【作者】 王敏；

【导师】 郭世伟；

【作者基本信息】 南京农业大学 ， 植物营养学， 2013， 博士

【摘要】 黄瓜枯萎病是一种真菌性土传病害,严重影响了黄瓜的产量和品质。其病原菌为半知菌亚门镰孢属的尖孢镰刀菌黄瓜专化型(Fusarium oxysporum f. sp. cucumerinum, FOC)。目前,黄瓜枯萎病致病机制的相关研究进展比较缓慢,争议最大的焦点是导管堵塞和毒素作用。导管堵塞学说认为病菌侵染后进入维管束,产生大量的菌丝和菌核,同时植物体会形成胼胝质、侵填体和胶质等,这些物质的形成会妨碍水分的运输,引起植株萎蔫。毒素作用理论认为病原菌毒素能够干扰植株的代谢活动,改变细胞膜透性,使水分直接从受伤害的细胞中扩散出去,破坏植株水分平衡,导致植株萎蔫。本研究通过探讨与黄瓜枯萎病发生相关的生理生化过程,以期从生理水平上阐述黄瓜枯萎病的致病机制,并为黄瓜枯萎病的防控提供理论基础。本研究采用土壤培养方式,研究了病原菌侵染对黄瓜植株水分、光合特性、毒素产生及作用方式等的影响,探讨了黄瓜枯萎病的发病生理机制。在发病机制的理论基础上,采用营养液培养,研究了不同形态氮素营养(铵态氮和硝态氮)对黄瓜枯萎病防控的影响,通过分析病原菌侵染对不同氮素形态下黄瓜植株体内水分关系、物质代谢过程、毒素的响应能力、分泌物组成及转录水平的影响,探讨了氮素营养的抗病机制。主要研究结果如下：1.病原菌侵染后黄瓜植株的蒸腾速率(E)、气孔导度(gs)、叶片含水量及叶片水势均显著降低;而E/gs显著上升,说明病原菌侵染促进了叶片非气孔水分散失。虽然病原菌侵染显著降低了导水率,但植株红墨水吸收结果表明,侵染植株叶片中红墨水的吸收量显著高于健康植株。此外,通过电镜观察侵染植株的叶片细胞膜,及分析叶片H+-ATPase酶活性及叶片电导率,发现侵染植株叶片细胞膜遭到严重破坏,说明尽管发病植株茎秆萎缩,但植株的水分吸收及运输仍能满足叶片的需求;病原菌的侵染导致叶片细胞膜受伤害,从而使水分直接以非气孔形式散失,叶片水分平衡遭到破坏,最终导致植株萎蔫。2.在侵染前期,病原菌的侵染导致叶片ABA含量升高、气孔关闭、叶片温度升高。叶片温度与蒸腾速率之间呈显著负相关关系。然而,植株叶片在光下和暗处呈现不同的响应：在光下,发病植株的叶片温度显著高于健康植株;在暗处,发病植株的叶片温度却显著低于健康植株,说明发病植株中叶片的水分散失不是通过气孔,而是从伤害细胞中扩散出去的。在侵染后期,侵染植株叶片水分平衡遭到严重破坏,叶片组织脱水坏死,从而导致植株叶片温度持续上升。上述结果说明,FOC的侵染能够导致叶片细胞膜伤害,使水分直接从受伤害的细胞中扩散出去,破坏叶片水分平衡,最终导致植株萎蔫。通过监测叶片在光下和暗处的温度响应,红外热像仪不仅可以直接非损伤的监测土传病害的发生过程,同时还能揭示植株体内与水分状况相关的生理过程。3.病原菌毒素(FA)处理后,叶片气孔关闭、叶片温度上升。在光暗交替环境中,FA处理植株在光下的叶片温度显著高于健康植株;在暗处,FA处理植株的叶片温度却显著低于健康植株。此外,FA处理后黄瓜植株叶片细胞膜遭到了严重的破坏。为验证FA处理后水分直接从受伤害的细胞中扩散出去,采用分根实验方法,对黄瓜植株的两侧根系分别供应0ppm和100ppm的FA。分根处理后,暗处叶片低温区域的FA含量及细胞伤害程度均显著高于高温区域。韧皮部烫伤对黄瓜植株FA的吸收以及分布无影响,FA主要通过木质部进行运输。由此可见,FA能够诱导植株叶片细胞膜伤害,使水分以非气孔途径散失,最终导致植株萎蔫。通过红外热像仪监测叶片在光下和暗处的温度变化,可以直接非损伤的监测病菌侵染时植株的萎蔫过程。4.与铵态氮相比,硝态氮营养显著降低了黄瓜植株枯萎病的发病率,并显著促进了植株的生长以及植株生物量的增加。铵态氮植株的光合速率、气孔导度、蒸腾速率、羧化效率及表观量子效率均显著高于硝态氮植株,然而铵态氮植株的叶片温度及水分吸收量均显著低于硝态氮植株。铵态氮植株中可溶性蛋白及可溶性糖含量均显著高于硝态氮植株,从而能够促进病原菌对铵态氮植株的侵染。FOC的侵染显著抑制了铵态氮植株的生长、叶片光合和水分吸收,干扰了植株体内的物质代谢,而病原菌的侵染却对硝态氮植株无显著影响。由此可见,硝态氮植株对黄瓜植株的生长更有益,能够更好的防控枯萎病的发生,因此在黄瓜的栽培中,应当适当增加硝态氮肥的施入而减少铵态氮肥的施用以抑制枯萎病的发生。5.FOC侵染后,硝态氮植株体内病原菌的数量显著低于铵态氮植株。与硝态氮相比,铵态氮植株的根系分泌物显著促进了病原菌孢子的萌发。经HPLC鉴定,黄瓜植株的根系分泌物主要含有草酸和柠檬酸两种有机酸,且铵态氮植株根系分泌物中柠檬酸含量显著高于硝态氮植株。FOC侵染显著促进了铵态氮植株根系分泌物中柠檬酸含量,而对硝态氮植株无影响。在柠檬酸浓度为5μ-50μM时,尖孢镰刀菌的孢子萌发率随柠檬酸浓度的增加而增加。此外,外源添加柠檬酸能显著增加黄瓜植株的病情植株及病原菌数量。由此可见,根系分泌物在硝态氮抗枯萎病的过程中发挥着重要作用;黄瓜植株在铵态氮营养下根系分泌物中柠檬酸含量增加,从而促进病原菌孢子的萌发,导致枯萎病发病率增加。6.FOC侵染后,在铵态氮植株和硝态氮植株体内均能监测到FA;铵态氮植株叶片和茎中的FA含量显著高于硝态氮植株,而根中的FA含量却与硝态氮植株无明显差异。FA处理后铵态氮植株迅速萎蔫,而硝态氮植株无明显萎蔫症状；FA对铵态氮植株叶片缅胞膜的伤害程度高于硝态氮植株。虽然铵态氮植株FA的吸收体积显著低于硝态氮植株,但铵态氮植株和硝态氮植株FA的吸收总量却无显著差异；硝态氮植株FA的平均吸收浓度显著低于铵态氮植株。FA处理后,硝态氮植株根系中FA的含量显著高于铵态氮植株,而叶片和茎中的FA含量却显著低于铵态氮植株。由此可见,FOC侵染后硝态氮植株中FA含量较少;硝态氮植株能够选择性的吸收FA,且吸收的FA主要积累在根中,限制FA向地上部的运输,减少对地上部的伤害；硝态氮植株对FA具有更强的耐毒性。7.通过对FOC侵染后不同形态氮素营养下黄瓜植株根系进行转录组水平分析,表明差异基因生物学过程主要富集在细胞过程、代谢过程、刺激响应和生物调控等中；细胞组分主要富集在细胞、细胞部分、细胞器和细胞膜等中；而分子功能主要富集在催化活性和结合中;差异基因所参与的KEGG通路主要富集在代谢途径、氮素代谢、植物-病原菌互作、次生代谢产物合成、苯丙素生物合成、苯丙氨酸代谢、类黄酮生物合成、黄酮及黄酮醇生物合成这8种通路中。通过比较病原菌侵染后不同形态氮素营养下黄瓜植株根系中与抗病相关基因表达的差异,表明FOC侵染后,硝态氮植株根系中大部分调控CERK1、BAK1、FLS2、Rboh、RPM1、PBS1和LOX的相关基因的表达均显著上调,从而诱导硝态氮植株的防御反应,增强硝态氮植株抗病性。更多还原

【Abstract】 Fusarium oxysporum f. sp. cucumerinum (FOC) is the causal agent of cucumber (Cucumis sativus L.) Fusarium wilt, a soil-borne disease that can cause severe losses in yield and quality of cucumber. Currently, the pathogenic mechanism of Fusarium wilt is attributed to plugging of xylem or systemic toxicity. However, the question of why F. oxysporum-caused wilt occurs remains unanswered. The plugging theory suggests that the vessels of the infected plant are plugged by fungal hyphae, callose, tylose, and gel. These substances can impair water transport which results in water deficiency of the infected plant. However, the toxin theory suggests that toxins produced by fungal pathogens disturb the metabolism of the infected plant, altering membrane permeability, and disturbing the water balance by uncontrolled water loss from injured cells, ultimately inducing wilt in the whole plant. In this current study, the physiological and biochemical processes of Fusarium wilt were investigated in cucumber seedlings to explore candidate mechanisms of Fusarium wilt. The major goal of our research is to provide the theoretical basis for prevention and control of Fusarium wilt in cucumber.As economic and living standards increase, more attention is paid to environmental sustainability and food security. These goals challenge the original model of agricultural production. Finding new agricultural techniques to meet the requirements of the sustainable agricultural development has become the focus in the research of agricultural production. The traditional chemical control measures which were harmful for environment and human health did not conform to sustainable agricultural development. With this background, adequate fertilizer has become the future for implementing sustainable plant protection. Appropriate fertilizer not only supplies the nutrition for plant growth, but also improves plant resistance to disease. As the nitrogen was largely used in commercial cucumber cultivation, increasing the resistance of cucumber to Fusarium wilt by adequate nitrogen supply plays an important role in cucumber cultivar development and production.In this thesis, soil culture experiments were conducted in greenhouse to illustrate the physiological mechanism of cucumber Fusarium wilt. The effects of FOC infection on water status, photosynthesis, toxin production and action of cucumber seedlings were investigated. On this basis, hydroponic experiments supplied with different nitrogen forms were conducted to control cucumber Fusarium wilt. The effects of FOC infection on water relations, substance metabolism, toxin response, root exudates and transcriptome level of cucumber seedlings supplied with different nitrogen forms were investigated to illustrate the resistant mechanism of nitrogen nutrition. The major results are listed as follows.1. The transpiration rate (E) and stomatal conductance (gs), leaf water content and water potential of cucumber seedlings were significantly reduced after FOC infection. However, E/gs ratio of infected plant was higher than that of healthy plants, indicating that non-stomatal water loss was occurring. In red ink absorption experiments, leaves of infected cucumber plants accumulated a higher amount of ink, although stem hydraulic conductance decreased markedly. Moreover, transmission electron microscopy, leaf H+-ATPase activity and electrolyte leakage determination revealed severe injury to leaf cell membranes of infected plants. In conclusion, although the stem of infected plant was wilted, leaf water supply was not restricted in infected plant. Leaf cell membrane injury caused by FOC infection induced uncontrolled water loss from damaged cells and destroyed the water balance in cucumber leaf, ultimately resulting in plant wilt.2. During the early stages of FOC infection, stomata closure was induced by ABA in leaves, resulting in a decreased transpiration rate and increased leaf temperature. A negative correlation between transpiration rate and leaf temperature existed. But leaf temperature exhibited a special pattern due to the dependence of the pathological process on light-dark cycling. Lightly wilted leaves had a higher temperature in light and a lower temperature in dark than healthy leaves. We identified that the water loss from wilted leaves was regulated not by stomata but rather by cell damage caused by pathogen infection. During the late stages of FOC infection, water balance in infected plants became disturbed and dead tissue was dehydrated, so leaf temperature increased again. These data suggest that membrane injury caused by FOC infection induces uncontrolled water loss from damaged cells and an imbalance in leaf water status, and ultimately leads to plant wilting. Combining detection of the temperature response of leaves to light-dark conditions, digital infrared thermography not only permits non-invasive detection and indirect visualization of the development of the soil-borne disease Fusarium wilt, but also demonstrates certain internal metabolic processes correlative with water status.3. During fusaric acid (FA) treatment, we found that the leaf temperature of cucumber plant was increased when stomata closure was induced by FA. Under the alternation of light and dark, FA-treated plant had a higher leaf temperature in the light and a lower temperature in the dark as compared with the untreated plant. Furthermore, leaf cell membrane of cucumber seedling was seriously damaged by FA. To confirm whether the uncontrolled water loss was from damaged leaf cells, as a result of FA treatment, and not from the stomata, an experiment was conducted using a split-root system in which spatially separated cucumber roots were each supplied by FA at0ppm or100ppm. In the split-root system, the low temperature areas of the leaves in the dark had higher FA concentration and more severe membrane injury than the high temperature areas. The FA uptake and distribution in cucumber seedlings were not affected by phloem scalding, demonstrating that FA is primarily transported through the xylem. We concluded that membrane injury caused by FA led to non-stomatal water loss and, ultimately, to wilting. Combining the response of the leaves under the light and dark conditions with the digital infrared thermography permitted noninvasive monitoring and direct visualization of wilting development.4. Nitrate nutrition significantly suppressed disease index of cucumber Fusarium wilt as compared to ammonium nutrition. Plant growth and biomass production were markedly increased in nitrate nutrition. Ammonium grown plant had higher net photosynthesis rate, stomatal conductance, transpiration rate, carboxylation efficiency and apparent quantum yield as compared to nitrate grown plant. However, leaf temperature and water uptake of ammonium grown plant were significantly lower than nitrate grown plant. Furthermore, soluble protein and soluble sugar contents of ammonium grown plant, which was favored by the pathogen, were markedly higher than nitrate grown plant. FOC infection significantly inhibited the plant grown, leaf photosynthesis, water uptake and disturbed the substance metabolism of ammonium grown plant while had no significant effect on nitrate grown plant. In conclusion, nitrate nutrition is favored by cucumber seedling and profitable for cucumber plants to defense FOC infection. We should increase the nitrate fertilizer and decrease ammonium fertilizer input in cucumber seedlings in order to improve the resistance to Fusarium wilt.5. The number of pathogen in nitrate grown plant was significantly lower than ammonium grown plant after FOC inoculation. Root exudates from ammonium grown plant significantly increased spore germination of FOC as compared to nitrate grown plant. Two organic acids, oxalic acid and citric acid, were identified from the root exudates of cucumber seedlings by HPLC. Citric acid in root exudates of ammonium grown plants was significantly higher than nitrate grown plants. FOC infection markedly increased the content of citric acid in ammonium grown plants, while had no effect on nitrate grown plants. Citric acid significantly stimulated the spore germination of FOC in concentrations ranging from5μM to50μM. Moreover, disease index and the number of pathogen were markedly increased in cucumber plants after exogenous application of citric acid. In conclusion, higher concentration of citric acid, which was preferable to FOC spore germination, was accumulated in the root exudates of ammonium grown plant resulted in the significant increase of disease incidence. Root exudate plays an important role in resistance of cucumber seedlings to Fusarium wilt under nitrate nutrition.6. FA could be detected in both ammonium and nitrate grown plants after FOC infection. FA content in leaf and stem of FOC-inoculated ammonium grown plants were significantly higher than nitrate grown plants, while no significant difference was found in roots. Ammonium grown plants were seriously wilted after FA treatment, while no visible wilt symptoms were found in nitrate grown plants after FA treatment. The injury degree of leaf cell membrane in ammonium grown plant was significantly higher than nitrate grown plant after FA treatment. Although the FA uptake volume in ammonium grown plant was significantly lower than nitrate grown plant, there was no significant difference in total uptake amount of FA between ammonium and nitrate grown plant. Nitrate gown plant had lower average uptake concentration as compared to ammonium grown plant. FA concentrations in root of nitrate grown plant was markedly higher than ammonium grown plant, whereas the FA concentrations in both leaf and stem were significantly lower than ammonium grown plant. In conclusion, nitrate grown plant produced less fungal toxin of FA in cucumber plants after FOC infection. Nitrate grown plant not only selectively absorbed FA in root, but also decreased FA transportation to shoot, consequently alleviating the toxic effect of FA on shoot. Nitrate grown plant was more resistance to FA.7. The effects of FOC infection on the transcriptome of cucumber roots under different nitrogen nutrition were investigated. The results showed that the biological process ontology was mainly enriched in the GO (Gene Ontology) terms of cellular process, metabolic process, response to stimulus and biological regulation. The cellular component was mainly enriched in the GO terms of cell, cell part, organelle and membrane. The molecular function was mainly enriched in the GO terms of catalytic activity and binding. The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway was mainly enriched in the metabolic pathways, nitrogen metabolism, plant-pathogen interaction, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, phenylalanine metabolism, flavonoid biosynthesis, flavone and flavonol biosynthesis. The effects of FOC infection on the expression of pathogen resistant genes in cucumber plants under different nitrogen nutrient were analyzed. It showed that expression of the most of the pathogen resistant genes, such as CERK1, BAK1, FLS2, Rboh, RPM1, PBS1and LOX were significantly increased in nitrate grown plant after FOC infection, which resulted in induction of defense response and increase of disease resistant.更多还原