| Verticillium wilt caused by Verticillium dahliae kleb is a vascular system disease, and is also the first major disease of cotton. To construct a convenient genetic transformant system, we construct a harboring a T-DNA binary vector which can expression GFP by Agrobacterium tumefaciens-mediated transformation (ATMT), we translate the GFP gene to Verticillium dahliae genome. When transfomants are excitated by blue exciting light, a steady green fluorescence singal can be observed. The difference or similitude on infection processes, colonization, resistant enzyme and resistant gene expression between the two cotton cultivars (resistance and susceptible cultivars) infected by two Verticillium dahliae (D5ss-24 or 1070) transformants which tagged by GFP were studied. The resesrch results of pathogenic mechanism of Verticillium dahliae on cytological level studies, to provides the theoretical basis of molecular and cytological for the control of cotton verticillium wilt.The major results are as follows:1. Constructed fungal expression vector pCL-sGFP, which contained sGFP gene promoted by gpd from Aspergillus nidulans, and selectible marker Hyg gene.54 (1070) transformants and 32 transgenic trnsformants D5ss-24 tagged by green fluorescent protein gene were obtained through Agrobacterium-mediated tranformation. According to colony morphology, growth rate, genetic stability and pathogenicity, we obtained two 1070-2 and D5-32 transformants, which were similar to their respective wild-type 1070 and D5ss-24. These transformants had not only strong fluorescent signal but also good genetic stability, and pathogenicity of two transformants was similar to their wilt-type, so the transformants D5-32 and 1070-2 were used to infect cotton.2. To identify the resistance of seven upland cotton cultivars to one V. dahliae and two V. dahliae fungi for their pathogenicity to one cotton cultivar, we selected two V. dahliae or seven cotton cultivars. Through root dipping inculating method, we identified the correlation between pathogen and the cotton host. The results showed that the seven cotton cultivars showed wilt, chlorosis and growth retardation from ten days to fourteen days after inculation. The pathogenicity of the D5ss-24 is stronger than that of 1070. The lowest disease index and AUDPC (area under the disease progress curve) were observed for Zhongmian41, infected by D5ss-24. The index and AUDPC of Zhongmian41 were 53.33 and 151.67, respectively; and 33.33 and 88.34 infected by 1070. While highest disease index and AUDPC were 98.33 and 324.16 for Sumian22 infected by D5ss-24, and were 81.67 and 272.49 infected by 1070, respectively. On the basis of phenotype, disease index and AUDPC, seven accessions were grouped into resistance, middle resistance and susceptibility. Inculated by 1070, Sumian9, Sumian16 and Sumian22 were susceptible to V. dahliae. Mian2 and Mian3 were middle resistant to V. dahliae. Zhongmian41and Mianl showed resistance to V. dahliae. Inoculated by D5ss-24, Sumian9, Sumian 16 and Sumian 22 were also susceptible, and the degree of the resistant to V. dehliae between ianland Mian2 were middle. However, Mian3 and Zhongmian41 showed resistance to V. dahliae.3. We observed the infection processes between susceptibility or resistance cotton cultivars inoculated by D5-32 or 1070-2 strains. There was no difference in conidium adsorbing on the surface of roots of two cotton cultivars before infection. But we found that the number of conidium on the roots of susceptibility was more than that of resistance cultivars. During infection process, two V. dahliae conidium adsorbing on the surface of roots began to genminate after 12 hours inculation. On Sumian22 infected by D5-32,3 days after inculation, hyphae expanded into cortex,9 days after inoculation, hyphae penetrated into vascular of root base and mass colonization,14 days after inculation, leaves wilt and chlorosis had been observated, some hyphae were found in xylem, at this time, we could separated 208 cfu from 100 mg base stem.1070-2 conidium on the surface of Sumian22 reached to cortex after 4 days inculation,9 days after inoculation, hyphae penetrated into vascular of root and expanded in vascular, meanwhile,141 cfu can be separated from 100 mg base stem. However, the time that two V. dahliae hyphae penetrated into vascular of Zhongmian 41 roots is later than Sumian 22, especially, hyphae was firstly observed into the vascular tissues of the Zhongmian 41 lateral roots, some hyphae could been observated in individual vasculars or a part of vascular region of main roots, but only 87 cfu (D5-32) and 57 (1070) could been separated from 100 mg base stem, while lots of hyphae still could be observated on the root tip. Two V. dehliae hyphae mainly penetrated through root cap, the site germinating lateral root and injury regions of roots. There was no appressorium observed when pathogens infected roost of cotton, but the tip of hyphae showed slightly swollen. Compared with the infection process in two cotton relative cultivars to two V. dehliae, The infection of V.dahliae in cotton roots, may activate signal pathways related to resistance and then induced physiological and biochemical resistance, resulted in accumulation of callose, lignin and gelatinous material, which could inhibit V. dahliae to expand and breed in root, Zhongmian 41 showed resistance to V.dahliae possibilly through this strategy.4. We study the difference of the change of defense enzyme in susceptive and resistive cotton cultivars infected by D5ss-24 or 1070, separately, The content of gossypol in both Zhongmian 41 and Sumian 22 treated with D5sss-24, was increasing significantly, but none difference appeared two cotton cultivars treated by 1070, in the second day after inoclution After inoclution, the content of CAT and H2O2 in susceptive cotton increased obviously than in resistance. Simultaneously, the more the content of CAT increasd, the less the content of H2O2 decreased. POD and PPO in Sumian 22 infected by D5ss-24 displayed a trend firstly decreasing then increasing, but in 10th day after 1070 or D5ss-24 inoculation, only POD in D5ss-24-zhongmian41 had the same trend. PAL increased rapidly in susceptive su22 after inoclution by D5ss-24 or 1070, and none difference was found between resistant Zhongmian 41 infected by two V. dehliae. The content of GLU and CHI in Sumian 22 infected by D5ss-24 or 1070 increased significantly. The increasing of CHI, and Glu were found after the plants were treated by D5ss-24 and 1070, respectively. The content of Lox in susceptive su22 infected by D5ss-24 or 1070, began to decreasing then increased significengtly, but in resistant zhongmian41 treated by V. dehliae appeared decreasing.We evaluated the various defense enzyme activities from initial inoclution, and found the change of defense enzyme in susceptive cotton was more obvious than that in resistant cotton.5. We study the difference of resistant gene expression between susceptive and resistant cotton infected by D5ss-24 or 1070. The level of CHI gene expression evidently increased, especially in the treatment inocluted by D5ss-24. Comparatively, Glu gene expression level of susceptive and resistant cotton cultivars treated by D5ss-24 increased obviously, however, that was higher inoculated by 1070. The hmg and Lox gene expression, in resistant cotton is higher than that in susceptive one. Compared with pod4 gene expression,6h after incoluted by D5ss-24, the pod4 gene of Sumian 22 is 150 times more than that of control (inoculated by water), atfter 12h inoculation, there was significantly increasing of pod4 gene expression in resistant cotton cultivar than that in susceptive cotton cultivars. |
PDF Full Text Request