Construction And Characterization Of Insertional Mutant Library In The Vascular Pathogen Of Verticillium Dahliae

Vascular wilt diseases caused by soil-borne pathogens are among the most devastating plant disease worldwide. It is mainly caused by Verticillium dahliae Kleb and is a globally severe disease on more than 400 plant species, including many crops which have economic significance. In China, more than 2 million ha of cotton crop is attacked by V. dahliae which causes about 10-15% yield loss due to such a disease, and is thus emphasized in this thesis. Firstly, we have developed and optimized ATMT system using defoliation strain of V. dahlia which is the causal agents of wilt on cotton. The donor plasmid named pTC-Hyg has been constructed for ATMT of V. dahliae. This plasmid contains the hph gene as a selectable marker in its transfer DNA element ?T-DNA? to transform the conidia of XJ2008. Two Agrobacterium tumefaciens ?AGL1 and EHA105? harboring the donor plasmid and V. dahliae ?XJ2008? were co-cultured under optimized conditions to achieve ATMT. As a result, we revealed that the frequency of transformation surpassed 100 transformants per 106 conidia. One hundred microlitre of fresh spore suspension ?106 spore/mL? was mixed with an equal volume of A. tumefaciens cell suspension ?OD600=0.8? and was spread onto cellophane placed on IM plates amended with 200?M of AS. After Co-Cultivation at 25? for 36h, the cellophane was transferred onto new PDA plates augmented with 200?g/mL of cefotaxime and 50?g/mL of hygromycin B that was a suitable marker for V. dahliae and cultivated for 5-7 days to select transformants. Strain AGL-1 yielded much more transformants. This optimization plays an important role to establish a large mutant library of a defoliating V. dahliae strain XJ2008 from cotton and search pathogenicity related mutant in the future through insertional mutagenesis.The 4890 T-DNA insertion mutants were generated. Two hundred and four transformants were randomly selected for the analysis. And we have illustrated the correlations between growth rate, Sporulation and Pathogenicity in V. dahliae mutants. The results showed that the mutants were single copies that could be stably inherited. Compared with wild-type strain, the mutants could be significantly grouped into three types by differentiation with to that of control strain,34.7% of mutants significantly reduced their colony growth rates among 194 mutants that were randomly selected, while 34.8% of mutants increased their colony growth rate,16.8% of mutants lost their ability to produce microsclerotia,30.0% of mutants decreased their sporulation ability, which meet that there were some mutants that were significantly different from those in the wild-type strain.In regression analysis, A highly significant correlations were found between the growth rate on the fresh PDA medium of each mutant and the disease index recorded at 21 dpi. They all were particularly useful for obtaining a linear relationship and explaining a reasonably high percentage of the variation in colony size indicating that the percentage of the variation in disease symptoms. The greatest amount of the sporulation quantity and percentage of the variation in colony size had a significantly power. Our comparison clearly indicates that the growth rate is the key factor for pathogenicity, especially when sporulation is lower than that of control strain.We then selected a root-dip method for screening each of mutant pathogenic defects and obtained 27.1% of mutants which reduced their pathogenicity. In this study, we have confirmed that the three pathogenic defects of mutant VdAG30, VdAG153 and VdAG151 plays several important roles during the growth rate and pathogenicity of V. dahliae. Sequencing of the TAIL-PCR product revealed the T-DNA element that was inserted into the gene annotation as the G-protein coupled receptor in VdL17 and the genome database numbered VDAG₀4611.1. The flanking sequence of T-DNA was compared with the Verticillium genome by BLAST, and The locus consists of a single 1386 bp and the gene is located on chromosome 8, encoding 462 amino acids, There are three introns size that were 55bp and 57bp,117bp. This may account for the reduction in pathogenicity. However, it is possible that certain plant defense responses. Our findings have increased our understanding of the regulatory mechanism of sporulation and hyphal growth during plant infection, also can lay a foundation for cloning candidate pathogenicity-related genes and development-related genes in V. dahliae in the future.