Preliminary Study On Apoplastic Ammonium Ion Affecting Tobacco-Pseudononas Syringae Pv.tabaci Interaction

2015-2017,through the pot experiment and field tests,the effect of tobacco leaf apoplastic ammonia on Pseudononas syringae pv.tabaci reproduction,toxin production and pathogenicity was researched in a simulation manner.Also,the nitrogen metabolic pathway-related enzymes and apoplastic ammonia of several flue-cured tobacco cultivars with different resistance to wildfire were analyzed and compared.And the relationship of glutamine synthetase(GS)to disease development under different growing stage,nitrogen application,transplanting period and rainfall was analyzed and discussed from the perspective of biochemistry.Theresults areas follows:1.By needleless injection to leaf,the leaf sensitivity of potted tobacco to ammonia solution under different growing stage and leaf position was studied..The results showed that the sensitivity was significantly different in different growing stages,that leaves in resetting stage and vigorous growing stage were strongly tolerant to ammonia stress,while that of matured leaves was sensitive obviously.The max limitation of ammonia content safe to tobacco in resetting stage and vigorous growing stage was 10-4mol/L,and 10-5mol/L in senescence stage.Thesensitivity descendingorder ofdifferentleafpositionwas:bottomleaf>midleaf>upperleaf.2.By the addition of ammonia to KB medium,the reproduction in vitro of P.s.pv.tabaci was inhibited significantly at 24 hpi,that the inhibition effect showed positive relation to dosage of ammonia added to medium.The highest content of ammonia solution(10-3mol/L)had highly significant inhibition to P.s.pv.tabaci,but lower content(10-6-10-4mol/L)showed a promoting effect on bacterial growth at 8-12 hpi.The further study indicated that the filtration of10-6-10-5mol/L ammonia activated pathogen with bacterium and protein removed could cause leaf chlorisis 12 hour early than non-ammonia activated control,and caused severe cell damage to tested leaf disc.It is suggested that the pathogenic bacteria can product more phytotoxin by the ammoniastimulation.3.In the leaf needleless-injection test with the resuspension of P.s.pv.tabaci culture undergoing 10-6-10-4mol/L ammonia activated 6h,12 h,respectively,the result showed that the more early leaf chlorisis and more sever necrosis by bacterium ammonia activated 12 h than that of 6h,and the stronger pathogenicity by 10-6and 10-5mol/L ammonia activated than the other treatments.To obtain further evidence of bacterial pathogenicity promoted by ammonia,the mixture of bacterial inoculum with ammonia solution added prior to injection was tested.The result proved again that the mixture could cause water-soaked and chlorisis at 48 hpi meaning a24 h early than that of non-ammonia activated pathogen.However,injection of ammonia solution alone showed not any symptom up to 108 hpi.Therefore,the infection of P.s.pv.tabaci was promoted by ammonia addition.Comprehensive consideration of toxin production and infection enhanced,it is suggested that the appropriate level of ammonia ion can promote the pathogenicity of P.s.pv.tabaci.This phenomenonwasfirst reportedin plant-bacteriainteraction.4.Comparison of the nitrogen metabolic pathway-related enzymes of several flue-cured tobacco cultivars with different resistance to wildfire,GS,nitrate reductase(NR),glutamate synthase(GOGAT),Invertase(INV),NH4-N content and apoplastic ammonia level were analyzed.The results showed that GS activity was closely related to cultivar disease resistance.The resistant leaf had higher GS activity at vigorous growing stage,but slower decline of GS during senescence.However,the GS of susceptible leaves reduced sharply during senescence.The resistant leaf had higher NH4-N content and higher apoplastic ammonia level because of higher GS activity and stronger ammonium assimilation.Also,GS of P.s.pv.tabaci inoculated leaves showed continuous increasement up to 48 hpi and then reduced.The GS of resistant leaf reduced quickly than that of susceptible leaf.When the GS of resistant leaf was specifically inhibited by phosphinothricin spray,the pathogen inoculum could result in a more rapid infection.Hence,all the forward and reverse experimental evidence stronger indicated that the changeof GSactivityplay oneimportant rolein P.s.pv.tabaci infectiontohost.5.Through leaf GS analysis of different nitrogen application,transplanting period and rainfall,the results showed that postponed transplant period had an effect of GS decreasement,and more nitrogen-fertilizer application caused GS increasement of bottom leaf,and rainfall could inhibit leaf GS activity.These results lay the foundation of exploring the biochemical mechanism in tobaccowildfireepidemiology.Based on the above analysis,it is concluded that the appropriate level of apoplastic ammonium ion can promote the pathogenicity of P.s.pv.tabaci,and can be regarded as an important factor affecting the interaction between tobacco and P.s.pv.tabaci.This preliminary study is the first systematic exploration on environmental ammonia ion affecting the hostplant-pathogenicbacteria interaction.