【作者】 薛高峰；

【导师】 梁永超；

【作者基本信息】 中国农业科学院 ， 植物营养学， 2009， 博士

【摘要】 前人对硅在抵御水稻病害中的研究报道多集中在稻瘟病、纹枯病等由真菌引起的病害,而有关硅对由细菌引起的水稻白叶枯病抗性的研究,国内外鲜见报道。本文采用水培试验,研究了在不同方式硅处理条件下,硅对水稻白叶枯病的抗病效果、硅吸收以及对水稻生长的影响;硅对抗氧化系统酶活性及多种与抗病相关的生理生化指标的影响;利用实时荧光定量PCR(Real-time fluorescence quantitative PCR)技术,在水稻与白叶枯病互作过程中,硅对相关防卫基因表达的调控。从生理生化和分子水平上系统而深入地研究了硅抗白叶枯病的机制,主要结论如下:(1)施硅能显著提高水稻对白叶枯病的抗性,减缓白叶枯病菌的危害。施硅处理感病植株病情指数显著降低,与对照相比降低11.83%-52.12%,对白叶枯病的相对防御效果达16.55-75.82%。后期施硅对水稻抗性提高的效果明显高于前期。接种前施硅处理(+Si-Si)的植株失去部分抗性,接种后施硅处理(-Si+Si)的植株与一直加硅处理(Si+)的植株具有较高的抗性;硅作为“机械屏障”作用在水稻抗白叶枯病中有一定作用,但不是主要作用。硅对水稻生长具有促进作用,施硅能明显提高植株硅含量和干物质累积量。接种白叶枯病菌后,植株地下部和地上部干物质均显著降低。施硅处理的水稻叶片组织迅速坏死,从而阻止了病菌的发展,而不施硅处理的水稻叶片表现为明显的失水、青枯、卷曲、萎蔫现象。(2)接种白叶枯病菌后48 h内,施硅处理的水稻植株,叶片中丙二醛(MDA)和过氧化氢(H2O2)含量显著升高;施硅能显著提高感病植株叶片中脂氧合酶(LOX)和超氧化物歧化酶(SOD)活性,降低过氧化氢酶(CAT)、过氧化物酶(POD)和抗坏血酸过氧化物酶(APX)活性,促进过氧化氢(H2O2)在植物体内积累,加强膜脂过氧化作用。因此,硅可通过参与植株体内代谢,调节抗氧化系统酶活性,激发机体过敏反应(HR),诱导植株对白叶枯病的抗性。(3)水稻感染白叶枯病后,施硅能使受白叶枯病菌侵染的水稻叶片中苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)活性以及总可溶性酚和木质素含量显著提高。结果表明,硅能够调控与抗病有关的酚类物质代谢过程,参与植物防卫反应而增强水稻对白叶枯病的抗性。(4)叶片感染白叶枯病菌后,β-1,3-葡聚糖酶活性和几丁质外切酶、内切酶活性均快速上升。β-1,3-葡聚糖酶活性在感染白叶枯病菌的8天内,施硅处理显著高于不施硅处理,接种后第8天达到最大值。整个试验过程中,加硅处理的水稻植株,叶片中几丁质外切酶、内切酶活性明显增加。施硅能提高病程相关蛋白β-1,3-葡聚糖酶和几丁质外切酶及内切酶的活性,从而提高水稻对白叶枯病的抗性。(5)感染白叶枯病菌后,施硅处理能激活Os03g0109600基因的表达,表达量也显著高于不施硅处理,有利于增强该转录因子的活性;在感病后期,施硅抑制转录阻遏物Os03g0126000基因的表达,有助于保持植物正常生理代谢和抗病反应。施硅能调控酚类物质代谢的关键酶PAL基因表达,在感病初期,施硅处理的PAL基因的表达量高于不施硅处理;施硅能诱导Pr1a和Rcht2基因更早更快表达,表达量也显著高于不施硅处理。施硅能显著提高Lox2osPil基因的表达量,同时调控CatA基因的表达,在感病前期能显著抑制CatA基因的表达。结果表明,在白叶枯病菌与水稻互作过程中,硅积极参与对相关防卫基因的诱导和调控,从而产生一系列生理生化抗病机制是硅抗白叶枯病的主要机制。综上所述,硅对水稻生长具有促进作用,施硅能显著提高水稻对由细菌病害引起的白叶枯病的抗性。硅能积极参与生理代谢活动,诱导和调控植物相关防卫基因,产生一系列的防御机制来阻止病原菌的入侵。本研究为防治植物细菌病害提供了既经济高效又安全环保的实用技术,对于发展新型病害综合防治措施,具有十分重要的理论和实际意义。更多还原

【Abstract】 Previous studies on silicon-enhanced disease resistance in rice were mainly focused on rice blast and sheath blight that were caused by fungi. To date, the effect of silicon on bacterial blight (Xoo) has never been reported. A series of hydroponics experiments were performed in the present study in a controlled rice-Xoo pathosystem to examine the silicon-enhanced disease resistance, silicon absorption and rice growth. The effects of silicon on activities of antioxidant defense enzymes in rice leaves in relation to induced resistance were also investigated. The defense-related genes regulated by silicon in rice-Xoo interaction pathosystem were quantified by using real-time fluorescence quantitative PCR technique. The comprehensive and systematic study of mechanisms of silicon-enhanced resistance to bacterial blight in rice was conducted. The main results are presented as follows:(1) Silicon addition could significantly enhance rice resistance to Xoo, thus alleviating damage resulting from infection by Xoo strain. The severity index of the infected plants was decreased by 11.83-52.12% by addition of silicon compared with the control with the relative immunization efficiency of 16.55-75.82%. The plants that were switched from Si+ (with Si added) to Si- (without Si added) nutrient solution lost partial resistance to Xoo, however, rice plants that were switched from Si- to Si+ nutrient solution exhibited the same high resistance as the plants treated continuously with silicon. Silicon played a role as a physical barrier in controlling bacterial blight in rice, although this was not a major mechanism. Application of silicon was beneficial for rice growth, resulting in an increased silicon content and total dry weight. The shoot and root dry weights were significantly decreased by inoculation with Xoo strain. Compared to the control plants, necrosis spots caused by the programmed cell death occurred rapidly in the leaf tissues of Si-fed plants infected by Xoo, thus preventing from further disease development. However, the leaves in rice plants grown in Si- nutrient solution appeared obvious symptoms of desiccation, immature death, curliness and wilt.(2) The contents of malondialdehyde (MDA) and peroxide hydrogen (H2O2) in Si-fed rice plants were significantly increased during the 48-h period after inoculation with Xoo strain. The activities of superoxide dismutase (SOD) and lipoxygenase activity (LOX) were significantly higher, while the activities of catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX) were lower in the Si-fed plants than in the Si-deprived Xoo-inefectd plants, resulting in accumulation of peroxide hydrogen (H2O2) accumulation in Si-fed plants and intensification of membrane lipid peroxidation intensification. It can be concluded that Si triggered hypersensitive reaction (HR) through mediating the activities of antioxidant defense enzymes, therefore inducing rice resistance to bacterial blight.(3) Silicon application significantly increased activities of phenylalanine ammonia-lyase (PAL) and polyphenoloxidase (PPO) , as well as contents of soluble phenolics and lignin in rice leaves infected by Xoo strain. The results demonstrated that silicon actively participated in metabolism of phenolics to accelerate accumulation of antimicrobial compounds, and therefore enhancing the resistance to bacterial blight in rice. (4) The activities ofβ-1,3-glucanase, exochitinase and endochitinase were all quickly increased after inoculation with Xoo strain. Theβ-1,3-glucanase activity was significantly higher in Si-supplied plants than that in Si-deprived plants within the eight days, and reached the maximum on the eighth day after inoculation with Xoo strain. The exochitinase and endochitinase activities in leaves of Si-fed plants significantly increased throughout the experimental period. The results showed that silicon application improved the enzyme activities ofβ-1,3-glucanase, endochitinase and exochitinase, thus enhancing the resistance to bacterial blight in rice.(5) Application of silicon could activate the expression level of the gene Os03g0109600, a transcript factor, after inoculation with Xoo strain. The expression quantity in Si-fed plants was significantly higher than in Si-deprived plants, which was benefical to increasing the gene’s activity, In the later stages of infection, the expression of Os03g0126000 gene was depressed, which was beneficial to maintaining normal physiological metabolisms and disease resistance. Silicon could regulate the expression of PAL gene. In the early stages of infection, its expression quantity was higher in Si-fed plants than in Si-deprived plants. Silicon induced expression of Pr1a and Rcht2 genes earlier and faster, and the expression quantity was also significantly higher in Si-fed plants than in Si-deprived plants. Application of silicon could significantly enhance the expression quantity of Lox2osPil gene and regulate the expression level of CatA gene. In the early stages of infection, silicon significantly depressed the expression of CatA gene. The results showed that silicon actively induced and regulated some defense-related genes in response to bacterial blight attack in rice-Xoo interaction and this was the main mechanism of Si-enhanced resistance to bacterial blight in rice.In conclusion, silicon is beneficial for rice growth. Silicon addition may significantly enhance the resistance to Xoo caused by bacilli. Silicon can actively participate in physiological metabolisms, induce and regulate defense-related genes of plant and induce a series of defense mechanisms to impede the invasion of pathogens. Using Si to control plant bacilli diseases is an economical, highly-effective and environmently-friendly technology. It is theoretically and practically important to develop alternative novel technologies for controlling plant diseases.更多还原