| Smoke-tree wilt disease is one of the most destructive vascular diseases, seriously affecting the beautiful red-leaf scenery on Beijing Fragment Hills, and its pathogenic fungus is Verticillium dahliae Kleb. Since the 1990s, a lot of research has been done on its occurrence, causes and prevention measures. Along with the increasing serious situation, especially in 2005, it caused a large area of smoke-trees’death on Beijing Fragment Hills. Though various control measures have been adopted, there is still no effective way to control or prevent V. dahliae. The main reason is the existence of microsclerotia and its wild range of hosts. Especially, microsclerotial formation plays a significant role in the infectious cycle and pathogenic cycle in V. dahliae. Microsclerotia is the main source of infection with a strong ability of adaptation to survive in the soil. Microsclerotial formation is proved to be the most difficult reason to prevent it effectively. It is accompanied by a large number of melanin, the relationship between them is not clear yet. It’s also the key and difficult point to prevent and reveal the mechanism. Therefore, in order to study the molecular mechanism of microsclrotial formation in smoke-tree wilt disease, this article aims to find out the function of VdHogl gene, the key component in fungal signal transduction pathway and the regulatory mechanism that how Hogl gene regulates microsclerotial formation, melanin systhesis and osmotic stress. The results will help to reveal the pathogenesis and mechanism of microsclerotial formation and to clarify the MAPK signal transduction pathway in the developmental growth, pathogenic process and stress response. The main results were as follows:(1) VdHogl gene regulated the stress response to high osmolarity conditions:After using Double-jiont method to build Hogl gene knockout vector with the resistance marker hygromycin B, four VdHogl mutants were successfully obtained by PEG-mediated protoplast transformation method. Via the phenotypic observation under various stress factors (NaCl, Sorbitol, hydrogen peroxide, CM-C, CM-N, Congo red, CuSO4 and fungicides carbendazim, thiophanate methyl, fludioxonil), the results showed that the deletion of Hogl gene led to the increase of its sensitivity to high osmotic stress NaCl and sorbitol, to the reduce to fludioxonil, but there was no big difference in other stresses.(2) The VdHogl gene regulated and the process of microsclerotial formation:After macroscopic observation, in 4 days wild type strain formed MS on BM while a few was seen on the mutant’s; microscopic observation results showed that the amount of MS on mutant was less than that on wild type in 5 days. It indicated that the deletion of Hogl genes led to the delay of the microsclerotial production and less in the amount.(3) VdHogl was involved in regulating the genes involved in DHN melanin synthesis:Using RT-qPCR to analyze the relative expression of five genes involved in DHN melanin pathway between wild type and Hogl mutant, the results indicated that relative expression of M2, M3, M4, and M5 genes in Hogl mutants respectively was significantly induced compared with wild type, demonstrating that VdHogl regulated four other genes except M1 related with the melanin pathway which positively connected with microsclerotial formation.(4) Silenced mutants of melanin genes were obtained by gene silencing and phenotypic analysis was done:After gene silencing method of smoke-tree wilt disease was established, the double promoter gene silencing carrier and transformation were done to get GFP silenced mutants to prove the gene silencing method was feasible. Then using the same method to obtain silenced mutants of five Melanin genes, to extract their RNA and analyze their relative expression indivisually by RT-qPCR, Melanin inhibitors (tricyclazole and carpropamid) were used to do the phenotypic analysis. Phenotypic results indicated that the inhibitory effect of carpropamid was better than that of tricyclazole. |
PDF Full Text Request