Using GFP Labeled Strains To Study The Colonization Of Watermelon Bacterial Fruit Spots Pathogen In The Host

The GFP segment was obtained by digesting plasmid PS4GFP with XbaⅠandHindⅢ, and it was connected with the large segment of plasmid T-gfp which wasrecycled by digesting with the same enzymes, then a recombinant plasmid T-GFP2011was established. The GFP segment was recovered by digesting T-GFP2011plasmidwith EcoR Ⅰ and Hind Ⅲand was attached to the big segment of plasmid PKK223-3which was digested by the same enzymes. Then a carrier, PKK-GFP, was obtained,which could express green fluorescent protein in Acidovorax citrulli and also hadAmp resistance.The preparation and conversation conditions of the pathogen competent cellswere optimized. The result showed that the transformation efficiency was muchhigher than any others as the procedure was followed, the NB medium was used toculture bacteria with vibration at28℃until the bacterial concentration to OD600=0.3-0.4, then competent cells were treated with precooled0.1M CaCl₂withMgCl₂.,and at last the GFP gene was labeled in Acidovorax citrulli strain A13successfully. The biometrics of the labeled strain showed that although the growthtendency of labeled strain was the same as the wild strain, the former entered thelogarithmic phase two hours earlier than the later. The genetic stability of the labeledstrain was high as its Amp resistance could keep100%and its florescenceconservation rate could reach to98%in the15th generation. And, its pathogenicitywas strong and was not significantly different from the wild strain.Strain A13-PK-GFP was further studied for colonization of watermelonseedlings. The result showed that when seedling roots were inoculated with the strainA13-PK-GFP by irrigation method, with a microscopy it could be found that thelabeled bacteria enriched and absorbed around lateral roots, then the bacteria invadedinto the surface layer of lateral roots and entered into endosexine and vascular bundles,then along the vascular bundles of main roots the bacteria moved to leaf stalks, stemsand leaves, mainly colonized in vascular bundles, junctions between roots and stems、surroundings of stomas. If the cotyledons were inoculated with labeled bacteria by spraying method, it could see that under a microscope the bacteria were found inyoung leaves, cotyledons upper stems and cotyledons. The bacteria concentration incotyledon lower stems was rather low and no labeled bacteria observed in roots. Itseemed that the labeled bacteria transferred upwards. In addition, the study result ofcolonizing dynamics showed the labeled bacteria mainly colonized in roots and leaves.When watermelon seedlings were inoculated with the labeled bacteria by irrigationmethod, the labeled bacteria could be re-isolated from roots in1d after inoculation.The bacteria concentration could reach as many as2.3×10⁵cfu/g in root, thendecreased and kept in10⁵cfu/g. But the labeled bacteria could be re-isolated fromleaves only in3d after inoculation, the concentration could arrive at its highestnumber,4.8×10⁵cfu/g, then it kept stable.