The Screening Of Microsclerotia Development And Pathogenicity Defective Mutants And Analysis Of The Flanking Sequence Of Verticillium Dahliae Instertional Site

The Verticillium Wilt of cotton caused by V.dahliae is a soil-borned vascular disease and has been one of the most devastating diseases in the cotton production.It’s difficult to control this disease for the lack of high-resistant cotton material sources and effective chemical fungicides on V.dahliae Microsclerotia are the main dormant structure of V.dahliae and primary inoculum in the soil,Currently,fewer studies of the pathogenicity and microsclerotia development mechanism of V.dahliae were reported.The pathogenicity-related genes and microsclerotia development related genes isolated and identified is helpful to reveal the mechanism of pathogenicity and microsclerotia formation about V.dahliae,which may provide the theoretical basis for controlling of cotton Verticillium wilt.Previously a T-DNA insertion mutant library of V.dahliae had been constructed in our laboratory.In this study,by the mutant phenotype and pathogenicity identification,we have obtained several mutants with defects about decreased microsclerotia formation,colony growth,sporulation and pathogenicity.The flanking sequence of T-DNA insertion defective mutants with microsclerotia formation and pathogenicity were obtained by high-efficiency TAIL-PCR technology.The main results are as follows:1.The colony morphology and microsclerotia formation of mutants were observed,and 5 mutants with more dense aerial mycelium didn’t not produce microsclerotia on the PDA,and 15 mutants showed significantly fewer microsclerotia.By testing colony growth rate of mutants,the growth rate of 7 mutants significantly reduced,while the growth rate of 7 mutants significently increased.2.Sporulation quantity was tested on PDA,and the sporulation of 62 mutants reduced significantly,accounting for 21.60%,while the sporulation of 7 mutants increased significantly,accounting for 2.44%.The sporulation of 5 hyphal type mutants and mutant V273-2 Further using of Czapek liquid medium for sporulation quantity tests showed that The 5 hyphal type mutants and mutant V273-2 were significantly reduced sporulation,which were confirm using of Czapek liquid medium for sporulation quantity tests,but the mutant V681-1 had no significant difference with wild type.3.The pathogenicity of V.dahliae mutants were tested by dipping cotton seedling root in spore suspension method.Two hyphal type mutants were no significant different with wild type in pathogenicity.Fourteen pathogenicity defective mutants were obtained,which including 3 hyphal mutants and 3 microsclerotia formation reduced mutants.4.The high-efficiency TAIL-PCR method was performed to amplify the flanking sequence of mutants,which 3 T-DNA insertional mutants with weakened pathogenicity and 5 hyphal type mutants.The flanking sequence were cloned,sequenced and alignmened.At last the T-DNA integration sites in V.dahliae genome of 4 mutants were identified.In mutant V229-2-1,the flanking sequence was 343 bp,T-DNA insertion site located in coding region of VDAG₀8333.1,was hypothetical protein gene.The mutant was hyphal type and no microsclerotia,the colony growth rate increased compared with wild type,sporulation quantity was reduced significantly,the pathogenicity was comparable to the wild type.In mutant V409-2,the flanking sequence was 422 bp,T-DNA inserttion site located in coding region of VDAG-06398.1,which was hypothetical protein gene.The mutant was ahyphal type and no microsclerotia,the colony growth rate was comparable to the wild type,sporulation quantity nd the pathogenicity were reduced significantly compared to the wild type.In mutant V430-2-1,the flanking sequence was 137 bp,the T-DNA insertion site located in the gene VDAG-01383.1（histone acetyltransferase type B subunit 2）downstream 54 bp,and VDAG-01384.1（5’-3’ exoribonuclease）479 bp upstream;the mutant was hyphal type,producted no microsclerotia,sporulation quantity and the pathogenicity significantly reduced were compared with the wild type.In mutant V424-2,the flanking sequence was 117 bp,the T-DNA insertion site located in the gene VDAG-06813.1（two-component syetem protein A）downstream 1574 bp,and VDAG 06814.1（hypothetical protein gene）upstream 1729 bp;the mutant was hyphal type,sporulation quantity significantly reduced,the pathogenicity reduced was weakened compared with the wild type.