| 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. |
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