| Fusarium wilt of banana, also known as Panama disease in foreign countries, is caused by Fusarium oxysporum f. sp. cubense (Foe), a devastating soil-borne fungal pathogen. Among4races of Foe, the race4(Focr4) seriously affected the development of banana industry due to its great destructive ability and wide host range. Both domestic and foreign scientists have contributed significantly to the study of pathology of banana wilt disease. The Focrl and Focr4genomes have been sequenced as a result of our collaborative research with other scientists and the mutant library of Focr4was also established. In addition, some genes related to the pathogenicy have been cloned. However, little is known about the pathogenic mechanism of Foc4. Several insertion mutant strains whose pathogenicity has been lost or seriously weakened were screened from the library. The copy and insertion sites of the inactivated gene were verified and the cDNA sequence was cloned. Thus, the insertion mutant Focr4-1366whose pathogenicity had been seriously weakened was selected for further research. The temperature of in vitro leaf inoculation was optimized, and the wild strain Foc4with enhanced pathogenicity was isolated and identified. The expression of insertion inactivation gene of Focr4was comfirmed by RT-PCR. The vector for knockout and complement was built. The pathogenicity correlation of this gene was identified by knockout and complement. The biological phenotype of the knockout mutantâ–³Focr4B-1366and complement mutantâ–³Focr4B-1366-cp was studied. The research achievements of this thesis include the following:(1) The possible variation in test results due to environment changes was avoided by optimizing the temperature of in vitro leaf inoculation. In turn, this led to the improvement of the stability and reliability of Foc pathogenicity.(2) The wild strain Focr4B with enhanced pathogenicity was obtained by isolation and identification.(3) The knockout and complement vector of Focr4-1366was built in order to carry out gene-knockout and gene-complement of Focr4B. The results showed that the pathogenicity was seriously weakened in the gene-knockout mutant strain AFocr4B-1366but restored in the complement strainâ–³Focr4B-1366-cp. The study revealed that the lost T-DNA gene of F. oxysporum was probably the pathogenicity-related gene. (4) The biological phenotypes of the T-DNA inserted mutant strain Focr4-1366, the gene-knockout mutant strainâ–³Focr4B-1366, the complementary mutant strainâ–³Focr4B-1366-cp and the wild strain Focr4B were studied. The results showed that sporulation and carbon source utilization of Focr4-1366andâ–³Focr4B-1366were lower than those of Focr4B andâ–³Focr4B-1366-cp, whereas there was no obvious difference in the morphology of mycelia and spores, optimal temperature and PH for fungal growth. The spore germination rate ofâ–³Focr4-1366-cp was slightly lower than that of the other three strains:Focr4-1366,â–³Focr4B-1366and Focr4B. The results showed that inserted inactivation gene affected spore production and carbon utilization but not mycelial morphology, spore morphology and spore germination rate.(5) The harmful effect of crude toxin produced by gene-knockout strainâ–³Focr4B-1366, and wild strain Focr4B on banana seedlings was confirmed, and the effect of cell wall degrading enzymes on four strains was determined. The results revealed that etiolation of banana leaves soaked in broth secreted by wild-type strain Focr4B was more serious than that of banana leaves soaked in broth secreted by knockout mutantâ–³Focr4B-1366. Therefore, the pathogenicity-related gene was related to toxin secretion, and this gene was lost in Focr4B. There was no obvious difference on the quantity of protoplast released by the mutant strain, wild strain and complement strain within the same digestion period. So the gene had no effect on the sensitivity to cell wall degrading enzymes. |
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