Bioprospecting of sweet potato (Ipomoea batatas) vines for the management of Cylas formicarius (Sweet potato weevil)

Introduction

Detrimental effects of synthetic insecticides on health, environment and organisms have necessitated the exploration of alternative strategies for insect control. The insect attractants represent an important link within these systems. Semiochemicals play an important regulative role in the life of insects in addition to other communication channels as optic, acoustic, or tactile communications (Howse et al., 1998; El-Shafie and Faleiro, 2017; Annie et al., 2017) [9,27] . In the field of sensory ecology, semiochemicals (info-chemicals) are classified as pheromones (intra specific) and allelochemicals (inter specific); the latter of which include kairomones (benefit the receiver), allomones (benefit the sender), and synomones (benefit both) (Dusenbery, 1992; Annie et al., 2017) [8] . Biologically active volatile phytochemicals represent an important means of communication within plants and between plants and other organisms in their environment (Hans, 1995). Phytochemicals also play critical roles in insect behaviour, modulating host finding, feeding, and oviposition responses for certain species where they may function as attractants, repellents, feeding and oviposition stimulants, deterrents, and toxicants (Tumlinson, 1988; Metcalf and Metcalf, 1992) [23,40] . Phytochemicals play critical roles in facilitating the development of host-plant resistance in long-run (Peshin and Dhawan, 2009) [28] . Sweet potato is the seventh most important crop in the world with a total production of 92 million metric tonnes in the year 2018 (FAO, 2019). Despite the crop's economic importance, widespread sweet potato weevil infestation results in losses of millions of dollars annually (Talekar, 1994) [39] . By being a major staple crop, sweet potato enjoys a wide literature support in its cultivation practice. (Hue and Low, 2015) [16] summarise most of the management Measures on the crop loss by C. formicarius. Even in this context, farmers are in big menace with this weevil attack. The level of infestation in the different sweet potato genotypes was reported to be related to the concentration of kairomones in the periderm of the tubers (Son et al., 1990) [35] . For example, Boehmeryl acetate-a kairomone identified in sweet potato tuber surface acts as an ovipositional stimulant for female weevils (Son et al., 1990) [35] . The present study aims at the extraction and isolation of phytochemicals from sweet potato vines for sweet potato weevil management.

Materials and Methods

Extraction techniques used for the isolation of kairomones from sweet potato

Sweet potatoes were grown in the year 2018 at the ICAR-Central tuber crops research institute (ICAR-CTCRI) using standard sweet potato production practices (Granberry and Kelley, 1987) [12] . Following techniques were employed for the extraction of possible kairomones from sweet potato (Ipomoea batatas Var. Sree Arun). All extractions were done from sweet potato plants maintained in an insect free condition (poly house) for eliminating the possible involvement of pheromones by the natural weevil population.

a. Solvent extraction from dried vines

Vines of fresh sweet potato were thoroughly cleaned and dried in hot air oven maintained at 50 °C for three days. It was then coarsely powdered using a conventional mixer grinder. It weighed approximately 450g. The powdered material was subjected to repeated extraction three times with hexane, acetone, methanol and double distilled water (dd_water) (Make: Merk) (500 ml × 3) at room temperature. The extracts were then concentrated under reduced pressure using a Buchi R-200 rotary evaporator (Buchi, Labortechnik, Switzerland).

b. Solvent extraction from fresh vines

Fresh vines of sweet potato, chopped in one cm length were used for repeated extraction three times with hexane, acetone, methanol and dd_water (Make: Merk) (500 ml × 3) at room temperature (30±2 °C). After each extraction, the solvent was removed by vacuum evaporation so as to obtain the crude extract in a concentrated form by using a Buchi R-200 rotary evaporator.

c. Cold extraction from fresh veins

For the study an ideal solvent with a mid-polarity need to be ascertained so that one can assure representative chemical species from both the ends of polarity scale. Fresh sweet potato vein cuttings (250 g) were kept in acetone (medium polar solution ̴ PI=5.1) for three days (500 ml × 3 at 25 °C) and was cold extracted. After extraction, the solvent was removed by using a lyophilized (iL Shin Bio Base TF05503 freeze dryer) so as to obtain the crude extract in a concentrated form. The crude extract was then analyzed by gas chromatography-mass spectrometry (GC-MS) for compound profiling.

d. Purification and fractionation of crude extracts by automated column-chromatography (acc)

The packing material silica gel (60-120 mesh size) is slurried with hexane and poured in column (30cm long and 2cm dia.) to get a homogeneous packing of the silica gel.

Application of Acetone concentrated crude extracts (10 ml), from all the three extraction technique were employed for further fractionation under ACC. When the respective crude extract has been absorbed on the top of the column, the space above it is filled with the first eluvent (hexane ̴ 50 ml) with no time for air entrainment in the silica. From the column run, the fractions thus obtained are collected in separate vials with a fixed time interval of 5 minutes using the time automation. Elution started using hexane and was followed by different solvents such as diethyl ether, dichloro methane, acetone, ethanol, methanol and dd_water. Different eluvents (50 ml) were continuously poured sequentially according to increasing order of their polarity and respective fractions were separated. There were two fractions for each solvent and were named as respective solvent fraction A and B. Throughout the experiment the fractions were flash evaporated or lyophilised until all the solvent evaporates or to a minimum quantity of 0.25 ml. All the liquid fractions isolated using the above given techniques were stored in-20 °C in air-tight 10 ml vials (Make: Tarson).

Rearing of cylas formicarius

Infested sweet potato tubers were collected from ICAR-CTCRI field and were maintained in bottles with spread muslin cloth as lid to ensure enough ventilation. We observed maximum emergence in 10±2 days. All the freshly emerged weevils in this period were reared in another set of well-ventilated bottles (rearing bottles) with uninfested tuber as food source (65 nos. per bottle; with 1:1 sex ratio) at 30 °C and 70±5 RH.

Bio activity of kairomones from sweet potato a. Olfactory studies

Both the crude liquid fractions (isolated by the method of solvent extraction from dried vines, solvent extraction from fresh vines & cold extraction from fresh vines) and the purified fractions from all the three extraction techniques were screened for olfactory response on C. formicarius. 'Y' shaped lactometer is used for the study (Fig. 1). From the crude fraction, 10 µl, 50 µl, 75 µl, 100 µl and from the purified fraction, 75 µl were used. Different fractions were applied in cotton balls (1g) and were kept on one of the two sample inlets, and the other inlet was considered as control with cotton soaked with dd_water. In extracts, were solvent fraction suspected even after the maximum possible flash evaporation, respective pure solvents were used as control. At time zero of the experiment, 52 weevils (26 nos. of male: 26 nos. of female) were released in the insect release chamber and were continuously monitored for their preference towards the fraction holding chamber (test). Numbers of weevils responded positively towards the tests and the average time taken were calculated for each sample. All experiments were replicated thrice. After each sample screening, the lactometer were assured to wash properly so that no residue will interfere the subsequent screening. For every trial fresh batches of weevils (from different rearing bottles) were used to eliminate any possible replication bias. Effective response of male and female weevils can be calculated by using the formula; Effective response (ER) value of male or female weevil = No. of male or female weevil preferring test-No. of male or female weevil preferring control b. Field studies In the field (10 × 20 m isolated fields of sweet potato) three replication traps (with same test extract-T1, T2 & T3) and one positive control (PC) trap with sex pheromone lure of C. formicarius (Make: Pherobio) where kept on all the four corners (Fig. 2). Attractions of weevil towards the traps were monitored for different time periods (1h, 2h, 5h, 8h, 12h and 24h). Every distinct extracts were tested in different such sweet potato fields of ICAR-CTCRI, Thiruvananthapuram (8.5241°N, 76.9366°E) on climatically compatible days. All the weevils collected in traps were preserved separately as batches in 70% ethanol and screened for male, female ratio.

Molecular characterization of acetone extract from sweet potato a. Gas chromatography (gc)-mass spectrometry (ms)

Gas chromatogram was generated for the acetone crude fraction by using cold extraction technique as suggested by Wang and Kays (2002) [45] . Gas chromatography (HP6890; Agilent Technologies USA Ltd) directly linked to a HP5973 mass selective detector (Agilent Technologies) operated in electron impact mode (source temperature 230 °C; transfer line 250 °C). The HP-5 MS phenyl methyl siloxane non polar capillary column (0.25 mm × 30 m × 0.25μ.) at a maximum temperature of 350 °C (Agilent part No 190915-433) was used for the separation of fractions. The mobile phase was helium with very high purity (Praxair India Ltd.) and was passed through the universal trap for removing the contaminants. The split inlet was used with split ratio of 50:1 and inlet temperature of 280 °C. The oven temperature program was set at 70 °C with 2 minutes hold and a ramp of 10 °C min -1 till 260 °C and held for 5 min with column flow of 1 ml/min. The mass spectral detector was maintained at a temperature of 280 °C with the interface temperature of 230 °C. The mass spectra created using the MS was compared with the Wiley mass spectral library powered by Wiley, 2012 and NIST, 2012 version.

b. Gas chromatography-mass spectrometry coupled with electro antennogram analysis (gc-ms-ms-eag)

Fractions that show high bioactivity both in laboratory and in field trial were recorded on the extirpated antennae of both sexes of 30 day-old virgin C. formicarius. Electroantennogram (EAG) recordings were made with Ag-AgCl glass microelectrodes filled with ringer solution (7.2 g NaCl, 0.37 g KCl, 0.17 g CaCl2, pH 7.3-7.4 in one litre water). The weevils were anaesthetized by chilling (at-20 °C for 1 minute and 40 seconds), and its antennae were excised from the head with a microscalpel. It was inserted into the electrode and signals were passed through a high impedance amplifier and analyzed using a customized software package (Syntech). In the coupled GC-MS-MS-EAG system, the effluent from the GC column is directly passed through the antennal preparation. Separation of volatiles was achieved in a GC (Agilent Technologies, 6890N) equipped with a cold on-column injector, a flame ionization detector (FID), and MS-MS (TQ_8030 SHIMADZU instrument & Syntech instrument) using an HP-1 column (50 m, 0.32 mm i.d. 0.52 μm f.t.). The oven was maintained at 30 °C for 2 min. and then programmed for increments at 5 °C min -1 to 250 °C. The carrier gas was helium. Outputs from the EAG amplifier and the FID were monitored simultaneously and analyzed using the MS-MS software package. Peaks eluting from the GC column were considered active if they elicited EAG response in three or more coupled runs. Authentic standards were tested using a delivery system which employed a filter paper strip in a disposable Pasteur pipette cartridge (Wad hams et al., 1982) [42] . The stimulus (2 s duration) was delivered into a purified airstream (0.5l/min) flowing continuously over the preparation using a stimulus controller (Syntech CS02). Samples (10 μl) of the standard solution of test compounds (1 mg/ml in redistilled methanol) were applied in the cartridge. Acetone (10 μl) was given for the control stimulus. Five replicates were performed for each sample using antennae from 5 different weevils. GC-MS-MS-EAG responses of both male and female C. formicarius were recorded.

Analysis

All statistical analyses were conducted using the software R Studio, version 3.3.2 (58) and by Microsoft Excel 2010. Names of statistical tests and levels of significance used are given in the appropriate figure legends. In general, the Duncan's Multiple Range Test (P ˂ 0.05) was used for the comparative analysis and for the significance studies. For the effective response comparison studies, trend lines were drawn and linear equations were generated since the response can be an expected continuum for further higher doses. Proportion of the variance were also provided in the legends.

Results and Observations

Solvent extraction from dried vines resulted, 0.75 ml of crude hexane extract, 1.25 ml of crude acetone extract, 2 ml of crude methanol extract and finally 3.25 ml of crude water extract. Bioactivity study on the olfactory response of both male and female C. formicarius over the crude fractions isolated by solvent extraction from dried vines shows high effective response (ER) values in highly polar fractions (Figure 3). By using the method of solvent extraction from fresh vines, 2.25 ml of crude hexane extract, 6 ml of acetone extract, 4.5 ml of methanol extract and 7 ml of water extract were obtained. Olfactory response of C. formicarius over the crude fractions by solvent extraction method from fresh veins shows an ER value of 16.7 by the male weevil for the acetone extract at a dose of 100 µl and the highest female ER value of 12.7 at a dose of 100 µl. (Figure 4). Cold extraction from fresh vines resulted, 1.75 ml of crude hexane extract, 3.25 ml of crude acetone extract, 4.25 ml of crude methanol extract and 5.50 ml of crude water extract. Study on olfactory response of the weevil over the crude fractions isolated by cold extraction from fresh vines shows the highest effective response vale for male weevils as 18.7 (Figure 5). After the fractionation through automated column chromatography a total of 14 fractions were result out when 7 different solvents were employed. All the fractions were scrutinized under bio assay for olfactory response of both male and female C. formicarius and the following results were obtained and were described in table 1. From the field trials total numbers of C. formicarius collected in response to different crude extracts over different time periods were represented in figure 6. Positive control yields the highest number of weevils (203±33.8 nos) on trap at the second hour of the experiment. Crude acetone extract from fresh veins at a doss of 100 µl shows the best result among test extracts by trapping 39±7.5 weevils with a time period of 24 hrs. (Figure 6). Crude extract profile (Figure 7) was generated for the acetone fraction from the cold extraction technique using gas chromatography and mass spectroscopy. Twenty four major compounds were detected, of which six are reported to have kairomonal activity (Table 2). From GC-MS-MS-EAG (Figure 8), five different compounds show clear response or stimuli in the antennae of C. formicarius irrespective of sex and three different compounds were observed as specific attractant to male weevils. No specific compound was screened with specific attraction to female weevils (Table 2).

Discussion

Even though 'pests' are also part of nature and is not a constant status as descried by (Krishnan et al., 2019) [21] , it is essential to check there population explosion through novel methods in commercially cultivated fields. Semiochemicals play an important role in this context either as repellents or as attractants. The sensillae present on the weevil antennae enables the insects for chemical as well as thermal reception (Visser, 1983; Krishnan et al., 2017a) [19,20,41] Technological advances in analytical instrumentation enable us to find out several hundred volatile compounds emanating from fruits and vegetables (Wang and Kays, 2000) [44] . Best yield was observed for many phytochemical extraction studies by employing the method of solvent extraction from dry plant materials. Waksmundzka et al., (2008) [43] and Arceusz et al., (2013) [2] reported a better response by this method from medicinal plants. (Sultana et al., 2009) [36] describes the importance of specific drying of plant materials in extraction technique for the functional activity of phytochemicals. The effective response of sweet potato weevil over the solvent extracts from dried vines in this background was not observed to be effective for the isolation of kairomone chemical species. By this method effective response value of + 3 and-3 was observed to be the [34] ; 1, 8-di bromo octane (Svirskaya and Leznoff, 1984) [38] ; 1-bromo do decane (Rajadhyaksha, 1982) [32] ; Phenylacetaldehyde (Wang and Kays, 2002) [45] ; Methyl geranate (Wang and Kays, 2002) [45] and Nerolidol iso butyrate (Aldrich et al., 1993) [1] are reported to have kairomonal activity. The compounds identified in this study included heterocyclic compounds, aldehydes, alcohols, ketones, aromatic hydrocarbons, terpenoids, and acids and included 18 compounds not reported previously from sweet potato.

Various saccharides with a caramel like aroma, was demonstrated previously to be an important component in the aroma of baked sweet potatoes (Sun et al., 2019) and the current study confirmed its importance even in fresh vines by the presence of compounds like 6-Acetyl-.beta.-dmannose; d-Glycero-d-ido-heptose; l-Gala-l-ido-octose . As described by (Tressl et al., 1981, 1989) [30,31] these simple sugars can act as an efficient attractant when they are heated up in vines with the exposure to direct sunlight and in the presence of amino acids. Phenylacetaldehyde gives a perfume aroma note and is formed via Strecker degradation of phenylalanine. It is found widely in many flavors including fruit, chocolate, and honey (Scarpellino and Soukup, 1993) [33] . Phenylacetaldehyde was also observed to be as a major flavouring factor in cooked beef (Gasser and Grosch, 1990; Münch et al., 1997) [11,25] . Methyl geranate, a relatively little known monoterpene ester in foods, had a pronounced influence on the aroma of baked sweet potato.

From our current study, compounds such as 3,4-Hexanediol, 2,5-dimethyl and 9-Acetoxynonanal were observed to be key components in sweet potato vines. The exponential growth in the population of a weevil is mainly facilitated by the availability of nutrition for a second reproductive cycle (Krishnan et al., 2016; Krishnan et al., 2017b) [18,19,20] For many species of weevils kairomones act as the response stimuli to direct the pest towards a new host or field. But the characteristic aroma of a number of crops are often derived from only a very small number or even a single volatile compound (Kays, 1991) [17] which might be a small fraction in a cocktail of chemicals, the correlation of volatile constituents with sensory analysis (such as GC-MS-MS-EAG) is essential to determine which odor-active compounds are important (Wang and Kays, 2000) [44] . Study by this technique was an explosion in the semiochemicals study and the legacy was still going on. Even though studies in all class of insect models are available in literature, most of them were from the group Lepidoptera (Hall et al., 1981; Nesbitt et al., 1985; Beevor et al., 1986, 1999; Cork et al., 1989) [3,4,7,14] , Orthoptera (Carlson et al., 1978; Bursell et al., 1988; Puri et al., 2006) [5, 6, 26, 29] and Coleoptera as described Inn. Mayer and McLaughlin (1991) [22] . All the eight compounds that we observe to be kairomone species where new and were first report, while the compound 6-Acetyl-beta-d-mannose was previously reported in Aspergillus fumigatus by Mohammad and Ghaidaa (2016)

The current study clearly indicates the presence of novel phytochemicals present in the sweet potato vines; this may potentially lead for the generation of an attraction lure in the near future. 5 out of the 8 compounds shows sex neutral attraction in the GC-MS-MS-EAG study. Specific purification and field standardisation studies on these five compounds will replace the existing sex pheromone attractants of C. formicarius.

Conflict of interest

Authors declare no conflict of interest as defined by this publishing group, or other interests that might be perceived to influence the results and/or discussions reported in this article.

Acknowledgement

AcknowledgementAuthors wish to acknowledge; Dr. Raghavendra Anjanappa, National Bureau of Agricultural Insect Resources (NBAIR), Bangalore, India for facilitating the EAG study Dr.Shashikumar, CSIR-National Institute for Interdisciplinary Science and Technology (NIIST), for his support in the GC-MS study

References

1. Aldrich JR, Waite GK, Moore C, Payne JA, Lusby WR, Kochansky JP. Male-specific volatiles from Nearctic and Australasian true bugs (Heteroptera: Coreidae and Alydidae). J Chem Ecol. 1993; 19:2767-2781.
2. Arceusz A, Wesolowski M, Konieczynski P. Methods for extraction and determination of phenolic acids in medicinal plants: a review. Nat Prod Common. 2013; 8:1821-1829.
3. Beevor PS, Cork A, Hall DR, Nesbitt BF, Day RK, Mumford JD. Components of female sex pheromone of cocoa pod borer moth, Conopomorpha cramerella. J Chem Ecol. 1986; 12:1-23.
4. Beevor PS, Youm O, Hall DR, Cork A. Identification and field evaluation of components of female sex pheromone of millet stem borer, Coniesta ignefusalis. J Chem Ecol. 1999; 25:2643-2663.
5. Bursell E, Gough AJE, Beevor PS, Cork A, Hall DR, Vale GA. Identification of components of cattle urine attractive to tsetse flies, Glossina spp. (Diptera: Glossinidae). Bull Entomol Res. 1988; 78:281-291.
6. Carlson DA, Langley PA, Huyton P. Sex pheromone of the tsetse fly: isolation, identification, and synthesis of contact aphrodisiacs. Science. 1978; 201:750-753.
7. Cork A, Murlis J, Megenasa T. Identification and field testing of additional components of female sex pheromone of African armyworm, Spodoptera exempta (Lepidoptera: Noctuidae). J Chem Ecol. 1989; 15:13491364.
8. Dusenbery DB. Sensory ecology: how organisms acquire and respond to information. New York: W.H. Freeman; 1992.
9. El-Shafie HAF, Faleiro JR. Semiochemicals and their potential use in pest management. In: Biological control of pest and vector insects. Rijeka: InTech; 2017. p. 322.
10. Food and Agriculture Organization. Sweet potato crop production. Rome: FAO; 2019.
11. Gasser U, Grosch W. Primary odorants of chicken broth. Z Lebensm Unters Forsch. 1990; 190:3-8.
12. Granberry DM, Kelley WT. Commercial sweet potato production. Athens (GA): University of Georgia Cooperative Extension Service; 1987. Bulletin No. 6.
13. Deutsch A, Guédot C. Utilizing insects' sense of smell for pest management. Madison (WI): University of Wisconsin Extension; 2017. p. 1-8.
14. Hall DR, Beevor PS, Cork A, Lester R. Female sex pheromone of the maize stalk-borer Busseola fusca (Fuller) (Lepidoptera: Noctuidae): identification and initial field trials. Zimbabwe J Agric Res. 1981;19:111122.
15. Linskens HF, Jackson JF. Plant volatile analysis. Berlin: Springer; 1995. Vol. 19.
16. Hue SM, Low MY. An insight into sweet potato weevils management: a review. Psyche. 2015;2015:849560.
17. Kays SJ. Postharvest physiology of perishable plant products. New York: Van Nostrand Reinhold; 1991.
18. Krishnan JU, et al. Stage and age specific life-table for Odoiporous longicollis Oliver (Curculionidae: Coleoptera) under laboratory conditions of minimum stress. Int J Zool Investig. 2016;2:17-23.
19. Krishnan JU, et al. Discovery of elytral thermo-receptor sensilla in three species of coleopterans: a comparative morphology study. Int J Entomol Res. 2017;5:11-16.
20. Krishnan JU, Jayaprakas CA, Ajesh G, Lekshmi NR, Sankar DS. Life-table of Odoiporus longicollis Oliver (Coleoptera: Dryophthoridae) under varying temperature ranges, an in-vitro study. Entomon. 2017;42:269-274.
21. Krishnan JU, Jayaprakas CA, Harish ER, Rajeswari LS. Banana (Musa spp.)-an unseen umbrella crop? Insect diversity on Musa spp. in the Indo-Pacific region. Orient Insects. 2019;53:1-13.
22. Mayer MS, McLaughlin JR. Handbook of insect pheromones and sex attractants. Uppsala: International Information System for the Agricultural Sciences and Technology; 1991.
23. Metcalf RL, Metcalf ER. Plant kairomones in insect ecology and control. London: Chapman and Hall; 1992.
24. Al-Jassani MJ, Mohammed GJ, Hassan IH. Analysis of bioactive chemical compounds of Aspergillus fumigatus and evaluation of antifungal activity using twenty-seven medicinal plants. Int J Pharm Bio Sci. 2016;7:191-204.
25. Münch P, Hofmann T, Schieberle P. Comparison of key odorants generated by thermal treatment of commercial and self-prepared yeast extracts: influence of the amino acid composition on odorant formation. J Agric Food Chem. 1997;45:1338-1344.
26. Nesbitt BF, Beevor PS, Cork A, Hall DR, Murillo RM, Leal HR. Identification of components of the female sex pheromone of the potato tuber moth, Scrobipalpopsis solanivora. Entomol Exp Appl. 1985;38:81-85.
27. Howse PE, Stevens IDR, Jones OT. Insect semiochemicals and communication. Dordrecht: Springer; 1998.
28. Peshin R, Dhawan AK. Integrated pest management. Dordrecht: Springer; 2009.
29. Puri SN, Mendki MJ, Sukumaran D, Ganesan K, Prakash S, Sekhar K. Electroantennogram and behavioral responses of Culex quinquefasciatus (Diptera: Culicidae) females to chemicals found in human skin emanations. J Med Entomol. 2006;43:207213.
30. Tressl R, Bahri D, Engel KH. Formation of flavor components in roasted coffee. In: Lipid oxidation in fruits and vegetables. Berlin: Technische Universität Berlin; 1981. p. 213-232.
31. Tressl R. Formation of flavor components in roasted coffee. In: Lipid oxidation in fruits and vegetables. Berlin: Technische Universität Berlin; 1989. p. 285301.
32. Rajadhyaksha VJ. Method for improved plant pest control. 1982. p. 1-30.
33. Scarpellino R, Soukup RJ. Key flavors from heat reactions of food ingredients. Washington (DC): American Chemical Society; 1993.
34. Shibata S, Kuwahara Y, Sato M, Matsuyama S, Suzuki T. Sex pheromone activity of 2-hydroxy-6methylbenzaldehyde analogs against males of two astigmatid mites, Aleuroglyphus ovatus and Acarus immobilis. J Pestic Sci. 1998;23:34-39.
35. Son KC, Severson RF, Arrendale RF, Kays SJ. Surface chemical differences between sweetpotato lines with varying levels of resistance to the sweetpotato weevil. J Am Soc Hortic Sci. 1990;115:696-699.
36. Sultana B, Anwar F, Ashraf M. Effect of extraction solvent/technique on the antioxidant activity of selected medicinal plant extracts. Molecules. 2009;14:21672180.
37. Sun JB, Severson RF, Schlotzhauer WS, Kays SJ. Identifying critical volatiles in the flavor of baked 'Jewel' sweetpotatoes [Ipomoea batatas (L.) Lam.]. J Am Soc Hortic Sci. 1995;120:468-474.
38. Svirskaya PI, Leznoff CC. Syntheses of unconjugated (Z,Z)-diolefinic insect pheromones on insoluble polymer supports. J Chem Ecol. 1984;10:321-333.
39. Talekar NS. World bibliography of sweetpotato weevils. Shanhua, Taiwan: Asian Vegetable Research and Development Center; 1994.
40. Tumlinson JH. Contemporary frontiers in insect semiochemical research. J Chem Ecol. 1988;14:21092130.
41. Visser JH. Differential sensory perceptions of plant compounds by insects. In: Bernays EA, editor. Insectplant interactions. Boca Raton (FL): CRC Press; 1983. p. 215-230.
42. Wadhams LJ, Angst ME, Blight MM. Responses of the olfactory receptors of Scolytus scolytus (F.) (Coleoptera: Scolytidae) to the stereoisomers of 4methyl-3-heptanol. J Chem Ecol. 1982;8:477-492.
43. Waksmundzka-Hajnos M. Effect of sample-preparation methods on the quantification of selected flavonoids in plant materials by high performance liquid chromatography. Acta Chromatogr. 2008;20:475-488.
44. Wang Y, Kays SJ. Contribution of volatile compounds to the characteristic aroma of baked "Jewel" sweetpotatoes. J Am Soc Hortic Sci. 2000;125:638-643.
45. Wang Y, Kays SJ. Sweetpotato volatile chemistry in relation to sweetpotato weevil (Cylas formicarius) behavior. J Am Soc Hortic Sci. 2002;127:656-662.