CENTRE FOR RESEARCH Centre for Research Development (CERAD) T he Federal University of Technology, Akure, Nigeria (www.jost.futa.edu.ng) JoST 2014 AND DEVE LOPMENT Ability of Rice Husk Husk Ash Powders to Protect Cowpea seeds against Callosobruchus Maculatus Fabricius (Coleoptera: Chrysomelidae: Bruchinae) Damage OFUYA, T. I.* ADLER, S. Institute of Ecological Chemistry, Plant Analysis Stored Products Protection, Konigin-Luise Strasse 19, 14195 Berlin, Germany. ABSTRACT: The ability of rice husk powder (RHP) rice husk ash (RHA) to protect cowpea seeds against Callosorbruchus Fabricius damage was investigated under controlled temperature relative humidity conditions. Lethality of RHP RHA to adults eggs of the beetle was determined. Each product was further tested at 0.05, 0.1, 0.2, 0.3, 0.4 0.5 g/20 g of grain respectively in glass Petri dishes against the beetle to determine prevention of seed weight loss. Effect of temperature on use of relatively low doses of RHA was also determined. RHA caused significant adult mortality at 0.5 g (100%) 0.4 g (83.3%) within 1 day post treatment whereas RHB caused 55% adult mortality at 7 days. Mean 2-day weight loss in adults exposed to RHA (2.40 mg) was significantly higher than for RHP (1.62 mg) control (0.19 mg). RHA was significantly lethal to egg the lethality increased with age of egg. Female beetles laid significantly fewer numbers of eggs on seeds treated with RHA than seeds treated with RHP the control, but F adult emergence from eggs laid was similar. There was no F adult mortality in 1 1 RHP treatments the control but significant mortality occurred in RHA treatments. Seed weight loss was significantly lowest when seeds were treated RHA ranging from 3.2% to 4.2%, but was highest in seeds in the control (13.5%) seeds protected with 0.1 0.2 g RHP (12.9%). When applied at relatively low dosages, RHA was significantly more lethal to C. adults at 30 C than at 25 K eywords: R ice husk, Ash, Callosobruchus, cowpea, mortality, damage JoST. 2014. 5(2): 70-79. Accepted for Publication, October 27, 2014 INTRODUCTION C allosobruchus (Fabricius) storage by often results in very (Coleoptera: Chrysomelidae: Bruchinae), also serious depredation making infested seeds unfit variously called the cowpea seed beetle, cowpea for human consumption farmers traders storage beetle, cowpea bruchid, or cowpea incur great financial losses (Ogunkoya weevil, is a principal pest of stored cowpea seeds O fuya, 2001; Badii et, 2011). In order to reduce ( Vigna unguiculata L. Walp.) many other the huge losses that can result from uncontrolled grain legumes in the tropics subtropics i nfestation damage to stored cowpea by (Ofuya, 2001). Grain legumes are the principal, synthetic insecticides such as sources of cheap affordable dietary protein pirimiphos-methyl aluminium phosphide for majority of the peoples in the developing have been effectively deployed by farmers countries of the tropics subtropics. traders in developing countries (Lale, 2010; Uncontrolled infestation of legume seeds in M agaji et, 2012). However, concerns over * Correspondence to: Ofuya, T.I.; inomisanofuya@yahoo.com Pp 70
Ability of rice husk husk ash powders to protect cowpea seeds against damage environmental human health impacts have led to increasingly restrictive regulation of chemical insecticides, particularly in North America, the European Union Japan, many other countries (Isman Miresmailli, 2011). Besides, increasing costs inconsistent supplies limit the use of these synthetic chemicals for stored products protection in many developing countries (Isman, 2006). Many insecticidally active plants have been investigated as alternative source of safer more readily available affordable stored products protection materials, against but with varying degrees of success ( Boeke et, 2001). Plant/wood ash is traditionally used for grain protection has b een investigated by some workers against m aculatus (Ofuya, 1986; Wolfson et, 1991; Apuuli villet, 1996). A prominent mechanism by which plant materials reduce damage to stored cowpea seeds by is inhibition of o viposition (Boeke et, 2001; Lale, 2010). Using no-choice tests, Ezekiel (2012) reported that females of the cowpea seed beetle did not oviposit on paddy rice amongst several crop seeds that were screened for susceptibility to the beetle. Therefore, the ability of paddy husk powder husk ash powder to protect cowpea seeds against C. damage was subjected to closer empirical verification is hereby being reported. MATERIALS AND METHODS The study was carried out in the Institute of Ecological Chemistry, Plant Analysis Stored (approximately 100 g) was then put in a separate plastic container with tight fitted lids. Products Protection, Berlin, Germany under controlled conditionsof 25 C 70% relative humidity except where otherwise indicated. Effect of paddy products on mortality Twenty newly emerged adults of culture cowpea variety The used in the study was a Nigerian strain of the beetle maintained at Institute of Ecological Chemistry, Plant Analysis Stored Products Protection, Berlin, Germany on blackeye cowpea. The culturing procedures have been described previously (Ofuya Reichmuth, 2002). were dusted by shaking with either rice husk or rice husk ash powders in clear glass Petri dishes (9.0cm diameter) containing 20g of cowpea seeds. Each product was tested 0.05, 0.1, 0.2, 0.3, 0.4 0.5g respectively. There was a control treatment with no paddy product. Adult mortality was observed daily for 7 days. Morphological changes in adults were noted. The experiment was replicated thrice. Rice variety paddy products Paddy husk was obtained from a processing mill in Emure in Ekiti State, Nigeria (7.4500 N, 5.4667 E) rice variety was Igbemo local grown by communities around the metropolis. Paddy husk E ffect of paddy products on weight of treated The weight of 20 newly emerged adults (mixed sexes) of placed inside a clear was first pulverized in an electric blender into plastic Petri plate (8.5cm diameter) was coarse powder. Part of the coarse paddy husk determined with an analytical balance (Sartorius powder was ashed in electric oven at 550 C for 0.001g). The insects were then dusted by three hours. The coarse paddy husk powder husk ash was each pulverized in a laboratory mill into a fine powder with particle size of < 300µm using a British stard sieve (Ofuya Dawodu, 2002). Each fine powder type shaking with 0.4g of either rice husk powder or rice husk ash powder in ten replications per paddy product. The weight of the Petri plate containing the ash insects was determined immediately after dusting the insects then Pp 71
T.I. Ofuya S. Adler 24 48 hours thereafter. There was a replicated control treatment consisting of Petri plate containing 20 newly emerged beetles but no paddy product added. This experiment was conducted in a climate chamber maintained at 30 C 70% relative humidity. Effect of paddy products on eggs on cowpea seeds A batch of clean cowpea seeds (500 g) contained in a clear glass bottle (20 cm high 10 cm diameter) was infested with numerous individuals of (< 4 days old) sieved out from a culture bottle. After 12 hours the introduced insects were sieved out. Seeds bearing 2 to 4 eggs were sorted out divided i nto lots with each group having 100 eggs on seeds. Each lot was made up to 20 g with clean seeds. The seeds in each lot were shaken with 0.4 g of either rice husk powder or rice husk ash powder in glass Petri dishes (9.0 cm diameter). This treatment was carried out with seeds bearing 1, 2, 3, 4-day old eggs, respectively. For each egg age there were three replicates as well as a replicated control with no paddy product added but shaken. Eggs subjected to the different treatments were observed under the microscope to observe changes in physical appearance 14 days post oviposition when they all should have hatched. Emergence of adult insects from the seeds was observed up to 35 days post oviposition the number recorded. Effects of paddy products on oviposition cowpea seed damage Paddy husk ash powders were tested separately as protectants against at different rates of application of 0.1, 0.2, 0.3, 0.4 g per 20 g of cowpea seeds in glass Petri dishes ((9.0 cm diameter). With each paddy product rate of application, two males t wo females of newly emerged adults of were used to infest cowpea seeds in a dish. Each dish was shaken such that each powder made contact with the seeds insects. There was also a control treatment in which the cowpea seeds insects were not exposed to the paddy products (i.e. 0.0 g rate of application). Each treatment including the control was replicated four times. Fourteen days after the application of treatments, the introduced insects (dead or alive) were removed, the number of eggs on the cowpea seeds in each replicate was counted recorded. Emergence of adult insects from the seeds was observed up to 35 days post oviposition the number found alive dead separately recorded. Seeds in each replicate were weighed to determine weight loss. Effect of temperature onuse of low dosage of rice husk ash Paddy husk ash powder was tested for lethality to adults at different low dosages rates of 0.01, 0.02, 0.03, 0.04 g per 20 g of cowpea seeds in glass Petri dishes ((9.0 cm diameter) at two different temperatures of 25 30 C relative humidity of 70% maintained in the climate chambers. Twenty newly emerged adults of were dusted by shaking with the seeds in each dish. There was a control treatment with no ash being mixed with the seeds introduced insects. Adult mortality was observed daily for 7 days. The experiment was replicated thrice. Data analysis Data was analyzed using the SigmaStat 3.5 software (Systat Software GmbH, Germany). Mortality data, where necessary, were corrected as recommended by Abbott (1925). Egg counts were subjected to square root transformation, percentage data arcsine transformed subjected to one-way analysis of variance (ANOVA. Where the ANOVA indicated significant difference between treatments, Student-Newman-Keuls method was used to separate means at 5% level of probability. Pp 72
Effect of paddy products on weight mortality of adults T he mean weight of the Nigerian strain of used in this study was 3.57 ± 1.22 mg. Mean 2-day weight loss in adults exposed to rice husk powder, rice husk ash powder the control was 1.62 mg, 2.40 mg 0.19 mg, respectively (Fig. 1). Adults showed deliberate dislike aversion for the presence of rice husk ash powder particles in the environment of the Petri dish. Some of the adults (mostly males) extended the membranous hind wings beyond the elytra at death. This was not very rampant with adults in treatment involving use of rice husk powder not at all in the control treatment All adults of were killed within 1 day post treatment (100% mortality) when rice hush ash powder was applied at 0.5 g/20 g of grain (Table 1). This was significantly higher (P < 0.05) than mortality recorded with each of the lower rates of application except 0.4 g where 83.3% mortality was recorded. As from 2 days post treatment all rates of application of rice husk a sh powder produced similar mortality in m aculatus adults ranging from 55 100%. Rice husk powder applied at 0.5 g/20 g of grain caused 26.7 55% mortalities at 5 7 days post treatment, respectively (Table 2). These mortalities were significantly higher (P < 0.05) D osage rate ( g/20 g) Ability of rice husk husk ash powders to protect cowpea seeds against damage 1 day RESULTS 4 3 2 1 0 1 2 3 Treatment 1 Rice husk ash powder Treatment 2 Rice husk powder Treatment 3 - Control Treatment F ig. 1. Mean2-day weight loss in adults of exposed to rice husk rice husk ash powders than what was recorded with other lower rates of application. The 0.4 g dosage also caused 18.3% 31.7% mortalities at 5 7 days post treatment, respectively, which were significantly higher than that observed with lower rates. Effect of paddy products on eggs on cowpea seeds Eggs in the control treatment those involving rice husk powder were wholesome developed normally with the milky white frass as a result of feeding by first instar larva (Giga Smith, 1987) highly pronounced. Majority Table 1: Mortality of adult after 1, 2, 3 4 days exposure to cowpea seeds treated with RHA at different dosages M ean % mortality (± S E) in: 2 days 3 days 4 days 0.05 10.0 ± 5.77d 55.0 ± 2.89a 81.7 ± 4.41a 0.1 33.3 ± 8.82cd 81.7 ± 1.67a 0.2 33.3 ± 4.41cd 90.0 ± 2. 89a 0.3 55.0 ± 5.77bc 0.4 83.3 ± 1.67ab 0.5 Column means followed by same letter (s) are not significantly different at the 5% level by Student- Newman-Keuls method. Pp 73
T.I. Ofuya S. Adler Table 2: Mortality of adult after 5 7 days exposure to cowpea seeds treated with RHP at different dosages D osage rate (g/20 g) M ean % mortality (± SE) i n: 5 days 7 days 0.05 1.7 ± 1.67a 5.0 ± 0.00a 0.1 3.3 ± 1.67a 6.7 ± 1.67a 0.2 3.3 ± 1.67a 10.0 ± 0.00a 0.3 6.7 ± 1.67a 13.3 ± 1.67a 0.4 18.3 ± 1.67b 31.7 ± 1.67b 0.5 26.7 ± 1.67c 55. 0 ± 2.89c Table 3: Ovicidal action of RHA RHP against. P addy powder type A ge of Egg (days) M ean % Egg Mortality ( ± SE) R ice Husk Ash 1 44.2 ± 3.19b 2 53.4 ± 2.20c 3 65.1 ± 1.85d 4 73.3 ± 1.47e R ice Husk 1 2.2 ± 1.10a 2 3.2 ± 1.10a 3 3.6 ± 0.40a 4 2.5 ± 1.94a Column means followed by same letter (s) are not significantly different at the 5% level by Student- Newman-Keuls method. Column means followed by same letter (s) are not significantly different at the 5% level by Student- Newman-Keuls method. C eggs Table 4: Means of variables measured when stored cowpea was protected with different dosage rates of RHP RHA, infested with P rotectant D osage rate Mean no. eggs % adult % of dead F1 % seed g /20 g grain l aid (± SE) e mergence a dults weight loss (± ( ± SE) ( ± SE) S E) R HP 0. 1 209.5 ± 3.92d 92.4 ± 2.07a 0. 0 ± 0.00a 12.9 ± 0.32cd 0.2 204.5 ± 8.87d 90.4 ± 2.38a 0. 0 ± 0.00a 12.9 ± 0.65cd 0.3 183.5 ± 2.26c 92.9 ± 1.21a 0. 0 ± 0.00a 12.3 ± 0. 57 c 0.4 145.3 ± 3.57b 92.0 ± 0.50a 0. 0 ± 0.00a 9.6 ± 0.21b RH A 0. 1 64.5 ± 8.13a 90.4 ± 0.86a 28. 8 ± 1.06a 3.6 ± 0.30a 0.2 66.3 ± 6.43a 92.9 ± 0.79a 33. 8 ± 1.32b 4.2 ± 0.62a 0.3 60.0 ± 8.65a 90.6 ± 2.46a 47. 0 ± 1.80b 3.5 ± 0.18a 0.4 51.5 ± 3.71a 92.0 ± 1.11a 52. 8 ± 0.83b 3.2 ± 0.15a C ontrol 0. 0 220.8 ± 11.2d 92.0 ± 1.28a 0. 0 ± 0.00a 13.5 ± 0.47d Column means followed by same letter (s) are not significantly different at the 5% level by Student-Newman-Keuls method. Pp 74
Ability of rice husk husk ash powders to protect cowpea seeds against damage of the eggs dusted with rice husk ash appeared shrunken some ruptured, were mostly free of frass. Where frass occurred, it was manifestly scanty. Percentage egg mortality as measured by adult emergence post treatment was significantly lower with rice husk powder than rice husk ash powder (Table 3). It increased significantly with age of egg in treatment involving the use of rice husk ash powder. Effects of paddy products on oviposition cowpea seed damage females laid significantly lower number of eggs on seeds treated with rice husk ash powder than seeds treated with rice husk powder the control (Table 4). Seeds treatment with rice husk powder at the dosage rates of 0.3 g 0.4 g also caused significant reduction in oviposition by the females in comparison with seed treatment involving lower dosages the control. Percentage emergence of F adults was not significantly different (P > 1 0.05) between treatments ranging from 90.4% to 92.9%. There was no F adult mortality in 1 treatments involving rice husk powder the control but significant mortality occurred in treatments involving use of rice husk ash powder. Seed weight loss was significantly lowest when seeds were treated with rice husk ash powder ranging from 3.2% to 4.2%. Seed weight loss was highest in seeds in the control (13.5%), but was not significantly higher than weight loss in seeds protected with 0.1 0.2 g of rice husk powder (12.9%). Effect of temperature onuse of low dosage of rice husk ash Mean percentage mortality of adults exposed to relatively low dosages of RHA was significantly higher under the temperature regime of 30 C than 25 C 1, 2 3 days post exposure respectively (Table 5). By 3 days post exposure, all the adults had been killed (100% mortality) irrespective of the dosage at 30 With the 25 C regime all adults were killed irrespective of dosage at 6 days post exposure (Table 6). Table 5: Mortality of adult after 1, 2, 3 days exposure to cowpea seeds treated with RHA at low dosages at two different temperatures T emperature ( C) 25 30 D osage g /20 g of seed M ean % mortality (± SE) in: 1 day 2 days 3 days 0.01 0.0 ± 0.00a 10.0 ± 2.89a 33. 3 ± 4.41a 0.02 3.3 ± 1.67a 26.7 ± 4.41b 46.7 ± 1.67b 0.03 6.7 ± 1.67a 31.7 ± 4.41b 55.0 ± 2.89b 0.04 11.7 ± 1.67a 41.7 ± 1.67c 66.7 ± 4.41c 0.01 31.7 ± 6.01b 71. 7 ± 1.67d 100.0 ± 0.00d 0.02 50.0 ± 10. 4c 80.0 ± 2.89d 100.0 ± 0.00d 0.03 68.3 ± 4.41d 90.0 ± 2.89e 100.0 ± 0.00d 0.04 88.3 ± 4.41e 100.0 ± 0.00f 100.0 ± 0.00d Column means followed by same letter (s) are not significantly different at the 5% level by Student- Newman-Keuls method. Pp 75
T.I. Ofuya S. Adler Table 6: Mortality of adult seeds treated with RHA at low dosages at after 4, 25 C 5, 6 days exposure to cowpea Dosage (g/20 g of M ean % mortality (± SE) in: s eed) 4 days 5 days 6 days 0.01 53.3 ± 4.41a 88.3 ± 1.67b 100.0 ± 0.00b 0.02 63.3 ± 4.41a 93.3 ± 4.41b 100.0 ± 0.00b 0.03 65.0 ± 5.00a 93.3 ± 3.33b 100.0 ± 0.00b 0.04 80.0 ± 2.89b 100.0 ± 0.00b 100.0 ± 0.00b Column means followed by same letter (s) are not significantly different at the 5% level by Student-Newman-Keuls method. DISCUSSION The results of this study have considerably substantiated reports by some earlier workers (Lee, 1981; Naito, 1999) that RHA is lethal to The lethal action of RHA on adult bruchids can be elucidated by various speculations some interesting indications. It is believed that inert storage bruchids so there may be substances generally cause a loss of body justification for its traditional use by farmers for storing legume seeds (Matsumoto, 1987; Naito, 1999). There are also some reports on the traditional use of other ashes to control bruchid moisture (Subramanyam Roesli, 2000). In this study, adult beetles exposed to RHA treatment manifested significant weight loss died. More than half of the body weight was lost by beetles attacking stored cowpea seeds (Ofuya, adults within 2 days. RHA has also been 1986; Wolfson e t, 1991; Apuuli villet, 1996). S ome mechanisms of action of RHA against have clearly been unraveled in this study. At the dosage of 1-2% (by weight) adults are killed within 3 days thus preventing them from full expression of their oviposition potenti Besides, adults bearing ash powder particles who are not immediately killed may lay significantly fewer eggs during their remaining observed to contain a large amount of needle- like particles presumably derived from the setae covering the outer surface of the rice husk (Naito, 1999). These needle-like particles may trigger a physical reaction on the integument of insects the resulting physical disturbance may help cause their death. They may also explain the manifest display of dislike aversion seen in the beetles extension of the membranous hind wings by many of them, life span (Ofuya, 1986). Thus, rice husk ash p resumably ready for take-off. Adult powder causes outright kill of adults significantly reduces oviposition. Irrespective may also have been weakened or even killed resulting from abrasion of insect o f dosage, RHA reduced oviposition by integument consequent dehydration. The by about 70% in comparison with major constituent (> 90 %) of RHA is silica (SiO 2 the control treatment. These observations are which may have a lethal effect on insects consistent with that reported by Naito (1999) on the effect of RHA on analis on soya beans chinensis on mung beans. Oviposition inhibition/reduction has also been observed with other ashes studied (Ofuya, 1986; Apuuli Villet, 1996), as well as many other i nsecticidal plant powders (Boeke e t, 2001; O fuya e t, 2007; Adesina Ofuya, 2011). including irritation desiccation of adult i nsects (Kalapathy e t, 2000; Anonymous, 2005; Ajay, et, 2012). Since RHA has similar silica content as diatomaceous earth (DE) (Naito, 1999), effective absorption of epicuticular lipids fatty acids being concluded as the primary mode of action of DE (Shah Khan, 2014) may also be operative with RHA. Pulverized Pp 76
Ability of rice husk husk ash powders to protect cowpea seeds against damage botanicals may also further physically impede movement of adult beetles hinder mating a nd oviposition (Wolfson et, 1991). This may be an unlikely mechanism of action in this study because at the highest rate of application of the paddy products (i.e. 0.5 g/ 20 seed) there was plenty of room between seeds for beetle movement activities. Unlike RHP, RHA appeared to be ovicidal when dusted on freshly laid eggs. Rice husk contains 50% cellulose, 25-30% lignin 15-20% silica (Siddique Khan, 2011). On thereafter. This residual lethal action of RHA will be an added advantage for its use in stored products protection against insects. Naito (1999) has also observed that the lethality of RHA on analis infesting soya beans seeds continued for three months after treatment. It is curious that females of the did not oviposit on paddy rice amongst several crop seeds that were screened for susceptibility to the beetle as reported by Ezekiel (2012). Neither RHP nor RHA was able to completely prevent oviposition by on cowpea seeds burning, cellulose lignin are removed protected with them in this study. It is leaving behind silica ash. The high noteworthy that in the study by Ezekiel (2012) concentration of amorphous silica RHA may explain its greater insecticidal efficacy than its parent RHP. Susceptibility of the eggs to RHA significantly increased with age. This may be due to changes in egg covering that could occur during incubation. The susceptible eggs may also have been desiccated or pierced by the needle like RHAparticles emptied. However, the husk the grain within may have acted in concert in the paddy against oviposition. It is probable that a much higher rate of application of each of the paddy products or a combination of both, for the protection of cowpea in storage may render the seeds less attractive to attack. This merits empirical verification. when females oviposited on seeds already treated with ash powder, ovicidal action was not observed; the eggs appear to develop normally. Abbreviated life span reduced oviposition caused by RHA on C. ultimately In majority of rice producing developing countries like Nigeria much of the husk produced from processing of rice is either burnt or dumped as waste poses a great environmental threat ( Ajay et, 2012). Commercial use of rice husk resulted in significantly reduced seed weight its ash is undoubtedly an alternative loss in comparison with RHP the control after the F1 adults have emerged from the seeds. It is interesting that unlike the control RHP t reatments, many F adults emerging from 1 treatments involving the use of RHA were found solution to the disposal problem. The results of this study show that RHA may have direct potential use in the control of storage insect pests. It may also serve as carrier for insecticides pesticides both of botanical chemical dead. They may have putatively enmeshed their origin. Studies in these directions are bodies in the ash after emergence died recommended for further consideration. REFERENCES A BBOTT, W. S. (1925). A method of computing the effectiveness of an insecticide. Journal o f Economic Entomology 18: 265-267. ADESINA, J.M. O FUYA, T. I. (2011). Evaluation of leaf vine powders of (Coleoptera: Bruchidae) in stored cowpea Vigna unguiculata (L.) Walp. South Asian Journal of Experimental Biology 1: 158-162. AJAY, K., KALY ANI, M., DEVENDRA, K. O M, P. (2012). Properties industrial Secamore afzelii (Schult) K. Schum for applications of rice husk: A review. control of C allosobruchus (Fab.) International Journal of Emerging Pp 77
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