The Study Of Pathogenicity Differentiation Of Sunflower Yellow Wilt And The Generation Of GFP Labeled Verticillium Dahliae

Sunflower yellow wilt is a systemic vascular disease caused by Verticillium dahliae. It became the most important disease in sunflower planting regions of China now. In this study, we analysis the pathogenicity differentiation with isolates collected from different geographic regions and isolates with different colonial morphology. This will provide a solid theoretical basis for sunflower yellow wilt genetic diversity study and also the resistant breeding in the future. At the same time, we generated GFP labeled V. dahliae isolates via optimized Agrobactium mediated transformation system. We selected19from111GFP labeled positive transformants to test the colonial and conidia morphology, spores production, spores germination, toxin production ability and also the pathogenicity. Our results listed as follows:1.12isolates collected from different sunflower planting regions such as Inner Mongolia, Ningxia, Shanxi province and Hebei province were tested on biological characteristics and pathogenicity. Our data indicated that the colonial morphology of tested strains are different. The conidia morphology of all tested strains is oval and conidiophores morphology is wheel branch. The data of pathogenicity of tested strains showed that the disease index of two isolates are higher than30, both of them showed the fast infection and early yellow symptom; disease index of six isolates are between20and30, the symptom are slow infection and less dead leaves; four isolates,which disease index was less than20, showed the delay infection and rather less infected leaves. The differentiation on pathogenicity among tested isolates do observed in this study.2. The pathogenicity differentiation of five different culture type V. dahliae isolates were studied also. Our results suggested that there do have significance difference on the wilting capability among tested isolates. Also, the strong correlation between the crude toxin concentration and the wilt index was observed. The concentration of toxin of V33(large number of microsclerotia), V21(no-production microsclerotia) and V27(less amount of microsclerotia and more hyphae) are0.36mg/mL,0.34mg/mL and0.23mg/mL respectively, the correspondence wilting index at72hpi (hour post inoculation) are58.33.52.08and37.50respectively. The pathogenicity and toxin production ability showed a strong positive correlation (R2=0.8169, P<0.05), but there was no correlation between pathogenicity and the culture type or the amount of microsclerotia.3. The optimal condition for the PEG-mediated transformation as follows:the optimal enzymes concentration is10mg/mL to15mg/mL; optimal enzyme digestion time is2h; transformation efficiency is1-2transformant/μg plasmids. The transformation efficiency of this method is low and transformants is not stable. The optimal condition for the Agrobacterium mediated transformation as follows:the optimal spores concentration for transformation is106/mL; optimal OD600value of Agrobacterium tumefacines is0.15-0.25; the transformation efficiency is7-13transformants/106spores. So, we used Agrobacterium mediated transformation method to transform sunflower Verticillium dahliae.4. By using Agrobacterium mediated transformation procedure, we transform plasmid containing GFP tag to sunflower Verticillium dahliae, we got111transforments which GFP flurescence can be detected under microscope.19transformants were selected for further study. Compared with control,2transformants have less microsclerotia formation; the conidia and conidiophores morphology of transformants is similar with wild type; no correlation was observed between spores number and the germination rate. The significant difference of toxin production ability among19transformants was detected.5transformants showed the higher toxin production ability than wild type and12transformants have the opposite effects. The pathogenicity among tested transformants showed the significant difference,5transformants showed the stonger pathogenicity and9transformants are less aggressive than the control. The positive correlation between toxin production ability and pathogenicity was observed in all tested transformants.5. In this study, Fusarium oxysporum was isolated from sunflower yellow wilt disease samples. Via Koch’s postulation, we verified that Fusarium oxysporum can infect sunflower and caused the symptom like wilting leave、leaves dropping or fading, the whole plant became dry, and the section of the vascular is brown color. This indicated that the Sunflower fusarium wilt and sunflower yellow wilt may occur on the same plant. We also tested the biological characteristics of Fusarium oxysporum, the results indicated that the colony diameter can reach8.96cm after cultured9d on PDA; optimal temperature for hyphae growth ranges from25℃to30℃; optimal PH value h is6-8. A variety of C-source and N-source can be used by Fusarium oxysporum.