Agrobacterium Tumefaciens-mediated Transformation Of Fusarium Proliferatum H10and T-DNA Insertionol Mutagenesis Of The Fungus

Apple Replant Disease (ARD), distributed in all over the apple producing areasaround the nationwide, is causing serious economic losses to farmers. It is also a seriousthreat to the sustainable development of apple industry. Our earlier investigation found thatFusarium proliferatum has never been reported as a causing agent of apple replant diseasebefore. Then strain H10was isolated, and showed strong virulence on radicle inoculationtests. In this study, we use F. proliferatum strain of H10as the original T-DNA insertionstrains. Before the establishment of mutant libraries, Agrobacterium-mediatedtransformation system was optimized including spore concentration, Agrobacterium OD600,the culture temperature and cultivation time. The results of hygromycin and PCRidentification show that exogenous T-DNA has successfully integrated into the genome ofthe pathogen. Aboat1109transformants of F. proliferatum strain H10have been obtainedby Agrobacterium tumefaciens-mediated transformation (ATMT), and all the mutants wereisolated and saved. Some mutants were selected randomly and analysed about the colonymorphologies, mycelium and conidium development, and pathogenicity. The followingresults have been achieved:1. Suitable sporulation media for strain H10were selected, including CYM, VBC,ATCC, BPY and TYG.. Results showed that sporulation reached102.33×105/mL96hafter inoculation on BPY medium, and reached306.83×105/mL240h after inoculation.BPY is satisfied for the demand of sporulation.2. H10hygromycin sensitivity test showed that hyphae and spores can not grow in themedium containing100μg/mL hygromycin, which could be used as screeningconcentration.3. Screening tests found that200μg/mL concentration of cephalosporins couldcompletely inhibit the growth of Agrobacterium.4. Before establishent of genetic transformation system on H10, factors affectingtransformation efficiency were optioized for the efficient transformation: AgrobaceriumOD600:0.3, spore concentration:107/mL, AS concentration:600μmol/mL, temperaturefor culture:26℃and culture time:48h.5. The results of hygromycin and PCR showed that mutants could grow in a medium containing100μg/mL hygromycin, and PCR can amplify the target band. Mutants werefully stable after five rounds successive culture indicating that foreign genes have beeninserted into the genome.6. The mutants randomly selected, were measured morphological, sporulation,pathogenicity, we found that for most mutants the colors changed, sporulation andmorphology changed, pathogenicity also weakened.