【作者】 薛华丽；

【导师】 毕阳；

【作者基本信息】 甘肃农业大学 ， 作物保护， 2015， 博士

【摘要】 干腐病是马铃薯块茎贮藏期间的重要病害,多种镰刀菌与该病有关,其中F.sulphureum,F.sambucinum和F.solani是我国西北地区的主要病原物。干腐病不仅导致块茎贮藏期间的巨大经济损失,而且会在块茎体内积累大量的真菌毒素,其中以单端孢霉烯族类毒素的积累量最大。本文以感病和抗病马铃薯品种(陇薯3号和陇薯6号)为材料,采用超高压液相色谱-三重四级杆质谱(UPLC-MS/MS)技术分析了感病块茎组织中的单端孢霉烯族毒素;研究了马铃薯品种、镰刀菌菌种和贮藏温度对单端孢霉烯族类毒素积累的影响;探讨了硅酸钠、壳聚糖和β-氨基丁酸3种诱抗剂处理对干腐病及单端孢霉烯族毒素的控制效果;通过蛋白组学等方法阐明了T-2毒素在诱导块茎抗干腐病的诱抗机理。结果表明:1.开发的UHPLC-MS/MS方法适合于同时测定F.sulphureum损伤接种块茎中4种单端孢霉烯族毒素(A型:T-2,DAS;B型:3ADON,Fus-X)的定性和定量测定。该法线性回归系数R≥0.9995、回收率113.28-77.97%、精密度RSD≤5.89、检出限(LOD)0.002-0.005μg/g;检出量(LOQ)0.005-0.015μg/g。分析结果表明,上述4种单端孢霉烯族毒素不仅存在于干腐病病部组织中,而且也存在于临近的健康组织。2.感病品种(陇薯3号)中上述4种单端孢霉烯族毒素的浓度显著高于抗病品种(陇薯6号)。F.sulphureum接种会导致块茎中较高Fus-X、3ADON和DAS浓度积累;而F.solani接种的块茎中,T-2毒素的浓度均高于F.sulphureum和F.sambucinum接种。室温较低温贮藏更有利于单端孢霉烯族类毒素在块茎体内的积累。从病部到健部,4种单端孢霉烯族毒素的积累呈逐渐递减的趋势。3.100mMβ-氨基丁酸、0.50%壳聚糖以及100mM硅酸钠处理显著抑制了F.sulphureum损伤接种的马铃薯块茎病斑直径的扩展。同时,随着贮藏时间的延长,干腐病组织中4种单端孢霉烯族毒素的积累水平呈现先增加后降低的单峰型变化趋势,Fus-X和3ADON的最高浓度出现在接种后第21d,而DAS和T-2毒素的最高浓度出现在接种后第14d。此外,β-氨基丁酸、壳聚糖和硅酸钠3种诱抗剂处理均可显著抑制干腐病中4种单端孢霉烯族毒素的扩散。这表明激发子处理可有效降低马铃薯块茎干腐病中单端孢霉烯族毒素的积累。4.iTRAQ的分析结果表明,T-2毒素处理共诱导了86个蛋白发生差异表达。这些蛋白主要涉及代谢、防卫反应、蛋白合成、能量途径、信号转导、转录及其它等七种功能,其中一些与防卫反应相关的蛋白,主要包括活性氧代谢相关蛋白(如:monodehydroascorbate reductase-like,peroxidase 72-like,peroxidase 12-like等)、苯丙烷途径代谢相关蛋白(如cinnamic acid 4-hydroxylase,suberization-associated anionic peroxidase,wound-induced basic protein-like等)和病程相关蛋白(如pathogenesis-related protein STH-2-like,pathogenesis-related protein P2-like precursor等)被鉴定发生不同程度上调表达,该类蛋白可能参与了块茎的抗病反应;同时一些与能量代谢相关蛋白也可能参与了马铃薯块茎抗病反应。综上所述,采用UHPLC-MS/MS可有效分析马铃薯块茎中的4种单端孢霉烯族毒素(A型:T-2,DAS;B型:3ADON,Fus-X)。同时单端孢霉烯族毒素的积累与马铃薯品种、镰刀菌菌种和贮藏温度密切相关;BABA、硅酸钠和壳聚糖具有抑制干腐病中单端孢霉烯族毒素积累的能力;同时T-2毒素诱导马铃薯块茎抗干腐病的机理与活性氧代谢、苯丙烷代谢及能量代谢途径的增强和病程相关蛋白的积累密切相关。更多还原

【Abstract】 Dry rot of potato is one of the most important postharvest diseases of potato tubers. There is a number of Fusarium species that are involved with the disease. Among them, F. sulphureum,F. sambucinum and F. solani play the most predominant role in casuing dry rot of potato in Northwese of China. In addition to the economic losses, they can also produce mycotoxins, trichothecenes are the main detected mycotoxins. In this study, tubers of the susceptible cultivar(cv. Longshu No.3) and resistant cultivar(cv. Longshu No.6) were chosen as materials,with the help of the UPLC-MS/MS, the accumulation and distribution of trichothecenes in the dry rot of potato tubes were analyzed, and the influence of cultivars, Fusarium strains and storage temperature on trichothecenes production was evaluated, and the treatment effects of three elicitors on controlling the trichothecenes penetrating and diffusing were investigated. Then, T-2, as elicitor, was used to treat slices of potato tuber. The mechanism of induced resistance against dry rot of potato was studied with the help of proteomics technology. The Main results were following:1. The method of UHPLC-MS/MS was developed and proved to be suitable for simultaneous determination four trichothecenes of two type-A of T-2 and DAS, and two type-B of 3ADON and Fus-X in potato tubers inoculated with F. sulphureum. The established method was further evaluated by determining the linearity(R≥0.9995), recovery(113.28-77.97%), precision(RSD≤5.89), and sensitivity(LOD, 0.002-0.005 μg/g; LOQ, 0.005-0.015 μg/g. In addition, it was found that T-2, DAS, 3ADON and Fus-X could be predominantly detected in the lesion, and the toxin could also be identified in tubers without any disease symptoms.2. The concentration of trichothecenecs was much higher in susceptible cultivar(cv. Longshu No.3) than in resistant one(cv. Longshu No.6). The susceptible cultivar infected with F. sulphureum had the maximum concentration of Fus-X, 3ADON and DAS. Among the three Fusarium strains, F. solani had the strongest ability to produce T-2 in both susceptible and resistant cultivars. Room temperature storage was more likely to accumulate trichothecenes than low temperature storage. Meanwhile, the trichothecenes were found not only in the lesion, but also in the adjacent asymptomatic tissue. Trichothecenes concentration showed a strong trend of decline with increase in distance from the infection point. 3. The lesion development in potato tubers inoculated with F. sulphureum were significantly supressed after treated with BABA at 100 m M and chitosan at 0.50% for 3d and sodium silicate at 100 m M for 2d. Meanwhile, the accumulation level of trichothecenes showed a single-apex model with incubation time extended, the highest concentrations for Fus-X and 3ADON appeared in the lesion at 21 d after inoculated, and for DAS and T-2 were at 14 d. In addition, three elicitors of BABA, chitosan and sodium silicate markedly prevented Fus-X, 3ADON, DAS and T-2 penetrating and diffusing in intercellular space of tuber tissue. These findings suggested that the elicitors effectively reduced trichothecenes accumulation in inoculated tubers.4. The results of a comparative proteomic analysis using isobaric tags for relative and absolute quantitation(i TRAQ) technology coupled with 2D LC-MS/MS(Two-dimensional liquid chromatography-tandem mass spectrometry) showed that: A total of 86 proteins were identified as significantly up- or down-regulated in response to T-2 induction in slices of tuber. After functional categorization, these proteins were attributed to metabolism, defense response, protein synthesis, energy pathway, transcription, signal transcription and other. Among them, defence response protein including ROS metabolism related proteins(monodehydroascorbate reductase-like, peroxidase 72-like, peroxidase 12-like), phenylpropanoid pathway related proteins(cinnamic acid 4-hydroxylase,suberization-associated anionic peroxidase, wound-induced basic protein-like) and pathogenesis-related protein(pathogenesis-related protein STH-2-like,pathogenesis-related protein P2-like precursor) were up-regulated in response to T-2 treatment, which suggested that they were involved in induced resistance directly or indirectly. Meanwhile, the energy metabolism related proteins were also involved in induce resistance of against dry rot of potato.In conclusion, UHPLC-MS/MS was a rapid, simple and sensitive method for simultaneous determination four trichothecenes of two type-A of T-2 and DAS, and two type-B of 3ADON and Fus-X in potato tubers inoculated with F. sulphureum. The acculmulation of trichothecenes clearly correlated with potato cultivars, Fusarium strains and storage temperature. BABA, chitosan and sodium silicate have the ability of suppressing the accumulation of trichothecenes in inoculated tubers. In addition, the induced resistance of potato tubers in response to T-2 treatment clearly was correlated with the metabolism of reactive oxygen species, phenylpropanoid pathway and energy. These findings not only have an important significance of theoretical guidance for potato tuber storage and protect the consumer from the risk of exposure to these trichothecenes, but also provide theoretical basis for the mechanism of induced resistance against dry rot in potato tubers by T-2 toxin treatment.更多还原