Production Of Verticillium Dahliae Kleb Tagged By Green Fluorescence Protein And Its Infection Process In Cottons

Verticillium wilt that is caused by Verticillium dahliae kleb is a vascular systemic disease and is also the first major disease of cotton, cultivating and planting for resisitance cultivars have been recognized as one of effective strategies to prevent against this disease.While screening and identifying potential germplasm resistance to Verticillium wilt are the basis of breeding resistance cotton cultivars.Wild cottons have excellent resistantance trait, obviously, it is extremely important to mine the potential resistant disease genes in wild cottons for breeding resistant cultivars. The first task is to ascertain if wild cottons are the host of V. Dahliae and identify resistant types, then studying infection processes of pathogen between resistant cotton and susceptible cotton to reveal inherent or induced resistant characteristics through plant pathology methods, which can provide basis for mining potential Verticillium resitance genes and studying pathogenic mechanism of V. dahliae.Eight wild cotton accessions were identified for their resistance to V. dahliae through root dipping.The results showed V. dahliae could infect eight accessions and colonies could be isolated from eight accessions, so wild cottons are the host of V. dahliae. Wild cottons began to wilt, chlorosis and growth retardation from ten days to fourteen days after inculation, while susceptible control Jun mian-1began to chlorosis after eight days inculation. Thirteen days after inculation, the first leaf began to defoliate.Three weeks after inculation, incidence rate was 100%. Resistant control Hai7124 began to wilt and necrosis after eight days inculation. Three weeks after inculation, incidence rate was 72%, but mainly on the cytyledon. Six weeks after inculation, the lowest disease index and AUDPC were Gossypium trilobum, its values were 13.33 and 25.56, respectively. Additonal, none were dead. The highest disease index and AUDPC were observed for G. davidsonii, its values were 88.33 and 256.97, respectively, its mortality rate was 68.89%.While disease index and AUDPC were 91.67 and 301.65 for susceptible control Jun mian-1, mortality rate was 71.11%. Disease index and AUDPC for resistant control Hai7124 were 58.33 and 170.56, respectively, but mortality rate was only 6.67%. Verticillium content analysis of stem showed the number of resistance colony from G. trilobum was 51 cfu/50mg, G. davidsonii was 331 cfu/50mg. While susceptible control Jun mian-1 and resistant control Hai7124 were 871 cfu/50mg and 97 cfu/50mg, respectively. On the basis of phenotype, disease index and AUDPC, eight accessions were grouped into high resistance, resistance and susceptibility. G. davidsonii was susceptible to V. dahliae. G. australe, G. aridum, G. thurber, G. somalense, G. anomalum and G.. longicalyx were resistant to V. dahliae. G. trilobum showed high resistance to V. dahliae.Constructed fungal expression vector pCH-sGFP, which contained sGFP gene promoted by GPD from Aspergillus nidulans, and selectible marker Hyg gene. 208 transformants tagged by Green fluorescent protein were obtained through Agrobacterium-mediated tranformation.According to colony morphology, growth rate, genetic stability and pathogenicity, we obtained transformant Vd-gfp77, which was similar to wild-type Vd991.This transformant had not only strong fluorescent signal but also good genetic stability, and its pathogenicity was similar to Vd991, Vd-gfp77 was used to infect cotton. Infection process of Vd-gfp77 in high resistance G. trilobum and susceptibility G. davidsonii were studied. There were no difference between G. trilobum and G. davidsonii before infection, conidispores randomly adsorbed on the surface of roots. During infection process, conidispores adsorbed on the surface of G. davidsonii began to genminate after 24 hours inculation, 5 days after inculation, hyphae expanded into cortex, 7 days after inculation, hyphae penetrated into vascular of root base, but only existed in some vessels, 14 days after inculation, leaves wilt and chlorosis had been observated, some hyphae were found in xylem, at this time, we could separated 147 cfu from 100mg stem base. Conidispores on the surface of G. trilobum began to genminate after 5 days inculation, hyphae mainly penetrated through root cap and injury regions of root. No appressorium was observed when pathogens infected root of cotton, but the tip of hyphae showed slightly swollen. 7 days after inculation, hyphae expanded into cortex, 10 days after inculation, hyphae penetrated into vascular of root, these hyphae were interwaved togather and expanded in vascular. 14 days after inculation, cotyledons began to chlorosis, but true leaves and vessels had no symptoms. some hyphae could been observated in vasculars, but only 10 cfu could been separated from 100mg stem base, while lots of hyphae still could been observated on the root tip. On the basis of infection process in G. trilobum, phenotype and stem Verticillium content, V. dahliae penetrated G. trilobum, which might activate signal pathways related to resistance and then induced to mainly form physiological and biochemical resistance, resulted in accumulation of callose, lignin and gelatinous material, which could inhibit V. dahliae to expand and breed in root, G. trilobum showed high resistance to V. dahliae through this strategy.