Plant Extracts as an Alternative to Control Leucoptera coffeella (Guérin-Mèneville) (Lepidoptera: Lyonetiidae)

PEST MANAGEMENT

Plant Extracts as an Alternative to Control Leucoptera coffeella (Guérin-Mèneville) (Lepidoptera: Lyonetiidae)

DS Alves^I; DF Oliveira^II; GA Carvalho^I; HM dos Santos Jr^II; DA Carvalho^III; MAI Santos^II; HWP de Carvalho^II

^IDepto de Entomologia

^IIDepto de Química,

^IIIDepto de Biologia. Univ Federal de Lavras, Lavras, MG, Brasil

^{Correspondence}

ABSTRACT

We evaluated the effects of crude extracts from the plantain Plantago lanceolata and the bitter gourd Momordica charantia on the oviposition preference and development of the coffee leaf miner Leucoptera coffeella Guérin-Mèneville & Perrottet under laboratory and/or greenhouse conditions. The ovicidal effects of these extracts were also studied in a greenhouse. Plantago lanceolata and M. charantia extracts also underwent fractionation directed by oviposition tests with the coffee leaf miner. The extracts of both plants reduced L. coffeella oviposition and egg hatching, apparently as a result of action of plant metabolites on the embryo. Adults originating from eggs treated with the extracts exhibited similar survival rates, but a higher female/male ratio. Fecundity was reduced for females obtained from eggs treated with the M. charantia extract. Partial chemical analysis indicated that both extracts produced polar fractions that reduced the oviposition of L. coffeella on coffee leaves under laboratory conditions. The extracts of P. lanceolata and M. charantia have potential for use in the development of new products to control the coffee leaf miner.

Keywords: Coffee leaf miner, Plantago lanceolata, Momordica charantia, natural product

Introduction

The coffee leaf miner, Leucoptera coffeella (Guérin-Mèneville), is an economically important pest of coffee crops (Pimentel 2007). It is mainly controlled by synthetic insecticides, which contaminate humans and the environment, and cause biological imbalances that lead to the increase of secondary pests and to the selection of insect populations resistant to insecticides (Sharma 2008).

A possible alternative to control the coffee leaf miner is the use of plant secondary metabolites that are active against herbivores (Biere et al 2004, Schaller 2008). These substances are intensively studied as an alternative strategy in insect pest management, and some of them are already commercially available, such as those derived from Azadirachta indica (Agboka et al 2009, Siddiqui et al 2009). However, the diversity of plants and their metabolites requires wider investigation into the role of other plant metabolites for insect control.

Some of the potential plants are the bitter gourd Momordica charantia (Cucurbitaceae), which acts as a repellent and a proteinase inhibitor (Boeke et al 2004, Telang et al 2003); and the plantain Plantago lanceolata (Plantaginaceae), which produces large amounts of glycosylated iridoids (Marak et al 2000, Tamura & Nishibe 2002) with deterrent or growth-reducing effects against insects (Bowers & Puttick 1988, Nieminen et al 2003). Based on the potential of M. charantia and P. lanceolata for use in the production of new insecticides, we evaluated their effect on the oviposition and development of L. coffeella, and conducted bioactivity-directed fractionation of the extracts.

Material and Methods

Plant extracts

Fresh leaves of P. lanceolata (1569.9 g) and M. charantia (617.8 g), which were in vegetative and reproductive states (with flowers), respectively, were collected in June, 2005 at the campus of the Universidade Federal de Lavras. The leaves were cut into small pieces and placed in a 1000 ml glass vial containing approximately twice the volume of methanol in relation to the amount of plants. After resting for 48h in methanol, the samples were filtered through cotton wool plugs. The plant material was re-extracted with methanol five additional times. The filtrates were dried in a rotary evaporator and freeze-dried for 24h to obtain 21.30 g and 44.24 g of M. charantia and P. lanceolata dry extracts, respectively, which were stored at -10°C.

Laboratory oviposition assay

Aliquots (18 mg) of the dry extracts were dissolved in 2.0 ml of an aqueous 1.0% (g/ml) Tween 80 solution, resulting in extract solutions at a concentration determined by preliminary tests. The extracts were spread onto the adaxial surface of coffee leaves, Coffea arabica cv. Catuaí, with a soft paintbrush. Control (no treatment) and treated leaves were set on a styrofoam board in a Petri dish. The bottom of the Petri dish was covered with wet cotton wool, and each dish was placed in a polyvinyl chloride cage covered with transparent plastic film. Inside the cage, two pairs (1-3 days old) of L. coffeella were released. These adults were reared according to Reis Jr et al (2000). Cotton wool soaked with a 10% sucrose solution was offered as food to the adults, which were allowed to oviposit for 72h under controlled conditions (25 ± 1ºC, RH 70 ± 10%, and 14h photoperiod). Then, the eggs on each leaf were counted. The experiment was carried out in a random design, using five replicates per treatment, with each replicate represented by an experimental cage containing one treated and one untreated leaf. Aqueous 1.0% (g/ml) Tween 80 and 0.2% (v/v) Lorsban® (chlorpyrifos, Dow AgroSciences, Indianapolis) solutions were employed as negative and positive controls, respectively. Data were expressed as a percentage [eggs on treated leaf x 100 / (eggs on treated leaf + eggs on untreated leaf)] and submitted to the χ2 test (P < 0.05).

Greenhouse oviposition assay

Six-month-old coffee plants (Coffea arabica cv. Catuaí IAC-99) were trimmed to four leaves and sprayed with extracts of M. charantia and P. lanceolata at 1.0% (g/ml) in 1.0 % (g/ml) Tween 80. The experiment was carried out in a randomized block design with five replicates, using water and aqueous Tween 80 as controls. Each block consisted of a wooden cage containing four coffee plants, in which each plant corresponded to a treatment. Twenty pairs of L. coffeella were released in each cage. The eggs were counted 48h and 72h after the moths were released, and their numbers were expressed as a percentage [eggs in each treatment x 100 / total number of eggs per cage] and submitted to the χ2 test (P < 0.05).

Greenhouse ovicidal assay

Coffee plants were exposed to L. coffeella for 12h, to allow oviposition. The plants were then removed from the cage and the number of eggs per leaf was counted, leaving five eggs on each leaf. The plants were then sprayed with solutions of M. charantia and P. lanceolata aqueous extracts 1.0% (w/v) Tween-80 until they were dripping. Each treatment comprised four replicates arranged in a random design. Aqueous 1.0% (g/ml) Tween 80 and 0.2% (v/v) Sumithion® 500 CE (Fenitrothion, IHARABRAS S.A., São Paulo) solutions were used as negative and positive controls, respectively. After seven days, nonviable eggs were counted, and the numbers were expressed as percentages and subjected to analysis of variance (ANOVA) and to the Scott-Knott test (P < 0.05) for comparisons.

Ultrastructural analysis of nonviable eggs

The nonviable eggs obtained from the greenhouse ovicidal trial were prepared for scanning electron microscopy analysis according to Alves (2004). Initially, pieces of coffee leaves containing nonviable eggs were kept in the modified Karnovsky fixative (2.5% glutaraldehyde, 2.5% formaldehyde, 0.05 M sodium cacodylate buffer at pH 7.2, 0.001 M calcium chloride) at 4°C for 24h, and later transferred to 1% osmium tetroxide solution (ml/ml) buffered with 0.05 M cacodylate for 4h at room temperature. Samples were washed with distilled water and dehydrated using a graded series of propanone (25, 50, 75, 90, and 100% - 10 min/each), and subjected to critical-point drying on a Balzers CPD 030 Critical-Point Dryer. Samples were mounted on aluminum stubs using double-sided carbon tape, sputter-coated with gold on a Balzers SCD 050 gold evaporator, and analyzed on a Leo Evo 40 scanning electron microscope using the Leo User Interface software.

Effect of plant extracts on L. coffeella development

This experiment was carried out as described for the greenhouse ovicidal trial. On the fifteenth day after the plants were sprayed with the extracts, leaves containing young miners were collected and maintained in Petri dishes under controlled conditions (25 ± 1ºC, RH 70 ± 10%, and 14h photophase) until pupation. Pupae were individually transferred to glass tubes, where they were kept until the adults emerged, to determine pupae survival and sex ratio. Each pair was released in a polyvinyl chloride cage containing a Petri dish with two coffee leaves. After 48h, the eggs were counted. All data were submitted to analysis of variance (ANOVA) and means were compared by the Scott-Knott test (P < 0.05).

Fractionation of P. lanceolata extract

A sample of 9.81 g of P. lanceolata extract was washed with hexane (Hex), ethyl acetate (AcOEt), and methanol (MeOH) (5 x 200 ml for each solvent). Each of the resulting solutions was concentrated to dryness in a rotary evaporator and freeze-dried to afford a hexane (2.0147 g), an ethyl acetate (0.3078 g), and a methanol (5.9264 g) soluble fraction, and an insoluble residue (0.7693 g). Aliquots (0.37%) of all fractions (including the residue) were dissolved in 2.0 ml of an aqueous 1.0% (g/ml) Tween 80 solution prior to use.

A sample (1.00 g) of the MeOH soluble fraction was eluted through a 3 x 20 cm column packed with silica gel (230-400 mesh, Merck), using MeOH, distilled water, and 0.1 M HCl (300 ml each) as eluents. After concentration of the resulting eluates, six new fractions were obtained: MeOH-I (0.0042 g), MeOH-II (0.7877 g), MeOH-III (0.0720 g), water-I (0.0678 g), water-II (0.0445 g), and HCl (0.2988 g). Aliquots (2.84%) of these fractions were dissolved in 2.0 ml of a 1% Tween 80 solution, and applied to coffee leaves to assess the effects on the oviposition of leaf miners under laboratory conditions.

Fractionation of the M. charantia extract

The M. charantia extract (10.2332 g) was treated similarly to the P. lanceolata extract, and four fractions were collected (Hex - 0.4398 g, AcOEt - 3.7300 g, MeOH-I - 4.6316 g, all colored dark green color; MeOH-II - 0.9153 g, colorless). A sample (1.0266 g) of the fraction MeOH-I was fractionated as described above, and three new fractions were obtained: MeOH (0.8453 g), water (0.1242 g), and HCl (0.2186 g).

Results and Discussion

Leaves treated with the extracts of Plantago lanceolata (χ² = 47.21, P < 0.05) and M. charantia (χ² = 63.71, P < 0.05) were less preferred than control leaves for oviposition by coffee leaf miner adults in a free-choice test, under laboratory conditions (Table 1). Forty-eight hours after the release of insects, the P. lanceolata extract at concentrations of 0.9% (χ² = 10.72, P < 0.05) and 1.8% (χ² = 14.81, P < 0.05) deterred oviposition by L. coffeella. In contrast, the M. charantia extract deterred oviposition only at 1.8% (χ² = 14.81, P < 0.05). By 72h after the release, only the P. lanceolata extract at 0.9% (χ² = 13.01, P < 0.05) and 1.8% (χ² = 14.48, P < 0.05) reduced oviposition by L. coffeella (Table 2).

The eggs of L. coffeella treated with extracts of P. lanceolata and M. charantia had lower viability than eggs under control treatments (Table 3). Scanning electron microscopy observations of nonviable eggs exposed to both extracts (Fig 1) showed alterations on their surfaces that were typical of those observed for nonviable eggs treated with commercial insecticides (Fig. 1 g, h). In all these cases, superficial fractures on the chorionic structure of the eggs could be easily observed.

The treatment of eggs of L. coffeella with extracts had no effect on adult emergence, although these treatments produced a skewed sex ratio towards females, which showed reduced fecundity (Table 4).

The methanol fraction from P. lanceolata (χ² = 24.93, P < 0.05) and the polar fraction MeOH I of M. charantia (χ² = 35.01, P < 0.05) reduced oviposition by leaf miner adults (Table 5). When fractionated by column chromatography, both fractions yielded new active fractions: MeOH I (χ² = 10.61, P < 0.05), MeOH II (χ² = 32.2, P < 0.05), MeOH III (χ² = 8.83, P < 0.05), Water I (χ² = 22.97, P < 0.05), and HCl (χ² = 37.98, P < 0.05) for P. lanceolata; and MeOH (χ² = 41.48, P < 0.05) and HCl (χ² = 29.6, P < 0.05) for M. charantia (Table 6).

The choice of the host plant for oviposition by the insect depends on a complex set of stimuli and responses (Reudler et al 2008, Städler & Reifenrath 2009) that may be mediated by the insect's sensory system, composed of receptors that can perceive plant metabolites, resulting in an increase or decrease in the number of eggs that females can lay on a plant (Schoonhoven et al 2005, Navarro-Silva 2009). Our data indicated that both extracts tested contain molecules that can affect the sensory system of L. coffeella females used to evaluate the quality of the host plant as a substrate for oviposition.

The non-preference of L. coffeella for oviposition on leaves treated with P. lanceolata extracts is probably due to the glycosylated iridoids aucubin and catalpol, which are produced by P. lanceolata and have shown deterrent activity against several insects (Talsma et al 2008). As observed with L. coffeella in the present study, the methanol extract of M. charantia has also reduced the oviposition of Liriomyza trifolii (Burgess) (Diptera: Agromyzidae) (Mekuria et al 2005), but no chemicals were identified by these authors.

The ovicidal effect observed for both extracts is very interesting, because ovicidal activity of both synthetic and botanical products is uncommon (Ofuya 1997, Martinez & Meneguim 2003). Although the mechanisms by which extracts of plantain and bitter gourd kill the embryos of L. coffeella are still unclear, our observations indicate that they may act as disruptors of the egg chorion, since the observed damage to the chorionic structure of the egg may favor dehydration and induce mortality.

Despite the lack of any report on the activity of M. charantia against L. coffeella, the effects here observed were similar to those reported elsewhere (Neraliya & Srivastava 1996), with the molecules momordicine I and II being associated with the noxious effects induced by extracts of this plant (Chandravadana 1987, Mekuria et al 2005, Ling et al 2008). Because of the polar nature of momordicine and because only the MeOH I fraction of M. charantia reduced the oviposition of L. coffeella, it is likely that momordicine I and II are also the molecules that affected this insect. Also the activity of P. lanceolata against some insects has been reported and attributed to the production of polar molecules such as aucubin and catalpol (Marak et al 2000, Fuchs & Bowers 2004, Harvey et al 2005). Furthermore, Bowers & Stamp (1993) demonstrated that catalpol concentration in plants increases under herbivore attack. Consequently, the reduction in the oviposition of L. coffeella on leaves treated with the MeOH polar fraction of the P. lanceolata extract may be induced by one of these molecules.

Our data indicate that M. charantia and P. lanceolata produce active compounds against L. coffeella, and may have potential for use in the development of new products for controlling the infestation level of this insect pest in coffee plantations. Therefore, further studies should be carried out to investigate the potential of these plants and their compounds for the control of L. coffeella.

Acknowledgments

To CNPq, CAPES, FAPEMIG, and EMBRAPA-CAFÉ for providing the necessary funds for this project.

Received 08 October 2009 and accepted 26 August 2010

Edited by Jorge B Torres - UFRPE

• Agboka K, Mawufe AK, Tamò M, Vidal S (2009) Effects of plant extracts and oil emulsions on the maize cob borer Mussidia nigrivenella (Lepidoptera: Pyralidae) in laboratory and field experiments. Int J Trop Insect Sci 29: 185-194.
• Alves E (2004) Introdução à microscopia eletrônica de varredura. Lavras, FAEPE, 43p.
• Biere A, Marak HB, Van Damme JMM (2004) Plant chemical defense against herbivores and pathogens: generalized defense or trade-offs? Oecologia 140: 430-441.
• Boeke SJ, Baumgart IR, Loon JJA, Huis AV, Dicke M, Kossou DK (2004) Toxicity and repellence of African plants traditionally used for the protection of stored cowpea against Callosobruchus maculatus J Stored Prod Res 40: 423-438.
• Bowers MD, Puttick GM (1988) Response of generalist and specialist insects to qualitative allelochemical variation. J Chem Ecol 14: 319-334.
• Bowers MD, Stamp NE (1993) Effects of plant-age, genotype, and herbivory on Plantago performance and chemistry. Ecology 74: 1778-1791.
• Chadravadana MV (1987) Identification of triterpenoid feeding deterrent of red pumpkin beetles (Aulacophora foveicollis) from Momordica charantia J Chem Ecol 13: 1689-1694.
• Fuchs A, Bowers MD (2004) Patterns of iridoid glycoside production and induction in Plantago lanceolata and the importance of plant age. J Chem Ecol 30: 1723-1741.
• Harvey JA, Van Nouhuys S, Biere A (2005) Effects of quantitative variation in allelochemicals in Plantago lanceolata on development of a generalist and a specialist herbivore and their endoparasitoids. J Chem Ecol 31: 287-302.
• Ling B, Wang GC, Ya JZ, Zhang MX, Liang GW (2008) Antifeedant activity and active ingredients against Plutella xylostella from Momordica charantia leaves. Agric Sci China 7: 1466-1473.
• Marak HB, Biere A, Van Damme JMM (2000) Direct and correlated responses to selection on iridoid glycosides in Plantago lanceolata L. J Evol Biol 13: 985-996.
• Martinez SS, Meneguim AM (2003) Redução do oviposição e da sobrevivência de ovos de Leucoptera coffeella causadas pelo óleo emulsionável de nim. Man Int Plagas Agroecol 67: 58-62.
• Mekuria BD, Kashiwig T, Tebayshi S, Kim S (2005) Cucurbitane triterpenoid oviposition deterrent from Momordica charantia to the leaf miner, Liriomyza trifolii Biosci Biotechnol Biochem 69: 1706-1710.
• Navarro-Silva MA, Marques FA, Duque L, Jonny E (2009) Review of semiochemicals that mediate the oviposition of mosquitoes: a possible sustainable tool for the control and monitoring of Culicidae. Rev Bras Entomol 53: 1-6.
• Neraliya S, Srivastava US (1996) Effects of plant extracts on post-embryonic development of the mosquito Culex quinquefasciatus J Adv Zool 17: 54-58.
• Nieminen M, Suomi J, Van Nouhuys S, Sauri P, Riekkola ML (2003) Effect of iridoid glycoside content on oviposition host plant choice and parasitism in a specialist herbivore. J Adv Zool 29: 823-844.
• Ofuya TI (1997) Effect of some plant extracts on two coccinellid predators of the cowpea aphid, Aphis craccivora (Hom.: Aphididae). Entomophaga 42: 277-282.
• Pimentel D (2007) Encyclopedia of pest management. New York, M. Dekker, 784p.
• Reis Jr R, Lima ER, Vilela EF, Barros RS (2000) Method for maintenance of coffee leaves in vitro for mass rearing of Leucoptera coffeela (Guérin-Méneville) (Lepidoptera: Lyonetiidae). Neotrop Entomol 29: 849-854.
• Reudler JHT, Biere A, Harvey JA, Van Nouhuys S (2008) Oviposition cues for a specialist butterfly-plant chemistry and size. J Chem Ecol 34: 1202-1212.
• Schaller A (2008) Induced plant resistance to herbivory. Netherlands, Springer, 462p.
• Schoonhoven LM, Jermy T, Van Loon JJA (2005) Insect-plant biology, USA. Oxford, Oxford University Press, 421p.
• Sharma HC (2008) Biotechnological approaches for pest management and ecological sustainability. Florida, CRC Press, 546p.
• Siddiqui BS, Ali ST, Rajput MT, Gulzar T, Rasheed M, Mehmood R (2009) GC-based analysis of insecticidal constituents of the flowers of Azadirachta indica A. Juss. Nat Prod Res 23: 271-283.
• Städler E, Reifenrath EK (2009) Glucosinolates on the leaf surface perceived by insect herbivores: review of ambiguous results and new investigations. Phytochem Rev 8: 207-225.
• Talsma JHR, Biere A, Harvey JA, van Nouhuys S (2008) Oviposition cues for a specialist butterfly-plant chemistry and size. J Chem Ecol 34: 1202-1212.
• Tamura Y, Nishibe S (2002) Changes in the concentrations of bioactive compounds in plantain leaves. J Agric Food Chem 50: 2514-2518.
• Telang M, Srinivasan A, Patankar A, Harsulkar A, Joshi V, Damle A, Deshpande V, Sainani M, Ranjekar P, Gupta G, Birah A, Rani S, Kachole M, Giri A, Gupta V (2003) Bitter gourd proteinase inhibitors: potential growth inhibitors of Helicoverpa armigera and Spodoptera litura Phytochemistry (Oxf) 63: 643-52.

Publication Dates

• Publication in this collection
  14 Mar 2011
• Date of issue
  Feb 2011

History

• Received
  08 Oct 2009
• Accepted
  26 Aug 2010