Identification Of Oviposition Signal Compounds From Tobacco For Helicoverpa Assulta Host Selection

The oriental tobacco bud worm,Helicoverpa assulta(Lepidoptera:Noctuidae),is an oligophagous insect,which damages seriously on the tender leaves,flowers,and capsules of tobacco,causing much loss of yield and quality.The common tobacco,Nicotiana tabacum,is the main species of cultivated plant for production of flue-cured tobacco,and it has been bred many varieties,while the aztec tobacco,Nicotiana rustica,is a sun-cured tobacco,being cultivated much less than N.tabacum in tobacco production.Previously,it has been reported that when N.rustica was intercropped with N.tabacum in the field,H.assulta females laid much more eggs on N.rustica than those on N.tabacum.The mechanism for this ovipositing attractiveness was preliminarily determined as olfactory cues emitted from N.rustica plants.However,three questions remained as follows:(1)the active volatiles have not been identified yet;(2)whether this ovipositional attractiveness is a species-level trait or variety-level trait has not been determined;and(3)it remains largely unknown that the ovipositional choice response of adults is in line with that of the larvae.Therefore,the author conducted three serials of experiments:(1)compared the olfactory preference and feeding preference of the larvae at different instars on these two tobacco species using leaf-disc method;(2)tested the ovipositional choice response of H.assulta ovipositing females to synthetic volatile blends or individual volatiles detected from the headspace of two tobacco species using gas chromatography-mass spectrometry(GC-MS).The main results are summarized as follows:(1)Olfactory preference and feeding preference of the larvae between two N.rustica varieties(Hanxiaoyan and Xianfengxiaolanhua)and one N.tabacum variety(Yuyan10)were measured.The results indicate that both the neonates and the 3rd instar larvae preferred N.tabacum to N.rustica after 1 h and 24 h exposure under the dual-choice condition of N.tabacum leaf discs and N.rustica leaf discs.Additionally,the feeding amount of the 3rd instar larvae on N.tabacum leaf discs was more than or significantly more than that on N.rustica leaf discs.Under the dual-choice condition of four leaf discs with each tobacco species two,mixed results were obtained from the fifth instar larvae:they did not show significant olfactory preference after 1 h exposure,and showed significant olfactory-and feeding-preferences to N.rustica leaf discs after 5 h exposure,while after 16 h exposure,they exhibited significant olfactory preference to one N.rustica variety(Hanxiaoyan)but showed significant feeding preference on N.tabacum.Within each instar,no significant olfactory preference was detected between different test environments(Hanxiaoyan vs.Yuyan 10 and Xianfengxiaolanhua vs.Yuyan 10),suggesting that H.assulta larvae could not recognize its appetite host plants via tobacco olfactory cues.(2)To investigate the differential ovipositional attractiveness of N.rustica and N.tabacum is whether a species-level trait or a variety-level trait,I arranged the following treatment groups:(1)(1)Hanxiaoyan leaves vs.K326 leaves;(2)Xianfengxiaolanhua leaves vs.K326 leaves;(3)Hanxiaoyan flowers vs.K326 flowers;(4)Xianfengxiaolanhua flowers vs.K326 flowers;(5)Hanxiaoyan leaves vs.Zhongyan 100 leaves;(6)Xianfengxiaolanhua leaves vs.Zhongyan 100 leaves;(7)Hanxiaoyan flowers vs.Zhongyan 100 flowers;(8)Xianfengxiaolanhua flowers vs.Zhongyan 100 flowers.The results show that this ovipositional attractive differentiation belongs to a species-level trait.(3)I tested the ovipositional choice response of H.assulta females to the extracts of different parts(leaves or flowers)from different tobacco species or warieties(N.rustica varities:Hanxiaoyan and Xianfengxiaolanhua;N.tabacum varieties:K326 and Zhongyan 100)extracted by different solvents(dichloromethane or n-hexane).The results show that all the leaf extracts from N.rustica varities were more attractive than all the leaf extracts from N.tabacum varieties.While the suitable solvents for extracting the flowers of N.rustica varities were different:dichloromethane was suitable for extracting Hanxiaoyan while n-hexane was suitable for extracting Xianfengxiaolanhua.(4)I collected the following headspace volatiles and identified their components and contents using GC-MS.The samples were collected from:(1)Hanxiaoyan vegetative plant;(2)Hanxiaoyan flowering plant;(3)Xianfengxiaolanhua vegetative plant;(4)Xianfengxiaolanhua flowering plant;(5)K326 vegetative plant;(6)K326 flowering plant;(7)Zhongyan 100 vegetative plant;and(8)Zhongyan 100 flowering plant.A total of 37 components were identified from all the samples.Twenty eight components were identified from N.rustica derived samples,and the shared components by the two varieties were:3-hexanol,(Z)-3-hexen-1-ol,benzaldehyde,benzeneactaldehyde,D-limonene,nonanal naphthalene,decanal,nicotine,β-elemen,dodecanal,and β-caryophyllene.During vegetative stage,only α-phellandrene was shared by these two N.rustica varieties.A total of 19 components were identified from the two N.tabacum varieties.3-carene,benzaldehyde,2-hydroxy,D-limonene,linalool,acetic acid,phenylmethyl ester,and naphthalene were shared by all the four samples(K326 vegetative plant,K326 flowering plant,Zhongyan 100 vegetative plant,and Zhongyan 100 flowering plant).The volatiles of the two tobacco species at different stages may be the reason for the different oviposition preference of H.assulta to N.rustica and N.tabacum.(5)The ovipositional choice responses of H.assulta females to the individual compound and their mixture in the natural ratio identified from GC-MS were tested(as indicated in total ion chromatogram).Synthetic volatile blends derived from N.rustica vegetative stage were more attractive than those derived from N.tabacum vegetative stage,but the inverse was true in the comparison of samples derived from the flowering stage.Bioassay of individual volatiles(at concentration of 0.001mol/L,and 0.01 mol/L,respectively)indicated that ovipositional attractiveness of these volatiles was dependent on both chemical structure and concentration.Among test samples,nicotine,y-terpinolene,and naphthalene(at concentration 0.001mol/L)and nonanal(at concentrations of 0.001mol/L and 0.01mol/L)showed significant ovipositional attractiveness.In summary,we concluded that these four compounds were responsible for the differential ovipositional preference of H.assulta females between these two tobacco species,regardless of variety.