Aphicidal Activity of Substances from Roots of Ferula foetida (Bunge) Regel. against Grain Aphid, Schizaphis graminum (Rondani) ELENA A. STEPANYCHEVA 1, ANARA SH. CHAKAEVA 2, ELENA I. SAVELIEVA 1, AND TAISIYA D. CHERMENSKAYA 1* 1 All-Russian Institute of Plant Protection, Podbelsky sh., 3, St.-Petersburg, Pushkin, 196608, Russia 2 Kyrgyz Research Institute of Livestock Breeding and Pastures, Dorozhnaya str., 12, Komsomolsky Village, 722128, Kyrgyz Republic Biopestic. Int. 8(1): 18-24 (2012) ABSTRACT Effects of a root extract of Ferula foetida on the grain aphid Schizaphis graminum have been studied. LC 50 of the extract was found to be 0.0224%, and concentrations = 0.06% caused complete mortality of aphids. Moreover, the extract of F. foetida had a fumigant effect upon females and young nymphs of the next generation. Plant substances with such properties could be useful for pest management, especially for greenhouse and storage products pests. Solvent partition of the F. foetida crude ethanol extract revealed that the hexane extract had the highest activity. An active principle of F. foetida was isolated by preparative TLC. Mass spectrum data indicated a formula of C 8 H 14 S 4 and the structure H 3 C-S-CH=CHCH 2 S 2 CH 2 CH=CH- S-CH 3, thus identified as (bis (3-methylthio-2E-propenyl disulfide). KEY WORDS : Botanical aphicide, Ferula foetida, fumigant activity, Schizaphis graminum INTRODUCTION Schizaphis graminum (Rondani) is an important pest of cereals crops in many areas of the world. This pest sucks juices from plants, hampering formation of vegetative and reproductive tissues. Aphids can also be vectors viral diseases from infected plants. The overuse of synthetic insecticides for many years has resulted in aphid resistance and environmental damage (Van Emden and Harrington, 2007). Botanical insecticides are attractive as an alternative means of pest management, since the substances of plant origin are generally safer for the environment. Many plant species produce secondary metabolites, which protect them from herbivorous arthropods. A glycoside from Solanum laxum Steud. (Soulé, 2000), 1,5-diphenyl-1-pentanone and 1,5- diphenyl-2-penten-1-one from Stellera chamaejasme L. (Gao et al., 2001), and the indole alkaloid gramine, isolated from barley (Ryan, 2002), have strong contact activities against S. graminum. In our investigation of the flora of Kyrgyzstan and search for plants containing substances with insecticidal and acaricidal activity, we have found the extract from roots of Ferula foetida (Bunge) Regel to have strong activity against S. graminum (Chermenskaya et al., 2010). The purpose of this study is to further assess effects of the extract and its constituents upon the development of the grain aphid S. graminum. * Corresponding author: E-mail: tchermenskaya@yandex.ru 0973-483X/12/18-24 2012 (KRF)
2012 Stepanycheva et al. : Aphicidal activity of Ferula foetida 19 MATERIALS AND METHODS Plant Ferula foetida (Bunge) Regel. (Umbelliferae) was collected from natural areas in the South of Kyrgyzstan (Alai Valley, near the confluence of the Ak-Suu and Kyzyl-Suu, the height of 2500 m above sea level, 21/08/2006) by an expedition of the Academy of Sciences of the Kyrgyz Republic, and deposited in the republican herbarium with voucher number 6109. Insect The grain aphid Schizaphis graminum (Rondani) (Homoptera: Aphididae) was reared on sprouting wheat (Triticum aestivum L.) grown in trays with moist filter paper at 22 ± 2 o C, 16:8 LD, and 50 ± 10 RH. Plant Substance Extraction, Isolation and Analysis The roots of F. foetida were extracted with ethanol as described previously (Chermenskaya et al., 2010). Aqueous emulsions for bioassays were prepared in 0.05% aqueous solution of Tween-60. A part of the crude extract (300 mg) was dissolved in 100 ml acetone water mixture (1:1), and acetone was evaporated under a stream of air. The residual 50 ml was partitioned in a separating funnel with hexane, methylene chloride, diethyl ether and ethyl acetate (three times with 30 ml of each solvent), sequentially. The pooled portions for each solvent were dried with anhydrous sodium sulfate. The hexane, methylene chloride, diethyl ether and ethyl acetate extracts were then evaporated to dryness and aliquots of each extract were redissolved in appropriate solvents to 0.25% concentration. The remaining aqueous layer was diluted to 0.25% and used for bioassays. The extracts were examined by analytical thin layer chromatography (TLC). For isolation of active principles, hexane, methylene chloride and diethyl ether extracts were combined and then separated into four fractions (I IV) with different Rf values using preparative TLC on plates (Silufol UV 254, Czech Republic) and a solvent system (hexane:diethyl ether 9:1, v/v). The Rf values for fractions I, II, III and IV were 0 0.14, 0.15 0.45, 0.5-0.75 and 0.79 1.0, respectively. Fraction IV was analyzed by gas chromatography mass-spectrometry (GC-MS). A GCMS QP 2010 Shimadzu instrument was equipped with a DB-5 MS capillary column of 30 m, 0.2 mm I.D. and 0.32 µm film thickness. GCMS solution workstation software was used for instrument control and data analysis. The following chromatographic conditions were applied: helium (99.999%) constant flow of 1.0 ml/min, inlet temperature 250 o C, injection volume 1 µl (splitless, sampling time 30 s); MS transfer line temperature 270 o C, detector temperature 200 o C; temperature program from 40 o C for 1 min, then a ramp of 15 o C min - 1 to 280 o C (held for 15 min). The mass detector was operated in full scan mode detecting ions between 33 m/z and 650 m/z. Ionization was performed with electron impact. Solid phase microextraction (SPME) of the components from equilibrium with chipped root vapor phase was performed as follows: Chipped root sample was heated at 50 o C for 30 min in a tightly screwed 4-ml vial mounted in a Manual SPME Sampling Stand (Supelco), after which a Carboxen/PDMS StableFlex 85 µm microfiber (Supelco) was inserted through the seal into the vapor phase over the sample and exposed for 10 min; thermal desorption of analytes from the microfiber in the GC injector was at 250 o C. Aphicidal Activity of Plant Substances To estimate insecticidal activity of the substances in laboratory bioassays, filter paper discs impregnated with test solutions (0.25 ml/disc) were placed in the bottom and lid of small Petri dishes (36 mm in diameter) (Corning Inc., USA), then a wheat leaf (3 cm in length) treated with the same test solutions and about 20 female aphids were added. The control was treated with a 0.05% solution of Tween-60. Twenty four hours later, live and dead aphids and their offspring were counted. There were ten replicates for each treatment. Efficacy was corrected by Abbott s formula (Abbott, 1925). For cage bioassays, wheat was germinated in the trays (12 21 cm). Forty females of S. graminum were placed on the wheat germs in each tray. Seven
20 Biopesticides International Vol. 8, no. 1 days later the aphids were sprayed with 0.5% crude extract of F. foetida. The control was treated with a 0.05% solution of Tween-60. There were four replicates for each treatment. The number of living aphids was counted 1, 5, 9, and 14 days after that in the 5 plants in each replicate. The efficacy was estimated by Henderson-Tilton s formula (Henderson and Tilton, 1955). Fumigant Activity of F. foetida Ethanol Extract To test for fumigant activity, 200-ml plastic containers were used. A wheat leaf was placed into each container on the wet filter paper, and 20 female aphids were placed on the leaf using thin brush. The amounts of ethanol extract of F. foetida corresponded to 26.4 10-3 µl/ml of air and were applied to filter paper attached to the underside of the lid. Control filter paper was treated with ethanol. This method is based on diffusion and evaporation of volatile components without direct contact with the test subjects (Stepanycheva et al., 2006). Live and dead females and their offspring were counted after 24 h. There were 10 replicates. The efficacy was corrected by Abbott s formula (Abbott, 1925). Statistical Analysis The LC 50 was determined by probit analysis (Finney, 1977). Data from each experiment were analyzed by one-way ANOVA (MicroCal Origin, version 3.01). Means were compared using Tukey s HSD test (P 0.05). Differences between means were considered to be significant at P 0.05. RESULTS Aphicidal Activity of Plant Substances The F. foetida (root) ethanol extract was toxic against adult female grain aphids, S. graminum. Concentrations = 0.06% produced 100% mortality (Table 1), and the LC 50 was estimated to be 0.0224% (224 mg/l). When sprayed at 0.5% on wheat plants, the extract did not influence the number of insects per plant at 1 or 5 days, compared to the control. After 9 days the number of insects/plant in the control was double than on treated plants though on the 14th day there were no significant differences between control and treated plants. Thus, the data obtained show a short-term decrease in the number of grain aphids after treatment with 0.5% F. foetida ethanol extract (Fig. 1). Fumigant Activity of F. foetida Ethanol Extract In a closed space the experiments showed that without direct contact with the aphids, the vapors from F. foetida extract have fumigant activity against female aphids and young nymphs of the next generation (Table 2). The effective concentration was 26.4 10-3 µl/ml air corresponding to a dose of 0.06% of the extract used in the Petri dishes (Table 1). Isolation and Analysis of Plant Substances The crude extract of the roots of F. foetida was partitioned successively with hexane, methylene chloride, diethyl ether and ethyl acetate to isolate active constituents. The remaining aqueous phase Table 1. Insecticidal activity of Ferula foetida (roots) extract against grain aphids S. graminum after 24 h of exposure in laboratory bioassays Concentration, N a Mortality of females Corrected mortality Mortality of the subsequent % (%) (mean ± SE) (%) nymphs, (%) (mean ± SE ) 0.06 200 100 a 100 100 a 0.03 204 78.1 ± 2.06 b 77.8 100 a 0.02 194 32.4 ± 0.92 c 29.9 36.1 ± 3.59 b 0.015 202 16.4 ± 1.69 d 15.2 11.1 ± 1.20 c Control 204 2.9 ± 1.05 e - 0.8 ± 0.8 d a N, number of aphids before treatment. * Means within columns followed by the same letter are not significantly different at the P = 0.05 (according to ANOVA and Tukey (HSD) test).
2012 Stepanycheva et al. : Aphicidal activity of Ferula foetida 21 Table 2. Fumigant toxicity of Ferula foetida extract (26.4 x 10-3 µl/ml air) against grain aphids Treatment N a Mortality of females Corrected mortality Mortality of the subsequent (%) (mean ± SE) (%) nymphs, (%) (mean ± SE ) Test 212 74.4 ± 3.76* 72.8 92.1 ± 2.89* Control 205 5.9 ± 1.61 0 0 a N, number of aphids before treatment. * The means are significantly different from control at the P = 0.05. was also used for bioassays. A 0.25% solution of the ethyl acetate extract or the aqueous phase had no activity against grain aphids. The hexane, methylene chloride and diethyl ether extracts (0.25%) were highly toxic to adult S. graminum (Table 3). The hexane extract at 0.03% was active (mortality rate of female aphids was over 85%, and that of their nymphs was over 90%), indicating the lipophilic nature of the active ingredients. Extract compositions were analyzed by TLC in hexane - diethyl ether solvent systems with gradually increasing content of ether. One compound was found in all active extracts. The percentage of this compound was highest in the hexane extract and was most active. The ethyl acetate extract (inactive) was free of this component. Thus, the active constituent of F. foetida was a volatile compound with Rf 0.26, 0.68 and 0.82 in hexane, hexane: diethyl ether = 9:1 and hexane: diethyl ether = 9:2, respectively. Evaluation of insecticidal activity of the fractions obtained by preparative TLC from combined active extracts showed that two fractions were effective against female grain aphids, but only fraction IV completely inhibited development of the nymphs (Table 4). To identify the substances, responsible for the insecticidal activity, gas chromatography - mass spectrometry (GC-MS) was used. Before analysis, the samples were concentrated in a stream of nitrogen. The principle peak was detected in masschromatograms of hexane, methylene chloride and diethyl ether extracts, which accounted for 77, 35 Fig. 1. Influence of the F. foetida (root) extract (0.5%) on populations of S. graminum in the cage bioassay. *Means significantly different at P = 0.05. Bars indicate means and standard error.
22 Biopesticides International Vol. 8, no. 1 Table 3. Insecticidal activity of different extracts from F. foetida (roots) against grain aphids S. graminum after 24 h of exposure Treatment Concentration N a Mortality of females Corrected mortality Mortality of the subsequent % (%) (mean ± SE) (%) nymphs, (%) (mean ± SE ) Hexane extract 0.25 105 100 a* 100 100 a 0.03 130 87.8 ± 1.25 b 87.0 94.7 ± 3.40 b Methylene 0.25 105 100 a 100 100 a chloride extract 0.03 106 18.3 ± 3.16 c 13.5 8.6 ± 3.03 c Diethyl ether extract 0.25 108 100 a 100 93.3 ± 0.85 b 0.03 116 30.3 ± 4.60 c 26.2 22.0 ± 3.81 d Ethyl acetate extract 0.25 104 5.4 ± 2.09 d 3.6 0 e Water residue 0.25 108 6.1 ± 2.04 d 4.3 0 e Crude (base 0.25 199 100 a 100 100 a ethanolic) extract 0.03 204 78.1 ± 2.06 e 77.8 100 a Control - 102 5.5 ± 1.11 d - 0 e a N, number of aphids before treatment. * Means within columns followed by the same letter are not significantly different at the P = 0.05 (according to ANOVA and Tukey (HSD) test). and 42% of the total ion current, respectively with chromatographic retention index (RI) 1623. After isolation of fraction IV from combined hexane, methylene chloride and diethyl ether extracts by TLC, significant enrichment of the potentially active component with RI 1623 was achieved. The masschromatogram of fraction IV is shown in Fig. 2. As can be seen after purification with TLC, the masschromatogram has essentially a single peak, which accounts for more than 90% of the total ion current. The mass spectrum in electron impact mode for the active component (Fig. 3) did not provide enough information to suggest the structure of the substance. To overcome the matrix effect and obtain a cleaner mass spectrum we analyzed equilibrium head space instead of liquid extracts. Headspace solid phase microextraction has the advantage of high purity of chromatograms. In the volatile profile of ferula root extract the principle component of the most active fraction was detected with good resolution from background noise. Weak but reproducible signals for ions with m/z 159, 139, 119, 104 were recorded in the mass spectrum. All of them, as well as the principle signals with m/z 87 (100) and 45 (40) correspond with the formula C 8 H 14 S 4 and structure H 3 C-S-CH=CHCH 2 S 2 CH 2 CH=CH-S-CH 3 Table 4. Insecticidal activity of fractions (0.06%) from hexane extract of F. foetida (roots) against grain aphid S. graminum after 24 h of exposure Treatment N a Mortality of females Corrected mortality Mortality of the subsequent (%) (mean ± SE) (%) nymphs, (%) (mean ± SE ) Fraction I 114 10.4 ± 6.35 a 1.54 0 a Fraction II 120 17.8 ± 7.94 a 9.67 0 a Fraction III 107 81.1 ± 2.37 b 80.2 25.7 ± 6.86 b Fraction IV 106 100 c 100 100 c Crude (base 200 100 c 100 100 c ethanolic) extract Control 108 4.5 ± 1.38 a - 0 a a N, number of aphids before treatment. *Means within columns followed by the same letter are not significantly different at the P = 0.05 (according to ANOVA and Tukey (HSD) test).
2012 Stepanycheva et al. : Aphicidal activity of Ferula foetida 23 Fig. 2. Mass-chromatogram of purified Fraction IV from F. foetida extract. (bis(3-methylthio- 2E-propenyl) disulfide). Unfortunately we could not get a signal for the molecular ion [M] + (m/z 238) required for unambiguous identification of this principle component. DISCUSSION The bioactivity of secondary metabolites of plants to arthropod pests had been well established. Secondary metabolites can be repellents, attractants, antifeedants and growth inhibitors (Akhtar and Isman, 2004a; Koul, 2005, 2008, 2012; Chermenskaya et al., 2010; Dubey, 2010; Pavela, 2010). For example, extracts from Lavandula spp. and Pinus spp. were highly toxic to the peach aphid Myzus persicae Sulz. (Homoptera: Aphididae) and the greenhouse whitefly Trialeurodes vaporariorum Westw. (Mateeva and Karov, 1983; Isman, 2000). Extract from Melia volkensii Gurke inhibited development of the noctuid moth larvae Pseudaletia unipuncta Haworth and Trichoplusia ni Hbn. (Akhtar and Isman, 2004b). Representatives of the genus Ferula, such as F. assafoetida L. are well-known medicinal plants (Ross, 2005), although an extract from F. assafoetida showed comparatively poor larvicidal activity to Fig. 3. Mass-spectrum of the major component of active Fraction IV from F. foetida extract.
24 Biopesticides International Vol. 8, no. 1 the yellow fever mosquito Aedes aegypti L. (Harve and Kamath, 2004). Previously we reported the aphicidal activity of an extract from F. foetida roots and its absence of deterrent activity (Chermenskaya et al., 2010). In the present study, we show that the activity of F. foetida extract (LC 50 = 0.0224%) is comparable to that of azadirachtin for some aphids (Lowery and Isman, 1994; Ekukole, 2006). The F. foetida extract exhibited toxicity to female aphids and a negative impact on the next generation. We have demonstrated the fumigant activity of a Ferula extract. Other researchers have shown fumigant properties of secondary plant metabolites to some greenhouse pests (Choi et al., 2003; Aslan et al., 2004; Choi et al., 2004). We have shown a disulfide to be the active principle in the crude ethanol extract of F. foetida through solvent partition with organic solvents. E-1-propenyl sec-butyl disulfide is the principal component of Ferula assa-foetida (Khajeh et al., 2005), and some other disulfides have similar properties (Hadavand Mirzaei and Hasanloo, 2009), probably because they are derivatives of the former compound (Kajimoto et al., 1989). No compounds described from Ferula assa-foetida extract corresponded with the mass spectrum shown in Fig. 3. The only probable candidate is bis(3- methylthio-2e-propenyl) disulfide, which was identified by Duan et al. (2002), although the insecticidal activity of this compound has not been investigated. Identification of this component appears to be a difficult task. Our study of fumigant activity of the ethanol extract from F. foetida revealed volatile compounds toxic to aphids. Plant substances having such properties may be safer for humans and environment than synthetic insecticides. They may be useful for management of agricultural pests (Tunc and Sahinkaya, 1998; Raja, 2000; Rajendran and Sriranjini, 2008). Acknowledgments This study was supported by ISTC project No. KR-1122.2. The authors are thankful to Georgy Lazkov (Biological-soil Institute NAS, Bishkek, Kyrgyz Republic) for collection and identification of plants. REFERENCES Abbott, W.S. (1925) A method of computing the effectiveness of an insecticide. J. Econ. Entomol., 18, 265 267. Akhtar, Y. and Isman, M.B. (2004a) Feeding responses of specialist herbivores to plant extracts and pure allelochemicals: effects of prolonged exposure. Entomol. Exp. Appl., 111, 201 208. Akhtar, Y. and Isman, M.B. (2004b) Comparative growth inhibitory and antifeedant effects of plant extracts and pure allelochemicals on four phytophagous insect species. J. Appl. Entomol., 128, 32 38. Aslan, I., Özbek, H., Çalmaur, Ö. and ªahin, F. (2004) Toxicity of essential oil vapours to two greenhouse pests, Tetranychus urticae Koch and Bemisia tabaci Genn. Ind. Crop Prod., 19, 167 173. Chermenskaya, T., Stepanycheva, E., Shchenikova, A. and Chakaeva, A. (2010) Insectoacaricidal and deterrent activities of extracts of Kyrgyzstan plants against three agricultural pests. Ind. Crop Prod., 32, 157 163. Choi, W.-I., Lee, E.-H., Choi, B.-R., Park, H.-M. and Ahn, Y.-J. (2003) Toxicity of plant essential oils to Trialeurodes vaporariorum (Homoptera: Aleyrodidae). J. Econ. Entomol., 96, 1479 1484. Choi, W.-I., Lee, E.-H., Choi, B.-R., Park, H.-M. and Ahn, Y.-J. (2004) Toxicity of plant essential oils to Tetranychus urticae (Acari: Tetranychidae) and Phytoseiulus persimilis (Acari: Phytoseiidae). J. Econ. Entomol., 97, 553 558. Duan, H., Takaishi, Y., Tori, M., Takaoka, S., Honda, G., Ito, M., Takeda, Y., Kodzhimatov, O.K., Kodzhimatov, K. and Ashurmetov, O. (2002) Polysulfide derivatives from Ferula foetida. J. Nat. Prod., 65, 1667 1669. Dubey, N.K. (2010) Natural Products in Plant Pest Management, CABI, Oxfordshire, UK, 312 pp. Ekukole, G. (2006) Some effects of an EC formulation of azadirachtin on important cotton pests in Senegal. J. Plant Prot. Res., 46, 313 323. Finney, D.J. (1977) Probit Analysis, 3rd edition.
2012 Stepanycheva et al. : Aphicidal activity of Ferula foetida 25 Cambridge University Press, Cambridge. Gao, P., Hou, T., Gao, R., Cui, Q. and Liu, S. (2001) Activity of the botanical aphicides 1,5-diphenyl- 1-pentanone and 1,5-diphenyl-2-penten-1-one on two species of Aphididnae. Pest Manag. Sci., 57, 307 310. Hadavand Mirzaei, H. and Hasanloo, T. (2009) Essential oil composition of root of Ferula assafoetida from two Iranian localities (Gonabad and Tabas). Asian J. Chem., 21, 6354 6358. Harve, G. and Kamath, V. (2004) Larvicidal activity of plant extracts used alone and in combination with known synthetic larvicidal agents against Aedes aegypti. Indian J. Exp. Biol., 42, 1216 1219. Henderson, C.F. and Tilton, E.W. (1955) Tests with acaricides against the brow wheat mite. J. Econ. Entomol., 48, 157 161. Isman, M.B. (2000) Plant essential oils for pest and disease management. Crop Prot., 19, 603 608. Kajimoto, T., Yahiro, K. and Nohara, T. (1989) Sesquiterpenoid and disulphide derivatives from Ferula assa-foetida. Phytochemistry, 28, 1761 1763. Khajeh, M., Yamini, Y., Bahramifar, N., Sefidkon, F. and Pirmoradei, M.R. (2005) Comparison of essential oils compositions of Ferula assafoetida obtained by supercritical carbon dioxide extraction and hydrodistillation methods. Food Chem., 91, 639 644. Koul, O. (2005) Insect Antifeedants. CRC Press, Boca Raton, Fl. Koul, O. (2008) Phytochemicals and insect control: An antifeedant approach. Crit. Rev. Plant Sci., 27, 1 24. Koul, O. (2012) Plant biodiversity as a resource for natural products for insect pest management. In: Geoff M. Gurr, Steve D. Wratten and Bill E. Snyder (eds.), Biodiversity and insect pests: Key issues for sustainable management. John Wiley & Sons Ltd., Australia, pp. 85 105. Lowery, D.T. and Isman, M.B. (1994) Insect growth regulating effects of neem extracts and azadirachtin on aphids. Entomol. Exp. Appl., 72, 77 84. Mateeva, A. and Karov, S. (1983) Studies on the insecticidal effect of some essential oils. Nauchni Trudove Vissha Selskostopanski Institute Vasil Kolarov, Plodiv, 28, 129 139. Pavela, R. (2010) Natural products as allelochemicals in pest management. In: N.K. Dubey (ed), Natural Products in Plant Pest Management, CABI, Oxfordshire, UK, pp. 134 148. Raja, N. and Ignacimuthu, S. (2000) Use of botanicals in bruchid control of stored pulses. J. Sci. Ind. Res. India, 59, 214 220. Rajendran, S. and Sriranjini, V. (2008) Plant products as fumigants for stored product insect control. J. Stored Prod. Res., 43, 126 135. Ross, I.A. (2005) Medicinal Plants of the World, Vol. 3: Chemical Constituents, Traditional and Modern Medicinal Uses, Humana Press Inc., Totowa, NJ. Ryan, M.F. (2002) Plant chemicals in pest control, In: M.F. Ryan (ed.), Insect Chemoreception. Fundamental and Applied, Kluwer Academic Publishers, New York, pp. 193 222. Soulé, S., Güntner, C., Väzquez, A., Argandoña, V., Moyna, P. and Ferreira, F. (2000) An aphid repellent glycoside from Solanum laxum. Phytochemistry, 55, 217 222. Stepanycheva, E.A., Shchenikova, A.V., Chermenskaya, T.D. and Pavela, R. (2006) Fumigant activity of plant essential oils to Tetranychus urticae Koch. (Acarina: Tetranychidae). VIIIth European Congress of Entomology, Izmir, Turkey, September 17-22, Abstract book, p. 128. Tunc, I. and Sahinkaya, S. (1998) Sensitivity of two greenhouse pests to vapours of essential oils. Entomol. Exp. Appl., 86, 183 187. Van Emden, H.F. and Harrington, R. (2007) Aphids as Crop Pests, CABI, Oxfordshire, UK. Accepted 10 March 2012