Insecticidal Activity Of Five Chinese Medicinal Plants Against Plutella Xylostella L. (Lepidoptera: Plutellidae)

Diamondback moth, Plutella xylostella (L.) (Lepidoptera:Plutellidae), is one of the most serious pests of cruciferous crops throughout the world, particularly in the tropical and subtropical countries. Crop damage is caused by larval feeding, or by the presence of insects (usually larvae) contaminating produce. The level of P. xylostella infestation varies according to locality, type of cabbage plants, the outlining plants, and the level of natural enemies. If no control measures are undertaken, feeding injury caused by this caterpillar may reduce production to zero. Farmers commonly use synthetic insecticides for P. xylostella control because chemicals have knock-down effect and are easily available in the local market. The indiscriminate use of synthetic insecticides leads to insecticide resistance in P. xylostella. Up to now, P. xylostella has developed resistance to all groups of conventional insecticides, some strains of Bacillus thuringiensis Berliner and newer insecticides, including abamectin, spinosad, indozacarb, emamectin benzoate. Widespread control failure of these synthetic insecticides and adverse environmental effects on the biotic and abiotic environment lead to search for effective and safer alternatives for P. xylostella control to decrease conventional insecticide application.The botanical insecticides have been suggested as alternative for the insect control because they are generally pest-specific and are relatively harmless to non-target organisms including humans. They are also biodegradable and harmless to the environment. Furthermore, unlike conventional insecticides that are based on a single active ingredient, plant-derived insecticides comprise an array of chemical compounds which act concertedly on both behavioral and physiological processes. Thus, the chances of pests developing resistance to such substances are less likely. The use of botanical insecticides in insect pest control is not only useful for suppression of insect pest population but also helps to maintain the sound ecological balance. Although some botanical insecticides are found on the market, new compounds with strong activity for P. xylostella control are always necessary either to prevent the insecticide development or to guarantee the ready availability of botanical insecticides through more widely distributed.Five Chinese medicinal plants, including Curcuma longa L., Kochia scoparia (L.) Schrad., Phytolacca americana L., Pseudolarix kaempferi Gord., and Veratrum nigrum L., are well-known medicinal plants with a long history of traditionally medicinal use in China. These plants have a variety of biological activities, for example, anti-inflammatory, anticancer, antitumor, antihypertensive antifungal activities. However, the insecticidal activity of these plants except C. longa against P. xylostella has never been investigated. Due to necessity in search for new botanical insecticide with strong insecticidal activity against P. xylostella and easy availability of these plants, thus, they were selected to investigation for the insecticidal activity against P. xylostella.The overall goal of this research is to search for new botanical insecticide for P. xylostella control in order to develop an economically and environmentally sound integrated pest management program. The specific goals of this research include the following:1) to assess the insecticidal activity of 5 Chinese medicinal plants against P. xylostella larvae; 2) to assess the insecticidal activity of fractions isolated from the extract that had the strongest insecticidal activity against P. xylostella larvae; 3) to isolate and identify the insecticidal active compounds from the plant; and 4) to compare the efficacy between the insecticidal active compounds isolated from the plant and synthetic insecticides against P. xylostella. Important research findings of my work are:1. Screening for insecticidal activity of 5 Chinese medicinal plants against P. xylostella larvaeDue to the advantage of botanical insecticides for insect pest control,5 Chinese medicinal plants, C. longa, K. scoparia, P. americana, P. kaempferi, and V. nigrum, were selected according to medicinal uses and easy availability to evaluate the insecticidal activity against P. xylostella larvae. The plant parts used in this study were the rhizome of C. longa, the seed of K. scoparia, the root of P. americana, the root bark of P. kaempferi, and the root and rhizome of V. nigrum. In the first step, the ground materials of these plants were separately extracted in one of five organic solvents (acetone,95%ethanol, ethyl acetate, petroleum ether, and distilled water). Twenty-five extracts were obtained and investigated for insecticidal activity against P. xylostella larvae by a leaf dipping bioassay method. Five extracts, the ethyl acetate, ethanol, and acetone extracts of V. nigrum root and rhizome, the ethyl acetate extract of P. americana root, and the petroleum ether extract of P. kaempferi root bark were effective against P. xylostella larvae. Among them, the ethyl acetate extract of V. nigrum root and rhizome showed the strongest insecticidal activity against the second and third instar larvae of P. xylostella, with LC50 values of 224.65 and 334.90 ppm, respectively,72 h after treatment. The LC50 values of the ethanol and acetone extracts of V. nigrun, the ethyl acetate extract of P. americana, and the petroleum ether extract of P. kaempferi for the second instar larvae were 365.20, 519.23,1217.00, and 1301.19, respectively, and those for the third instar larvae were 417.39,667.90,1585.52, and 1632.31 ppm, respectively. The other 20 extracts gave little or no insecticidal activity against P. xylostella. These results indicated that the ethyl acetate extract of V. nigrum root and rhizome may be considered as a potent source for P. xylostella larval control, and that the organic solvent used for plant extraction can affect its insecticidal activity.2. Determination for insecticidal activity of fractions isolated from the ethyl acetate extract of V. nigrum root and rhizomeThe ethyl acetate extract of V. nigrum root and rhizome which had the strongest insecticidal activity against P. xylostella larvae was further fractionated using silica gel column chromatography. A total of 109 fractions were obtained and then monitored with TLC. Based on the band appearance on TLC, the 109 fractions were pooled into 29 fractions. In preliminary test, the leaf dipping bioassay method was conducted to evaluate the insecticidal activity of 29 fractions against the second instar larvae of P. xylostella. Seven fractions, the fourth, fifth, tenth, thirteenth, twentieth, twenty-fourth, and twenty-seventh fractions showed strong insecticidal activity with 80.00-97.50%mortality. Moderate insecticidal activity (60.00-77.50% mortality) was obtained from the second, third, sixth, seventh, and twenty-sixth fractions. Weak insecticidal activity was produced from the ninth, twelfth, fourteenth, nineteenth, twenty-third, and twenty-ninth fractions (32.50-47.50% mortality). The other 11 fractions exhibited little or no insecticidal activity (<30.00 %mortality) against P. xylostella larvae. Among 7 fractions with strong insecticidal activity, the further studies were conducted to compare the toxicity of these fractions against P. xylostella larvae by two bioassay methods, the leaf dipping bioassay and topical application. The fifth fraction showed the strongest insecticidal activity against larvae. At 72 h after treatment, its LC50 values for the second and third instar larvae in leaf dipping bioassay method were 81.53 and 160.52 ppm, respectively, and those for the third instar larvae in topical application were 561.19 ppm. All active 7 fractions had both ingestion and contact toxicities to the insect. However, the ingestion toxicity was higher than the contact toxicity. These results indicated that the fifth fraction contained predominately insecticidal active compounds and there were some compounds that had insecticidal activity in other 6 fractions.3. Isolation and identification of the insecticidal active compounds from the ethyl acetate extract of V. nigrum root and rhizomeIn order to identify the insecticidal active compounds from the plant, the ethyl acetate extract of V. nigrum root and rhizome was further isolated by chromatographic procedures. The fifth fraction was obtained and monitored by TLC. Due to the strongest insecticidal activity against P. xylostella larvae and the presence single spot on TLC using petroleum ether-ethyl acetate (10:1.5), petroleum-methanol (9:1), hexane-ethyl acetate (10:1) and chloroform-methanol (7:3) as developing systems, the fifth fraction was recrystallized with methanol. The compound 1 was obtained as white powder and its melting point was 130.9℃. It gave positive responses to Liebermann-Burchard and Salkowski reactions, indicating the presence of steroid skeleton. The compound 1 was further identified asβ-sitosterol (24-ethyl-5-cholestene-3-ol). Its structure was elucidated by means of IR, NMR, and MS spectroscopic analysis. The compound 1 was identified by comparison of its IR,'H-NMR,13C-NMR and mass spectra with the literature. The present study was the first report to identify the insecticidal active compound from V. nigrum root and rhizome against P. xylostella asβ-sitosterol. 4. Comparison on the efficacy ofβ-sitosterol, isolated from the ethyl acetate extract of V. nigrum root and rhizome, and two synthetic insecticides against P. xylostellaStudies were conducted to investigate the insecticidal activity ofβ-sitosterol, isolated from the ethyl acetate extract of V. nigrum root and rhizome, and two synthetic insecticides, abamectin and cypermethrin to larvae, pupae, and adults of P. xylostella. These synthetic insecticides were selected according to common uses for insect pest control in cruciferous production areas. Leaf dipping bioassay and topical application for larvae, direct dipping bioassay for pupae, and residual bioassay for adults of P. xylostella were used to evaluate mortalities. Abamectin showed the strongest insecticidal activity against all three stages of P. xylostella, followed by cypermethrin andβ-sitosterol, respectively. At 72 h after treatment, the LC50 values of abamectin for the second and third instar larvae in leaf dipping bioassay method were 0.03 and 0.05 ppm, respectively, and those for the third instar larvae in topical application were 0.19 ppm. In the direct dipping and residual bioassays, the LC50 values of abamectin were 19.98 and 39.69 ppm to pupae and adults, respectively.β-sitosterol gave high efficacy for P. xylostella control with LC50 values of 34.72 and 55.95 ppm for the second and third instar larvae in leaf dipping bioassay method, respectively, and 279.67 ppm for the third instar larvae in topical application. The LC50 values ofβ-sitosterol were 2767.94 and 4662.89 ppm to pupae and adults, respectively. The larval stage of P. xylostella was the most susceptible to all insecticides while the adult stage was the most tolerant. The results of this study clearly showed that V. nigrum may be a promising source and P-sitosterol as a new naturally occurring agent for P. xylostella control.