| This paper seeked to explore a simple routine analytical method to separate and determine four major alkaloids in tobacco and cotinine of metabolism of nicotine, and apply this methodology to analyze alkaloids and cotinine of larvae, frass, and tobacco, in order to determine the adaptation mechanism of noctuid caterpillars to tobacco alkaloids. Herbivore feeding or mechanical wounding can activate some chemical defense system of tobacco, which induce a rapid increase of nicotine in roots and transport to damaged and adjacent parts. On the basis of the interaction between tobacco and insects, a bionics signal molecule which has a gainst responses to would signal of tobacco was selected to treated tobacco. The research on the interaction effects between bionics signal molecule and the cultivation factors of varieties, fertilizer, and topping time were studied. The contents of tobacco alkaloids and composition were controlled in a appropriate range by optimizing the application conditions of BSM. To provide a new way in the control of alkaloids content in tobacco leaves.Four main aspects were included in the dissertation:1. A novel, simple, quick and reliable method for analysis of nornicotine, anatabine, anabasine, nicotine and cotinine has been developed utilizing reverse high performance liquid chromatography. A series of optimization were done to find the most effective extraction solvent and technology for tobacco sample, and application of solid-phase C18 column for purification of samples. The best determination conditions were chosen after a series of optimization, and the method was validated by linearity, percision, repeatability and recovery. RSD value of five reference materials were from 0.89 to 3.56%, and the overall recovery was above 90%, all these datas showed that this improved method was feasible for tobacco analysis system.2. Tobacco is a common host plant of Helicoverpa armigera (Hübner), Helicoverpa assulta (Guenée) and Spodoptera litura (Fabricius) (Lepidoptera, Noctuidae). Larvae, frass and tobacco were analyzed by HPLC to determine if alkaloids were assimilated, sequestered, metabolized and excreted. In order to confirm the adaptation mechanism of this three noctuid caterpillars to tobacco alkaloids. The results of this investigation strongly suggest that only a small amount of alkaloids are sequestered and detoxified into cotinine by three noctuid insects, and alkaloids are excreted unmodified. Furthermore, H.assulta and H.armigera have stronger ability of nicotine metabolism and alkaloids excretion capacity than S.litura. Cotinine concentration in storaged frass of S.litura were increased with the growth of storage time, and alkaloids content were declined in this conditions. It is showed that alkaloids contained in the frass naturally degrades to cotinine in the presence of environmental conditions such as air , or microorganism. Three noctuid insects which have become adapted to tobacco as a host plant mainly because of an efficient excretory mechanism.3. The application of bionics signal molecule (BSM) from tobacco feeding oligophagous insect oral secretion to diverse types of tobacco after topping in different conditions. The major alkaloids concentrations in tobacco leaves treated with BSM and untreated control were analyzed by HPLC, and the impact of BSM on the alkaloids under different conditions were discussed. Results indicated that BSM could inhibit the alkaloids accumulation after topping in various tobacco leaves at different levels. Applied BSM on 9717 could markedly reduced the level of nornicotine in middle and upper leaves by 55.78% and 63.40% respectively, while alkaloids in upper leaves decrease more in comparison with middle leaves, there were 25.49% and 26.28% decline of nicotine and total alkaloids in upper leaves. The concentrations of BSM also have inhibitory effects on alkaloids accumulation in burley, with the biggest effect on nicotine by 0.1 mol/L and nornicotine by 0.2 mol/L. Applied 0.1 mol/L BSM to burley displayed a decrease of nicotine by 22.99% and 42.45%, total alkaloids by 22.21% and 37.73% in middle and upper leaves respectively ,While treated with 0.2 mol/L BSM showed a decrease of nornicotine by 62.94% and 58.53 % in middle and upper leaves respectively. Furthermore, there are some influence of nitrogen content on the regulating effect of BSM in K326 . The total alkaloids concentrations of upper leaves were lowest with 9 kg/667 m2 N applied , an decrease of 34.76 % compared with the control.4. The effects of the topping time in the budding, early-flowering, medium- flowering and full-blooming stage treated with BSM at nitrogen rate of 7kg N/667m2 and 9kg N/667m2 on the content of alkaloids in flue-cured tobacco yunyan 85 were studied, which indicated that both the early and late topping treated by BSM could inhibit the alkaloids accumulation in tobacco leaves. Tobacco treated by BSM aftertopping at budding stage were favorable for reducing the nicotine accumulation and the topping at medium-flowering stage could dramatically decrease the three minor alkaloids accumulation in tobacco leaves. The content of alkaloids in different topping time treated with BSM at nitrogenous fertilizer of 9kg N/667m2 has greater decline compared to 7kg N/667m2.
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