Efficacy of essential oil of Ocimum basilicum L. and O. gratissimum L. applied as an insecticidal fumigant and powder to control Callosobruchus maculatus (Fab.) [Coleoptera: Bruchidae]

Journal of Stored Products Research 37 (2001) 339–349

Efficacy of essential oil of Ocimum basilicum L. andO. gratissimum L. applied as an insecticidal fumigantand powder to control Callosobruchus maculatus (Fab.)

[Coleoptera: Bruchidae]

Sekou Moussa Keitaa, Charles Vincentb,*, Jean-Pierre Schmita,John Thor Arnasonc, Andre Belangerb

aDepartment of Chemistry, University of Quebec at Montreal, P.O. Box 8888, Downtown Station, Montreal,Quebec, Canada H3C 3P8

bHorticultural Research and Development Centre, Agriculture and Agri-Food Canada, 430 Gouin Blvd.,

Saint-Jean-sur-Richelieu, Quebec, Canada J3B 3E6cDepartment of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada K1N 6N5

Accepted 30 June 2000

Abstract

Essential oils from sweet basil, Ocimum basilicum, and African basil, O. gratissimum, (Labiatae) grown inGuinea were obtained by steam distillation. Following exposure of newly emerged adult beetles(Callosobruchus maculatus) to 12 h of fumigation using pure essential oils at a dose of 25 ml/vial, 80%mortality was recorded for O. basilicum, 70% for O. gratissimum and 0% in the control. A significantdifference was observed between the responses of males and females with males exhibiting greatersensitivity. When 1 g of aromatized powder was applied to adults, a 50% lethal concentration at 48 h wasfound to be 65 ml/g for O. basilicum and 116 ml/g of O. gratissimum oils. The essential oils from the two plantspecies exhibited a significant effect both on the egg hatch rate and on the emergence of adults. The egghatch rate was reduced to 3% with O. basilicum and 15% with O. gratissimum using an essential oilconcentration of 30 ml, whereas the egg hatch rate for the control was 95%. When compared with thecontrol (97%), adult emergence dropped to 0% with O. basilicum and to 4% with O. gratissimum. Storagebioassays were run to assess the long-term effect of powders aromatized with essential oils of Ocimum.Complete protection was observed over 3 months starting at a dose of 400 ml in the case of both oils. Froma germination test, it was concluded that aromatized powders have no significant effect on the seedgermination rate. After 5 d, a rate of 88% germination was seen in seeds treated with aromatized powder

*Corresponding author. Tel.: +1-450-346-4494 ext. 202; fax: +1-450-346-7740.

E-mail address: [email protected] (C. Vincent).

0022-474X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.

PII: S 0 0 2 2 - 4 7 4 X ( 0 0 ) 0 0 0 3 4 - 5

and protected from insects, compared with 97% for untreated seeds that were not exposed toinsects. # 2001 Elsevier Science Ltd. All rights reserved.

Keywords: Ocimum basilicum; Ocimum gratissimum; Bioinsecticide; Callosobruchus maculatus; Essential oil;

Steam distillation

1. Introduction

The flora of Guinea includes plant species that have insecticidal properties and that have longbeen used for a variety of ethnobotanical purposes. However, effective and economical methodsfor using the plants have not been commercially developed. In the present study, essential oils ofsweet basil, Ocimum basilicum L., and African basil, O. gratissimum L., (Labiatae) were evaluatedeither alone or in combination with kaolin powder, as control agents for the cowpea beetle,Callosobruchus maculatus (Fab.) (Coleoptera: Bruchidae). The findings, which complement thoseof Keita et al. (2000), represent an ongoing effort to provide rural people with effective and simplemethods to protect stored cowpeas. In Guinea, O. basilicum and O. gratissimum are well studiedand used in cosmetics, medicine and agriculture (e.g. Berhaut, 1975; Nianga et al., 1993; Keita etal., 1999). Gas chromatography analysis of Guinean Ocimum spp. revealed significantly differentphytochemical profiles for the two species used here (Keita, 2000).Natural compounds of plant origin are biodegradable, often of low mammalian toxicity, and

pose low danger to the environment if used in small amounts. Recent research has focused onnatural product alternatives for pest control in developing countries. Various studies havedemonstrated the efficacy of neem (Azadirachta indica A. Juss.: Meliaceae) as a protectant(Makanjuola, 1989; Lale and Abdurahman, 1999; Golob and Gudrups, 1999). Root bark powderfrom Zanthoxylum zanthoxyloides (Lam.) (Rutaceae) caused a significant reduction in the bruchidpopulation in cowpea stocks (Ogunwolu and Idowu, 1994; Ogunwolu and Odunlami, 1996), andsimilar results have been achieved with groundnut oil (Singh et al., 1978); Citrus peels (Don-Pedro, 1985), and extracts of Boscia senegalensis (Pers.) Lam. Ex Poir. (Capparidaceae)(Seck et al., 1996). Essential oil of Ocimum canum Sims, whose major constituent is linalool, hasbeen employed successfully against some insects (Weaver et al., 1991). In addition, the successfuluse of cotton and peanut oils as well as shea butter suggests that local African plant species haveconsiderable potential for the protection of stored products (Dabire, 1993). Certain simple post-harvest storage methods may also be helpful for reducing bruchid infestations of cowpea in anIntegrated Pest Management approach (Prevett, 1962). The goal of the present study was to assessthe effect of applying pure essential oil from two species of Ocimum as a fumigant and applying(by sprinkling) kaolin powder aromatized with the same oils, on bruchid eggs, larvae and adults.

2. Materials and methods

2.1. Bioassay

We conducted storage bioassays in Gueckedou (a prefecture of the forest region in southernGuinea) to assess in situ the activity of the essential oils of O. basilicum and O. gratissimum on

S.M. Keita et al. / Journal of Stored Products Research 37 (2001) 339–349340

C. maculatus. The bioassays were conducted on adult cowpea beetles using different essential oilconcentrations, applied both by fumigation and by sprinkling a powder aromatized with thoseoils. Some infested cowpea was purchased at the Madina market in Conakry (Guinea). Adultbruchids were sifted out of the cowpea and removed in order to obtain an emerging generation.Newly emerged individuals were obtained within a month and used in the bioassays. The beetleswere isolated from the stocks using the methods described in Keita (2000) during which they wereimmobilized for about 30 s under a stream of CO2 and quickly counted into groups for the desiredreplications. The CO2 stream, even when applied for about 60–90 s, has no significant (P50.05)effect on bruchid survival (Dawson, 1995; Keita, 2000). Uninfested chick peas (Cicer arietinum L.,Fabaceae) bought in Canada were also used during the tests (control of oviposition and egghatch).The kaolin powder (see Keita et al., 2000 for methodology) was aromatized using different

concentrations of essential oils from the two species of Ocimum. Four types of bioassays, twotypes of storage bioassays, and a germination test were conducted.

2.2. Fumigation of adult beetles with pure essential oils

Newly emerged 1-week-old adults (unsexed individuals) were counted into bottles, which wereimmediately covered with a piece of fine steel gauze. Four-millilitres bottles were used forfumigation by superposition (Keita et al., 2000). Ten pairs of bottles (20 bottles) were prepared foreach concentration. Five insects were placed into the bottles, with 10 replications. Volumes of 0, 5,10, 15, 20, and 25ml of essential oil from each plant species were placed in similar bottles,matching the number of replications established for the insects. The bottles containing the insectswere superimposed on the bottles, giving a total volume of 8ml: the bottles were sealed togetherwith HighlandTM 203 tape. Mortality counts of the insects were made 3, 6, 9, and 12 h after thetreatment. Control treatment consisted of an identical apparatus but without any essential oil.

2.3. Treatment of adult beetles with aromatized powder

A gram of each of the seven stocks of kaolin powder (fine white clay) aromatized with differentessential oil concentrations (i.e. 0, 10, 20, 40, 60, 80, and 100ml) was sprinkled onto the adultinsects in polyethylene bags (13� 30 cm), which had previously been filled with air to facilitatehandling. An identical number of bags with insects but non-aromatized powders were employedas a control under the same temperature (28� 18C) and relative humidity (70� 5%) conditions.Five replications were carried out for each concentration, with 10 insects per replication. Thenumber of dead individuals was counted after 24, 48, and 72 h.

2.4. Treatment of male and female beetles with aromatized powder

Insects were isolated using plastic bags (Keita, 2000). Five beetle pairs (in five replicates), sexedaccording to Bandara and Saxena (1995), were placed in 4ml bottles with screwcaps. Six stocks ofpowder aromatized with essential oils of O. basilicum and O. gratissimum were prepared, using 0,10, 20, 30, 40, and 50 ml of essential oil per gram of kaolin powder. One milligram from each stockwas applied to each beetle pair, and mortality rate was recorded after 48 h.

S.M. Keita et al. / Journal of Stored Products Research 37 (2001) 339–349 341

2.5. Treatment of eggs with aromatized powder

Two chick peas were deposited into each of 60 glass bottles. A pair of newly emerged beetles(i.e., a male and female, sexed as adults: Bandara and Saxena, 1995) was placed in each bottle (60pairs in all). After 3 d, the insects were removed, the eggs were counted and 20 eggs were left ineach bottle (the others were removed using a needle). The eggs were then sprinkled with 0.5mg ofpowder aromatized with essential oil of O. basilicum and O. gratissimum at concentrations of 0,10, 20, 30, 40, and 50ml/g. Each concentration was applied to the chick peas in 10 bottles (e.g., fivereplicates per treatment), which were then screwed shut. The control consisted of eggs that werenot sprinkled with the powder but were held under the same conditions (number, replications, andenvironment). The eggs were examined after 5 d to distinguish the ones that had developed(opaque, yellowish) from those that had not (translucent and yellow). The bottles were coveredagain and checked again after 30 d to compare the number of emerged insects with the number ofeggs that had been deposited.

2.6. Storage bioassays in burlap bags

Powder from seven different stocks (each containing 50 g) of kaolin powder, aromatized with0, 100, 200, 400, 600, 800, and 1000ml of the different essential oils, was mixed into 60 g of infestedseeds (from which the adult insects had been removed with a sieve). Five replicates of eachconcentration were placed in burlap bags. The burlap bags were fastened with a cord and placedin a large plastic bag, which was stored in a traditional farmer’s storage facility protected againstrodents and cockroaches (which are capable of destroying the bags and eating the seeds). Checkswere made after each month over a 3-month period. Five days after the checks, live and deadinsects were removed and the stock weighed.These data were used to compute the percentage weight loss. To calculate the seed weight loss

index (WL), we used the equation:

%WL ¼ ðIW � FWÞ�100=IW ;

where IW is the initial weight and FW is the final weight.

2.7. Storage bioassays in polyethylene bags

Fifty uninfested chick peas (purchased in Montreal, Canada) were placed in plastic bags, foreach of five replications. Five grams of kaolin powder which were aromatized with the fiveessential oil concentrations (i.e., 0, 50, 100, 150, 200, 250, and 300ml) from the two species ofOcimum was applied to 50 peas in a plastic bag. Two young male beetles and five young femaleswere placed in each bag, and the bags were closed after making sure that they contained enoughair. The bags were hung on a line (to avoid damage by mice) in a traditional cowpea granary usedby rural people. After 2 months, the number of perforated seeds was counted and the percentageperforation was calculated.

2.8. Cowpea seed germination test

Cowpea pods were harvested shortly before drying in anticipation of a possible infestation(since females lay eggs only on dry pods). They were dried in a rattan basket (Ancystrophyllum

S.M. Keita et al. / Journal of Stored Products Research 37 (2001) 339–349342

secundiflorum [P. Beauv.] Wendl.; Araceae) coated with cow dung (to seal any openings). Thebasket opening was covered with a piece of fine steel screen to prevent infestation. After 3 d ofdrying, the seeds were removed from the pods, and the sorted seeds were further dried under thesame conditions as for the pods. These healthy, uninfested seeds were used in the tests. Four lotseach weighing 250 g were placed in polypropylene containers (rearing chambers). The first lot wasstored without any treatment in a traditional facility exposed to beetles (control). The second lotwas sprinkled with kaolin powder aromatized with essential oil of O. basilicum, and the third lotwas sprinkled with powder aromatized with oil of O. gratissimum (these two lots were storedunder the same conditions as the control). The fourth lot was covered with a lid containing an airhole covered with a piece of fine steel screen (to keep insects out). After 6 months of storage, theseseeds were assayed for germination. They were sowed in some moist sand using five containerswith 20 seeds per treatment (i.e., 100 seeds). Observations were made after 3, 4, and 5 d.

2.9. Statistical procedures

The data were processed in Polo-PC (LeOra Software, 1994) using analysis of variance followedby Scheffe’s tests to compare effects among the treatments (SPSS for Windows 7.0, 1995).

3. Results and discussion

3.1. Fumigation of adult beetles with pure essential oils

After 12 h of fumigation, mortality of adult insects was found to increase as the essential oilconcentration increased (Fig. 1). At the highest dose of 25 ml of essential oil per 8ml vial, 80%mortality was recorded with O. basilicum, 70% for O. gratissimum and 0% in the control. LC50values of the essential oils were 0.66ml/ml (minimum=0.19 and maximum=1.00) for O. basilicumand 1.06ml/ml (minimum=0.45 and maximum=1.89) for O. gratissimum.

3.2. Treatment of adult insects with aromatized powder

There was a concentration-dependent increase in mortality of adults treated with the twopowders from which the LC50 and LC90 values at 24, 48, and 72 h were calculated. The resultsshow that O. basilicum (LC50=65ml/g and LC90=107 at 48 h) is more effective thanO. gratissimum (LC50=116ml/g and LC90=1573). In both treatments the LC50 and LC90decrease with time (Table 1).

3.3. Treatment of male and female insects with aromatized powder

There was a significant difference (P50.05) between the responses of males and females, withmales exhibiting steeper slopes. The t and g values indicate that the tests were reliable (Robertsonand Preisler, 1992), and this is corroborated by a comparison of the LC50 and LC90 values whichare, respectively, for O. basilicum (male and female) and O. gratissimum (male and female) 18.7;32.6, and 25.1; 35.2ml/g (Fig. 2).

S.M. Keita et al. / Journal of Stored Products Research 37 (2001) 339–349 343

3.4. Treatment of eggs with aromatized powder

The essential oils from the two plant species had a significant (P50.05) and concentration-dependent effect on both egg hatching and on adult emergence (Fig. 3). As a practical controlagent, this is a useful property, since in addition to killing the adult beetles (which died just after

Table 1

Probit analysis (concentration vs. probit model) of results following treatment with 1 g of kaolin powder aromatizedwith different concentrations of Ocimum gratissimum and O. basilicum applied on Callosobruchus maculatus (10 adults/replication� five replications)

Duration (h) O. basilicum O. gratissimum

LC50 (ml/g) LC90 (ml/g) Slope LC50 (ml/g) LC90 (ml/g) Slope(Min–max) (Min–max) (SD) (Min–max) (Min–max) (SD)

24 75 473 1.60 147 2048 1.12(67–85) (353–694) (0.11) (117–203) (1092–5161) (0.11)

48 65 107 6.07 116 1573 1.1

(58–71) (98–124) (0.8) (87–158) (783–5790) (0.2)72 51 82 6.18 60.0 127 3.9

(44–55) (76–90) (0.70) (51–66) (111–160) (0.62)

Fig. 1. Mortality of weevils following fumigation with the essential oil of O. gratissimum and O. basilicum for 12 h in4-ml bottles covered with a piece of fine steel mesh.

S.M. Keita et al. / Journal of Stored Products Research 37 (2001) 339–349344

hatching or shortly before emergence), the oils serve to break up the generation resident in cowpeastocks, thus promoting longer seed storage. A similar result was obtained by Keita et al. (2000)with essential oil of Tagetes minuta L., Hyptis suaveolens Poit., Ocimum canum L., Ocimumbasilicum and Piper guineense Schum.

3.5. Storage bioassays in burlap bags

Insect emergence was observed in the control within a month, whereas in the treatments withO. basilicum emergence was noted solely with concentrations of 100 ml and with O. gratissimumemergence was observed only at concentrations of 100 and 200ml.After a 3-month period low (i.e., 58%) percentage weight losses were observed starting at a

dose of 600 ml for O. gratissimum and 400 ml for O. basilicum oils (Table 2). Lower percent weightlosses were consistently obtained at 800 and 1000ml oil concentrations of O. gratissimum andO. basilicum.

Fig. 2. Effect of concentration of the essential oils of O. gratissimum and O. basilicum on male and female beetlemortality.

S.M. Keita et al. / Journal of Stored Products Research 37 (2001) 339–349 345

3.6. Storage bioassays in polyethylene bags

Using the percentage of protection of cowpeas seeds as an index (Fig. 4), O. basilicum reduceddamage more than O. gratissimum. In both cases the perforation rate declined as the essential oilconcentration increased in the kaolin powder.

Table 2Percentage weight loss of grains treated with kaolin powder aromatized with differentessential oil concentrations of Ocimum gratissimum (Og) and O. basilicum (Ob)

Treatment (ml) Percentage weight loss

1 month 2 months 3 months

Og Ob Og Ob Og Ob

0 (Control) 33 67 80

100 33 25 42 32 47 33200 17 5 25 8 33 10

400 8 1 15 3 20 3600 5 0 8 0 8 0800 5 0 5 0 5 0

1000 0 0 0 0 0 0

Fig. 3. Effect of the concentration of essential oils of O. gratissimum and O. basilicum on beetle eggs and adult

emergence.

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Fig. 4. Protective effect of the powders aromatized with essential oils of O. gratissimum and O. basilicum in storagebioassays tests conducted in traditional storage facilities.

Fig. 5. Effect of the essential oils on the germination capacity of cowpea seeds after exposure for 3–7 d (Error bars are

standard deviations). (Tr-Ex: Treated and Exposed; Tr-Uex: Treated and Unexposed; Ut-Ex:Untreated and Exposed;Ut-Uex: Untreated and unexposed).

S.M. Keita et al. / Journal of Stored Products Research 37 (2001) 339–349 347

3.7. Cowpea seed germination test

The experiment showed that the treatment using essential oil from the two species of Ocimumdoes not have a significant (P>0.05) effect on the seed germination. Indeed, after 5 d, thefollowing germinations were recorded: 88% for seeds treated with aromatized powder andprotected from insects; 79% for seeds that were treated and exposed to the insects; 15% foruntreated seeds and exposed to the insects; and finally 97% for seeds that were neither treated norexposed to the insects. This is an important finding since cowpea seed must be protected frominsects over a period of many months and eventually sown (Fig. 5).

4. Conclusion

Earlier results demonstrated the efficacy of kaolin powder aromatized with different types ofessential oils (Keita et al., 2000). Our results with Ocimum could be of importance in a WestAfrican context because the two species are abundant in Guinea as well as being easy to grow.Although seeding is the traditional propagation method, we recommend coppicing(i.e., systematic cutting of buds) instead, especially for O. gratissimum, which is a shrub. Thetwo species could be maintained for a long time by coppicing, combined with regular manureapplications, watering and hoeing. Distillation technology is readily affordable by peasants orIPM practitioners and the yield of oils are 1 and 1.8%, respectively, for O. gratissimum and O.basilicum (Keita, 2000).

Acknowledgements

We are grateful to the Government of Guinea, the University of Quebec at Montreal (UQAM)and the Canadian International Development Agency (CIDA) for providing funding to theCentre d’Etude et de Recherche en Environnement (University of Conakry) through the projectUQAM- CIDA No. 436/18051. Sekou Moussa Keıta received a doctoral stipend under theproject. Our thanks also go to the Biology Department and the Chair in Plant Ecology at theUniversity of Conakry and the agronomic research centre in Foulaya (Centre de RechercheAgronomique) for allowing us to work on their premises and for the assistance provided bycolleagues Alpha Kabine Traore, Koıkoı Kalivogui, Nyankoye Camara and Dr. Lancine Traore.

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