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植物保護學會會刊 47：281-281, 2005 281

* Corresponding author. E-mail: [email protected]

Bioassays of the ovipositional responses of the tomato fruitworm, Helicoverpa armigera (Lepidoptera: Noctuidae), to Solanum viarum

leaf extracts

Aliou Diongue1, Po-Yung Lai1*, Chieh Lin2, and Jenn Sheng Hwang3

1 Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, Neipu, Pingtung 912, Taiwan (ROC)

2 Department of Environmental Science and Engineering, National Pingtung University of Science and Technology, Neipu, Pingtung 912, Taiwan (ROC)

3 Applied Toxicology Division, Taiwan Agricultural Chemicals and Toxic Substances Research Institute (TACTRI), Wufong, Taichung 413, Taiwan (ROC)

(Accepted for publication: Oct. 24, 2005)

ABSTRACT

Diongue, A., Lai, P. Y.*, Chieh, L., and Hwang, J. S. 2005. Bioassays of the ovipositional responses of the tomato fruitworm, Helicoverpa armigera (Lepidoptera: Noctuidae), to Solanum viarum leaf extracts. Plant Prot. Bull. 47: 281 – 292

Effects of Solanum viarum leaf extracts on stimulating the oviposition of Helicoverpa armigera (Hubner) were evaluated in 2-choice I-tubes bioassay (250 cm long x 30 cm in diameter). Different types of solvent-extracts were placed on filter paper (90 mm) and compared their oviposition stimulation against the check with only the respective solvent in each treatment. Oviposition was evaluated by the mean number of eggs laid on both net and cylinder. There were significant difference in the mean number of eggs laid on the net containing the leaf extracts with acetone and hexane airtight acrylic, hexane and methylene chloride dipping. However, the

282 植物保護學會會刊第 47 卷第 4 期 2005

aqueous extract trapped in diethyl ether from the steam distillation appeared to inhibit the oviposition of H. armigera. From the mean number of eggs laid on the cylinder, it showed that only the dipping in hexane and methylene chloride significantly stimulated the oviposition of H. armigera but no significant difference was observed in the extracts from the other methods.

(Key words: Helicoverpa armigera, Solanum viarum, leaf extract, oviposition stimulant)

INTRODUCTION

The selection and acceptance of host-plants as substrate for oviposition or as a food source for most herbivorous insects are based on chemical stimuli from the plants(19). Similarly, Honda(8) and Schoonhoven et al.(22) stated that there is growing evidence that host-finding in moths was largely guided by secondary plant metabolites. Nowadays, several studies using laboratory bioassays have demonstrated the role of plant volatiles in the orientation, host acceptance and/or rejection and the oviposition of various moths to their host plants. The number of compounds identified as oviposition stimulants or as components of a stimulatory blend has recently increased. Several studies have focused on surface chemicals affecting oviposition choice of the genus Heliothis. In the case of the corn earworm, Helicoverpa zea (Boddie), three structurally related sesquiterpene carboxylic acids were identified as stimulants from a wild tomato accession. Mitchell & Health(16) have also tested the H. subflexa oviposition response to methanolic extracts of ground cherry. The female of H. virescens was found to respond positively to cuticular extracts of tobacco leaves, and two isomeric duvane tripenes were identified as active

constituents(10). Females of the moth, Acrolepiopsis assectella, were attracted to leek volatiles in an olfactometer bioassay(23). In this study we investigated, through different extractions methods, the stimulatory effect of Solanum viarum leaf extract on the oviposition of H. armigera.

Solanum viarum is an herbaceous plant largely described in the Florida as a noxious weed and ironically called “the plant from the hell” because of its high potential of invading many perennial grasses pastures(18) and cultivated fields crops: such as in sugarcane as well as in vegetable fields and citrus plantations(17). Described as a medicinal crop, its roots served as tooth medicine in Chad(5). There are reports of the deliberate cultivation of the plant for the production of solasodine, a precursor in the industrial production of steroid hormones useful in the treatment of cancer(1). However, the potential of this plant to be used as a trap crop for H. armigera (TFW) female (Talekar, pers. comm.) leads to our investigation of S. viarum leaf extract on the oviposition of H. armigera.

MATERIALS AND METHODS

Helicoverpa armigera Rearing Helicoverpa armigera eggs were

Oviposition of Helicoverpa on Solanum viarum 283

initially obtained from the Taiwan Agricultural Chemicals and Toxic Substances Research Institute (TACTRI), Wufeng, Taichung, Taiwan. The colony was maintained at the Department of Tropical Agriculture and International Cooperation (DTAIC) laboratory, in the National Pingtung University of Science and Technology (NPUST), Taiwan, in continuous culture. Moths were grouped into 15 pairs each and placed in 33-cm-high x 15-cm-diameter acrylic cylinders, the inner surface of which was lined with rough tissue paper to facilitate oviposition. A cotton plug, soaked in a solution containing 400 ml honey, 60 ml Taiwan beer, 20 g ascorbic acid, 12 g methyl para-benzoic acid, and 1000 ml distilled water, was hung from the top of the cylinder to serve as food and a mating stimulant. The cylinders were placed in a laboratory under ambient light. Eggs laid on the tissue paper were collected daily and disinfected with 7 % formaldehyde for 25 min and rinsed in clean water for 2 h. After disinfection, eggs were maintained at 27 ± 2 °C, 68 ± 2 % RH and a 12: 12 (L: D) photoperiod. Newly hatched larvae were reared on a wheat germ-based artificial diet

(12). From the 3rd instar onwards, larvae were reared individually in separate containers. The containers and diets were changed every 2 days. Pupae were sorted by sex, and male and female pupae were placed in separate containers. Adults were allowed to emerge, and the emergence date was recorded. Two newly emerged females were paired with 2 newly emerged males for mating for 2 days in a cylinder as described above, and 2 day-old gravid females along with males were used for the experiments.

Solanum viarum production Solanum viarum seeds were obtained

from the Asian Vegetable Research and Development Center (AVRDC), Taiwan. Seeds were sown individually in trays containing a standard greenhouse mixture of 1:3 by volume of vermiculite (South Seavermiculite and Perlite Co., The Netherlands) and peat moss growing media (Know-You Seed Co., Taiwan). After 2 months, the seedlings were transplanted to plastic pots of 25 cm in diameter and 20 cm in height. The plants were fertilized once a week with Nitrofoska N: P: K: Mg (20:19:15: 0.5), (BASF, Germany) at 3g / 1000 ml. New plants were sown every month to ensure a sufficient supply of leaf material for chemical extraction.

Solvents Hexane (n-hexane) and acetone (85%,

HPLC/spectro HS-2722) used in the extraction were purchased from Tedia company Inc., USA. Methylene chloride (84 %, HPLC/spectro) and diethyl ether were purchased from Baker analyzed®, J. T. Baker, Inc. Phillipsburg, USA.

Volatile collection 1. Airtight acrylic chamber

To collect the volatile from 55 days old S. viarum at vegetative stage, an airtight acrylic chamber (31×31×41 cm) was used (Fig. 1.). Two small fans were attached inside the acrylic walls of the chamber. There were two holes (inlet and outlet) on the top of the chamber for the flow of air moving in and out. A glass tube was fixed in the inlet hole that opened half way inside the chamber. The outlet tube of the acrylic chamber was


Fig. 1. Airtight acrylic extraction method used in AVRDC to extract Solanum viarum volatiles (A & B; Aspirator containing respectively 600 ml hexane and 600 ml acetone)

connected by Tygon tubing in the inlet of the first aspirator containing 600 ml hexane. The outlet of the first aspirator was connected to the inlet of the second aspirator containing 600 ml acetone. The outlet of the second aspirator was connected to a vacuum pump. Volatile extracts were collected from potted S. viarum with 8 leaves. The whole pot was covered with aluminum foil to avoid volatile from the vermiculite being extracted in our system. The volatile sucked by vacuum, was first dissolved in the hexane column and, then, in the acetone column. This procedure was continued for 24 h. After 24 h, the extract were concentrated to 5 ml in the rotary evaporator under 28 ± 2 °C. The extract was refrigerated at -20 °C until needed.

2. Dipping methods

The methodology, modified from Jackson et al.(10), was aimed at extracting the cuticular leaf components. Solanum viarum plants, 55 days (8 leaves) after being

transplanted, were cut at 10-15 cm above the ground. The leaves were dipped separately 4 times in each of the two 2-liter beakers containing 600 ml of hexane or methylene chloride. The duration of dipping was 5 sec each. The solution was filtrated through folded filter paper containing Sodium sulfate (Na2SO4) and then concentrated by rotary evaporation to 5 ml. The extract was refrigerated at -20 °C until needed.

3. Steam distillation

The distillation methodology was modified from Konstantopoulou et al.(13). Solanum viarum leaves (200 g, of vegetative stage) were steam distilled for 50 min in a steam distillation with water-cooled oil receiver. The volatiles carried by water vapors were condensed and trapped in a layer of diethyl ether. The ether layer was dried over magnesium sulfate to remove residual water, and concentrated in a fume hood to 5 ml under a gentle stream of


nitrogen, and stored at -20 °C.

The bioassay set-ups The behavioral response of mated H.

armigera adults toward S. viarum leaf extract was observed in an I-tube olfactometer. The I-tube in a clear acrylic cylinder in the length of (250 cm×30 cm) was used during the test. An amount of 0.25 ml for the steam distillate or 0.6 ml for the others extracts corresponding to 1 leaf equivalent (leq) of extract was used per test. Extract was coated on a 90 mm filter paper (Toyo Roshi Kaisha No73171175, Japan). After 5 min of evaporating the solvent, the filter papers containing an equal volume of either solvent was hung on nylon net to cover one side of the I-tube while the filter paper containing the solvent control covered the opposite side. Both nylon nets were attached with rubber band. In our set up; airflow (0.5 m/s), regulated by a speeder, was pumped up into the 2 end of the tube. Each replicate was bioassayed separately, employing a clean I-tube. The bioassays were conducted under the room temperature at 26 ± 2 °C with a relative humidity of 70 ± 2 %. During the bioassay 2-day old mated adults were individually released into the I-tube at its middle portion. The tube was covered with black cloth to avoid light interference. We recorded the number of eggs laid on the net, (which indicated the contact property of the extract) and on the cylinder (distant from the stimulant source which indicated olfactory ability of an extract used) at a distance of 20 cm from the bottom of the I-tube after 2 days. Each bioassay was replicated 4 times. After each test, the apparatus was cleaned with

detergent, acetone and distilled water.

Statistical analysis Data on the ovipositional preferences of

the female H. armigera were subjected to unpaired t-test analysis using SAS(21). Data were square root (SQRT)-transformed before analysis. The oviposition active index (OAI)(14, 25) also described as oviposition index (OI) or preference value was used to characterize the ranking preference of the female towards the extract. The OAI of different concentrations of different extract was calculated as following [OAI = (Nt - Ns) / (Nt + Ns)]. Where, Nt is the number of egg laid on filter paper containing the test extract and Ns is the number of egg laid on the filter paper served as the control. As suggested by Kramer and Mulla(14), compounds with an OAI of +0.3 and above are considered as attractants, while those with –0.3 and below are considered as repellents.

RESULTS

Results of the oviposition of H. armigera on the net are summarized in Figs. 2, 3, 4, 5 and 6. On their mean ovipositional responses, H. armigera females showed, significantly, varying preference degree on the paper bearing the extract of the acrylic extraction. The mean (± SE) number of eggs laid on the hexane extract was (15.07 ± 0.40) compared to the control (10.85 ± 0.58) (t = 5.53, df = 3, p = 0.011) while on the acetone extract we recorded 14.36 ± 1.04 compared to the control (9.69 ± 0.60) (t = 8.03, df = 3, p = 0.004, respectively). The methylene chloride (12.20 ± 0.36) and the hexane dipping (10.11 ± 0.32) extracts showed significant oviposition


Fig. 2. Net’s ovipositional responses of female

H. armigera on steam distillate from Solanum viarum leaf (*= p<0.05).

Fig. 3. Net’s ovipositional responses of

female H. armigera on acetone acrylic extract (Ace.A.E) from Solanum viarum leaf (**= p<0.01).


H. armigera on hexane acrylic extract (Hex.A.E) from Solanum viarum leaf (**= p<0.01).


H. armigera on methylene chloride dipping extract (Me.Cl.D.E) from Solanum viarum leaf (*= p<0.05).


H. armigera on hexane dipping extract (Hex.D.E) from Solanum viarum leaf (*= p<0.05).


stimulation effect of the female compared to their control (8.18 ± 0.94) (t = 3.44, df = 3, p = 0.041) and (7.12 ± 0.68) (t =3.45, df =3, p = 0.040), respectively. However the steam distillation extract seems to deter the egg-laying of the female as there were significantly more eggs laid on the control filter papers (14.22 ± 1.51) which were soaked with an equal volume of distillated water rather than on the filter papers bearing the steam distillate (6.95 ± 0.53) (t = 3.9, df = 3, p = 0.029). When we ranked the female H. armigera preference based on the OAI index described above; there were such descending preference of the female on acetone acrylic extract (+0.37), methylene chloride dipping extract (+0.36), hexane dipping extract (+0.33), hexane acrylic extract (+0.31) and the steam distillate (-0.6) which lasted with a negative value; thus acting as a repellent.

The mean (± SE) number of eggs laid on the cylinder (Figs. 7, 8, 9, 10 and 11) showed no significant difference for the steam distillate (2.56 ± 1.24) compared to its control (3.29 ± 1.01) (t = 0.47, df = 3, p = 0.667). Similarly, hexane (4.85 ± 1.57) and acetone (4.58 ± 1.15) acrylic extracts showed no significant difference on their mean number of eggs laid compared to their control (3.85 ± 2.14) (t = 0.34, df = 3, p = 0.753) and (4.00 ± 0.85) (t = 0.30, df = 3, p = 0.743), respectively. Only the extracts from the methylene chloride and the hexane dipping significantly stimulated the egg-laying of H. armigera (t = 4.89, df = 3, p = 0.016) and (t = 14.19, df = 3, p = 0.0008), respectively. The rank order of preference, as similarly described on the net-ovipositional responses, showed their OAI in descending order of: hexane dipping extract (+0.26) >

methylene chloride extract (+0.16) > hexane acrylic extract (-0.4) > steam distillate (-0.11) > acetone acrylic extract (-0.06). According to the mean percentage of eggs in response to the extracts, female H. armigera deposited significantly more eggs on net (74.97 %) compared to the cylinder (25.01 %).

DISCUSSION

The methods tested to extract oviposition stimulants from S. viarum leaves have produced several results that, to varying degrees, stimulated the oviposition of H. armigera. In contrast, steam distillation, may have extracted inhibitory (repellent or deterrent) and/or non-stimulatory substances from leaves of S. viarum. The acetone acrylic cylinder method and the dipping method were more effective in extracting chemical cues (semiochemicals) that elicit ovipositional response than the hexane acrylic chamber or the steam distillation methods. If we consider that the ovipositional response of the female towards an extract may indicate a triggering effect from the extract to the female H. armigera; therefore such behavioral evaluation demonstrated that, except the steam distillation, both extractions triggered the female on laying eggs. The evaluation of our extracts on H. armigera showed an egg-laying according preference on the net (4 in 5 extracts were tested significant) rather than on the cylinder which resulted in 2 significant extracts from the 5 tested in the bioassay apparatus. Our results demonstrated that S. viarum emits semiochemicals from their leaves which stimulated the oviposition of H. armigera females. This may show that


Fig. 7. Cylinder’s ovipositional responses of

female H. armigera on steam distillate from Solanum viarum leaf (NS= p>0.05).


female H. armigera on acetone acrylic extract (Ace.A.E) from Solanum viarum leaf (NS= p>0.05).


female H. armigera on hexane acrylic extract (Hex.A.E) from Solanum viarum leaf (NS= p>0.05).


female H. armigera on methylene chloride dipping extract (Me.Cl.D.E) from Solanum viarum leaf (**= p<0.01).


female H. armigera on hexane dipping extract (Hex.D.E) from Solanum viarum leaf (**= p<0.01).


leaf waxes chemical could be involved in the process of oviposition, as female of H. armigera, before accepting any substratum for oviposition may explores the surface chemicals by its contact senses. The large amount of eggs laid on the net which was in contact with the filter paper bearing the extract, compared to the lesser amount found in the cylinder could explain the contact-stimulatory properties of our extracts. Such preference could involve both contact chemoreception and olfactory reception of the female moth prior to oviposition, therefore we may not exclude that the different extracts used may also have oviposition-attractant properties as previously theorized in phytophagous insects(4, 9, 26). It is likely that the chemicals responsible for the attractiveness were emitted from the leaf surface of the tropical soda apple plant or underneath its leaf epidermis(5) as the short extraction time of 20 sec in the dipping method stimulated the oviposition of H. armigera. It is reported by Degen et al.(3) that an ideal extraction method should selectively target active compounds present in the uppermost wax layers while avoiding leakage of substances from the leaf interior as much as possible. Therefore, our fast hexane and methylene chloride dipping methods were aimed as extracting those leaf surface chemical acting as oviposition stimulant; even though Riederer & Schneider(20) argued that there was no evidence or theoretical reason supporting the assumption frequently made that any apolar solvent does not enter the cuticle during short dippings or rinsings.

Several papers have reported the presence of oviposition-stimulating

phytochemicals in leaves solvent extracts(2, 3,

6, 7, 11, 15, 24, 25). These bioassay results of the extracts - demonstrating that S. viarum emitted volatile from their leaves stimulating oviposition of H. armigera - and that leaf waxes chemical could be involved from the contact oviposition as previously reported by Diongue et al.(5) using Sep-Pak fractioned extracts in I-Tube bioassay. Similarly, the rank preference-value given by the OAI of the 3 extracts showed positive attractiveness in the net.

We also have suspected that it may have temperature induced-inhibitory effect from the hot extract, steam distillate, or a low concentration of oviposition stimulatory semiochemicals which showed no stimulatory effect in our two choice assays. In contrast, microwave assisted extract (using hexane as solvent and lasting 60 sec duration time) (Georgievska, unpublished data) showed stimulatory effect of S. viarum leaves on the oviposition of H. armigera in I-tube bioassay. Therefore, even that the steam distillation was not a good method to extract egg-laying stimulation substance from the leaf of S. viarum; heating the leaves may not destroy the oviposition-mediating semiochemicals in the case of S. viarum. The repellent effect from the steam distillate could be probably due to the long duration time (50 min) required by the used apparatus. This implies that the solubility of the oviposition stimulants present in the leaf surface of S. viarum was affected by duration time of the extraction and not the heating effect. In the airtight acrylic extraction method, both hexane and acetone extracts stimulated H. armigera oviposition on the net while there was no significant


stimulation of H. armigera based on those eggs laid in the cylinder. Such preference on the filter paper bearing the extract jointed to the net might explain induced oviposition by sensory contact (tarsal or ovipositor). The placement of either solvent (the hexane was 40 cm ahead of the acetone) in the extraction processes seems did not influencing the results. Further test will confirm if measurable I-tube behavioral effects might be evoked with concentrations of the extracts.

Based to our findings, H. armigera females preferred the extracts of both dipping method and acrylic cylinder method which demonstrated good results in terms of stimulatory power of oviposition. The airtight acrylic extraction method is suitable in cases where the semiochemicals are either non polar or of medium polarity. The dipping method also offers a rapid extraction time and a good stimulatory effect without the need to heat the solvent. This experiment showed that different extractions methods may have efficiency in extracting compounds mediating oviposition preference. However the chemical composition and structure which may trigger the variation of stimulatory among different extracts need to be investigated for better optimization of the extraction methodology.

ACKNOWLEDGMENTS

We are grateful to Dr. Y. F. Chang from the Asian Vegetable Research and Development Center (AVRDC) Tainan, Taiwan, for his comment on an earlier version of this manuscript and to Dr. N. S. Talekar for the air-tight acrylic apparatus.

Thanks are due to Dr. S. S. Kao from the Taiwan Agricultural Chemicals and Toxic Substances Research Institute (TACTRI), Wufeng, Taichung, for his endless and kind technical support. Special thanks to Dr. R. Kou of Institute of Zoology, Academia Sinica, Taiwan for her valuable advices during this study.

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摘要

Diongue, A.1、賴博永 1*、林傑 2、黃振聲 3 2005 Solanum viarum 葉片萃取物對番茄夜蛾（Helicoverpa armigera）刺激產卵之生物檢定植保會刊 47：281 – 292 （1 屏東縣內埔鄉國立屏東科技大學熱帶農業暨國際合作系；2 屏東縣內埔鄉國立屏東科技大學環境科學暨工程系；3 台中縣霧峰鄉農業藥物毒物試驗所應用毒理組）

利用雙選 I 形管生物活性測定法（長 250 cm×直徑 30 cm）檢測 Solanum viarum葉片萃取物對番茄夜蛾刺激產卵的影響。將不同溶劑萃取物滴於 I 形管一端網子之濾紙（9 cm）上，另一端僅滴溶劑做為對照組，比較二者對產卵刺激之差異。結果顯示，比較 I 形管二端在網子上及圓筒管壁上之平均產卵量，以網子上含有來自密閉壓克力箱內 Solanum viarum 葉片氣味之丙酮或正己烷萃取物，及浸泡於正己烷或二氯甲烷之葉片萃取物等之平均產卵量，較對照組有顯著性差異；然而以乙醚蒸餾出的葉片萃取物則可明顯抑制番茄夜蛾產卵。比較圓筒管壁上之平均產卵量，則顯示只有浸泡於正己烷或二氯甲烷之葉片萃取物較對照組具顯著刺激番茄夜蛾產卵，但以其他萃取方法獲得之葉片萃取物對番茄夜蛾則無明顯刺激產卵作用。

(關鍵詞：番茄夜蛾、Solanum viarum、葉片萃取物、產卵刺激物)

*通訊作者。E-mail: [email protected]

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