Studies On Mechanisms Of Different Nitrogen Nutrition On Effecting Cucumber Fusarium Wilt Occurance

Cucumber Fusarium wilt,a fungal disease caused by the soil-borne pathogen Fusarium oxysporum f.sp.cucumerinum(FOC),which greatly affects the yield and quality of cucumber.resulting in a huge economic loss.Up to present,the pathogenic mechanism of Fusarium wilt was ascribed to plugging or systemic toxicity.The plugging theory suggested that the vessels of infected plants were plugged by fungal hyphae,callose,tylose and gel.These substances could disturb water transport,and finally caused the water deficiency of infected plants.However,the toxin theory suggested that the phytotoxin produced by pathogens could interfere the water metabolism of the infected plants,alter the membrane permeability and disturb the water balance by uncontrolled water loss from injured cells,finally inducing the wilt of whole plants.Nitrogen,a necessary element for plant growth and development,which influences plant diseases by regulating growth and physiology.affecting pathogen growth and virulence,as well as modifying the rhizosphere environment,playing an important role in the disease development.In the previous studies of our lab,we found that the nitrate nutrition reduced the occurrence of cucumber Fusarium wilt compared with the ammonium nutition in the hydroponic experiments.However,cucumber Fusarium wilt is a soil-borne fungal disease,in this thesis we further study the effect of nitrate nutrition on the cucumber Fusarium wilt with the soil culture.The effects of' different nitrogen forms(NH4+ vs.NO3-)on the cucumber plants growth,FOC population,toxin production,rhizosphere pH and microbial grop were investigated to discuss the resistance mechanism of the NO3--fed plants.Based on the results of pot experiments,to further study the effects of different nitrogen nutrition on the interaction of plant-pathogen,we conducted the hydroponic experiments to study the effects of different nitrogen forms and supply on the cucumber plants growth,photosynthetic characteristics,FOC population,toxin production,chancges of xylem sap composition,toxin response and carbon and nitrogen metabolism.The effects of different nitrogen nutrition on the cucumber Fusarium wilt and the underlying physiological resistance mechanism were investigated,and the main results are listed as follows:1.The results of pot experiments demonstrated that plants supplied with NNO3-were more resistant to cucumber Fusarium wilt.In addition.NO3-significantly increased the cucumber growth.After FOC infection,compared with NH4+-fed plants,the FOC number in roots and stems of NO3--fed plants were reduced about 17%and 30%,respectively,and the FA content in the stems and leaves of NO3--fed plants were decreased about 49%and 76%,respectively.In addition,FOC infection significantly repressed the cucumber growth and increased the leaf cell membrane injury of NH4+-fed plants,but it had no effects on the NO3--fed plants,which suggested that the resistance of NO3--fed plants to disease was related with the FA production,However,NO3-significantly increased the FOC growth and FA production in vitro culture,which predicated that the effect of nitrogen on the disease development was attributed to plant-pathogen-environment interaction.After FOC infection,compared to NO3-treatment,the lower pH and FOC number of rhizosphere soil in the NH4+-fed plants may contribute to the more susceptibility of NH4+-fed plants to the disease.Besides,FOC infection significantly increased the fungi number and decreased the actinomycete number of the rhizosphere soil under NH4+nutrition,which may result in the susceptibility of the NH4+-fed plants.2.The results of hydroponic experiments showed that the disease index of cucumber plants decreased as the increase of NO3-supply but increased with the increased NH4+supply.Compared to different NH4+ treatments and low NO3-treatment,the plant growth was enhanced in the high-NNO3--fed plants.Under non-inoculation conditions,compared with high-NO3--fed plants,the photosynthetic rate,stomatal conductance,intercellular CO2 concentration and transpiration rate in high-NH4+-fed plants were significantly increased by 23%,29%,10%and 18%respectively,accompanied by lower water uptake and leaf temperature.After FOC inoculation,the photosynthetic characteristics and water uptake of plants with low and high NH4+ were significantly inhibited,and the leaf temperature was significantly increased,especially in the high NH4+-fed plants;while no significant difference was found between plants supplied with high NO3-.The content of nitrogen,soluble sugar and protein in roots,stems and leaves of high-NH4+-fed plants were significantly higher than those of high NO3--fed plants.After FOC inoculation,the soluble protein in the high NH4+-fed plants was inhibited to transport to shoot,and the soluble sugar was inhibited to transport to the roots,suggesting that the material metabolism was changed after FOC infection,the NH4+-fed plants might provide much nutrition,which can promote FOC growth and increase disease.In addition,compared with high NH4+ nutrition,the colony diameter,spore number and FA production of FOC under high NO3-significantly increased by 21%,4-fold and 106-fold respectively,which further suggested that the effect of different nitrogen on the disease development was contributed to plant-pathogen interaction.3.Compared to plants with different NH4+,the flow rate of xylem sap in high NO3--fed plants was significantly higher.After FOC inoculation,compared to the high NO3--fed plants,the content of nitrogen,soluble sugar,amino acid,malic acid,citric acid and succinic acid in high NH4+-fed plants were significantly increased by 19-fold,78%.2.3-fold,1.1-fold,15-fold and 5-fold respectively,in addition,the citric acid of xylem sap was positively related with disease index,which suggested that the xylem sap of high NH4+plants might be beneficial to the FOC growth.The results of FOC distribution showed that the FOC number in the NH4+-fed plants was much higher than NO3--fed plants.And the FOC number in the stems of high-NH4+-fed plants was increased by 80%compared with low-NH4+-fed plants,while the FOC number in the leaves of high-NO3--fed plants was reduced by 1.4-fold than that of low-NO3--fed plants.In addition,the FA content in the stems and leaves of NO3--fea plants was significantly reduced compared with NH4+-fed plants.Based on the results that the FOC number in the roots and stems was positively related with the NH4+ content but negatively related with the NO3-content,as well as positive relation between FOC number and FA content or disease index,we induced that lower FA may induce the resistance of high NO3--fed plants to disease.After FOC inoculation,the cell membrane was significantly impaired in the high-NH4+-fed plants,while no changes in the NO3--fed plants,and the cell membrane injury was positively related with FA content,further suggesting that lower FA caused the resistance of high NO3--fed plants to disease.4.After FA treatment,as the N supply increased,the wilt symptoms of plants supplied with NH4+ were obviously aggravated,while those of NO3--fed plants were markedly alleviated.And the water uptake was significantly inhibited,and the leaf temperature and cell membrane damage were significantly increased in the NH4+-fed plants compared with NO3--fed plants,these changes were much obvious as the NH4+ supply increased.Additionally,there was no significant difference in total uptake amount of FA between NH4+-fed plants and NO3--fed plants,while the average FA uptake concentration was much lower in high NO3--fed plants,which implied that high-NO3--fed plants could selectively absorbed FA.Compared with high-NH4+-fed plants,the FA content of high-NO3--fed plants was increased by 58%in the roots and reduced by 37%in the leaves,which suggested that high-NO3--fed plants could accumulate FA in roots and decrease FA transportation to shoot.Thus,the lower FA content in the leaves alleviated the cell membrane injury,suppressing the Fusarium wilt disease.5.Without FOC inoculation,compared to NH4+-fed plants,the content of oxalic acid,malic acid,citric acid,succinic acid,fumaric acid were significantly higher in N03--fed plants,and most amino acid content in the NO3--fed plants were much higher.FOC infection significantly increased the alanine and glutamic acid contents and decreased the threonine,aspartic acid,serine and arginine contents in the leaves of NH4+-fed plants,but decreased the leaf alanine,threonine,lysine and tyrosine contents in NO3--fed plants.Additionally,FOC infection increased the content of all amino acid in the roots.Transcriptome analysis showed that genes related to carbon metabolism were mostly up-regulated in NO3--fed plants,whereas genes related to primary nitrogen metabolism were mostly up-regulated in NH4+-fed plants.The expression of most amino acid-related genes in the roots of both NH4+ and NO3--fed plants significantly increased after FOC infection.In conclusion,organic acid metabolism and related gene expression increased under NO3-nutrition,whereas NH4+ nutrition increased the amino acid level and the expression of related genes;the varied organic acid and amino acid levels resulted in different resistances to FOC infection between different forms of nitrogen.