Environmental Protection Agency, Chemicals Control and Management Centre, P.O. Box M. 326, Accra, Ghana. 2

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1 Available online at Journal of Dynamics in Agricultural Research Vol. 3(1), pp.12-18, August 2016 Article ID: JDAR/JDYA Author(s) retain the copyright of this article Copyright 2016 ISSN: Original Research Paper Laboratory investigations of candlewood Zanthoxylum Xanthoxyloides as a grain protectant against Sitophilus Zeamais Motschulsky and Callosobruchus Maculatus (f.) Koomson, C.K. 1*, Owusu, E.O. 2, Ayertey, J.N 3 and Obeng-Ofori, D 4 1 Environmental Protection Agency, Chemicals Control and Management Centre, P.O. Box M. 326, Accra, Ghana. 2 Department of Animal Biology and Conservation Science, University of Ghana, Legon, Ghana. 3,4 Crop Science Department, University of Ghana, Legon, Ghana. *Corresponding Author. charleskoomson@yahoo.co.uk. Received 10 May, 2016; Accepted 12 July, 2016 Open Access article distributed under the terms of the Creative Commons Attribution License permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. ABSTRACT Zanthoxylum xanthoxyloides is a plant that is traditionally used for the treatment of ailments like tooth aches, leprous ulceration and ulcers, syphilitic sores, fever, post-delivery pains while the leaves are fed to ruminants. The insecticidal effects of Z. xanthoxyloides tablets formulations made from extracts of a combination of plant parts were assessed in the laboratory. The extracts were 2.2ml leaves and 1.8ml roots; 2.4ml leaves and 1.6ml roots; 2.6ml leaves and 1.4ml roots; 2.8ml leaves and 1.2ml roots; 2.2ml leaves and 1.8ml bark; 2.4ml leaves and 1.6ml bark; 2.6ml leaves and 1.4ml bark; 2.8ml leaves and 1.2ml bark. The target insects were the maize weevil, Sitophilus zeamais Motschulsky and the cowpea beetle, Callosobruchus maculatus (F.). Five tablets of each preparation were randomly placed in 5kg of grains to assess toxicity or grain protection by fumigation, persistency and repellency. The effective tablet formulations were extracts made up of 2.2ml leaves and 1.8ml roots; 2.4ml leaves and 1.6ml roots; 2.2ml leaves and 1.8ml bark and 2.4ml leaves and 1.6ml bark. These caused nearly 50% mortality, offered about 92% protection to both maize and cowpea in terms of weight loss and was repellent to both insects. There was however, a rapid loss of repellency and toxicity activities after 7 days following treatment, irrespective of the dosage applied. Z. xanthoxyloides tablets could be prepared for resource-poor farmers to protect grains against stored-products infestation. Key words: Zanthoxylum xanthoxyloides, Sitophilus zeamais Motschulsky, Callosobruchus maculatus (F.), grain protectant. INTRODUCTION Sitophilus zeamais Motschulsky (Coleoptera:Curculionidae) and Callosobruchus maculatus (F.) (Coleoptera:Bruchidae) cause serious post-harvest losses to grains in the tropics leading to food

2 Koomson et al. 13 insecurity (Dobie et al., 1991). FAO production figures in 1980 showed that 1, million metric tons of major grains were produced worldwide and using the oftenquoted conservative minimum overall food grain loss figures of 10% (Ayertey, 1986) estimated the total loss to mankind of these cereal grains to be million tones. Efforts to develop insecticide-based techniques for protecting grains in small traditional farm stores have only been partially successful because of problems such as high cost of synthetic insecticides and erratic supply due to foreign exchange constraints (Obeng-Ofori et al., 1997). Furthermore, the overuse of synthetic chemicals have led to serious problems, including development of insect resistant strains to insecticides, toxic residues on stored grains, health hazards to grain handlers, food poisoning, environmental pollution (Champ and Dyte, 1976; White, 1985; Zettler and Coperus, 1990). These problems have stimulated research into plants with insecticidal properties grown locally that are readily available, effective, affordable, less poisonous and less detrimental to the environment (Tierto, 1994). Most plants are rich sources of compounds that have insecticidal properties (Schmutterer, 1995; Bekele et al.; 1997; Obeng-Ofori et al.; 1998). Melia volkensi seeds (Rembold, 1994), Ocimum species (Kumar, 1987), Adzadirachta indica A. Juss (Addae-Mensah, 1998),Cassia sophera (Koomson, 2000) and many others have been successfully used to control insect pests. Z. xanthoxyloides is a shrub of about 1.25 m high and 0.13 m girth (Irvine, 1961). The seeds are used for making necklaces in Senegal (Irvine, 1961) and ripe seeds serve as pepper and for food. The dried and pulverised leaves are sometimes used to flavour food in La Cote d Ivoire (Dalziel, 1937) and to feed sheep in certain Ga villages in Ghana (Irvine, 1961). Various parts of the plants have long been used to treat many ailments such as stomach aches, ulcers, sores, rheumatism, paralysis and post delivery pains. Z. xanthoxyloides, therefore, appears to be quite safe for both man and domestic animals. Dry roots, stems, leaves and seeds of the plant proved effective against S. zeamais, C. maculatus and Tribolium castaneum under laboratory conditions (Osafo, 1999; Udo, 2000) and field conditions (Koomson, 2003). Though the dust proved effective in controlling the insect pests, such dust-treated grains are often rejected by consumers because of discolouration and loss of quality (Koomson, 2003). According to Udo (2000), the bark and roots caused 100% mortality after 72 hours while the leaves only gave about 64% mortality. But using either the root or bark alone would mean destroying and wasting the leaves. The research was thus aimed at combining the leaves with the roots and bark in different proportions to determine how best to maximize the use of the plant to control S. zeamais and C. maculatus. This paper reports the results of laboratory investigations on the bioactivity of Z. xanthoxyloides tablets against S. zeamais and C. maculatus in the laboratory. MATERIALS AND METHODS Insects Adults S. zeamais and C. maculatus were collected from a stock of maize and cowpea, respectively maintained in the Food Security Laboratory at the Zoology Department, University of Ghana, Legon and cultured in a controlled environment room at 28± 2 o C, 68±2% RH and 12L: 12D photoperiod. Whole maize and cowpea grains were used as food for S. zeamais and C. maculatus, respectively and mixed sex adults of each species were used for the respective bioassays. Preparation of Z. xanthoxyloides tablets The tablets were prepared in the Food Security Laboratory of the Zoology Department of the University of Ghana, Legon. Roots, bark and leaves of Z. xanthoxyloides were collected from the University of Ghana farms at Legon, Accra, chopped into smaller pieces and 250 g of each part soaked in 500 ml glass jar of 70% methanol and left in darkness for three days. The methanol was evaporated at 30 C-40 C using a rotary evaporator with rotary speed of 3-6 rpm for 8 hours following the procedure of Godefroot et al., (1981). The extracts were stored in a refrigerator at 8 C (Ofuya and Okuku, 1994). Some 3% of agar was prepared to hold and mould the extracts in a tablet form. The preparation was done as follows: 3g of agar was boiled with 100ml of distilled water in a water bath for 30 minutes. The type of agar used was LAM M TM Agar No.1 MC 2 manufactured by the International Diagnostic Group Plc. The plant part extracts were thus formulated as follows: 2.2ml leaves and 1.8ml roots; 2.4ml leaves and 1.6ml roots; 2.6ml leaves and 1.4ml roots; 2.8ml leaves and 1.2ml roots; 2.2ml leaves and 1.8ml bark; 2.4ml leaves and 1.6ml bark; 2.6ml leaves and 1.4ml bark; 2.8ml leaves and 1.2ml bark. The various formulations were poured into airtight containers with 3.5cm diameter and 3.0cm height. One millimeter of hot agar solution was mixed with each of the formulations in the airtight containers. The mixtures were left to cool at room temperature into solid tablets with the containers tightly closed. The weight of each tablet was 10.0g and this was made up of 8.0g of Z. xanthoxyloides parts extracts and 2.0g of agar. Toxicity of Z. xanthoxyloides tablets Each test tablet was placed at the base of 5kg of either maize or cowpea containing 20 mixed adults of S. zeamais or C. maculatus respectively in jute sacks. Each treatment was replicated five times. Mortality was assessed daily for 7 days to determine the most effective

3 14 J. Dyn. Agric. Res. tablet formulation. The assessment was done by sieving out the insects using an impact test sieve, and counting the number of dead or live insects. Insects were considered dead if they do not respond to probings of a blunt forceps (Udo, 2000). Percentage mortalities were calculated and corrected using the Abbott s (1925) formula: P= O - C X 100% C where, P = corrected mortality O = observed mortality C = controlled mortality The antifeedant activity of the Z. xanthoxyloides tablets was assessed using the count and weigh formula. (Anonymous, 1985). % Wt loss = (UNd DNu) x 1000 U(Nd + Nu) Where U = weight of undamaged grains D = weight of damaged grains Nd = No. of damaged grains Nu = No. of undamaged grains Persistency Test Five kilograms of maize and cowpea in jute sacks were treated with the most effective tablet formulations which were tests tablets containing extracts of 2.2ml leaves and 1.8ml roots; 2.4ml leaves and 1.6ml roots; 2.4ml leaves and 1.6ml bark; 2.6ml leaves and 1.4ml bark. The treatments were replicated five times and left in the controlled experimental room. Twenty S. zeamais and twenty C. maculatus were put one set of the replicates containing maize and cowpea, respectively. These were left in the controlled experimental room for one week after which the insects were sieved out and the number of dead and alive insects recorded. Another set of twenty S. zeamais and twenty C. maculatus were put in the second set of replicates after the first week and the insects were sieved out after another one week. The numbers of dead and alive insects were also recorded. The same procedure was repeated for the third set of replicates after the second week, until such period that the treatments were no longer effective in controlling the insects. This period was recorded. Repellency Test Repellency test was carried out in a choice bioassay method (Udo, 2000) using grains treated with Z. xanthoxyloides tablets. Ten grams of grains were treated with the tablets and a control of another 10g of grains of grains was placed on a clean filter paper adjacent to each other. This was replicated twice. Ten adult S. zeamais and C. maculatus were introduced separately onto each experimental set. The number of insects present on the control (Nc) and treated (Nt) grains were recorded after one hour. Percentage repellency (PR) was computed using the following formula: PR= Nc-Nt x 100 Nc+Nt (Obeng-Ofori et al., 1997). The data were analysed using ANOVA after transforming the percentages into arcsine values. All negative PR values were treated as zero (Obeng-Ofori et al., 1997). RESULTS Toxicity of the various tablet formulations of Z. xanthoxyloides to S. zeamais and C. maculatus. Table 1 shows the percentage mortalities of S. zeamais and C. maculatus in grains treated with the different formulations of Z. xanthoxyloides tablets. All tablets gave significant mortalities against both insect. Toxicity increased with increasing concentrations of the bark and root extracts. Tablets made up of extracts of 2.2 ml leaves and 1.8 ml roots, 2.4 ml leaves and 1.6 ml roots, 2.2 ml leaves and 1.8 ml bark and 2.4 ml leaves and 1.6 ml bark were the most effective. The 2.2 ml leaves and 1.8 ml roots extract tablets caused the highest mortality of nearly 50% and the 2.8 ml leaves and 1.8 ml bark gave a mortality of almost 10 percent. Protective Potential of Z. xanthoxyloides tablets. The protection provided by the Z. xanthoxyloides tablets are shown in Table 2. There were significant differences (P< 0.01) among the treatments in reducing the damage caused by the two stored product pests. The tablets with extract formulation of 2.2 ml leaves and 2.8 ml root tablets provided the highest protection of 96% and those containing extract formulation of 2.8 ml leaves and 1.2 ml bark provided the lowest protection of 91%. Persistency Tests of Z. xanthoxyloides tablets The toxicity and protection provided by the Z. xanthoxyloides tablets persisted during the first week of application irrespective of the dosage. The effectiveness of the tablets to kill the insects or provide any protection to the grains were significantly (P< 0.01) reduced to approximately zero during the second and third week of application irrespective of the dosage (Table 3). Repellent action of Z. xanthoxyloides tablets to S. zeamais and C. maculatus. The various formulations of the Z. xanthoxyloides tablets showed different levels of repellence to both S. zeamais

4 Koomson et al. 15 Table 1. Mean percentage mortalities of S. zeamais and C. maculatus exposed to Z. xanthoxyloides tablets made from different proportions of leaves, roots and barks. Tablets S. zeamais C. maculatus 2.2 ml L, 1.8 ml R ± ± ml L, 1.6 ml R ± ± ml L, 1.4 ml R ± ± ml L, 1.2 ml R ± ± ml L, 1.8 ml B ± ± ml L, 1.6 ml B ± ± ml L, 1.4 ml B ± ± ml L, 1.2 ml B 9.60 ± ± 1.80 Control 0.20± ± 0.40 LSD (P < 0.01) ml L, 1.6 ml R= tablet formulation made up of extracts of 2.4 ml leaves and 1.6 ml root. Table 2. Mean percentage protection (weight loss) of Z. xanthoxyloides tablets on maize and cowpea. Tablets S. zeamais C. maculatus 2.2 ml L, 1.8 ml R 4.83 ± ± ml L, 1.6 ml R 5.70 ± ± ml L, 1.4 ml R 7.08 ± ± ml L, 1.2 ml R 7.95 ± ± ml L, 1.8 ml B 5.35 ± ± ml L, 1.6 ml B 6.50 ± ± ml L, 1.4 ml B 8.03 ± ± ml L, 1.2 ml B 7.90 ± ± 0.2 Control ± ± 0.2 LSD (P < 0.01) ml L, 1.6 ml R= tablet formulation made up of extracts of 2.4 ml leaves and 1.6 ml root. and C. maculatus (Table 4). S. zeamais were less repelled with the highest repellence of 32% in the tablets made up of extracts of 2.2ml leaves and 1.8ml roots and the lowest of 9% in the tablets made up of extracts of 2.8ml leaves and 1.2ml bark. C. maculatus were more repelled recording the highest repellence of 69% in the 2.2ml leaves and 1.8ml bark tablet formulations and lowest of 19% in the tablet formulations made up of extracts of 2.8ml leaves and 1.2ml roots. DISCUSSION The low levels of toxicity obtained for Z. xanthoxyloides dust (Koomson, 2003) as compared with the Z. xanthoxyloides tablets could be attributed to loss of some toxic volatile constituents through volatilization during hardening of the Z. xanthoxyloides tablets (Bekele et al., 1996). According to Udo (2000), the reduced susceptibility of S. zeamais to the toxic effect of the Z. xanthoxyloides tablets may depend on the size of the insect, adaptive physiology and mode of action of the active component of the fumigant. The reduced susceptibility may also be attributed the sex, size, age and physiological status of the insects (Busvine, 1971). Beetles killed in Z. xanthoxyloides tablets treated grains had unfolded metathoracic wings and outstretched elytra. This according to Obeng-Ofori et al., (1997), suggests that toxicity was not due to only ingestion of treated grains but also to inhalation of toxicants. The Z. xanthoxyloides tablets provided different levels of grain protection against insect attack. The protection of the grains coupled with the mortalities observed may suggest that the plant has antifeedant properties (Udo, 2000). The low protection offered by Z. xanthoxyloides tablets as compared to the dust (Koomson, 2003) could be attributed to the loss of toxic volatile secondary constituents during drying and extraction process of the

5 16 J. Dyn. Agric. Res. Table 3.Persistency of Z. xanthoxyloides tablets. S. zeamais Tablets First week Second week Third week 2.2 ml L, 1.8 ml R 9.00 ± ± ± ml L, 1.6 ml R ± ± ± ml L,1.4 ml R ± ± ± ml L, 1.2 ml R ± ± ± ml L, 1.8 ml B 7.33 ± ± ± ml L, 1.6 ml B 8.67 ± ± ± ml L, 1.4 ml B ± ± ± ml L, 1.2 ml B ± ± ± 0.00 Control ± ± ± 0.70 LSD (P < 0.01) C. maculatus Tablets First week Second week Third week 2.2 ml L, 1.8 ml R 7.00 ± ± ± ml L, 1.6 ml R 8.67 ± ± ± ml L,1.4 ml R ± ± ± ml L, 1.2 ml R ± ± ± ml L, 1.8 ml B 7.33 ± ± ± ml L, 1.6 ml B 8.33 ± ± ± ml L, 1.4 ml B ± ± ± ml L,1.2 ml B ± ± ± 0.00 Control ± ± ± 0.00 LSD (P < 0.01) ml L, 1.6 ml R= tablet formulation made up of extracts of 2.4 ml leaves and 1.6 ml root Table 4. Mean % repellence (pr) values for the tablets against S. zeamais and C. maculatus in the choice test. Tablet Formulation S. zeamais C. maculatus 2.2ml leaves and 1.8ml roots 32 ± ± ml leaves and 1.6ml roots 30 ± ± ml leaves and 1.4ml roots 30 ± ± ml leaves and 1.2ml roots 16 ± ± ml leaves and 1.8ml bark 23 ± ± ml leaves and 1.6ml bark 19 ± ± ml leaves and 1.4ml bark 10 ± ± ml leaves and 1.2ml bark 9 ± ± 1.68 LSD (P< 0.05) ml L, 1.6 ml R= tablet formulation made up of extracts of 2.4 ml leaves and 1.6 ml root. tablets (Bekele, 1994). Though the dust offered a much higher protection than the tablets, it would not be much preferred by consumers since it will lead to qualitative losses (Koomson, 2003). Thus, the toxicity of the Z. xanthoxyloides tablets could further be improved by further toxicology research. The various tablets repelled S. zeamais and C. maculatus, S. zeamais adults being less repelled than C. maculatus. The deference may be attributed to the fact that S. zeamais are always found within the grains whiles C. maculatus are confined to the surface of the grains. The repellent effect of the tablet may be attributed to the presence of secondary metabolites in Z. xanthoxyloides (Owusu, 2000). The repellent action coupled with its inhibition to development of eggs and immature stages taking refuge within the grains increases the protectant

6 Koomson et al. 17 potential of the plant against insect pests damage during storage (Talukder and Howse, 1995). Effect of the Z. xanthoxyloides tablets persisted for only one week. The highly significant loss of potency after the first week may be attributed to rapid evaporation and or degradation of the chemical constituents of the tablets. This may be due to the physico-chemical properties of the compounds on the grains. Most unprocessed grains and legumes, such as maize and cowpea, lack polar surfaces to which chemical may be bound to reduce their volatilization (Weaver et al., 1994a). Combining the tablets with natural plant oils could enhance persistency of the tablets in grain (Weaver et al., 1991). 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