Studies On Autotoxin-and Fusarium Wilt-induced Disturbance In Roots And The Regulation In Cucumis Sativus

Greenhouse horticulture is an important integrative part of modern agriculture,however, product yield and quality are severely reduced by monocropping obstacle due to repeatedly cultivation of a single vegetable species in the same land over several years. Understanding the mechanism of monocropping obstacle is of great importance to improve resistance of greenhouse vegetables.In this study,we chose two genotypes of cucumber(Cucumis sativus L.)and another cucurbit crop figleaf groud(Cucurbita ficifoli Bouché),to investigate the effects of autotoxicity and soil-born Fusarium wilt on reactive oxygen species(ROS)metabolism,gene expression and rhizospheric microorganism community.Also,experiments were carried out to examine the possible role of 24-epibrassinolide(EBL)in the control of Fusarium wilt in cucumber.The results are as follows:1.To provide insight into the mechanism of interspecific interactions mediated by allelochemicals,cucumber and figleaf gourd seedlings were compared on the response to cinnamic acid(CA),an autotoxin from root exudates of cucumber. Cucumber and figleaf gourd had different sensitivity to CA.Exposure to CA resulted in oxidative stress in roots of cucumber but not figleaf gourd due to differential activation of NADPH oxidase.The cucumber roots produced significant amount of ROS immediately after CA treatment,consequently inducing membrane peroxidation, inactivation of membrane H⁺-ATPase activity and reduced root viability.2.In the response to CA,cucumber had a high rate of CA uptake by the roots,leading to reduced root growth,however,figleaf gourd was resistant to CA.The CA treatment resulted in an intracellular release of Ca²⁺from vacuole to cytoplasm, suggesting that the[Ca²⁺]_cythomeostatic disturbance is one of the primary triggers for CA phytotoxicity in cucumber.3.Two genotypes of cucumber with difference in the resistance to Fusarium oxysporum f.sp.cucumerinum(FO)were grown hydroponically with or without FO inoculation. For resistant plants,FO-inoculation induced expression of a series of defence genes and transient activation of ROS scavenging enzymes,leading to resistance of whole plant and suppressed FO-growth.For susceptible plants,however,FO-inoculation induced ROS accumulation and membrane peroxidation,leading to Fusarium wilt at the end of experiment.4.Microbial community on root surfaces was affected by a complex interaction between plant genotypes and pathogens.Compared to the resistance genotype,FO inoculation resulted in a higher FO population on root surfaces and a lower population in nutrient solution in the susceptible genotype.Root exudates from FO-inoculated susceptible plants promoted FO spore germination and germ-tube elongation.Pathogen introduction resulted in overall increases of bacterial,fungal and actinobacterial population on root surfaces of the resistant plants.PCR-DGGE analysis showed that FO-inoculation induced significant changes of the bacterial structure on root surfaces of susceptible plants but not in resistant plants.5.EBL pretreatment either to roots or shoots significantly increased resistance to FO in susceptible genotypes.Infection of FO induced oxidative stress in roots as shown by induction of antioxidant enzymes and changes in flavenoids and phenolics levels. However,no such changes were observed in EBL-treated plants.There was no apparent difference between these two EBL application methods.The results demonstrated that EBL-conferred resistance to Fusarium wilt was established via a mobile secondary messenger rather than by a direct increase in antioxidant activities.6.We also investigated the effects of EBL on changes of microbial population and community in roots of cucumber plants after FO inoculation.EBL applications decreased FO population on root surfaces and in nutrient solution,but increased population of fungi and actinobacteria.EBL applications also alleviated the changes of bacterial community.Overall,these results suggest that microbial community on root surfaces was affected by a complex interaction between phytohormone induced resistance and plant pathogens.7.To provide insight into the molecular mechanism by which EBL reduced incidence of Fusarium wilt,we investigated the effects of foliar EBL application on gene expression and ROS metabolism after FO inoculation.EBL application could induce expression of a set of resistance-related genes and activities of corresponding enzymes.Moreover,EBL treatment induced a transient H₂O₂ accumulation at the beginning of the experiment.Overall,these results suggest that EBL induced resistance to Fusarium wilt by inducing H₂O₂ generation at the early stage of FO inoculation.8.A mobile secondary messenger is supposed to transmit from the EBL application site to distant organs and increase stress tolerance in these sites.We have confirmed that EBL induced systemic resistance to oxidative stress in upper and lower leaves.EBL induced H₂O₂ accumulation and resistance-related gene expression in treated leaves. In addition,EBL could also induce H₂O₂ accumulation and gene expression in systemic leaves.However,the timing of this H₂O₂ accumulation was slower than that of treated leaves and genes expression was lower than that of treated leaves.All results suggest that in the processes of EBL-induced resistance,H₂O₂ might play an important role in signal conduction.