas syringae pv. actinidiae （Psa） is the causal agent of bacterial canker ofkiwifruit, which is one of the most important destructive diseases in kiwifruit production. Thedisease is very difficult to control because of extensive distribution, severe harm, broad hostand high pathogenicity and so on, which causes serious yield and economic losses. Becauselittle is known about natural host, infection process and colonization sites of the pathogen, so itis still not clear what the pathogenic mechanism is, which results in high cost and poorefficience of control. In this paper, in order to analyze the infection process of the pathogen inkiwifruit, the Psa strain was tagged with GFPuv through genetic transformation technology.We systematically researched the infection way, movement speed and colonization site of Psaby plate dilution, fluorescence microscope, light microscope and transmission electronmicroscope technology, to provide theoretical basis for elucidating the pathogenic mechanismof the disease and formulating prevention and control measures. The mainly results were asfollows:1The Psa strain was tagged with GFPuv by electroporation through a pDSK-GFPuvplasmid vector containing the GFPuv gene. The transformants showed strong greenfluorescence under UV-light indicating GFPuv expression, and the electroporation methodhad a transformation efficiency of1.78×10⁵CFU·μg^-1. A700bp fragment of the GFPuvgene was amplified from the genome DNA of the transformants. There were no differencesbetween the engineered PSAmx7-GFPuv1strain and wild-type strain including morphology,optimum temperature, pH and pathogenicity. The green fluorescence could be kept until20times of subcultures.2The in vitro and potted kiwifruit were inoculated artificially with the GFPuv-labeledstrain PSAmx7-GFPuv1to detect and monitor the infected dynamic of the pathogen by usingplate dilution method and fluorescence microscope techniques. The results indicated that thepathogen can infect stems by entering wounds, leaf scar and lenticels, followedly thepathogen could migrate up and down and the number of colonies increased gradually. Thepathogen migrated faster in stems tissue under4℃culture condition when compared with both16℃and25℃. The pathogen could migrate up and down to lateral veins and the base ofpetiole. Besides, the pathogen stopped expanding and growing in the leaf and vein when theculture temperature reached24℃. Still, after watering inoculation, the pathogen could infectand colonize in root. In addition, the pathogen also could survive in sterile soil for threemonths, and three weeks or so in non-sterile soil.3Light and transmission electron microscope were used to detect the colonization sitesof the GFPuv-labeled strain PSAmx7-GFPuv1after aritificial inoculation in kiwifruit and thePsa in infected samples of the orchard. The results showed the pathogen could penetratethrough cortex from lenticels, and phloem from intercellular spaces of phloem fibre afterinoculating lenticels. After oblique cut inoculation, the pathogen extensively colonized cortexcells, phloem cells, xylem vessel and intercellular spaces, which resulted in markedalterations of cell structure including cell plasmolysis and cell distortion. After smearingbacterium suspension on the leaves, a larger bacterium colonized intercellular spaces ofmesophyll cell and xylem vessel and cortex of veins, which finally caused cell distortion andcell disruption. Still, at3day post watering inoculation, the pathogen absorbed on theepidermis, colonized epidermis cell and cortex cell of roots, then gradually expanded intovascular bundle. About the infected field samples, the Psa not only mainly colonized cortex,phloem and xylem vessel in stem, but it also colonized upper and lower epidermis andintercellular spaces of mesophyll cell in leaf.