Histological Observation Of Watermelon Root Infected By Fusarium Oxysporum F. Sp. Niveurmand Functional Study Of Pathogenicity-Related Gene Fovel

Fusarium wilt of watermelon caused by Fusarium oxysporum f. sp. niveum is one of the most important diseases of watermelon. In order to further develop and explore the pathogenesis of the Fusarium oxysporum f. sp. niveum, we optimized conditions for protoplast production of the F. oxysporum f. sp. niveum strain FO1022, and the two plasmids respectively carried gus gene and hph gene were used to co-transform F. oxysporum f. sp. niveum FO1022 by PEG-mediated protoplast transformation. The biological characteristics and gus gene expression of the transformants were studied. The result showed that the yield of the protoplast could reach 2.48×108 protoplast/mL when fresh mycelium was digested for 3 h in 10 mg/mL lywallzyme solution with 0.8mol/LNaCl as osmotic stabilizer. The efficiency of co-transformtion of two genes was more than 40%. Detection of gus gene and its activity showed that the gus was transformed into the genome of F. oxysporum f. sp. niveum and can be expressed in the transformed strains. After subcultured on PDA 7 times, the gus activity of all transformants remained stable. By artificial inoculation with the transformant, the difference of the infection in nine watermelon cultivars with different resistance levels was clearly observed by staining.The physiological race type of F. oxysporum f. sp. niveum strain FO1022 was dentified using the root-dip technique, and the green fluorescent protein was transformed into this strain. The histopathological observation was carried out after the inoculation of watermelon root with GFP-FO1022. The result showed that FO1022 was FON race 2. After inoculation in the susceptible cultival’Sumi No.1’for two days, conidia of FO1022-GFP were found to attach the watermelon root epidermis and germinated, the preferential colonization sites on the root surface were the grooves along the junctions of adjacent epidermal cells. On the fifth day after inoculation, massive hyphae and macroconidia were observed in xylem vessels of the watermelon root and massive chlamydospores were observed in the root epidermis. Only a few hyphae were observed in xylem vessels of the watermelon stem.48.6% of the seedlings appeared wilt. On the nineth day after inoculation, all xylem vessels of the watermelon stem were clogged by massive hyphae, and 91.7%of the seedlings appeared wilt. Compared to the infection process in susceptible cultivar, this pathogen infected more slowly in the middle-resistant or resistant cultivar.The fusion PCR was used to get Fovel gene homologous recombination fragment, and this fragment were used to transform Fusarium oxysporum f. sp. niveum strain FO1022 by PEG-mediated protoplast transformation. Through this transformation we obtained Fovel disruption mutant and complemented strains. The morphology, virulence and secondary metabolite production of the mutants and wild type were compared to study the function of Fovel. The results showed that disruption of Fovel significantly reduced aerial hyphae and increased hyphae branching. The conidia of knockout mutants become narrow and production increased significantly. The yield of chlamydospores of mutants significantly reduced. The mutants almost lost the ability to produce the pigment. The virulence of mutants was much lower than the wild strain. The production of secondary metabolites (Fusarium acid, Beauvericin etc.) of mutants was also destroyed. However, the complementation of this mutant with the Fovel gene restored the wild type phenotypes. These results suggested that these differences were due to the lack of Fovel gene rather than the random insertion of fragments.