【作者】 马波；

【导师】 张俊华；

【作者基本信息】 东北农业大学 ， 作物栽培学与耕作学， 2020， 博士

【摘要】 水稻立枯病是一种主要由尖孢镰孢菌（Fusarium oxysporum）等真菌引起的土传病害,也是水稻旱育秧苗的主要病害,发病后会造成秧苗根部腐烂,导致营养吸收受阻,心叶枯黄,致使秧苗成片干枯死亡,给水稻生产带来极大危害。目前生产中主要采用杀菌剂防治水稻立枯病,但杀菌剂防治存在药效持续期短、易产生抗药性、污染环境等众多问题。植物病害防治的另一种重要途径是利用植物免疫诱导剂诱导植物自身产生抗病性,因其具有持效期长、作用广谱、不产生抗药性,以及使用浓度低、对人体无害、不污染环境等众多优点,近年来在植物病害的防治上得到了广泛应用,现已成为植物病害防治研究的热点。然而有关利用诱导剂防治水稻立枯病的系统研究鲜有报道,其诱导水稻抗立枯病的生理及分子机制尚不清楚。因此,开展植物免疫诱导剂诱导水稻抗立枯病的全面系统研究,可以为植物免疫诱导剂防治水稻立枯病的大面积应用和丰富水稻抗立枯病生理及分子基础提供技术支持和理论依据,对建立水稻病害绿色防控及农药减施技术体系具有重要意义。本试验对364份水稻品种资源进行立枯病抗性评价,从中选择6个不同抗性类型的品种对黑龙江省采集分离得到的129个尖孢镰孢菌（F.oxysporum）菌株进行了致病力测定,明确364个品种的抗病类型以及F.oxysporum菌株间的致病力差异;以高感立枯病品种齐粳2和强致病力菌株QA09为供试材料,采用三因素的正交设计L₈（4×2²）优化诱导方案,评价12种诱导剂对水稻立枯病的诱导抗病效果;分析了F.oxysporum胁迫下氟唑活化酯（FBT）和壳寡糖（COS）分别对水稻秧苗干物质积累、根系形态、细胞膜透性、抗氧化系统及防御系统关键酶活性的影响,从形态和生理角度阐述了其对水稻立枯病的诱导抗病作用;并进行转录组学和蛋白质组学分析,分别从转录和翻译水平揭示其可能的诱导抗病分子机制。主要研究结果如下:（1）供试品种立枯病抗性存在显著差异（P<0.05）,2年的病情指数分别在4.2～78.8之间和5.6～81.3之间。364份供试品种中,2年抗病类型一致的品种共有220份,其中高抗立枯病品种仅有7份;中抗立枯病品种有26份;中感立枯病品种有59份;感病级品种有101份;高感立枯病品种有27份。高抗和中抗品种共占9.06%,而感病和高感品种共占35.17%,说明感病品种明显多于抗病品种。（2）从黑龙江省13个水稻产区采集的病样中共分离得到312个立枯病菌株,根据F.oxysporum的形态学特征及EF-1α基因序列分析,共鉴定得到F.oxysporum菌株129个。以鉴选的高抗、中感、高感6个品种作为鉴别品种测定F.oxysporum菌株致病力,结果表明,菌株间致病力存在明显差异,病情指数差异幅度在13.4～43.1之间。通过聚类分析将129个菌株划分为4个致病类型群,其中Ⅰ型菌群为强致病类型群,平均病情指数41.5,占总菌株的9.3%。Ⅱ型菌群为较强致病类型群,平均病情指数33.5,占总菌株的28.7%。Ⅲ型菌群为较弱致病类型群,平均病情指数17.8,占总菌株的27.9%。Ⅳ型菌群为中等致病类型群,平均病情指数25.3,占总菌株的34.1%,是菌株数量最多的类群。（3）12种诱导剂在不同浓度下对F.oxysporum均无明显的杀菌作用。通过不同诱导浓度、诱导次数、诱导方式三因素的正交试验,优化了诱导方案。在此方案下,12种诱导剂对水稻立枯病的诱导抗病效果存在显著差异（P<0.05）,FBT和COS防效最高,分别达到82.2%和80.3%,2种诱导剂之间差异不显著,但都显著高于其它10种诱导剂（P<0.05）。（4）未进行诱导处理的水稻植株接种F.oxysporum后,秧苗根系生长受到明显抑制,地上部和根部干物质增长缓慢,根冠比下降;而FBT处理和COS处理的水稻秧苗接种F.oxysporum后均能够缓解根系生长受到的抑制,增加了秧苗根长、根表面积及根体积,促进了地上部和根部干物质积累;同时提高了根冠比,平衡了地上部与地下部的物质积累与分配,促进秧苗健康生长。（5）FBT处理和COS处理均降低了F.oxysporum胁迫下秧苗根系中MDA含量和相对电导率,减轻了细胞膜受到的损伤。FBT处理和COS处理均可以提高F.oxysporum胁迫下秧苗根系POD、SOD、CAT、PPO和PAL的活性,增强水稻根系抗氧化能力和防御能力,提高水稻对立枯病的抗性。（6）转录组学分析表明,在F.oxysporum胁迫下,FBT诱导水稻差异表达基因6988个,COS诱导水稻差异表达基因6599个;KEGG通路富集分析表明,FBT处理的差异基因参与了125条pathways,COS处理的差异基因参与了122条pathways。实时定量PCR验证结果表明转录组测序数据真实可靠。本研究发现,在FBT和COS分别处理的pathways中,有多条共有的通路参与了水稻对立枯病的诱导抗性。FBT和COS都可以提高水杨酸（SA）通路的多个PR-1基因表达,激活SA信号转导途径,诱导水稻产生系统获得抗性（SAR）,抵御F.oxysporum的侵染。FBT处理和COS处理中AUX1、SAUR、GH3和BRI1基因均上调表达,从而激活生长素（auxins）和油菜素内酯（BR）信号转导通路发挥抗病性。此外,FBT和COS均可以提高角质ω-羟化酶、脂肪酸-羟基脱氢酶和脂肪醇形成酰基辅酶A还原酶编码基因的表达水平,促进角质、栓质和蜡质的生物合成来增强水稻对立枯病的抗性。（7）蛋白质组学分析表明,在F.oxysporum胁迫下,FBT处理鉴定到922个差异蛋白,其中150个差异蛋白富集到9个KEGG通路中;COS处理鉴定到1323个差异蛋白,其中187个差异蛋白富集到14个KEGG通路中。实时定量PCR验证结果表明蛋白质组测序数据真实可靠。本研究发现,有多个FBT处理和COS处理共有的差异蛋白参与了水稻对立枯病的诱导抗性。FBT处理和COS处理中2个反向-柯巴基焦磷酸(CPS_ent)合成酶（Os CPS1ent和Os CPS2ent）均上调表达,都能够促进水稻合成柯巴基焦磷酸进而合成植物抗菌素抵抗F.oxysporum侵染。同时,FBT和COS均可以提高稻内酯合成酶的表达水平,促进稻内酯的生物合成,通过大量积累稻内酯来提高水稻对立枯病的抗性。更多还原

【Abstract】 Rice seedling blight is a soil-borne disease mainly caused by fungi such as Fusarium oxysporum,and it is also the main disease of dry rice seedlings.After the onset of disease,the root of rice seedling will rot,resulting in obstruction of nutrient absorption and yellowing of the heart leaves,causing the seedlings to dry up and die,bringing g reat harm to rice production.At present,fungicide is mainly used to control rice seedling blight in production,but there are many problems such as short duration of drug effect,easy to develop drug resistance and environmental pollution.Another important way of plant diseases control is to induce plant disease resistance by plant immune inducers.It has been widely used in the prevention and control of plant diseases in recent years due to its many advantages such as long duration,broad-spectrum effect,no drug resistance,low concentration,no harm to human body,and no pollution to the environment.It has become a hot topic in plant disease control research.However,systematic studies on the use of inducers to control rice seedling blight have been seldom reported,and the physiological and molecular mechanisms of inducing resistance to rice seedling blight are still unknown.Therefore,a comprehensive and systematic study on rice resistance to seedling blight induced by immune inducers can provide technical support and theoretical basis for the large-scale application of immune inducers to control rice seedling blight and enriching the physiological and molecular basis of resistance to rice seedling blight.It is of great significance to establ ish the technical system of green control and pesticide reduction of rice disease.In this study,364 rice varieties were evaluated for resistance to rice seedling blight,and 6 varieties with different resistance types were selected to determine the patho genicity differentiation of 129 F.oxysporum strains which were collected and isolated in Heilongjiang Province.The disease resistance types of 364 varieties and the pathogenicity differences among F.oxysporum strains were identified in our study.The high-sensitivity seedling blight variety Qijing 2 and the highly pathogenic strain QA09 were used as experimental materials,the three-factor orthogonal design was adopted to optimize the induction scheme and evaluates the induction control effect of 12 kinds of inducers on rice seedling blight.The effects of fluoro-substituted benzothiadiazole derivatives(FBT)and chitosan oligosaccharide(COS)on dry matter accumulation,root morphology,membrane permeability,key enzyme activity of antioxidant system and d efense system in rice seedlings under F.oxysporum stress were analyzed.The induced resistance to rice seedling blight were elucidated from morphological and physiological perspectives.Transcriptome and proteomic analyses were used to reveal the possible molecular mechanisms of inducing disease resistance at the transcriptional and translational levels respectively.The main research results were as follows:(1)There were significant differences in the resistance of the tested varieties to rice seedling blight(P<0.05),and the two-year disease index was between 4.2～78.8 and 5.6～81.3 respectively.Among the 364 tested varieties,there were 220 varieties with the same type of disease resistance in 2 years,among which only 7 varieties were high resistant varieties,26 varieties were moderate resistant varieties,59 varieties were moderate susceptible varieties,and there are 101 susceptible varieties and 27 high susceptible varieties.The high and moderate resistant varieties accounted for 9.06%,while the susceptible and high susceptible varieties 35.17%,indicating that the susceptible varieties were significantly more than the resistant varieties.(2)A total of 312 seedling blight strains were isolated from disease samples collected from 13 rice producing areas in Heilongjiang Province.According to the morphological characteristics of F.oxysporum and EF-1α gene sequence analysis,a total of 129 F.oxysporum strains were isolated and identified.The pathogenicity of F.oxysporum strains was determined by selecting 6 varieties of high-resistance,medium-sensitivity and high-sensitivity varieties.The results showed that there were significant differences in pathogenicity between the strains(P<0.05),and the difference of disease index ranged from 13.4 to 43.1.Cluster analysis was used to divide 129 strains into 4 pathogenic type groups,among which type Ⅰ F.oxysporum group was a strong pathogenic type group with an average disease index of 41.5,accounting for 9.3% of the total strains.Type Ⅱ F.oxysporum group was a pathogenic type group with an average disease index of 33.5,accounting for 28.7% of the total strains.Type Ⅲ F.oxysporum group was a weak pathogenic type group,with an average disease index of 17.8,accounting for 27.9% of the total str ains.Type Ⅳ F.oxysporum group was a medium pathogenic type group with an average disease index of 25.3,accounting for 34.1% of the total strains,which was the group with the largest number of strains.(3)There was no significant bactericidal effect of 12 inducers on F.oxysporum at different concentrations.The induction scheme was optimized by orthogonal experiment of three factors: different induction concentration,induction frequency and induction mode.Under this scheme,there was a significant difference in the control effect of 12 inducers against rice seedling blight(P<0.05),FBT and COS had the highest control effect(82.2% and 80.3%),which was significantly higher than the other 10 inducers.(4)After inoculation with F.oxysporum,rice plants without FBT and COS treatment showed obvious inhibition of seedling root growth,slow growth of overground and root dry matter,and decreased root-shoot ratio.For rice seedlings,FBT and COS alleviated the inhibition of root growth,significantly increased root length,root surface area and root volume,and promoted the accumulation of dry matter in overground and root.At the same time,the root-shoot ratio was improved,and the accumulation and distribution of matter in overground and underground parts were balanced to promote the healthy growth of seedlings.(5)FBT and COS treatments significantly reduced MDA content and relative conductivity of seedling roots under F.oxysporum stress,and alleviated the damage of cell membrane.FBT and COS treatments could significantly improve the activity of POD,SOD,CAT,PPO and PAL in seedling roots under F.oxysporum stress,enhance the antioxidant ability and defense ability of rice roots,and improve the resistance of rice to seedling blight.(6)Transcriptome analysis showed that 6,988 differentially expressed rice genes were induced by FBT and 6,599 differentially expressed rice genes were induced by COS.KEGG pathway enrichment analysis showed that the differential genes treated by FBT and COS participated in 125 and 122 pathways,respectively.Real-time quantitative PCR verification results showed that transcriptome sequencing data were true and reliable.In this study,it was found that a number of pathways with the same function of FBT and COS were involved in inducing resistance to rice seedling blight resistance.Both FBT and COS treatments could improve the expression of multiple PR-1 genes in the salicylic acid(SA)pathway,activate the SA signal transduction pathway,induce the production of system acquire resistance(SAR)in rice to resist the infection of F.oxysporum.The AUX1,SAUR,GH3 and BRI1 genes were up-regulated in FBT and COS treatment groups,so as to activate auxins and brassinolide(BR)signal transduction pathway to play the role of disease resistance.In addition,both FBT and COS treatments could increase the expression level of genes encoding CYP86A4,fatty acid omega-hydroxy dehydrogenase and alcohol-forming fatty acyl-Co A reductase,thereby promoting the biosynthesis of cutin,suberine and wax to enhance the resistance of rice to seedling blight.(7)Proteomic analysis showed that 922 differentially expressed proteins were identified by FBT treatment,among which 150 differentially expressed proteins were enriched into 9 KEGG pathways.And 1323 differentially expressed proteins were identified by C OS treatment,among which 187 differentially expressed proteins were enriched into 14 KEGG pathways.The real-time quantitative PCR verification results showed that the proteome sequencing data were true and reliable.In this study,it was found that a number of proteins common to FBT and COS were involved in inducing resistance to rice seedling blight.In the FBT and COS treatment groups,two ent-copalyl diphosphate(CPSent)synthase(Os CPS1 ent and Os CPS2ent)were significantly up-regulated,which promoted synthesis of ent-copalyl diphosphate in rice to synthesize plant antibiotics against F.oxysporum infection.At the same time,both FBT and COS could improve the expression level of momilactone synthase,promote the biosynthesis of momilactone,and enhanc e the resistance of rice to seedling blight by accumulating a large amount of momilactone.更多还原