【作者】 李梅婷；

【导师】 张绍升；

【作者基本信息】 福建农林大学 ， 植物检疫， 2010， 博士

【摘要】 本文对香蕉枯萎病菌(Fusarium oxysporum f.sp. cubense)毒素和细胞壁降解酶(cell wall-degrading enzymes,CWDEs)的致病性进行系统研究,并探讨了枯萎病菌毒素在香蕉(Musa spp.)抗病品种选育和抗病性鉴定中的应用,主要研究结果如下:系统研究了香蕉枯萎病菌毒素和CWDEs的体外诱导技术,建立了诱导香蕉枯萎病菌高产毒素镰刀菌酸(fusaric acid,FA)、纤维素酶(cellulase)和多聚半乳糖醛酸酶(polygalacturonase,PG)的方法。香蕉枯萎病菌4号小种(F. oxysporum f.sp. cubense race 4)FOCAAA315菌株在改良Czapek B培养液中培养第15 d时FA产量最高,达669.2 mg/L。在改良Czapek培养液中添加麸皮诱导香蕉枯萎病菌产生纤维素酶效果明显,而添加果胶则能有效诱导产生PG。β-D-葡萄糖苷酶(BG)和PG具有典型的酶动力学特性。在反应体系底物P-硝基苯-β-D-吡喃葡萄糖苷(pNPG)浓度为4.0 mmol/L,温度为60~65℃,pH5.0条件下反应10 min,β-D-葡萄糖苷酶(BG)活性最高;而以柑橘果胶为底物其浓度达到0.5%,反应体系温度50℃,pH5.0条件下反应20 min,PG活性最高。明确了香蕉枯萎病菌毒素与CWDEs的致病作用。用香蕉枯萎病菌粗毒素和CWDEs接种于香蕉组培苗,引起的病害症状与病菌孢子侵染引起的病害症状相似。香蕉枯萎病菌的混合代谢物接种比单独使用粗毒素接种对香蕉组培苗表现更强的致萎作用,说明香蕉枯萎病(Fusarium wilt of banana)发生是病原菌多种致病物质共同作用的结果。试验结果表明,不同的香蕉品种对病菌致病物质的敏感性存在差异。香蕉枯萎病菌毒素是一类非专化性毒素,对不同抗性的香蕉品种均具有明显的致病作用,不同香蕉品种对毒素的敏感性程度与田间真实的抗病性无显著相关。香蕉枯萎病菌CWDEs对香蕉假茎组织具有降解破坏作用,其中PG对香蕉假茎组织的降解能力与香蕉的抗病性有关。对于抗病的香蕉品种,PG降解其假茎的能力较弱,降解后释放出还原糖的量较低;PG降解感病香蕉品种假茎的能力较强,还原糖的释放量较高。利用病菌孢子悬浮液在香蕉苗期接种,可能作为香蕉品种抗病性鉴定和抗病品种筛选的一种便捷的方法。采用枯萎病菌孢子在香蕉苗期进行接种,不同香蕉品种苗期的抗感性与田间抗感性一致。通过利用病菌人工接种和组织病理观察发现,病原菌在抗病品种导管中扩展距离显著小于感病品种,表明其抗病性为抗病原菌的扩展。构建了利用香蕉枯萎病菌毒素诱变和筛选抗枯萎病香蕉突变体的技术体系。试验结果证明,以巴西蕉(Musa AAA group Brazil)为材料,香蕉枯萎病菌毒素为选择压,通过多重诱变筛选,巴西蕉不定芽的耐毒性逐步提高并从中获得耐毒素诱变株。耐毒素诱变株经苗期人工接种测定和重病田种植试验均表现出良好的抗病性。通过3年的田间种植试验和示范,获得适应性好,抗病性强的香蕉品种。引进品种中,抗枯1号(Musa AAA group Kangku No.1)、抗枯5号(Musa AAA group Kanku No.5)和广粉2号(Musa AAA group Guangfen No.2)表现出良好的抗性、优良的农艺性状和果品质量,有望成为本地大面积推广种植的高产优质抗枯萎病香蕉品种。采用RAPD技术对12个供试香蕉品种基因组DNA的遗传多样性进行分析,依据基因型和地理来源可将12个香蕉品种分为3个主要类群,6个亚群。对8个供试香蕉品种的rDNA ITS全序列(含5.8S)进行PCR扩增,均得到分子量大小在650 bp左右的扩增产物。将ITS序列进行多重比较表明,供试香蕉品种的ITS序列全长为633~642 bp;经比较分析发现,品种间的遗传变异与ITS的GC含量相关,GC含量低则遗传变异大。RAPD引物扩增产物多态性分析结果表明,巴西蕉抗病诱变株与母本巴西蕉的相似系数仅为0.755,基于ITS序列构建的进化树与RAPD聚类结果基本一致,诱变株与抗枯1号和抗枯5号的同源性达到92%以上,与母本巴西蕉亲缘关系较远,说明枯萎病菌毒素诱变会引起巴西蕉基因组DNA的变异,使诱变株表现出抗病性。从RAPD引物扩增图谱和ITS序列中发现抗病品种存在某些特征性差异条带和特异性位点,与其他供试品种有明显区别。本研究结果为香蕉品种的遗传多样性分析和遗传变异研究提供依据,为进一步开发香蕉种质和抗病性鉴定的分子标记奠定基础。更多还原

【Abstract】 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.更多还原