VDHl And GPCR Function In The Development Of Verticillium Dahliae Microsclerotium

Verticillium dahliae Kleb. is a widely distributed soilborne pathogen causing vascularwilt diseases on more than660plant species in38families. It causes serious damage to cotton,eggplant, sunflower, and so on. Microsclerotium produced by V. dahliae is the main primarilyinoculum of Verticillium wilt disease. It can survive in soil for more than20years. Up to now,few reports were found on microsclerotium germination-related genes. In order to clarify themolecular mechanism and indentify genes related with microsclerotium germination, geneknockout system of V. dahliae was developed and optimized. VDH1gene related withmicrosclerotium development and GPCR gene related with microsclerotium germination wereselected as candidate genes to verify the system. The main results of this research wereshowed in following:1. Gene knockout vector pOSCAR-SVDH1and pOSCAR-SGPCR were achieved usingfusion PCR method. At the same time, pOSCAR-VDH1and pOSCAR-GPCR were alsoachieved through one step method. One-step method was simpler and more efficient thanfusion PCR method. It would be an ideal method for gene knockout system of V. dahliae.2. One VDH1transformant and two GPCR transformants were achieved. Fourty GPCRtransformants and nineteen VDH1were achieved by Agrobacterium-mediated T-DNAinsertion in V. dahliae XJ2008for VDH1and GPCR gene, respectively. The transformantswere screened through hygromycin resistance and PCR detection. Finally, one VDH1deletionmutant (vdh1), and two GPCR deletion mutants (gpcr1, gpcr2) were achieved.3. The gene funtion of VDH1and GPCR of V. dahliae were evaluated through myceliumgrowth, production and germination of microsclerotium, and pathogenicity. VDH1gene wasrelated with mycelium growth and microsclerotium formation of V. dahliae. However, GPCRgene was only involved in mycelium growth, no relationship with microsclerotium formationand germination.