[advances in phytomedicine] naturally occurring bioactive compounds volume 3 || chapter 15...

Rai and Carpinella (eds.)

Naturally Occurring Bioactive Compounds

r 2006 Elsevier B.V. All rights reserved.

379

CHAPTER 15

Promissory botanical repellents/deterrents formanaging two key tropical insect pests, thewhitefly Bemisia tabaci and the mahoganyshootborer Hypsipyla grandella

LUKO HILJE, GERARDO A MORA

Introduction

In the past 60 years, conventional chemical pesticides have been the predominantmethod for controlling pests worldwide. Nonetheless, the recognition and docu-mentation of many unwanted agroecological, environmental, social, and economicproblems resulting from pesticide overuse, has led scientists to look for alternatives,among which integrated pest management (IPM) has stood out. IPM tactics includeplant breeding and cultural practices, as well as physical, biological, and selectivechemical control.

In fact, in recognition of the concept of co-evolution between herbivorous insectsand their host plants, as well as its practical implications, IPM also promotes thesearch and utilization of natural active principles (insecticides, repellents, attract-ants, etc.) which could be helpful in dealing with insect pests of crops and forestplantations.

Historically, some of the insecticides first used in agriculture and forestry werederived from plants (Stoll, 2000), such as nicotine from tobacco (Nicotiana tabacum,Solanaceae) leaves; rotenone, from the roots of ‘‘timbo’’ (Derris spp.), ‘‘chaperno’’(Lonchocarpus spp.), yam bean (Pachyrhizus spp.) and other leguminous plants;quassinoids, from bitterwood (Quassia amara, Simaroubaceae) wood; azadirachtin,from neem (Azadirachta indica, Meliaceae) seeds; and pyrethrum, from Chrysanthemum

cinerariifolium (Asteraceae) flowers; other plants well known for having substances withinsecticidal properties include ryania (Ryania speciosa, Flacourtiaceae) and ‘‘sabadilla’’(Schoenocaulon officinale, Lilliaceae). However, their use in agriculture, and even intraditional tropical systems, vanished in the 1950s, as a result of the appearanceand widespread use of synthetic insecticides, as their rather simple molecules lendthemselves for these materials to be manufactured at an industrial scale and a relativelylow cost.

Naturally occurring bioactive compounds380

Today, as a consequence of the wide promotion and acceptance of the sustainabledevelopment paradigm among conservation and farmer organizations, govern-ment policy- and decision-makers, and international donor agencies, environment-friendly and highly profitable production schemes and practices, such as organicagriculture, have gained a lot of adoption and support in many countries.

This new scenario opens better opportunities and possibilities for agrichemicalcompanies involved in manufacturing non-conventional insecticides (bioinsecticidesor biorationals) and other types of pesticides (Rodgers, 1993; Hall and Menn, 1999)harmless to non-target organisms, generally perceived as environmentally benign,and best suited for IPM programs within those production schemes. Such bio-insecticides can be obtained by extracting the natural active principle per se, as itcurrently occurs with neem, or by using them as leads to synthesize their analoguemolecules (Pillmoor et al., 1993), as it happens with pyrethroid insecticides.

Tropical biodiversity as a source of bioactive substances against insects

The issue of biodiversity has received a lot of attention in recent years, because of theworldwide concern about the high rates of destruction of some of the last majorforest masses on Earth, which are mainly located in the neotropics (Wilson, 1988).These forests contain many organisms not yet described, some of them potentiallyuseful for humankind.

Nevertheless, few resources have been allocated to search for biodiversity appli-cations to agriculture, including IPM programs, despite biodiversity and IPM beingclosely interrelated (Hilje and Hanson, 1998); new products (genes, natural pesti-cides, and beneficial organisms) for IPM programs can be obtained from tropicalspecies and, conversely, implementation of IPM programs can have beneficial effectson both terrestrial and aquatic biodiversity.

One of the best ways to take advantage of the remarkably high tropical bio-diversity is to explore, identify, and utilize plant-derived substances in IPM programsaimed at key insect pests of crops and forest plantations (Hilje and Hanson, 1998).For instance, secondary metabolites with defensive properties against insects arerather common in plants, including alkaloids, non-protein amino acids, steroids,phenols, flavonoids, glycosides, glucosinolates, quinones, tannins, and terpenoids(Harborne, 1977; Panda and Khush, 1995).

An excellent example along these lines is the highly successful and widespread useof neem-seed derivatives, a very well-known tree from India, Pakistan, Indonesia,and Thailand (Schmutterer et al., 1982; Walter, 1999). But it would also be possibleto exploit a large number of tropical plants with an untapped potential as sources ofactive principles against insect pests (Grainge and Ahmed, 1988; Stoll, 2000), someof which are present in pristine ecosystems (Wilson, 1988) and even in man-madeenvironments (such as agroforestry systems) and in other disturbed habitats.

Insecticides or repellents/deterrents?

In addition to insecticides, plants may contain a wide array of substances actingagainst insects, including several types of allomones (those providing an adaptive

Promissory botanical repellents/deterrents for managing two key tropical insect pests 381

advantage to the emitter), such as repellents, suppressants, deterrents, antibiotics,and anorexigenics (Warthen and Morgan, 1990). Substances belonging to the firstthree categories mentioned above act by interfering with the ability of insects inlocating, feeding or ovipositing on their host plants. Even though there are long listsof plant species claimed as having compounds with such properties (Grainge andAhmed, 1988; Warthen and Morgan, 1990; Stoll, 2000), many references are ratheranecdotal, thus requiring scientific confirmation.

For those substances commonly named as repellents, there are several possiblelevels of response from an insect to a plant, which are not always easy to differ-entiate. A true repellent is a substance which acts at a distance, causing orientedmovements away from the source, i.e. a plant (Matthews and Matthews, 1978),whereas other substances cause an effect once an insect has got in contact with theplant structure emitting such substances. Thus, for instance a suppressant inhibitsthe initiation of feeding or oviposition, a deterrent impedes the continuation of suchprocesses, and an anorexigenic causes a loss of appetite (Warthen and Morgan,1990).

It is very difficult to exactly differentiate and recognize such effects, unless highlysensitive and sophisticated equipment and tools are available, such as electronicfeeding monitor, which provides electrical penetration waveforms or graphs (EPG)when a wired insect feeds or oviposits (Walker and Perring, 1994). Therefore, for thepurpose of this chapter, the terms repellent and deterrent will be the only ones usedhere, defining repellents as those substances that keep an insect away before itlands on a plant, and deterrents as those substances inhibiting feeding or ovipositiononce an insect has landed and got in contact with the plant structure emitting suchsubstances.

The acceptance and spread of IPM as a sound strategy to deal with pest problemshas made a growing number of agrichemical companies to develop commercial bio-insecticides (Hall and Menn, 1999), for which there has been a renewed interest inrevisiting ethnobotanical knowledge to formally pursue screening of crude extractsfor biological activity. But, unfortunately, the discovery, characterization, and ex-ploitation of natural products against insects have proceeded in a biased trend to-wards bioinsecticides, disregarding repellent and deterrent substances. Perhaps this isbecause their production process is as complex as that for an insecticide, whereastheir effects under field conditions do not eliminate a pest problem, but rather couldtransfer it to neighboring farmers (Schoonhoven, 1982).

Nevertheless, for key insect pests showing a very low damage threshold, as it willbe discussed in the next section, semiochemicals (repellents and deterrents) couldplay an important role as a component of IPM programs based upon a preven-tive approach. This is the case of the whitefly Bemisia tabaci and the mahoganyshootborer Hypsipyla grandella, on which the authors have conducted research inrecent years.

The target pests: why these ones?

Currently, B. tabaci (Gennadius) (Homoptera: Aleyrodidae) and H. grandella Zeller(Lepidoptera: Pyralidae) can be considered as the main agricultural and forest pests


in Latin America, respectively, so that their management is relevant in both eco-nomic and social grounds for agricultural and forest producers in the continent.

On one hand, B. tabaci is a cosmopolitan insect and a key pest in many tropicaland subtropical cropping systems (Brown and Bird, 1992; Brown, 1994). Currently,it is causing serious problems all the way from the U.S. throughout Argentina,including Caribbean countries, as well as in many African, Asian and Europeancountries, and in Australia. Estimated economic losses amount to several hundredsor even thousands of millions of dollars a year, worldwide (Oliveira et al., 2001).Crop damage occurs directly through excessive sap removal, or indirectly by pro-moting the growth of sooty mold, inducing systemic disorders (syndromes) throughfeeding, or by vectoring plant viruses.

It is a highly polyphagous pest, which can develop or reproduce on over 500different plant species, belonging to 74 families (Greathead, 1986). It can attacksome 30 crops worldwide, including both cash and staple crops (tomato, peppers,melon, watermelon, soybean, cotton, beans, and cassava). However, in Mesoamericaand the Caribbean, as well as in paleotropical areas, it acts mainly as a virus vector ina number of such crops. Moreover, B. tabaci has at least 19 well-documented racesor biotypes (Perring, 2001) which may vary in their degree of association with par-ticular host plants and the induction of specific syndromes, as well as their repro-ductive potential and response to climatic changes.

On the other hand, H. grandella is one of the two shootborer species (Lepidoptera:Pyralidae) which attack precious wood plants of the Meliaceae family (Schabel et al.,1999) worldwide, the other being H. robusta, restricted to paleotropical areas.H. grandella (Zeller) is distributed throughout the neotropics, where it damages some17 species of mahoganies (Swietenia spp.), cedars (Cedrela spp.) and related species.Its larva mainly bores the terminal shoots of trees, which causes forking of the stems,which has prevented attempts to establish commercial plantations of such species inLatin America and the Caribbean.

Even though B. tabaci and H. grandella are very different insects both taxonomi-cally and biologically, they are treated together here, as they have some common-alities in regards to their damage and possible management approaches.

Considerable research aimed at managing each pest species has been conductedand summarized in Naranjo and Ellsworth (2001) and Newton et al. (1993), re-spectively. Nevertheless, management tactics, including chemical control by noveland effective insecticides, have not been successful enough due to a number ofreasons, the main one being that both H. grandella and B. tabaci as a virus vectorhave a extremely low damage threshold. For H. grandella, damage caused by asingle larva normally results in severe economic losses, as forking of the main stemsince early stages of tree development renders them unmarketable. For B. tabaci, inthe case of geminiviruses affecting tomato – which is probably valid to other virusesand crops – viral disease incidence can reach 100% with an average vector density aslow as 0.3 adults/plant (Hilje, 2001).

Then, since in both cases damage caused by even a single insect can result inirreversible losses, it would be worthless to kill either H. grandella larvae or B. tabaciadults once damage is done. Therefore, a preventive management scheme would bewell in place, with semiochemicals (repellents and deterrents) precluding these pests


from causing serious damage. Hopefully, such a scheme should also be environment-friendly, cost-effective, and fully compatible with other IPM tactics.

Methodological approaches

In order to detect the biological activity of plant extracts on B. tabaci andH. grandella herewith reported, a number of methodological approaches were used,some of them so far unpublished. Hydroalcoholic extracts were prepared atCIPRONA (Research Center on Natural Products), and laboratory and greenhousebioassays carried out at CATIE.

Extract preparation

Each type of plant material was collected from a single location and at the same time,in order to avoid undesirable variability due to geographic or seasonal differences.

Crude extracts

For extracts prepared from leaves, corresponding to the majority of the speciestested (Table 1), as well as those from seeds, bulbs, flower buds, and fruits (exceptingwild ‘‘tacaco’’), plant material was dried in an oven at 40 1C, ground and placed in70% methanol in a suitable flask for 24 h; the solvent was drained and the residuewas treated again with methanol for 24 h. The pooled extracts were filtered through aWhatman No. 4 filter paper, and concentrated at 40 1C using a rotary evaporator.The final residue was freeze-dried to eliminate any water remaining in the crudeextract. The same procedures were followed for preparing extracts from woodytissue, such as bitterwood (Quassia amara), which started by drying wood chips. Inthe case of leaves from mother-of-cocoa (Gliricidia sepium), the crude extract wasdefatted with hexane before freeze-drying it.

Extract fractions

Fractions were obtained only for the most promising of all plant extracts studied.A column 31 cm high and 4.5 cm diameter was prepared with 100 g of the syntheticresin Diaion HP-20 (Mitsubishi Chemical Industry, Yokohama, Japan). The resinwas washed with water, water/methanol (1:1), methanol and diethyl ether. A maxi-mum of 10 g of the crude freeze-dried extract was placed on the column and elutedwith 1 l each of the solvents, starting with water and finishing with diethyl ether. Thecolumn was used as many times as necessary to completely process one batch ofcrude extract. The solvents were evaporated and freeze-dried, if necessary, to havethe weight of each fraction in order to establish the proper dose to be used in thebioassay.

In the case of extracts prepared from fruits, such as wild ‘‘tacaco’’ (Sechiumpittieri), which was prepared for other kind of experiments (Castro et al., 1997), freshfruits were extracted with methanol and the extract was concentrated in vacuo to givean aqueous suspension which was passed through the Diaion HP-20 column. The

Table 1Approximate degree of feeding deterrence caused by crude plant extracts to Bemisia tabaciadults (Bt) and Hypsipyla grandella larvae (Hg)

Common name Scientific name Family Structure tested Deterrencea

Bt Hg

Allspice Pimenta dioica Myrtaceae Essential oil (leaves) + �

Balsam pear Momordica charantia Cucurbitaceae Leaves +++ 0

Bitterwood Quassia amara Simaroubaceae Leaves + ++

Bitterwood Quassia amara Simaroubaceae Wood +++ +++

Cardamomo Elettaria cardamomum Zingiberaceae Essential oil (seed) + �

‘‘Chile muelo’’ Drimys granadensis Winteraceae Leaves +++ �

‘‘Chile muelo’’ Drimys granadensis Winteraceae Essential oil (leaves) ++ 0

Clove Syzygium aromaticum Myrtaceae Flower buds ++ 0

Common rue Ruta chalepensis Rutaceae Leaves + +++

Coriander Coriandrum sativum Apiaceae Leaves ++ 0

Eucalyptus Eucalyptus deglupta Myrtaceae Leaves ++ 0

Fish bean Tephrosia vogelii Fabaceae Leaves +++ �

Garlic Allium sativum Alliaceae Bulb 0 0

Gumbo-Limbo Bursera simaruba Burseraceae Essential oil (fruit) 0 �

Hot pepper Capsicum frutescens Solanaceae Fruits + 0

Jackass bitters Neurolaena lobata Asteraceae Leaves ++ 0

Lemongrass Cymbopogon citratus Poaceae Leaves 0 0

Marigold Tagetes jalisciensis Asteraceae Leaves ++ 0

Marigold Tagetes microglossa Asteraceae Leaves ++ 0

Mexican oregano Lippia graveolens Lamiaceae Leaves + 0

Mother-of-cocoa Gliricidia sepium Fabaceae Leaves +++ 0

Onion Allium cepa Alliaceae Bulb + �

Peppermint Satureja viminea Labiatae Leaves ++ 0

Portugal cedar Cupressus lusitanica Cupressaceae Essential oil (leaves) + �

Spiked pepper Piper aduncum Piperaceae Spikes (essential oil) ++ �

Spiny coriander Eryngium foetidum Umbelliferae Leaves + 0

Sweet lime Citrus limetta Rutaceae Essential oil (fruit

skin)

++ �

Sword bean Canavalia ensiformis Fabaceae Leaves +++ �

Sword bean Canavalia ensiformis Fabaceae Seeds ++ �

Wild ‘‘tacaco’’ Sechium pittieri Cucurbitaceae Fruits +++ T

Wild sunflower Tithonia diversifolia Asteraceae Leaves +++ �

Worm-seed Chenopodium

ambrosioides

Chenopodiaceae Leaves +++ 0

Sources: Cubillo et al. (1994, 1997, 1999); Gomez et al. (1997); Mancebo et al. (2000a, 2000b,2001); Soto (2000); Hilje and Stansly (2001), Aguiar et al. (2003); Flores (2003).Note: The word ‘‘approximate’’ is used because it is not possible to compare results fromdifferent sources and even different methodologies.aResponse: Non-tested (�), nil (0), weak (+), mild (++), strong (+++) and toxic (T).


column was washed with water, water: methanol (1:1), methanol, and ethyl acetateor diethyl ether.

Essential oils

These were obtained by hydro-distillation (Ciccio, 1996). The plant material, nor-mally 500—800 g, was placed on a round bottomed flask and water was added upto a volume of 3 l. The essential oil was collected by means of a Clevenger-type


apparatus in the lapse of 2.5 h. The oil obtained was dried on anhydrous sodiumsulfate and then filtered through a cotton plug. The sample was kept under refrig-eration at 10 1C until it was used.

Laboratory and greenhouse tests

B. tabaci adults andH. grandella larvae for the experiments were taken from colonieskept at the Entomology Laboratory at CATIE.

Whiteflies

So far, 32 candidate crude plant extracts have been tested, including samples fromleaves, seeds, bulbs, flower buds, fruits, and essential oils (Cubillo et al., 1994, 1997,1999; Gomez et al., 1997; Hilje and Stansly, 2001; Aguiar et al., 2003). They wereselected based on ethnobotanical references, as well as on their low or nil taxonomicaffinity with the most common hosts of B. tabaci (Greathead, 1986).

Each plant extract was tested individually, at the following doses: 1, 5, 10, and15ml/l water (0.1%, 0.5%, 1.0%, and 1.5%, v/v). They were compared with aninsecticide (endosulfan), a control treatment (distilled water), of either Volck 100Neutral or Sunspray 9E, which are agricultural oils that strongly deter whiteflyadults (Hilje and Stansly, 2001), and the emulsifier Citowett. Endosulfan (Thiodan35% CE; Hoechst, Germany) (350 g a.i./l) was used at its commercial dose (2.5ml/lwater), and Volck (Chevron Chemical Co., CA) or Sunspray (Sun Co., Philadelphia)at 1.5% v/v. Citowett (BASF, Germany) (0.25ml/l) was applied in all treatments, atits commercial dose (0.025%).

Treatments were applied to tomato plants (var. Hayslip) with two true-leaves. Thiswas done with a hand-sprayer DeVilbiss 15, with an adjustable tip (The DeVilbiss,Somerset, PA), which was connected to an air pump, under a constant pressure(10 kg/cm2). Plants from each treatment were separately sprayed with each substancein an isolated room, for which they were placed on a table and thoroughly sprayed torun-off. Treated plants were introduced into sleeve cages (30� 30� 45 cm, with wallsmade of wood, a fine net, and glass) 30min after being sprayed.

A randomized complete block design with four replicates was used for the threeexperiments. For these restricted choice experiments, two pots with a tomato plant ineach one were placed in a sleeve cage. One of them has been sprayed with a givensubstance (either an extract or the control treatments), whereas the other plant wastreated with distilled water. For the absolute control treatments, one of the pottedplants was sprayed with Citowett and the other with water. The experimental unitwas represented by each potted plant receiving a given treatment.

Fifty B. tabaci adults were collected with a hand aspirator from a greenhousecolony reared on tomato, and released into each cage. Release took place between8:30 and 10:30 h; 2min later, the aspirator flask was checked, in order to count andrelease additional adults for replacing those which had died because of handling.

In addition to these experiments, unrestricted choice experiments were performed,by exposing potted plants to flying whiteflies inside a greenhouse where their colonyis maintained. Plants were placed on a bench and arranged in a randomized completeblock design. Treatments included the best doses from the previous experiments, andwere compared to the same controls, except endosulfan, in order not to disturb


colony development. Also, the experimental unit was represented by each pottedplant receiving a given treatment.

Moreover, in order to gain insight into more specific groups of substances re-sponsible for causing phagodeterrence, testing of fractions (water, methanol: water,methanol, and ether) of some promising extracts was carried out by means of bothtypes of experiments just described, at the same doses tested for the crude extracts(0.1%, 0.5%, 1.0%, and 1.5%, v/v).

For all experiments described above, counts were made on the foliage of the wholeplant. The criterion to appraise feeding deterrence was the number of landed adultsat 48 h, in combination with the number of those surviving within such interval.Oviposition response was appraised by counting the number of eggs laid up to 48 h.Also, mortality was determined by counting the total number of living adults in eachcage (in both plants) at 48 h, and subtracting the product from 50; in this case, theexperimental unit was represented by each cage containing two potted plants. Eggswere counted under a stereo-microscope.

Mahogany shootborer

Thus far, 20 out of the 32 crude plant extracts prepared for the experiments onwhiteflies have been tested (Table 1) on this pest (Mancebo et al., 2000a).

H. grandella larvae were taken from colonies, where they were initially reared ontender foliage of Spanish cedar (Cedrela odorata) and later transferred to an artificialdiet (Vargas et al., 2001). Third-instar larvae, which had been fed exclusively oncedar foliage, were selected because their size allowed easy handling. Experimentswere carried out in environmental chambers (Percival I-35L) set at 22 1C, 80%–90%RH, and 12:12 (L:D) photoperiod.

Bioassays included treatments with both wood and leaf extracts of bitterwood, aswell as leaf extracts of mother-of-cocoa and wild ‘‘tacaco,’’ at five increasing con-centrations of each extract (0.10%, 0.312%, 1.00%, 3.16%, and 10.00%) mixed witha surfactant (Nu film 17, at 0.03%). They were compared to two relative controls(70% methanol, and Nu film 17 at 0.03%), and an absolute control treatment(distilled water). All dissolutions were prepared just before the experiment was setup, with distilled water as a carrier.

Disks of cedar tender foliage (2.3 cm in diameter) were cut with a cork-borer,dipped in the selected treatment for 10 s, and allowed to dry for 30min. Treated diskswere placed individually in 30ml glass flasks, along with a third-instar H. grandella

larva which had been deprived of food for 3 h. A piece of paper towel was fastenedwith the lid of each flask and was moistened periodically, in order to retain leafturgor.

A randomized complete block design, with four replications, was used. The ex-perimental unit consisted of seven larvae, except in the control (14 larvae). Blockswere represented by plastic trays, and flasks representing each treatment were ran-domized within each tray.

After being exposed to the treatment for 24 h, each larva was transferred to a flaskcontaining about 6ml of artificial diet (Vargas et al., 2001), where it was allowed tocomplete its development; larvae were transferred to other flasks in cases where itwas judged that the diet was not suitable for their development.


Such procedures were also used to test fractions (methanol, water, methanol: water,and ether) of the wood of Q. amara. To prepare the bitterwood test solutions, theequivalent amount for each fraction, in accordance to the yield of the fractionationprocess, was weighted and dissolved into 100ml of the respective solvent. So, because10% w/v was the highest concentration of crude extract tested at which feedingdeterrence was observed (Mancebo et al., 2000b), treatments corresponded to thefollowing concentrations (weight/volume) for each fraction: 2.3% (water), 0.625%(methanol), and 0.14% (ether). The absolute control treatment corresponded to a10% bitterwood crude extract (in water), obtained from a 21.2% solution, which wasthe concentration at which it was obtained in the process of extraction. Relativecontrol treatments corresponded to each one of the solvents (water, methanol, anddiethyl ether).

In addition, greenhouse experiments were carried out, where first-instar larvaewere carefully placed on the main shoot of potted cedar plants previously treatedwith each one of such fractions.

Three types of variables were measured in response to the bitterwood, mother-of-cocoa, and wild ‘‘tacaco’’ extracts: food consumption, mortality, and develop-mental effects. Food consumption was assessed for each disk, by recording thepercentage of leaf area which was consumed in 24 h. This was done by means ofa visual scale of the program Distrain 1.0 (Tomerlin and Howell, 1988). Mortalitywas determined for each larva every 24 h, and the instar at which mortality occurredwas recorded; cessation of movement and color change to black were the cri-teria used for judging mortality. Developmental effects included developmentaltime for each larval instar and the pupa, as well as pupal weight on the day afterpupation; dates for larval moulting, conversion into pupae and adult emergence wererecorded.

For the greenhouse experiments with Q. amara fractions, variables included thenumber of orifices, sawdust mounds, and tunnels made by larvae, as well as thenumber of wilt and fallen shoots.

Moreover, since bitterwood and common rue showed promise as deterrents, andeven wild ‘‘tacaco’’ showed biological (insecticidal) activity to the mahogany shoot-borer (Mancebo et al., 2000a, 2000b, 2001), they were assessed for their systemicactivity. Therefore, they were tested at a high enough concentration (10%), in orderto clearly detect any possible systemic effects, and were compared to carbofuran(Furadan, FMC Corp. and Mobay Chem. Corp.) and Azatin EC (AgriDyne Tech-nologies Inc., Salt Lake City, UT). The former is a very effective systemic insecticideagainst H. grandella larvae when it is applied as a granular material under fieldconditions (Wilkins et al., 1976), whereas the latter was shown to act as a strongtoxicant to H. grandella (Mancebo et al., 2002).

Plant extracts and the control treatments were dissolved into distilled water andmixed with a basic tissue culture media (Murashige and Skoog, 1962), supplementedwith sucrose and solidified with agar. Two-month old plantlets grown from micro-cuttings or apical shoots of 45-day old cedar plants, were individually transplantedinto 250ml glass flasks containing the culture media. Systemic activity was assessed3 days, a week and 2 weeks later by excising folioles from any of the upper leavesfrom each plant and exposing them to first or second-instar larvae which had beendeprived of food for 3 h, inside 30ml glass flasks (Soto, 2000). Variables measured


included leaf area consumed as well as mortality, according to the procedures pre-viously described.

Achievements

Whiteflies

The majority (29) of the crude extracts tested thus far have induced some degree ofresponse by B. tabaci adults (Table 1), meaning that they have substances actingas feeding deterrents on them (Cubillo et al., 1994, 1997, 1999; Gomez et al., 1997;Hilje and Stansly, 2001; Aguiar et al., 2003).

The following nine species have stood out for their ability to deter adult whiteflies:bitterwood, ‘‘chile muelo,’’ fish bean, mother-of-cocoa, ‘‘sorosı,’’ sword bean, wildsunflower, wild ‘‘tacaco,’’ and worm-seed. Such an effect has been detected atdoses as low as 0.1% v/v (1ml/l water) for bitterwood, 0.1% (wild ‘‘tacaco’’), 0.5%(‘‘sorosı’’), 1% (‘‘chile muelo’’), 1% (mother-of-cocoa), 1% (fish bean), 1% (swordbean), 1% (wild sunflower), and 1% (worm-seed).

Phagodeterrence is revealed by the reluctance of whitefly adults to remain on thetomato plant treated with a given extract once they have landed on it and, presum-ably, made contact with the deterrent substances present in the extract, so that overtime they tend to accumulate in the untreated plant. This is illustrated here with theirresponses to the mother-of-cocoa extract (Figure 1A). In general, the same patternholds for oviposition response (Figure 1B), as the latter is a direct expression of thenumber of females present on the tomato plants.

In all cases there is a clear-cut deterrence effect by the mineral oil (Figures 1A and1B), as well as a toxic effect by endosulfan (Figure 1C). Mortality is revealed by thetotal number of living adults inside the cage at the end of each experiment, regardless of the plant where they are located. In fact, sometimes deterrence itself is sostrong that it causes a high degree of mortality (Figure 1C), statistically similar(p>0.05) to that of the insecticide control, endosulfan.

Spoiled by these findings, the authors purposely decided to concentrate researchefforts on three promising extracts: bitterwood, mother-of-cocoa, and wild ‘‘tacaco’’.This is so because they represent three ‘‘prototypes,’’ considering a number of factorssuch as plant habit (tree, shrub, vine, etc.) and life cycle (perennial or annual);temporal availability and operational difficulties in harvesting the specific plantstructures from which they are obtained (wood, foliage, and fruits, respectively);degree of harmfulness of their chemical components to farmers, consumers, andwildlife; and easiness to establish rather large-scale plantations of these species,either in open areas, within common tropical agroforestry systems (associated withcoffee and cacao) or in enriched forests, for the industry to count on a permanentsupply of raw material for preparing such extracts.

Therefore, in order to gain insight into more specific groups of substances re-sponsible for causing phagodeterrence, the next step was to test fractions of thesepromising extracts. Results are encouraging (Table 2), showing that methanol andmethanol: water fractions provoked a stronger response by whitefly adults, exceptfor wild ‘‘tacaco,’’ for which the response to the methanol fraction was weak,

Treated Untreated

Water Citowett Endos Oil 0.1% 0.5% 1.0% 1.5%

N° adults

0

5

10

15

20

25

30

Water Citowett Endos Oil 0.1% 0.5% 1.0% 1.5%

a

a

a

a

aa

a

a aa

aa

b

b

b

A

N° eggs

0

10

20

30

40

50

60a

aa

a

ab

b

a

a a

aa

a

a

b

b

B

Water Citowett Endos Oil 0.1% 0.5% 1.0% 1.5%0

5

10

15

20

25

30

35N° adults

aab

ab

abab

aa

b

C

a

Fig. 1. Average number of landed B. tabaci adults (A) and deposited eggs (B) at 48 h after themother-of-cocoa (G. sepium) extract was applied to tomato plants, as well as the averagenumber of surviving adults (C) in that interval. Means followed by the same letter in each pairof bars for A and B, and between individual bars for C, are not significantly different(p ¼ 0.05) (Hilje and Stansly, 2001). Abbreviations: Endos (endosulfan), Oil (Volck 100Neutral).


Table 2Minimum concentration of either the crude extracts of three plant species or their fractions atwhich feeding deterrence to Bemisia tabaci adults was detected, using the assays described inthe text

Plant species and fractions Deterrence

Bitterwood (Q. amara)

Crude 0.1%Methanol 0.1%Water 1.0%Methanol: water 0.5%Ether No

Mother-of-cocoa (G. sepium)

Crude 1.0%Methanol 0.1%Water NoMethanol: water 0.5%Ether 1.5%

Wild ‘‘tacaco’’ (S. pittieri)

Crude 0.1%Methanol NoWater 0.5%Methanol: water 0.5%Ether 0.1%

Source: Flores (2003).


whereas the ether one showed the strongest response. This could be explained con-sidering that the more polar components of this extract are glycosides of bayogenin(saponins) (Castro et al., 1997) and the ether fraction could contain some of theaglycone bayogenin as a product of partial decomposition of the saponins. So,eventually, a further experiment has to be performed to obtain some of the bay-ogenin itself and test the pure compound on whiteflies.

Results are illustrated here with adult responses to the bitterwood methanol frac-tion, showing strong deterrence at a dose as low as 0.1% (Figure 2A), the samepattern holding for oviposition (Figure 2B). Also, deterrence is so strong that itcauses a high degree of mortality (Figure 2C), statistically similar (p>0.05) to thatof endosulfan. Likewise, the methanol fraction mother-of-cocoa, as well as theaqueous fraction of wild ‘‘tacaco’’ caused deterrence at doses as low as 0.1 and 0.5%,respectively.

Deterrence by bitterwood could be explained by the presence of quassinoids, suchas quassin and neoquassin, which are common in this species (Polonsky, 1973). Forexample, Leskinen et al. (1984) found that a type of quassin from Q. amara detersfeeding by Epilachna varivestis (Coleoptera: Coccinellidae). In the case of mother-of-cocoa, its foliage contains a wide array of compounds, including terpenoids,flavonoids, anilpropanoids, and isoflavonoids (Lopez, 1995), some of which mayhave deterrent activity.

0

20

40

60

80

100

120

140

160

a b

a a a

b

a a

a

a

a

bb

bb

b

N° eggs

B

Treated Untreated

0

10

20

30

40

50

60

Water Methanol Endos Oil 0.1% 0.5% 1.0% 1.5%


a

b

b

a a a a

a a

bb b

b

b

a a

N° adults

A

0

10

20

30

40

50

60

a

ab

b

abab

aab

ab

N° adults


C

Fig. 2. Average number of landed B. tabaci adults (A) and deposited eggs (B) at 48 h after themethanol fraction of bitterwood (Q. amara) was applied to tomato plants, as well as theaverage number of surviving adults (C) in that interval. Means followed by the same letter ineach pair of bars for A and B, and between individual bars for C, are not significantly different(p ¼ 0.05) (Flores, 2003). Abbreviations: Endos (endosulfan), Oil (Sunspray).


In regards to the wild ‘‘tacaco’’ extract, chemicals causing the observed effectsremain unknown, although probably they are a series of glycosides known as ta-cacosides, which are very bitter and irritating. Six of these bayogenin saponinshave been isolated from fruits and aerial parts of S. pittieri and S. talamancense


(Castro et al., 1997), in an effort to look for antiproliferative principles in neotropicalplants. Cucurbitacins, which have several kinds of activities, including toxicity andfeeding deterrence (Mabry and Gill, 1979), are not found in this plant.

When tomato plants treated with all bitterwood fractions were exposed to flyingwhiteflies, the methanol fraction performed better than the rest, closely followed bythe ether one, but none of them did better as well as the mineral oil (Figure 3A), atrend that lasted for only 48 h (Figure 3B). Within a week (Figures 3C and 3D), noneof these fractions performed better (p>0.05) than the absolute control (water),which suggests that the deterrent principles decompose under the experimental con-ditions. Quassinoids, which are possibly responsible for such effects, are not volatile,but probably are decomposed by the air and/or light. Other components of Q. amara

which could have a similar activity are some indole alkaloids of the canthin-6-onetype, mainly present in the leaves (Saenz and Nassar, 1970) but which can be foundin the wood (Barbetti et al., 1990; Coe and Anderson, 1996), especially if the prepa-ration of the sample included some bark. These compounds can also be decomposedby air and/or light.

Mahogany shootborer

Some 20 plant extracts, from the 32 ones also tested for B. tabaci, have been assessed(Table 1), and three of them have shown some type of biological activity againstlarvae: bitterwood, common rue, and wild ‘‘tacaco,’’ the first two acting as feedingdeterrents and the latter as a powerful insecticide (Mancebo et al., 2000a, 2000b,2001).

Mortality by the wild ‘‘tacaco’’ extract, which kills larvae within a few hours ordays, depending on its concentration, is probably due to tacacosides (Castro et al.,1997). Both foliage and wood methanol extracts of bitterwood, as well as that ofcommon rue have substances acting as feeding deterrents, so that larvae refuse toeat treated leaf disks but, once they are placed into flasks containing an artificialdiet they continue feeding and complete their development, normally reaching theadult stage.

In terms of leaf disk consumption, there were very large differences between woodand leaf extracts of Q. amara (po0.0001), the former showing far higher antifeedantactivity than leaf extracts (Figure 4). Such activity was detected at a concentration aslow as 0.32% for the wood extract and as high as 3.16% for the leaf extract, with theresponse curve for the extract concentrations and leaf disk consumption being bestfitted by a potential model in both cases.

Such differences are probably explained by the specific chemical constituents ineach plant structure, and especially by the concentration of quassinoids. For in-stance, bitterwood foliage contains substances toxic to insects such as larvae of themosquito Culex quinquefasciatus (Diptera: Culicidae) (Evans and Raj, 1988), someof which could be quassinoids, which appear in low concentrations (Robins andRhodes, 1984). Also, quassin and neoquassin concentrations vary within the treebranches, and increase with branch diameter (Villalobos, 1995). In addition, thewood contains some beta-carboline related indole alkaloids (Barbetti et al., 1987).

In the case of the common rue extract, the lower consumption averages wereattained at the 3.16%, followed by 0.32%, 1.0%, and 10% concentrations, which did

0

50

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150

200

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300 N° adults

A

c

ab

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d

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350N° adults

Ba

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80

100

120

140N° adults

Da

ab

aa

b b

WF MF MWF EF Oil Water

WF MF MWF EF Oil WaterWF MF MWF EF Oil Water

WF MF MWF EF Oil Water

Fig. 3. Average number of B. tabaci adults on tomato plants at intervals of 1 (A), 2 (B), 8 (C), and 15 days (D), in response to four fractions (water,methanol: water, methanol, and ether) of bitterwood (Q. amara) and two control treatments (Sunspray oil and water). Means followed by the sameletter are not significantly different (p ¼ 0.05) (Flores, 2003). Abbreviations: WF (water), MF (methanol), MWF (methanol: water), and EF (ether)fractions, and Oil (Sunspray).

Promisso

rybotanica

lrep

ellents/d

eterrents

formanagingtwokey

tropica

linsect

pests

393

y = 1.9126 x -1.0395

R2 = 0.93

0

10

20

30

40

50

60

A

y = 13.993 x -0.6381

R2 = 0.90

0

10

20

30

40

50

60

0 1 2 3 4 8 9 10

B

Concentration (%)

Co

nsu

mp

tio

n (

%)

765

0 1 2 3 4 8 9 10 765

Fig. 4. Average cedar leaf disk consumption (% area) by third-instar H. grandella larvae at24 h, in response to increasing concentrations of wood (A) and leaf extracts (B) of bitterwood(Q. amara). The continuous line depicts the predicted response curve (Mancebo et al., 2000b).


not differ among themselves (p>0.05), the response curve also being best fitted by apotential model (Figure 5). Common rue extracts can deter Colorado potato beetle,Leptinotarsa decemlineata (Coleoptera: Coccinellidae) larvae and adults (Hough-Goldstein, 1990), and repel the cat flea, Ctenocephalides canis (Siphonaptera:Pulicidae) (Cox, 1980). Common rue foliage contains a number of chemicals, such asbenzenoids (moskachans), quinoline alkaloids (arborinine and several acridone de-rivatives), terpenoids (elemol and beta-eudesmol from the essential oil), flavonoids(rutin), and coumarins derivatives (coumarin, bergapten) (Torres, 1950; Vasudevanand Lukner, 1968; Kong et al., 1984), but it remains unknown if any of them areresponsible for causing either antifeedant or repellent activities.

Results were encouraging regarding systemic activity of the extracts, which wouldbe an asset for using them or their analogs in IPM programs. Foliole consumptionby H. grandella larva was nil for those plantlets grown on culture media treatedwith carbofuran, as expected (Soto, 2000), being followed by Azatin, bitterwood,common rue, and wild ‘‘tacaco’’ all of them differing from the control (po0.05)(Figure 6). Therefore, substances causing either deterrence or toxicity can be trans-located within the plant, reaching the leaves.

Afterwards, once the crude extract showed this valuable asset, bitterwood frac-tions were analyzed to gain insight into more specific groups of substances respon-sible for causing phagodeterrence, as previously described.

Co

nsu

mp

tio

n (

%)

0

24

6

810

12

14

1618

20

Water Carbofuran Bitterwood Azatin Commonrue

Wild tacaco

d

a

c

cd

bc

b

Fig. 6. Average foliole consumption (% area) by first-instar H. grandella larvae at 24 h, inresponse to cedar plantlets grown in vitro in culture media treated with different crude plantextracts, neem (Azatin) and two control treatments (carbofuran and water). Means followedby the same letter are not significantly different (p ¼ 0.05) (Soto, 2000).

0

5

10

15

20

25

30

0 2 3 4 5 6 7 8 9

Co

nsu

mp

tio

n (

%)

Concentration (%)

1 10

y = 9.0044 x -0.3615

R2 = 0.75

Fig. 5. Average cedar leaf disk consumption (% area) by third-instar H. grandella larvae at24 h, in response to increasing concentrations of a leaf extract of common rue (R. chalepensis).The continuous line depicts the predicted response curve (Mancebo et al., 2001).


Methanol and ether fractions stood out and performed as well as the crude bit-terwood extract in precluding larvae from feeding on leaf disks (Figure 7A). Thisfinding was corroborated when larvae were exposed to shoots treated with suchfractions under greenhouse conditions (Figure 7B); in both cases the water fractionperformed very poorly, probably because the water solubility of the quassinoids atroom temperature is scarce and the effect of the very small amount of these com-pounds present in the water fraction could be arrested by the presence of othercomponents.

Results with the bitterwood methanol extract are used here to illustrate theconcentrations at which phagodeterrence to H. grandella occurs (Figure 8). Whenexposed to disks impregnated with increasing concentrations of that fraction,leaf disk consumption by larvae was significantly lower (po0.05) at doses as lowas 0.02%.

Consumption (%)

0

5

10

15

20

25

d

a

cd d

ab

bcdbc

A

N° holes

0.0

0.5

1.0

1.5

2.0

2.5

Crude WF MF EF Water Methanol Ether

Crude WF MF EF Water Methanol Ether

c

ab

a ab

b

cc

B

Fig. 7. Response of H. grandella larvae to four fractions (water, methanol: water, methanol,and ether) of bitterwood (Q. amara) and the respective solvents, expressed as: the averageconsumption (% area) by third-instar larvae in 24 h to leaf disks treated with them (A), and theaverage number of holes in cedar plants treated with them, two days after their exposure tofirst-instar larvae I (B). Means followed by the same letter are not significantly different(p ¼ 0.05) (Soto, 2000). Abbreviations: WF (water), MF (methanol), and EF (ether) fractions.


Concluding remarks

Even though findings herewith reported are still preliminary, they clearly show thata rather wide range of tropical plant crude extracts – some of them not reported inthe literature yet – contain substances that can act as strong feeding deterrents ofB. tabaci and H. grandella. It has been shown that, prior to insert their stylet orsucking tube into plant tissue B. tabaci adults rub or tap the apex of their labium onthe plant surface, where they have several pairs of sensilla whose ultrastructuresuggests that they can act either as chemoreceptors or mechano-chemoreceptors(Walker and Gordh, 1989). Likewise, H. grandella larvae possess deterrent recep-tors in the medial and/or lateral sensilla styloconica located on the maxillae(Schoonhoven, 1980).

Consumption (%)

0

2

4

6

8

10

12

Water Methanol 0.00625% 0.02% 0.0625% 0.2% 0.625%

a a

ab

bc

cc c

Fig. 8. Average cedar leaf disk consumption (% area) by third-instar H. grandella larvae, in24 h, in response to increasing concentrations of the methanol fraction of bitterwood(Q. amara). Means followed by the same letter are not significantly different (p ¼ 0.05) (Soto,2000).


In fact, 20 out of 32 as well as three out of 20 plant extracts tested on B. tabaci andH. grandella, respectively, showed deterrence or toxicity. This general trend is kind ofunexpected, since B. tabaci is quite polyphagous, whereas H. grandella is ratheroligophagous, as its host range is restricted to members of the Meliaceae family.

At any rate, in order for the agrichemical industry to get involved into develo-ping commercial deterrents based upon plant extracts, as it currently occurs withneem derivatives (Walter, 1999), a number of questions and concerns ought to beaddressed.

First of all, there should be a demand strong enough as to justify their involvementin such a business. However, nowadays market considerations are closely tied toother issues related to the concepts and practices of sustainability, aimed at recon-ciling both agricultural production and economic development with environmentalconservation.

On one hand, since IPM practitioners are continuously striving for developingsuch sustainable production systems, commercial deterrents would fit very well intothese systems, provided that they are compatible with the conservation of water, soil,and wildlife, as well as with farmer and consumers’ health.

On the other hand, since the only discrepancy between IPM and organic farming isthat the former approach allows a rational use of some synthetic inputs (fertilizersand pesticides), safe and botanical-based deterrents would also fit well into organicsystems, as well as in certified forest systems and products. Since both systems allowproducers to take advantage of some economic benefits, such as particular niches ininternational markets associated with a premium value compared to the price paidfor conventional products, they would be willing to adopt environment-friendlysemiochemicals, provided that they are cost-effective.

This situation represents a unique opportunity for local small and medium-sizecompanies in developing countries. For instance, in Mesoamerica, both micro-bial and botanical pesticides have been widely accepted, which explains the everincreasing number of companies involved in biopesticide production, such as neem


products, entomopathogens, botanical products, and pheromones (Hilje et al., 2003).Local companies could not only have a rather easy access to tropical biodiver-sity resources, but also add value to their products, favoring local economies andcommunities.

Secondly, toxicity to wildlife and people is a major concern not only for conven-tional insecticides, but also for natural products, including botanical ones. Of course,toxicological aspects for the majority of plant materials, as well as for those reportedhere, remain unknown. Nevertheless, some of the latter seem not to pose risks tohumans and other mammals.

For instance, common rue and bitterwood are commonly used as natural medi-cines in some neotropical countries and elsewhere. Likewise, leaves and shoots ofmother-of-cocoa are normally used as fodder for ruminants (CATIE, 1991). As forwild ‘‘tacaco,’’ most of what is known about it responds to ethnobotanical knowl-edge but, being very bitter, this is normally a plant which people regard as a weed.

In the case of bitterwood, it was one of the botanical insecticides widely usedbefore synthetic insecticides were developed (Metcalf et al., 1951). Also, Q-assia isthe brand of a new pharmaceutical product for digestive problems recently releasedinto the market by Lisanatura, a local pharmaceutical company in Costa Rica.

It has been shown that, on rats, the aqueous extract of the dry wood of Q. amara

was capable of preventing the formation of ulcers as induced by indomethacin,ethanol, or stress (Badilla et al., 1998). In a previous study, no sign of acute toxicitywas observed at any oral dose of an aqueous extract of the wood; however, theintraperitoneal administration of 500mg/kg, presented acute toxicity signs with a24 h recovery, but the 1000mg/kg dose was lethal to a 100% within 24 h (Garcıaet al., 1996). The crude methanol extract of the stem wood significantly caused areduction in the weight of the testis, epididymis, and seminal vesicle, but an increasein that of the anterior pituitary gland. Quassin produced similar biological actions asthe crude extract while the effects of 2-methoxycanthin-6-one did not seem to differfrom those of the control (Raji and Bolarinwa, 1997). Quassin was also shown toinhibit the synthesis of testosterone in rat Leydig cells in a dose-dependent fashion(Njar et al., 1995).

Thirdly, the desired degree of industrialization for plant semiochemicals is a highlyrelevant matter in practical terms. In fact, some of these would lend themselves to beapplied as crude extracts, as it normally occurs with many plant extracts in tropicalrural communities (Stoll, 2000). Moreover, some of them could be further processedas to obtain semi-rustic products based on the most active fractions against B. tabaciand H. grandella, in which farmer organizations may participate, as it has happenedwith IPM projects aimed at other pests, such as the small-scale processing plantsfor the production of Beauveria bassiana, an entomopathogenic fungus attackingthe coffee berry borer (Hypothenemus hampei), in Colombia and elsewhere. In suchcases, farmers could require periodical support from governmental or academicentities to guarantee adequate standards of quality control for their products.

Otherwise, farmer organizations could participate in the initial steps of industrialprocesses leading to the production of well-elaborated materials by the agrichemicalindustry, which has shown a real interest in developing bioinsecticides (Hall andMenn, 1999). For instance, there is a growing interest in promoting the utiliza-tion of the bitterwood tree as an economic resource for indigenous communities in


Mesoamerica, so that several of its ecological, silvicultural, and marketing aspectshave been researched in recent years (Ocampo, 1995).

Currently, Bougainvillea S.A., which is a new Costa Rican company, is starting aneffort to produce an industrial extract of bitterwood for insecticidal purposes (RafaelOcampo 2005, pers. comm.). This initiative begins with the domestication of theplant. Until the present day the plant has been managed – not cultivated – by thenative population of Talamanca. The domestication of the plant includes a studyof the best conditions for cultivation and observation and control of its pests.Harvesting practices compatible with the conservation of the species and assuranceof a high content of quassinoids have been contemplated, as well as the optimizationof the extraction conditions.

Also, now that it has been determined the most active fractions of bitterwood,mother-of-cocoa, and wild ‘‘tacaco’’ against B. tabaci and H. grandella, agrichemicalcompanies interested in developing commercial products based upon either naturalsubstances or use them as leads to obtain their synthetic analogues (Hall and Menn,1999) could proceed in identifying and characterizing the specific substances re-sponsible for the phagodeterrent effects and eventually manufacture them in for-mulations well fitted to meet farmer’s needs.

And, last but not least, there is the concern about the availability of raw materialin large enough amounts to supply industry on a continuous and reliable basis,which could be a hindrance when dealing with tree species. Fortunately, in the caseof bitterwood, mother-of-cocoa, and wild ‘‘tacaco’’ this is not a serious problem, asall lend themselves to be planted in different types of schemes for commercial pur-poses, outside natural forests.

The bitterwood tree, Q. amara L. ex Blom (Simaroubaceae) is a native tropicalshrub, whose range extends from Mexico to Ecuador, including the Caribbean(Ocampo, 1995). It normally grows in the forest understory, where it reaches up to9m in height and 10 cm in diameter. However, it can also grow easily in disturbedareas and can be reproduced readily by vegetative cuttings. Due to the interest inutilizing it for pharmaceutical and agricultural purposes, silvicultural aspects havebeen researched in recent years, including pruning regimes and resprout responses, asto guarantee a continuous harvest of wood for quassinoid extraction (Brown, 1995).

Mother of cocoa, G. sepium (Jacquin) Kunth ex Walpers (Fabaceae) is a perennialshrub that occurs in tropical seasonal lowlands, from Mexico to Panama. It reachesup to 15m in height and 40 cm in diameter, and is commonly used in agroforestrysystems to provide shade to cacao and coffee plantations. The trees are also usedas living supports for growing black pepper and vanilla, as living fences, in alleycropping systems, and as fodder (CATIE, 1991).

Wild tacaco, Sechium pittieri (Cogn.) C. Jeffrey (Cucurbitaceae) is a perennialvine that occurs at a very wide altitudinal range (100–2500m) from Nicaragua toPanama, where it grows on both wild and disturbed habitats, usually near rivers orcreeks, and even in flooding areas (Lira, 1995). Its fruits, 4–6 cm long and 3–4 cmwide, are green, kind of ovoid or fusiform, very bitter, and can appear all year round;the fruits of its congeneric species S. tacaco are edible.

In case of eventually counting upon formulated deterrents to use against B. tabaciand H. grandella in the field, their use could be optimized by deploying them onlyduring certain times in the crop life (critical period), aimed at minimizing contact


between the insect and the host plant. For instance, since the impact of viral diseaseson yields is higher at earlier stages of plant development (Hilje, 2001), any man-agement scheme for B. tabaci should focus on this critical period (60 days aftergermination), by spraying the product on the plant. For H. grandella, the criticalperiod corresponds to the first 5–8 years of tree development, depending on theregion (Cibrian et al., 1995), and the product could be delivered into trees as aslow-release formulation through an implant or a microinjection.

However, in both cases, during such a period the deterrents could be comple-mented with other IPM tactics, as semiochemical by themselves very seldom providerobust pest control (Pickett et al., 1997). Tactics like plant breeding, cultural prac-tices, and biological control, along with semiochemicals, should be aimed at rec-onciling production with environmental conservation, in accordance to the paradigmof sustainable development, for the economic benefit of farmers and of society asa whole.

Acknowledgments

The authors thank their students Fernando Mancebo, Francisco Soto, GuillermoFlores, Alana Aguiar, and Paul Gomez, as well as their assistants Douglas Cubillo,Manuel Carballo, Guido Sanabria, and Arturo Ramırez, who have supported theseefforts over the years at CATIE. To Juan Carlos Brenes (CIPRONA), for preparingthe extracts for the experiments described here, and to Vıctor Castro and JoseFrancisco Ciccio (CIPRONA) for providing samples of wild ‘‘tacaco’’ and essentialoils of some plants, respectively. To Bernal Valverde, Phillip J. Shannon, and PhillipA. Stansly, for their valuable contributions at early stages of our research devel-opment. To Francisco Soto, for his valuable help in preparing the illustrations.

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