Abstract: The toxicity and repellent effects of steam distilled oils of Ocimum suave, Piper guineese, Syzgium aromaticum and Xylopia aethiopica were evaluated against the cowpea bruchid (Callosobrochus maculatus) infesting stored cowpea seeds under prevailing storage conditions in middle belt of Nigeria. In toxicity test C. maculatus adults were exposed to 10 g of cowpea seeds (Vigna unguiculate) admixed with five dosages of each oil. While in the repellency tests adult bruchids were introduced onto test arenas (filter paper discs) treated with different dosages of each of the oil. Insecticidal oil were ranked in the order of decreasing toxicity as Syzgium aromaticum oil (LC50 = < 0.1 mg/10 g seed) > Xylopia aethopica oil (LC50 = 0.485 mg/10 g seed > Piper guineese oil (LC50 = 0.510 mg/100 g seed) > Ocimum shave oil (LC50 0.660 mg/10 g seed). Significantly higher proportion of C. maculates adults were repelled from filter paper discs treated with all plant oils with average means repellent order of 60.24, 77.87, 80.23 and 86.66% for Xylopia aethiopica, Piper guineese, Syzguim aromaticum and Ocimum suare, respectively.
INTRODUCTION
The World Health Organization (WHO) estimates that there are 20,000 unintentional deaths and 3 million poisoning caused by synthetic pesticides misuse in the third world each year (Nigeria included). The region has been used as a dumping ground for synthetic pesticides banned from Europe and United States. Due to lack of training, lack of money of illiteracy, farmers apply inappropriate synthetic pesticides to crops, families consume treated seeds during lean periods, deaths occur because of someone uses an empty pesticide tin for food storage or as a cooking pot (Lowel, 1998). As part of effort to bring this into barest minimum, we have conducted an investigation of naturally occurring substance that possess insect repellent and toxic activity to pest and harmless to man.
In the past few years, several studies have focused on the potential use of essential oil applications in control of different insect pests (Yadava, 1971; Su et al., 1972; Su, 1976a; Osisiogu and Agbakwuru, 1976; Don-petro, 1985; Lale, 1987, 1991; Ibrahim and Zarida, 1998; Marrio et al., 2002). The essential oil may be more rapidly degraded in the environment than synthetic compounds and some have increased specificity that favours beneficial insects (Pillmoor et al., 1993). Recent research has demonstrated their larvicidal and antifeeding effect (Bathal et al., 1993); Larocque et al., 1999; Lajide et al., 1995), their capacity to delay development and adult emergence and cause egg mortality (Marimutu et al., 1997), their deterrent effects on oviposition (Naumann and Isman, 1995) and their arrestant and repellent action (Moretti et al., 1998).
Ofuya (1990) and Okonkwo and Okoye (1996) have in different time examined the efficacy of Xylopia aethiopica powder against Callosobrochus maculatus. Ivbijaro and Agbaje (1986), Olaifa and Erhum (1988) have revealed the insecticidal activities of powder and solvent (hexane) extract of Piper guineese. Biological effect of oil and powder of Syzgium aromaticum have shown that it could serve as natural insecticides that may be employed in the protection of stored cowpea from infestation by C. maculatus (Lale, 1991). Further studies using steam distilled oil of these plants was investigated here in this work.
Cowpea, being one of the prominent agricultural products found in this area (Ohiagu, 1986; Agboola, 1979), which serves as a major source of protein to the people and Callosobruchus maculatus (F) which is the major pest of cowpea in storage. The bruchid commence infestation in the field once cowpea has been harvest and left for dry, reducing the quantity and quality of the grain duping storage (Prevett, 1986; Dike, 1994). To reduce the damage during storage and to maintain the quality a locally available natural product was investigated to see their effectiveness, also our environment was taken into consideration as the oils are biodegradable and it is cheaper than synthetic insecticide and remove the risk of toxic residues in foods and ensures the continued availability of insect free cowpea for food, planting and trade.
MATERIALS AND METHODS
Insect rearing and oil extraction: The culture of C. maculatus used for the study was established from infested cowpea purchased from a local market in Keffi, middle belt of Nigeria. The colony of C. maculatus was maintained subsequently on cowpea under prevailing conditions (32-36°C and 55-67% RH). Experiment were conducted under these conditions in the faculty of Agriculture, Nasarawa State University, Keffi (Lafia Campus) Nigeria.
Fresh leaves of Ocimum suave (wild), dry seed of West Africa black pepper (Piper guineese), dry fruit of clove (Syzgium aromaticum) and dry fruit of Negro pepper (Xylopia aethiopica) were crushed to coarse powder and steam distilled for 2 h for Ocimum suave and 5 h for other plant materials in the chemistry laboratory of the University in August 2005.
Insecticidal efficacy of the essential oils: In this bioassay, five concentrations of each oil (0.025, 0.050, 0.075, 0.100 and 0.125 mg) were dissolved separately in 0.5 mL of analytical grade of acetone. Each of the concentrations for each oil was admixed with 10 g of cowpea contained in 50 mL glass jar. The admixture was stirred thoroughly with a glass rod to ensure adequate coating of seeds with oil and until the acetone completely evaporated according to the method of Lale (1991). Forty mixed sex adults of C. maculatus (3-5 days old) were introduced into each jar and the lid was replaced. Control seeds were treated with 0.5 mL of pure acetone. Each treatment and control were replicated four times. Mortality counts was taken 24 h after introducing insects on treated seeds. Insects which did not respond to the gentle touch of a small probe were considered dead (Su, 1976b). Mortality data were expressed as percentages of the total number of beetles in each replicate and subjected to probit analysis of Finney (1971). Toxicity of the insecticidal spice oils was compared using the control lethal of 50% of test insects (LC50) as index.
Repellent effect of the essential oils: The method used for testing the repellency or the essential oil was based on the area preference test described by McDonald et al. (1970) and modified to varying degrees by several workers (Jilani and Malik, 1973; Malik and Maqvi, 1984; Sighamony et al., 1984; Ojimelukwe and Adller, 1999).
Test areas consisted or 9 cm Whatman No.1 filter papers cut in half. Five dosages of each essential oil were prepared by dissolving 0.025, 0.050, 0.075, 0.100 and 0.125 mg of oil in 0.2 mL of analytical grade of acetone. Each dosage was applied to a half filter paper disc as uniform as possible by means of a pipette. The other filter paper halves were left untreated. In the control chambers, one paper half was treated with 0.2 mL of pure acetone and the other half was left untreated. The oil-treated or in the case of the control chamber, the acetone-treated half discs were air-dried to evaporate the solvent completely. The treated and untreated half discs were rejoined using clear adhesive tape and placed in a glass Petri dish of 9 cm diameter. Thirty (3-5 days old) mixed sex of C. maculatus adults were released at the centre of each repellency chamber and then covered. Each treatment and the control were replicated four times. The number of insects present on untreated and treated discs were recorded after 30 min (Obeng-Ofori and Reichmuth, 1997). Percentage repellency was calculated for each replicate according to the method of Sighamony et al. (1984) as follows:
| Where, | C = | Number of insects of untreated paper |
| T = | Number of insects on treated paper |
Repellency data were subjected to two way analysis of variance (ANOVA) and means were compared using the Least Significant Difference (LSD) test of p = 0.05, repellency was then classified according to McGovern et al. (1977) as follows:
Class O = negative repellency: Class 1 = 0-20% repellency
Class II = 20.1-40%; Class III 40.1-60%, Class IV = 60.1-80;
Class V = 80.1-100%.
RESULTS AND DISCUSSION
The toxicity result shown in Table 1 revealed that all the steam distilled oil used were toxic to C. maculates. Clove, cause a significantly higher mortality to adult C. maculatus than other three oils. With respect to the bioassay technique the oils showed a ranked order of toxicity with significant differences as their LC50s indicated in the sequence Syzgium aromaticum > Xylopia aethiopica > Piper guineese > Ocimum sauve, with LC50 of <0.10, 0.485, 0.510 and 0.660 mg/10 g seed, respectively.
| Table 1: | Acute toxicity of steam-distilled oils from four edible spices to Callosobruchus maculatus |
In toxicity studies involving products obtained from obtained from other species, it has been reported that mortality was due to the biologically active components in the plant products. Schanenberg and Pans (1977) reported that the seed oil of S. aromaticum contain eugenol, a sesquiterpene and caryophylline as major constituents. Eugenol has been shown in previous studies to possess high insecticidal efficacy against stored product coleoptera (Lale, 1987; Obeng-Ofori and Reichmuth, 1997). It appears, however, that eugenol contained in clove oil as its major constituent has a higher insecticidal potency than the other active components of other plants.
Significantly higher proportions of beetles were repelled from filter peper discs treated with Ocimum sauve than other three plants (Table 2) the order of their repellency is as follows:
Ocimum sauve > Syzgium aromaticum > Piper guineese > Xylopia aethiopica. The repellent effect of the essential oil indicate that they contain active principles responsible for the repellency activity. The major components of the oils when in combination with other compounds of diverse structure in the oil could exhibit different mode of action against the test organism, contributing towards their repellency activity. Various spices have been found to be effective in controlling insect pest of cereal grains (Jilani and Su, 1983). It has been reported that some plant oils contain irritant and foul smelling chemicals that have the ability to strongly repel stored product insect pest (Malik and Maqvi, 1984).
The repellency was concentration-dependent (Table 2), increasing with increasing concentration.
At the concentration all the plants evoked classes IV and V repellency (Table 3). Toxicity and repellency characteristics of these plant varied, Ocimum suave has the least toxicity potential to other plants but have more repellent activity than then other three plants against C. maculatus.
Assastyasih and Madden (1986) reported the successful use of oil clove, black pepper and ginger dissolved in alcohol as repellents against house flies (Musca domestic). In previous investigation, Sighamony et al. (1984) reported that the seed oil of Piper nigrum was significantly more repellent (80-100% repellency) than the synthetic repellent dimethyl phthalate which evoked 40-80% repellency in Tribolium castaneum.
| Table 2: | Mean number of Callosobruchus maculatus adults repelled from filter papers treated with four insecticidal oils |
SED = 4.63, LSD = 9.26 (Interaction of oil x Dosage rate),
SED = 1.89, LSD = 3.78 (Essential oil), SED = 2.32, LSD = 4.64 (Dosage rate) |
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| Table 3: | Classification of repellent action of four insecticidal essential oils against Callosobrochus maculatus |
| a = Repellency class 1 = 10-20%, Class II = 20.1-40%, Class IV = 60.1-80% and Class V = 80.1-100% | |
The present results strongly support using these plants volatile oils for repellency and toxicity against cowpea brochid in addition to the work that has been carried out on the hexane extract and powder of these plants against insect. These plants volatile oils shows that it can be a substitute to synthetic insecticide because of its efficacy and to remove the problems that is associated with synthetic pesticide. Further studies is needed in the formulation of these volatile oils, since its release rate will determine how long it can preserve cowpea brochid.
ACKNOWLEDGMENT
The authors would like to thank Dr. F.A. Ajayi of the Faculty of Agriculture, Nasarawa State University for his technical assistance.