Volatile Terpenes-Mediated Indirect Defense Against Spodoptera Litura Larvae By Attracting Natural Enemies In Chrysanthemum Morifolium

Chrysanthemum(Chrysanthemum morifolium Ramat)is a perennial root herb or subshrub of the genus Chrysanthemum in the family Compositae.Studies have found that the noctuid pests that harm C.morifolium Hangbai and ‘Jinba’ mainly include Spodoptera litura and S.exigua.According to research reports,there are many secondary metabolites in the flowers,leaves,and stems of C.morifolium.In other plants,such secondary metabolites have the function of resisting insects and/or attracting natural enemies of insects.However,little is studied on the roles of secondary metabolites of C.morifolium in defense against insects.Therefore,this thesis mainly studies the terpenoids released from C.morifolium leaves after being damaged by herbivorous insects for attracting natural enemies of the insects,and the composition and biosynthetic mechanism of terpenoids in C.morifolium.The detailed research results are as follows:1.The Y-tube olfactometer bioassay was used to study the behavioral response of S.litura(SL),the predatory natural enemy,Arma chinensis,and the parasitic natural enemy Trichogramma pretiosum to healthy plants and plants after SL infestation.The results showed that the plants at 24 h and 48 h after SL infestation had a very significant attracting effect to A.chinensis and T.pretiosum,indicating indirect defense is functioning in C.morifolium.Next,seedling ’Jinba’ plants with 18-20 real leaves were subject to mechanical injury,SL infestation and methyl jasmonate(MeJA)treatment.These and healthy plants were then used for volatile profiling.Volatile compounds were collected at 4 h,24 h,48 h and 72 h,and analyzed by GC-MS.Compared to mechanical injury,the types and amount of terpenoids released by the SL infestation continued to increase.The release of monoterpene β-pinene reached the highest level during 48 to72 h of SL-infestation.After 24 h,the emission rates of(E)-β-caryophyllene,γ-cadinene and Aromandendrene were significantly higher than other sesquiterpenes.Compared with mechanical damage,β-phellandrene,1,8-cineol,β-copaene,2-epi-β-funebrene andα-patchoulene were detected only from SL-infested plants.Compared with SL infestation,MeJA treatment included the emission of more terpenoids within 72 h,but the types of terpenoids were similar to those produced by SL infestation.This suggest that volatile terpene production in response to insect herbivory is regulated through the jasmonate pathway.2.Transcriptome sequencing was performed on the C.morifolium leaves with SL infestation for 72 h.Through volcano graph analysis,5071 genes up-regulated after SL infestation were screened from the transcriptome.All up-regulated genes were classified by GOC function,and they were classified into 23 functional categories.Upregulated genes are mostly involved in the biosynthesis,transport,catabolism and defense mechanisms of secondary metabolites.Using Blast2 GO to perform genetic GO classification analysis on all up-regulated genes,the six main GO terms identified are:catalytic activity,binding,membrane,metabolic process,membrane part,cellular process.The results of KEGG functional analysis marked 5071 up-regulated genes as119 different pathways.The significant changes in SL infestation include alphaLinolenic acid metabolism,phenylpropanoid biosynthesis,plant-pathogen interaction and MAPK signaling pathway-plant.The Venn diagram of 66 terpene synthase(TPS)genes enriched in the KEGG pathway and all the up-regulated genes showed that a total of 19 TPS genes were up-regulated after the SL infestation.Through transcriptome analysis,we screened 11 full-length TPS genes from C.morifolium leaves,and analyzed the expression of genes in the two pathways that are responsible for terpene biosynthesis in C.morifolium leaves.The results showed that the expression levels of key genes in response to insect herbivory were higher in the MVA pathway and GPPS in the treatment group than in that in the control group.3.In the second chapter of this study,11 full-length TPS genes from ’Jinba’ leaves were analyzed through transcriptome.These genes have a significant response to Spodoptera litura infested.We obtained the protein sequences of 11 CmjTPS genes through transcriptome data,and analyzed the conserved domains to further verify that they are terpene synthase.Using q RT-PCR,all CmjTPS genes except CmjTPS10 were up-regulated,validating the transcriptome data.All 11 genes showed up-regulation after MeJA treatment.In addition,we used the terpene synthase genes identified in the representative plants of the Compositae and model plants such as Arabidopsis(Arabidopsis thaliana)and grapes(Vitis vinifera)to construct a phylogenetic tree with11 CmjTPS,which lead to useful prediction about their biochemical functions.