| A research on pathogenicity of cell wall-degrading enzymes(CWDEs) and toxin from Fusarium oxysporum f.sp. cubense was carried out systematically in this study. Application of toxin to resistant varieties breeding and disease resistance identification was also investigated. The main results were indicated as follows:A technique for effectively inducing toxin and CWDEs in vitro from the causal agent was studied. The results showed that toxin, fusaric acid(FA), production of the strain FOCAAA315 can reach 669.2 milligram per liter after fifteen days'fermentation in modified Czapek Dox-B medium. The optimal carbon source in modified Czapek Dox medium for inducing cellulase and polygalacturonase (PG) in vitro was wheat bran and citrus pcetin respectively.The results showed that bothβ-D-glucosidase(BG) and PG produced by the causal agent had typical kenetic characteristics. BG showed the highest activity reacted with pNPG at the concentration of 4.0 mmol/L under the conditions of reaction temperature 60~65℃, buffer pH 5.0 and reaction time 10 minutes. PG showed the highest activity reacted with citrus pectin at the concentration of 0.5% under the conditions of reaction temperature 50℃, buffer pH 5.0 and reaction time 20 minutes.Pathogenicity of toxin and CWDEs from the causal agent to banana plantlets was defined. Pathogenicity tests indicated that disease symptom caused by crude toxin or CWDEs was similar to that of the causal agent. Wilting effect of mixture inoculation was more serious than that of toxin inoculation, so that Fusarium wilt was caused by the interaction of different pathogenic substances from F. oxysporum f.sp. cubense.The tests showed that there were differences in sensitivity among different banana varieties to pathogenic substances from the causal agent. Toxin of the causal agent, which resulted in obvious wilting of different resistant banana variteties, was a kind of non-specific toxin. Sensitivity of different varieties to toxin had no significant correlation to their resistance in the field. It was indicated that CWDEs from the causal agent can degrade pseudostem tissues of different banana varieties, and the degradability of PG was related to resistance. After being treated with crude PG solution, reducing sugar content released by susceptible varieties was higher than resistant ones.Seedling inoculation test with conidial suspension may be the convenient and rapid method for identifying resistance and screening resistant varieties of banana. Resistance and susceptibility of banana to the causal agent in seedling test was consistent with that in field test. Observation based on histopathology indicated that the causal agent can move longer distance in vessels of susceptible varieties than that of resistant ones, so that resistance of banana was due to its prevention of the causal agent from expending.A technology for mutation screening was developed using toxin from F. oxysporum f.sp. cubense in this study. The results proved that the toxic tolerance of adventitious buds of Musa AAA group Brazil increased under toxin stress in multiple mutation screening, then the toxin tolerant mutant was successfully obtained. The mutant had good disease resistance in artifitial seedling test and disease field assessment.Some banana varieties with high adaptability and disease resistance were obtained through field test and demonstration for 3 years. Musa AAA group Kangku No.1, Musa AAA group Kanku No.5 and Musa AAA group Guangfen No.2 showed good disease resistance, agronomic characteristics and fruit quality, therefore, they expected to be planted in large areas in locus.The genetic diversity analysis of 12 banana individuals was conducted using RAPD technology. The cluster result showed that 12 individuals can be divided into 3 groups and 6 subgroups according to genotype and geographic origin.Amplification of the inter transcribed spacer(ITS) produced a 650 bp fragment in all banana individuals used in this study, and the length ranges from 633 to 644 bp obtained by sequencing. The multiple alignment result showed that the GC content of ITS was negatively related to genetic variation. The polymorphism analysis of random amplified polymorphic DNA (RAPD) showed that the similarity coefficient between the toxin tolerant mutant and maternal plant was 0.755. Cluster analysis result of ITS sequences was consistent with that of RAPD. The mutant showed 92% identity compared to Musa AAA group Kangku No.1 and Musa AAA group Kanku No.5, while the mutant and maternal plant in relationship were far with each other. It was indicated that induced mutation screening resulted in genomic DNA variation and disease resistance of the mutant.Some specific bands and sites were found in RAPD electrophoresis maps and ITS sequences. The results offered new foundation for genetic diversity and variation analysis of banana, and provided a basis for searching molecular markers of banana germplasms and resistance identification. |
