The rice weevil (Sitophilus oryzae) is a serious stored product pest which attacks several economically important crops. In an attempt to find alternative to control this insect, extracts of seven plant species (Bauhinia purpurea, Caesalpinia gilliesii, Cassia fistula, Cassia senna, Chrysanthemum frutescens, Euonymus japonicus and Thespesia populnea acutiloba) were evaluated under laboratory conditions for their ability to protect rice grains against Sitophilus oryzae insect. The efficacy of the tested plant extracts was evaluated on whole rice grains with respect to mortality and emergency of S. oryzae adults. Moreover, gas chromatography-mass spectrometry (GC-MS) analysis was carried to identify the chemical components of the most effective plant extract against S. oryzae. Furthermore, the safety of the most effective plant extract was evaluated with respect to biochemical and histological changes in treated rats relative to control. The results revealed that, the tested botanical extracts showed high efficiency against S. oryzae and C. gilliesii extract was the most effective plant extract against S. oryzae. The GC-MS analysis showed the presence of different bioactive chemical components that known by its insecticidal activity. The most effective plant extract showed low toxicity on rats relative to control with respect to biochemical and histological changes. The results suggest the ability of using these plant extracts in rice grains protection as a safe alternative to insecticides. Moreover, the presence of mixture of components with, apparently, different mechanisms of insecticidal activity of the tested plant extracts may be delay the resistance development by insect relative to the insecticide.
PDF Abstract XML References Citation
How to cite this article
Pest insects affect food output directly by reducing the quality and quantity of the crop produced, or indirectly by serving as vectors of plant diseases. The use of synthetic compounds to control insect pests has lead to several adverse effects, including water and soil contamination, insect resistance and toxicity to non target species (Zettler and Cuperus 1990). Thus effective and safe alternatives to fungicides for controlling plant disease are in demand. Plant derived materials for potentially useful products as bio-insecticides are a source of major concern (Regnault-Roger and Hamraoui, 1994; Regnault-Roger, 1997; Oparaeke and Kuhiep, 2006; Sathyaseelan et al., 2008; Khorram et al., 2011; Mulungu et al., 2011). Moreover, resistance by pests and vectors against hese bio-insecticides has not been reported and likely to be difficult (Regnault-Roger, 1997).
Furthermore, these alternatives (bio-insecticides) supposed to have low persistence in the environment than chemical fungicides (Koul and Dhaliwal, 2001).
Essential oils derived from plants received much attention for using it as potential components of integrated pest management (IPM) due to its efficacy against pests and its environmental compatibility (Ikbal et al., 2007; Katz et al., 2008; Akinkurolere, 2007). Recently, many studies have focused on the possibility of using it for control of stored grains pests (Collins, 2006; De Carvalho and Da Fonseca, 2006).
In spite of the efficacy of botanical extract in pest's control, there are lack studies on the safety of these botanical extracts on human health. Although, the assessment of enzymes activity in the blood is generally a more sensitive measure of compounds toxicity than histopathological changes and can be assessed within a shorter time. The tissue alterations considered a confirmatory and supporting diagnostic role in the case of certain abnormalities in blood sampling (Massoud et al., 2010).
The use of plant materials can lead to the identification of new bio-insecticides for the benefit of agriculture production and human health. Therefore, this study attempted to evaluate insecticidal activity of some newly used plant extracts (Bauhinia purpurea, Caesalpinia gilliesii, Cassia fistula, Cassia senna, Chrysanthemum frutescens, Euonymus japonicus and Thespesia populnea acutiloba) against Sitophilus oryzae in rice grains with the respect to progeny and mortality of the insect adults. Also to identify the chemical components of the most effective plant extract against S. oryzae. Furthermore, to evaluate the toxicity of the most effective plant extract on rats with respect to biochemical and histological changes in treated rats relative to control.
MATERIALS AND METHODS
The insect rereading: S. oryzae (Egyptian strain) was obtained from the Department of Stored Products Pests Control, Research Institute of Plant Protection, Sakha, Kafr-El-Shiekh. This strain was continuously reared free of insecticidal contamination for several years at 30±2°C and 70±5% R.H. The cultures were maintained under the same conditions in the Pesticide Department, Faculty of Agriculture, Kafr-El-Shiekh University, Egypt and 200-400 adults from the pervious culture were added in 1000 mL glass jars containing 400 g of rice as a culture medium. The mouth of the jars was covered with muslin cloth. Then, 7-14 d old adults were used for experimental work.
The stored product: Rice grains were used to culture S. oryzae and to evaluate the efficacy of the tested plant extracts against the same insect as well. Rice grains stored in airtight tins until required for experiments. The experiments were carried out in a room kept at a constant temperature of 25°C and 70% RH.
Plants and preparation of its crude extracts: The leaves of seven medicinal plant species (C. senna, C. gilliesii, T. populnea var. acutiloba, C. frutescens, E. japonicus, B. purpurea and C. fistula) were collected from a local nursery at Kafr El-Sheikh, Monofia, Gharbia and Alexandria Governorates, Egypt. C. senna (Alexandrian senna) belonging to the family Fabaceae and cultivated in Egypt and Sudan. C. gilliesii (bird of paradise) belonging to the family Fabaceae, is native to tropical America, mainly Argentina and Uruguay. T. populnea var. acutiloba (Portia tree) belonging to the family Malvaceae, is native to South Africa. C. frutescens (marguerite daisy) belonging to the family Asteraceae, is native to the Canary Islands. E. japonicus (Japanese spindle) belonging to the family Celastraceae, is native to Japan, Korea and China. B. purpurea (purple camel's foot) belonging to the family Fabaceae, is native to South China. C. fistula (cassias) belonging to the family Fabaceae, is native to southern Asia. The different leave samples were oven dried for 24 h at 70°C and then, finely powdered using a blender. Each sample (25 g) was extracted twice with 300 mL of methanol at room temperature for 2 days. The extracts were filtered through filter paper (No. 15, Whatman, Inc., Piscataway, NJ, USA) and the combined filtrates from the twice-extracted leaves were concentrated to dryness by rotary evaporation at 40°C.
Effect of tested plant extracts and malathion on progeny of S. oryzae: The tested plant extracts at concentration levels of 100, 200 and 300 ppm were used to evaluate its efficacy against S. oryzae. Malathion was used as recommended compound against S. oryzae at concentration levels of 10, 20 and 30 ppm. Each concentration was applied in three replicates and in each replicate there were 20 g of rice grains. The treatment of rice grains was carried out by dipping rice grains in water solution of malathion and botanical extracts at tested concentration levels twice consecutively for 5 sec and subsequently spread on top of plastic sheets to dry for 90 min. The control treatment was carried using water only and replicated three times. Then, 10 adults of S. oryzae were transferred to treated rice grains which putted in a 85x75 mm plastic jar and kept at 30±2°C and 70±5% RH according to the method described by Kestenholz et al. (2007). The emerged adults from the hatched eggs were recorded after 6 weeks of treatment. These adults were used to calculate the reduction percentages in S. oryzae progeny from the use of the tested plant extracts as well as malathion compared to the control as shown in Eq.1 as described by El-Lakwah et al. (1992).
MNEC = Mean No. of those which emerged in the control
MNET = No. of those which emerged in the treatment
Efficiency of the tested plant extracts and malathion on adults of S. oryzae by mean mortality: Efficiency of the tested plant extracts and malathion on adults mortality of S. oryzae was evaluated according to the method described by Kestenholz et al. (2007) as mentioned before. The number of dead insects in each jar was counted after one and two weeks and the percentage of insect mortality was recorded and corrected using the Abbott formula (Abbott, 1925).
Chemical composition of the most effective plant extract: GC/MS analysis was carried to identify the components of the most effective plant extract (C. gilliesii) according to the method described by Duarte-Almeida et al. (2004).
Animal treatment: Toxicity assessments were performed using 8-week-old 80-100 g Wistar male rats (Rattus norvegicus) obtained from Faculty of Medicine, Tanta University. Wister rats were housed in wire cages under standard conditions with free access to drinking water and food. The rats were kept in temperature-controlled room with 14 h light and 10 h dark cycles. The rats were given a standard diet as describe by Romestaing et al. (2007). Before treatment, rats were left two weeks during feeding for adaptation. The animals were randomly divided into two groups each comprising of three animals. One groups for the treatment with the most effective plant extract (21 days) and the second group for control. The most effective plant extract (C. gilliesii) against the tested insect were administered to rats orally at concentration level of 500 mg kg-1 body weight. Control group rats were orally administrated with equal amount of almond oil. After 21 days the rats were sacrificed under anesthesia. Then, the blood samples were taken under anesthesia by cardiac puncture in vials containing heparin. Moreover, specimens from kidney and liver were taken from each treatment and kept in neutral buffered formalin 10% for histopathological tests.
Enzymes assays: Blood samples were centrifuged at 4500 rpm for 15 min at 4°C and the blood serum was used to determine Creatinine, Alkaline Phosphatase (ALP) and Glutamate Pyruvate Transaminase (GPT) according the methods described by Reitman and Frankel (1957), Wilkinson et al. (1969) and Barham and Trinder (1972), respectively.
Histopathological test: The histopathology test was carried out at Histopathology laboratory, Department of Histopathology, Faculty of Veterinary Medicine, Kafr El-Sheikh University according to the method described by Bancroft et al. (1996).
Statistical analysis: Data from the experiments were statistically analyzed using one-way repeated measurement analysis of variance. Duncans multiple range test (Duncan, 1955) were used to separate means using SAS program (Version 6.12, SAS Institute Inc., Cary, USA).
Effect of tested plant extracts and malathion on progeny of S. oryzae: The numbers of emerged adults of S. oryzae were significantly decreased after treatments compared to the untreated control, as shown in Table 1.
|Table 1:||Effect of the tested plant extracts and malathion on progeny of S. oryzae|
|Letters in columns indicate separation of means according to the Duncan's multiple range test (p<0.05)|
Increasing the concentration level of all tested treatments reduced the emergence of S. oryzae even more (concentration dependent) malathion (86%) followed by C. gilliesii (79%) extract and C. frutescens (73%) were the most effective treatments on reduction of S. oryzae progeny while C. senna extract was the lowest effective one.
Efficiency of tested plant extracts and malathion on S. oryzae adults determined by mortality values: The efficacy of the tested plant extracts and malathion against S. oryzae adults by means mortality are presented in Table 2. The results showed that, C. gilliesii (100%) was the most effective treatment against S. oryzae adults followed by C. frutescens (95.6%), T. populnea var. acutiloba (88%), E. japonicus (85%) , B. purpurea (75%), C. senna (80%), and C. fistula (70%), respectively. Malathion (100%) and C. gilliesii (100%) extract were the most effective treatments on adult's mortality of S. oryzae after two weeks while C. fistula extract (70%) was the lowest effective one. The morality percentages of S. oryzae were significantly increased in the second week as compared to the first week at all tested treatments. Increasing the concentration level of all tested treatments increased the mortality of S. oryzae adults even more (concentration dependent).
|Table 2:||Effect of the tested plant extracts and malathion on adult's mortality of Sitophilus oryzae|
|Letters in columns indicate separation of means according to the Duncan's multiple range test (p<0.05)|
|Table 3:||The main constituents of Caesalpinia gilliesii plant extract under GC-MS analysis|
Composition of the most effective botanical extract: The identified chemical components of the most effective botanical extract against S. oryzae (C. gilliesii) presented in Table 3. Twenty six compounds were identified from C. gilliesii extract separately with different percentages as shown in Table 3. The identified compounds belonged to different fatty acids and it derivatives (aldeyhydes, esters and alcohols). Some compounds such as tetradecanoic acid (4.65%); pentadecanoic acid (4.62%); loliolide (3.51%); octadecanoic acid (2.17%); n-hexadecanoic acid (1.39%) and phytol (1.03%) were detected with high percentages relative to other detected compounds (Table 3).
Effect of the most effective plant extract on liver enzymes: The ALP and GPT activities are known as cytosolic marker enzymes reflecting hepatocellular necrosis as they are released into the blood after cell membrane damage. In the present study, therefore, both enzyme activities were used as indicators of hepatic damage. The obtained data in Table 4 show that, there were no significant differences in the activity of ALT and GPT after 21 days of rats administrated with the tested plant extract at dose level of 500 mg kg-1 body weight relative control treatment. The normal ALP and GPT level in rats treated with the most effective plant extract relative to control treatment assumed to be the normal liver functions.
Effect of the most effective plant extract on kidney function: Regarding to the kidney function, there were no significant differences in creatinine level in rats administrated with the most effective plant extract at dose level of 500 mg kg-1 relative to control one (Table 4). The normal creatinine in rats treated with the most effective plant extract relative to control treatment assumed to be the normal kidney function. Moreover, the histology of kidney tissue treated with the most effective plant extracts relative to control confirms this explanation. The normal creatinine level in rats treated with the most effective plant extract relative to control treatment assumed to be the normal kidney functions.
The histopathological changes in the kidney: The normal structure of kidney tissue was shown in Fig. 1a. For the rats treated with C. gilliesii extract at dose level of 500 mg kg-1, the tissue was some what like control with a small vacuolation in renal tubules (Fig. 1b). These results were in agreement with the creatinine level in treated rats mentioned before in this study (Table 4).
The histopathological changes in the liver: The normal structure of liver tissue was shown in Fig. 2a. For the rats treated with C. gilliesii at dose level of 500 mg kg-1, blood vessels were observed to be engorged with blood and hepatocyte contained vacuolated cytoplasm (Fig. 2b). Moreover, there was a necrotic area recorded (Fig. 2b). These results were in agreement with the GPT and ALT levels in treated rats in this study (Table 4).
|Table 4:||Effect of the most effective plant extract (Caesalpinia gilliesii) on serum GPT, ALT and creatinine activities of rats at dose level of 500 mg kg-1 body weight|
|*SE: Standard Error, *IU L-1: International Unit per Liter, mg dL-1: Milligram per deciliter*|
|Fig. 1(a-b):||Sections from kidney of rats after 21 days of treatment with Caesalpinia gilliesii (b) November 2, 2011at dose level of 500 mg kg-1 relative to control (a)|
|Fig. 2(a-b):||Sections from liver of rats after 21 days of treatment with Caesalpinia gilliesii (b) at dose level of 500 mg kg-1 relative to control (a)|
The results of the present study implied that, the tested plant extracts were effective against S. oryzae in stored rice with the respect to progeny and adults mortality. The efficacy of plant extracts against S. oryzae insect in stored rice with the respect to progeny and adults morality have been reported by many researchers (Tapondjou et al., 2002; Ketoh et al., 2005; Kestenholz et al., 2007; Iboudo et al., 2010; Derbalah and Ahmed, 2011). However, the efficacy of the tested plant extracts especially the most effective ones, has not been reported against S. oryzae and is considered first report.
Among the identified compounds from C. gilliesii extract, some compounds such as tetradecanoic acid; pentadecanoic acid; n-hexadecanoic acid; phytol; loliolide and octadecanoic were detected with high percentages relative to other detected compounds. The antifungal activity of C. gilliesii extract against S. oryzae may be due to the presence of the previous these fatty acids and its derivatives (Negahban et al., 2006; Rozman et al., 2007; Lopez et al., 2008; Ogendo et al., 2008; Derbalah and Ahmed, 2011).
Although, the insecticidal activity of tested plant extract is attributed mainly to its major compounds mentioned before, the synergistic or antagonistic effect of some compounds in the mixture has to be considered (Ragasa et al., 2002). Each of the plant extract components has its own contribution on biological activity of the extract against the tested insect. For example loliolide detected with low percentage but it is known to possess diverse biological properties such as insect repellant (Gordon et al., 1982).
The mode of action of the bioactive natural monoterpinoids (hydrocarbons, alcohols and ketones) isolated from plant extracts oils may be due to inhibition of acetylcholinestearase (Miyazawa et al., 1997; Lee et al., 2000; Derbalah and Ahmed, 2011). Since, Lee et al. (2000) reported that, 1,8-cineole was the most potent inhibitor of AChE among the monoterpenes tested. This inhibition may be a mode of action for essential oils and monoterpenes against stored grain insect pests as well. Also, the mode of action of the tested botanical extracts may be largely attributable to its fumigant action (Shaaya et al., 1997; Park et al., 2003).
The botanical extracts as pest control agents present two main characters: the first is their safety to the people and the environment and the second is, the less resistance development against it by the tested insect. Regarding to the safety, the toxicity evaluation of the most effective plant extract revealed that, there were some slight variations occurred sporadically in treated rats relative to control with the respect to enzymes markers and histopathology of treated organs. Moreover, the observed changes in the tissues were mostly uncorrelated with a dosage which potentially indicates the safety of the plant extracts in the context of human health. Also, the rat tests are often more sensitive and may not reflect human sensitivity. Moreover, the exposure levels may be far greater than what would actually be experienced or detected in rice grains after they grow and are processed. With the referring to resistance development, it is believed that, it is difficult for the insect to develop resistance to such a mixture of bioactive components with, apparently, different mechanisms of insecticidal activity (Liu et al., 2008).
This study considered the first step toward more investigation and concern about using these effective botanical extracts as alternative for controlling of stored products pests. This will be help to reduce the environmental pollution and the adverse effect on human health resulted from insecticides using. Since, these botanical extract revealed non significant toxicity relative to the high dosage that given orally and will not reached to human by this dose as a residue under any conditions.3
The insecticidal activity of the tested plant extracts against S. oryzae indicated the potential of some plant species as a natural source of insecticidal material. Insecticidal activity was confirmed in all the tested plant species, although the results showed variation in their effectiveness against S. oryzae. The ability of using botanical products as alternative of chemical control of S. oryzae is possible if the problem of cost-effective commercial production can be solved. Moreover, some of these botanical extracts could find a place in IPM strategies, especially where the emphasis is on environmental, food safety and on replacing the more dangerous toxic insecticides. Also, work in this regards should continue to obtain information regarding its practical effectiveness under natural conditions to protect the stored products without any side effects.
- Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol., 18: 265-267.
- Duarte-Almeida, J.M., G. Negri and A. Salatino, 2004. Volatile oils in leaves of Bauhinia (Fabaceae Caesalpinioideae). Biochem. Syst. Ecol., 32: 747-753.
- Collins, D.A., 2006. A review of alternatives to organophosphorus compounds for the control of storage mites. J. Stored Prod. Res., 42: 395-426.
- Duncan, D.B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42.
- De Carvalho, C.C.C.R. and M.M.R. Da Fonseca, 2006. Carvone: Why and how should one bother to produce this terpene. Food Chem., 95: 413-422.
- Derbalah, A.S. and S.I. Ahmed, 2011. Oil and powder of spearmint as an alternative to sitophilus oryzae chemical control of wheat grains. J. Plant Prot. Res., 51: 145-150.
- Gordon, W.P., A.J. Forte, R.J. McMurthy, J. Gal and S.D. Nelson, 1982. Hepatotoxicity and pulmonary toxicity of pennyroyal oil and its constituents terpenes in the mouse. Toxicol. Appl. Pharmacol., 65: 413-424.
- Katz, T.M., J.H. Miller and A.A. Hebert, 2008. Insect repellents: Historical perspectives and new developments. J. Am. Acad. Dermatol., 58: 865-871.
- Kestenholz, C., C.P. Stevenson and S.R. Belmain, 2007. Comparative study of field and laboratory evaluations of the ethnobotanical Cassia sophera L. (Leguminosae) for bioactivity against the storage pests Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) and Sitophilus oryzae (L.) (Coleoptera: Curculionidae). J. Stored Prod. Res., 43: 79-86.
- Ketoh, G.K., H.K. Koumaglo and I.A. Glitho, 2005. Inhibition of Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) development with essential oil extracted from Cymbopogon schoenanthus L. Spreng. (Poaceae) and the wasp Dinarmus basalis (Rondani) (Hymenoptera: Pteromalidae). J. Stored Prod. Res., 41: 363-371.
- Lee, S., W. Choi, H. Lee and B. Park, 2000. Cross-resistance of a chlorpyrifos-methyl resistant strain of Oryzaephilus surinamensis (Coleoptera: Cucujidae) to fumigant toxicity of essential oil extracted from Eucalyptus globules and its major monoterpene, 1,8-cineole. J. Stored Prod. Res., 36: 383-389.
- Lopez, M.D., M.J. Jordan and M.J. Pascual-Villalobos, 2008. Toxic compounds in essential oils of coriander, caraway and basil active against stored rice pests. J. Stored Prod. Res., 44: 273-278.
- Miyazawa, M., H. Watanabe and H. Kameoka, 1997. Inhibition of acetylcholinesterase activity by monoterpenoids with a p-menthane skeleton. J. Agric. Food Chem., 45: 677-679.
- Ogendo, J.O., M. Kostyukovsky, U. Ravid, J.C. Matasyoh and A.L. Deng et al., 2008. Bioactivity of Ocimum gratissimum L. oil and two of its constituents against five insect pests attacking stored food products. J. Stored Prod. Res., 44: 328-334.
- Park, I.K., S.G. Lee, D.H. Choi, J.D. Park and Y.J. Ahn, 2003. Insecticidal activities of constituents identified in the essential oil from leaves of Chamaecyparis obtusa against Callosobruchus chinensis (L.) and Sitophilus oryzae (L.) J. Stored Prod. Res., 39: 375-384.
- Ragasa, C.Y., J.G. Hofilena and J.A. Rideout, 2002. New Furanoid Diterpenes from Caesalpinia pulcherrima. J. Nat. Prod., 65: 1107-1110.
- Reitman, S. and S. Frankel, 1957. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol., 28: 56-63.
- Regnault-Roger, C., 1997. The potential of botanical essential oils for insect pest control. Integ. Pest. Manage. Rev., 2: 25-34.
- Rozman, V., I. Kalinovic and Z. Korunic, 2007. Toxicity of naturally occurring compounds of Lamiaceae and Lauraceae to three stored-product insects. J. Stored Prod. Res., 43: 349-355.
- Romestaing, C., M.A. Piquet, E. Bedu, V. Rouleau and M. Dautresme et al., 2007. Long term highly saturated fat diet does not induce NASH in Wistar rats. Nutr. Metab., 4: 4-4.
- Shaaya, E., M. Kostjukovski, J. Eilberg and C. Sukprakarn, 1997. Plant oils as fumigants and contact insecticides for the control of stored-product insects. J. Stored Prod. Res., 33: 7-15.
- Tapondjou, L.A., C. Adler, H. Bouda and D.A. Fontem, 2002. Efficacy of powder and essential oil from Chenopodium ambrosioides leaves as post-harvest grain protectants against six-stored product beetles. J. Stored Prod. Res., 38: 395-402.
- Liu, W.W., W. Mu, B.Y. Zhu, Y.C. Du, F. Liu, 2008. Antagonistic activities of volatiles from four strains of Bacillus spp. and Paenibacillus spp. against soil-borne plant pathogens. Agric. Sci. China, 7: 1104-1114.
- Zettler, J.L. and G.W. Cuperus, 1990. Pesticide resistance in Tribolium castaneum (Coleopteran: Tenebrionidae) and Rhyzopertha dominica (Coleoptera: Bostrichidae) in wheat. J. Econ. Entomol., 83: 1677-1681.
- Akinkurolere, R.O., 2007. Assessment of the insecticidal properties of Anchomanes difformis (P. Beauv.) powder on five beetles of stored produce. J. Entomol., 4: 51-55.
- Khorram, M.S., N.T. Nasabi, S. Jafarnia and S. Khosroshahi, 2011. The toxicity of selected monoterpene hydrocarbons as single compounds and mixtures against different developmental stages of colorado potato beetle, Leptinotarsa decemlineata say (Coleoptera: Chrysomelidae). J. Entomol., 8: 404-416.
- Mulungu, L.S., B. Ndilahomba, C.J. Nyange, M.W. Mwatawala, J.K. Mwalilino, C.C. Joseph and C.A. Mgina, 2011. Efficacy of Chrysanthemum cinerariaefolium, Neorautanenia mitis and Gnidia kraussiana against larger grain borer (Prostephanus truncatus Horn) and maize weevil (Sitophilus zeamays Motschulsky) on maize (Zea mays L.) grain seeds. J. Entomol., 8: 81-87.
- Oparaeke, A.M. and G.C. Kuhiep, 2006. Toxicity of powders from indigenous plants against Sitophilus zeamais motsch on stored maize grains. J. Entomol., 3: 216-221.
- Sathyaseelan, V., V. Baskaran and S. Mohan, 2008. Efficacy of some indigenous pesticidal plants against pulse beetle, Callosobruchus chinensis (L.) on green gram. J. Entomol., 5: 128-132.
- Ikbal, C., B.H.K. Monia and B.H.M. Habib, 2007. Development perturbation of cotton leave noctuid with green cestrum extracts. J. Entomol., 4: 121-128.
- Barham, D. and P. Trinder, 1972. An improved colour reagent for the determination of blood glucose by the oxidase system. Analyst, 97: 142-145.