Physiological And Biochemical Responses Of Cucumber Seedlings To Fusarium Wilt And The Regulation Mechanisms Of Ammonium And Nitrate Nutritions

The Fusarium wilt of cucumber(Cucumis sativus L)was a fungal disease caused by Fusarium oxysporum f.sp.cucumerinum(FOC).This disease is widespread and difficult to control,which would result in irreparable loss on the yield and quality of cucumber plants.During the process of infecting the host,FOC would affect the normal physiological activities through vessel blockage or toxin substance.Previous studies have illustrated that FOC could interfere plant metabolism through fusaric acid generation,which induced the disorder of physiological process and plant immunity and eventually caused membrane injury,cell death and leaf wilt.However,it’s still unclear whether the Fusarium leaf wilt was related to the vessel blockage induced water shortage.Meanwhile,during the process of nutrition-regulation of plant disease,we have discovered that N forms(NH4+ and NO3-)have influence on Fusarium wilt of cucumber plants,with significantly improved disease resistance under nitrate condition.However,little researches were focused on the relationship between NH4+/NO3-and plant defense and the inherent mechanisms were still unclear.In our present study,hydroponic experiments were conducted,with the control of water stress simulated by PEG caused xylem blockage.We mainly investigated the water physiological responses,photosynthetic responses,redox balance changes and the injury on chloroplast after pathogen infection,to further explore the physiological and biochemical responses after FOC infection and pathogenic mechanism of Fusarium wilt.In addition,the regulation mechanisms of N4+ and NO3-on cucumber defense were also studies through hydroponics experiments,Through analyze the influence of NH4+ and NO3-on leaf temperature variation,cell damage,defense responses,carbon and nitrogen metabolism changes and photosynthetic responses after FOC infection,we investigated the relationship between nitrogen forms and plant disease resistance,which could provide theoretical basis for the future prevention and control of Fusarium wilt.The main results were listed below:1.Both the FOC infection and water stress would induce the decrease of water loss and hydraulic conductivity,while under water stress the water loss reached a relative stable level at last but FOC infection induced continuously decreased water loss.Through experiments,we discovered that the responses of cucumber leaves to FOC and PEG differed in leaf regions(leaf centre and leaf edge):under water stress,the leaf edge was more seriously injured,with a higher leaf temperature and disrupted leaf water status(leaf water potential was significantly decreased while the osmolytes like proline were significantly accumulated);under FOC-infection,a higher temperature was observed in leaf centre while abscisic acid and proline did not varied with water potential.Fusarium-infected cucumber exhibited swelled chloroplasts and accumulated starch grains in leaf centre,while the shrink chloroplasts were observed under water stress without starch grain.The different physiological responses under FOC infection and water stress demonstrated that the Fusarium wilt of cucumber plants was not caused by vessel blockage.2.Both the FOC infection and water stress would cause leaf membrane injury,reactive oxygen species(ROS)accumulation and redox imbalance of cucumber leaves,while the responses were different.Water stress caused accumulated ROS and improved antioxidant enzymes activities and antioxidant substance contents in leaf edge,while the leaf centre was not influenced;after FOC infection,the redox imbalance was primarily occurred in leaf centre.We further studied the redox variations of cucumber leaves under stresses,through the foundation of integrated redox index(IRI)and oxidative disturbance index(ODI).The significantly positive correlation was discovered between IRI and leaf membrane injury,regardless of leaf regions(leaf centre and edge)and stress types(FOC infection and water stress).Meanwhile,the ODI further verified the spatial difference between FOC infection and water stress.3.NH4+ and NO3-nutrition would significantly influence cucumber resistance to Fusarium wilt.Under NH4+ condition,FOC infection would induce significantly improved leaf temperature,decreased photosynthetic and chlorophyll fluorescence parameters,accumulated ROS as well as the injury on cell membrane and chloroplast;while the FOC induced injury was alleviated under NO3-condition.Under NO3-condition,the defense responses of cucumber leaves to FOC infection was improved,including the improved antioxidant enzymes activities,enhanced nitric oxide signal and the unregulated defense genes expression including PR-1,MAPK1,MYB and MYC,which were significantly decreased under NH4+ condition.The physiological changes and defense responses under FOC infection together demonstrated that NO3-nutrition could improve cucumber resistance to FOC infection.4.NH4+ and NO3-nutrition could influence cucumber leaf photorespiration.Compared with NH4+ nutrition,NO3-supply induced significantly increased photorespiration rate,photorespiration metabolic substances and enhanced plant resistance to FOC infection.We assume that the enhanced plant resistance was related to leaf photorespiration and we further altered the leaf photorespiration rate through changing of light density and spraying of photorespiration inhibitor(Isoniazid,INH).We found that the decreased photorespiration rate would resulted in decreased plant resistance(the increased leaf temperature,decreased photosynthetic capacity and leaf wilt),and the significantly negative correlation was observed between photorespiration rate and membrane injury(malondialdehyde accumulation),which further indicated the importance of photorespiration in plant-disease.Under FOC infection,the significantly positive correlation was observed between photoreception rate and nitrate reductase or catalase activities,indicated that the improved resistance by photorespiration may resulted from the energy consumption of nitrate assimilation and the generation of hydrogen peroxide signal.5.Pathogen infection would induce the alteration of plant carbon and nitrogen metabolism.Our results showed that compared with non-infection condition,FOC infection resulted in significantly increased free amino acids and soluble protein contents in cucumber roots and leaves as well as leaf soluble sugar content.Meanwhile,the carbon and nitrogen metabolic substances of cucumber plants were influenced by NH4+/NO3-ratio.With the increased NH4+ supply in nutrient solution,the increased free amino acids,soluble protein and soluble sugar were increased while the organic acids in tricarboxylic acid cycle were decreased.The increased amino acids and soluble sugar contents could promote pathogen growth while the plant organic acids have antibacterial effect.The altered cucumber carbon and nitrogen composition under NH4+ and NO3-nutrition would influence plant resistance and resulted in the higher leaf temperature under ammonium condition.In conclusion,the alteration of cucumber leaf water,temperature,photosynthetic and redox balance under FOC infection and water stress were differed in leaf regions,the leaf wilt caused by Fusarium infection was not caused by vessel blockage.Compared with NH+4 nutrition,NO3-nutrition could significantly enhance cucumber resistance to Fusarium infection and activate the defense responses;the relationship between NP4+ and NO3-nutrition and cucumber resistance may related to the altered leaf photorespiration rate as well as plant carbon and nitrogen metabolism.