Study On Physiological And Biochemical Mechanisms Of Three Chrysanthemum Cultivars Of Hangbaiju Against Leafhopper

Chrysanthemum morifolium Ramat cv ’Hangbaiju’ could be used as food and medicine,whose original production area was Tongxiang City of Zhejiang Province.As one of the famous Chinese herbal medicine "Zhebawei",it have been widely introducedat home and abroad.The chrysanthemum green leafhopper(Empoasca flavescens F.)preferred to suck and pierce chrysanthemum leaves and the petal.Based on its population dynamics investigation and its feeding behavior on chrysanthemum foliage monitored by EPG techniques,the differences in the resistance of different cultivars against the leafhopper were discussed.?Zaoxiaoyangju?,?Wanxiaoyangju?,and ?Yizhongdabaiju? were the three elite cultivars,and their resistance mechanism to pests was little reported.In the present work,the leafhoppers were introduced on the leaves.Then the dynamic of activity of defense enzymes,the expression of defense enzyme genes,and the change in volatiles from leaves after injured by the leafhopper were deeply investigated,so as to roundly evaluate the resistance differences among three elites Chrysanthemum cultivars of Hangbaiju and explore the physiological and biochemical defense mechanisms.The main research contents and results were as follows.1.Physiological responses of three elite cultivars of Hangbaiju to leafhopper injured stress.The chrysanthemum leaves were injured by leafhoppers at different time(0 h,12 h,24 h,48 h,96 h)and injured degrees(10 heads/plant,30 heads/plant),and the dynamics activity of CAT,POD,PPO,LOX and PAL were detected.The results showed that(1)The activity of five defense enzymes in response to leafhopper injured stress was all increased,and the response duration and intensity were different.Among them,POD and LOX displayed higher enzyme activity after injuring 12 h-24 h,PPO disclosed higher enzyme activity at 24 h-48 h,PAL had higher enzyme activity at 48 h-96 h,while CAT had higher enzyme activity at 0 h-12 h,and fluctuated from 24 h to 96 h;(2)The main effect analysis showed that the cultivars,injured time and injured degree had a significant impact on the activity of five defense enzymes(P <0.05).Meanwhile,the injured time and injured degree had a reciprocal reaction for each defense enzyme,among which CAT,PPO and LOX existed a significant level(P <0.05);(3)The enzyme activity of the leaves under severely injured was generally higher than that lightly injured,and there was a significant positive correlation between the dynamic changes of [PPO and POD,PAL,LOX],and[LOX and POD](P <0.05),showing a synergistic insect resistance.2.Study on expression of defense enzyme genes of Chrysanthemum “Hangbaiju” in response to leafhopper injured stress.Three elite cultivars were injured by leafhopper stress,and samples were fetched after 0 h,6 h,12 h,24 h,48 h,96 h,then the quantitative expression of defensive enzyme genes pod,ppo,and pal in different treated samples were detected by q RT-PCR.The results showed that:(1)Leafhopper injured stress could effectively stimulate the expression levels of pod,ppo,pal in the leaves of “Hangbaiju”,which were higher than control,but there are differences in the expression rules for the three cultivars;(2)There was a positive correlation between the gene expression and its corresponding enzyme activity,such as pod gene of cultivar ?Zaoxiaoyangju?,pal gene of ?Wanxiaoyangju? and ?Yizongdabaiju?,and the correlation coefficients were 0.621,0.639,and 0.620,but there was no significant correlation between other genes and changes in enzyme activity.Therefore,the defense enzyme gene regulates the dynamic changes of the defense enzyme in some scope.3.Analysis of volatile changes in leaves of “Hangbaiju” induced by leafhopper injured.The volatiles of leaves were collected by dynamic adsorption method.After GC-MS detection,we found that the three cultivars of Hangbaiju changed the species and their proportions of volatiles released after they were infested by leafhoppers.The results are as follows:(1)The injured leaves of Zaoxiaoyangju have newly detected,including phyllol,β-myrcene,β-basalene,2-alcohol,isoryl citral,myrtenal,verbenone,extrinsine,isophorone,cubene,rhubarbene,hopene,(+)-herumene,2,6,6-trimethyl-1,3-cyclohexadiene-1-Formaldehyde,15-heptadenal;the injured leaves of Wanxiaoyangju have newly detected including nectarine,trans-2-hexenal,n-hexyl formate,isopiperin,(1R)-myrtenal,cubene,cyclopentanol,α-ginseng Ene,β-ginsene;The injured leaves of Yizhongabaiju have newly detected including n-hexanal,trans-2-hexenal,1-octen-3-ol,folate acetate,α-terpinene,2-(1,1-di Methylethyl)-phenol,15-heptadenal;(2)The volatiles released from the leaves of“Zaoxiaoyangju”、“Wanxiaoyangju” and “Yizhongdabaiju” were measured to be 49,39,and 54,and the percentages relative to the internal standard were 2887%,2072%,and 2052%.The volatiles released from the infested leaves were measured to be 80,50,and 65,and the percentages relative to the internal standard were 3317%,2581%,and2367%;(3)Terpene volatiles were the main components,followed by alkane,and the content of volatiles such as aldehydes,alcohols,and esters in the infested leaves has increased.To sum up,the comprehensive analysis suggests that the three elite cultivars of ?Hangbaiju? occupied different resistance levels to leafhopper.The defense enzyme system of ?Yizhongdaibaiju? showed higher activity than ?Zaoxiaoyangju? and?Wanxiaoyangju? when responding to leafhopper-damadeg stress.The fingerprint volatile components and content released by ?Zaoxiaoyangju? plants were significantly higher than those of ?Wanxiaoyangju?,which showed positive control measures against leafhopper and had stronger resistance than ?Wanxiaoyangju?.Therefore,it is preliminarily considered that the resistance from strong to weak is?Yizhongdabaiju?、?Zaoxiaoyangju?、 ?Wanxiaoyangju?.This study can provide a reference for the physiological and biochemical mechanisms of resistance to leafhopper injured stress,and provide a reference for comparison of insect resistance among the three cultivars.