Full Length Research Paper. Pretheep-Kumar, P. 1*, Balasubramanian, A. 1 and Mohan, S. 2

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1 Sky Journal of Agricultural Research Vol. 4(4), pp , June, 2015 Available online ISSN Sky Journals Full Length Research Paper Efficacy of extracts of pea (Pisum sativum L.) in the management of saw-toothed beetle, Oryzaephilus surinamensis (L.) infesting stored neem (Azadirachta indica A. Juss) seeds Pretheep-Kumar, P. 1*, Balasubramanian, A. 1 and Mohan, S. 2 1 Forest College and Research Institute, Tamil Nadu Agricultural University, Mettupalayam , Tamil Nadu, India. 2 Tamil Nadu Agricultural University, Coimbatore , Tamil Nadu, India. Accepted 20 May, 2015 Acceptability of neem (Azadirachta indica A. Juss) seeds by the saw-toothed beetle, Oryzaephilus surinamensis (L.) for the purpose of feeding decreased significantly when admixed with protein-rich pea fractions. Among the pea extracts evaluated, protein-rich fractions of pea (Pisum sativum L.) at 1% concentration was highly effective in its repellent, toxic and progeny-reduction effect than whole-pea flour. Key words: Azadirachta indica, Oryzaephilus surinamensis, Pisum sativum, food preference, progeny. INTRODUCTION Neem (Azadirachta indica A. Juss) is a fast growing evergreen medicinal plant native to the Indian subcontinent. Every part of the neem tree has been found to have multidirectional uses and enormous application making it a Green treasure (Girish and Bhat, 2008). Neem is a natural source of eco-friendly insecticides, pesticides and agrochemicals (Brahmachari, 2004). More than 195 species of insects are controlled by neem extracts. The most useful and valuable products of neem tree are the seeds that yield up to 40% of deep yellow oil called Margosa oil. The isolation of azadirachtin from neem seeds has gained worldwide attention in view of the insect antifeedant and growth inhibiting properties (Jacobson, 1989). Azadirachtin, a tetratriterpenoid present in neem seeds to the extent of % is highly potent at low concentration against more than 200 agricultural pests and is eco-friendly (Mordue and Blackwell, 1993). Although these seeds are reported to have insecticidal properties, the chemical does not appear to be toxic to the saw-toothed beetle, Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae). It is a pest of stored foods including cereals, nuts and dried fruits and found all over * Corresponding author pretheepkumar_phd@yahoo.co.in. the world. Most stored grain pests are susceptible to neem s effect except the saw-toothed beetle, which breeds well on neem. Legume seeds contain a wide range of allelochemicals with toxic and deterrent effect against insect pests (Bell, 1978). Peas and pea extracts has been found to be toxic to the stored product insects (Grenier et al., 1997). A bioactive protein fraction extracted from pea (Pisum sativum L.) has been found to be effective in controlling insect pests (Bodnaryk et al., 1999; Pretheep-Kumar et al., 2004). Considering the importance and wide application of neem seeds in various pharmaceutical, cosmetic and pesticide manufacturing companies, finding a viable solution to protect neem seeds from insect pest infestation during storage assume significance. Hence, this study aimed at assessing the efficacy of pea extracts in reducing damage by the saw-toothed beetle to stored neem seeds. MATERIALS AND METHODS Test materials Pea seeds (Pisum sativum var. Bonneville) of wrinkled nature were procured from Ooty, Tamil Nadu State, India. Whole-pea flour was obtained by grinding the pea seeds

2 68 Sky. J. Agric. Res. in a mixer grinder. The whole-pea flour was passed through different sized sieves in an electronic sieve shaker to obtain the fine protein-rich pea fractions (53 ) by following the air classification process of Wright et al. (1984). Total protein content of whole-pea flour and protein-rich pea fractions were 16% and 60%, based on the method of Lowry et al. (1951). Local strains of Oryzaephilus surinamensis (L.) adults mass cultured on dry grapes and maintained at ambient laboratory temperature of C and 75-80% RH were used. Food preference Food preference of Oryzaephilus surinamensis adults to different concentrations of whole-pea flour or protein-rich pea fraction treated neem seeds were assessed by multiple choice bioassay. Neem seeds were treated with pea extracts at concentrations of 0%, 0.001%, 0.01%, 0.1%, 0.5% and 1% w/w basis. The treated seeds were kept in food preference chamber, a plastic container of 20 cm diameter and 7 cm high partitioned into 6 chambers. One hundred grams (100 g) of neem seeds treated with different concentrations were placed in each of the partitioned chamber. After placing the test seeds, 100 unsexed adults of O. surinamensis were released in the circular chamber at the centre and the entire chamber was covered with a transparent glass. Adults found in each of the chamber were counted after 48 h of introduction to assess the repellent or antifeedent property of the whole-pea flour or protein-rich pea fraction treated neem seeds at different concentrations. The experiments were conducted at a room temperature of 28 C with 75% RH. Four replications were maintained for each set of experiments. whole-pea flour or protein-rich pea fraction treated and untreated neem seeds. Neem seeds of 1 kg were treated with 10 g of whole-pea flour or protein rich pea fractions separately which resulted in 1% concentration of wholepea flour or protein-rich pea fraction treated seeds. Concentration of 1% was chosen since it was found very effective among various concentrations tested by multiple choice bioassay and progeny studies. One kilogram (1 kg) of neem seeds without any treatment served as untreated control. Both the treated and untreated neem seeds were kept in plastic containers of 20 cm high and 17 cm diameter. Fifty (50) unsexed adults of O. surinamensis were released into the containers and probe traps of 2 mm perforations were inserted into both the untreated and treated seeds. The number of adults which got trapped in the probe traps were recorded at 0.5, 1, 6, 12, 18 and 24 h after the placement of traps and the results were expressed in per cent insects trapped. The experiments were conducted at a room temperature of 28 C and 75% RH. Four replications were maintained for each treatment. Toxicity Whole-pea flour or protein-rich pea fractions were mixed with 100 g of neem seeds at 1% w/w basis as this concentration was found effective in multiple choice bioassay and progeny studies. Untreated neem seeds served as control. Twenty unsexed adults of O. surinamensis were introduced into whole-pea flour or protein-rich pea fractions treated neem seeds placed separately in plastic containers of 10 cm high and 7 cm diameter and the containers were covered with a muslin cloth. Mortality of adults were recorded at 1, 2, 3 and 4 weeks after introduction of the insects. Progeny For evaluating the effect of pea extract in reducing the progeny, both the whole-pea flour or protein-rich pea fraction were mixed with neem seeds at concentrations of 0%, 0.001%, 0.01%, 0.1% and 1% w/w basis. The treated neem seeds were placed separately in transparent plastic containers (10 cm high and 7 cm diameter). Ten unsexed adults of O. surinamensis were released inside each container and covered with a muslin cloth. Released adults were removed after 2 weeks and the adult offspring were counted after a further period of 5 weeks. The experiments were conducted with four replications at a room temperature of 28 C and 75% RH. Probe trap The trapping efficiency of probe traps (Mohan and Gopalan, 1992) were studied by placing the traps in Statistical analysis The observations were analyzed in a completely randomized design and the mean values were separated using Duncan s Multiple Range Test (Gomez and Gomez, 1984). RESULTS AND DISCUSSION Food preference The study revealed that O. surinamensis had the least preference for neem seeds treated with 1% protein-rich pea fraction. No significant difference was observed in the food preference behavior of O. surinamensis adults exposed to neem seeds treated with whole-pea flour under multiple choice bioassays. Multiple choice bioassay is the most common method for screening the

3 Kumar et al. 69 Table 1. Food preference of Oryzaephilus surinamensis adults exposed to neem seeds treated with pea extract for 48 hours under multiple choice set up. Concentration % adults of total population (mean±s.e.) Whole-pea flour Protein-rich pea fraction 0% 17.0±2.1 Aa 21.0±1.1 Ac 0.001% 16.3±2.5 Aa 21.3±1.8 Ac 0.01% 16.5±1.9 Aa 20.5±1.2 Ac 0.1% 17.8±2.3 Aa 19.0±0.8 Ac 0.5% 15.3±1.3 Aa 11.7±0.5 Ab 1% 17.1±1.7 Ba 6.5±1.0 Aa In a column, means followed by the same letter (lower case) and in a row, means followed by the same letter (upper case) are not significantly different by Duncan s multiple range test (P=0.05). Table 2. Progeny of Oryzaephilus surinamensis developed from neem seeds treated with pea extract. Concentration Number of adult offspring after 7 weeks (mean±s.e.) Whole-pea flour Protein-rich pea fraction 0% 126.3±3.3 Ae 126.3±3.3 Ae 0.001% 103.8±3.0 Bd 83.5±1.9 Ad 0.01% 93.5±3.1 Bc 74.3±1.7 Ac 0.1% 81.5±3.7 Bb 41.0±2.2 Ab 1% 33.0±2.9 Ba 7.8±1.0 Aa In a column, means followed by the same letter (lower case) and in a row, means followed by the same letter (upper case) are not significantly different by Duncan s multiple range test (P=0.05). effect of plant products as it facilitates testing several concentrations at a time (Jilani and Saxena, 1990). Only 6.5% of the total population of O. surinamensis adults was found in the section containing neem seeds treated with protein-rich pea fraction at 1% concentration as against 21.0% of the population in untreated control (Table 1). It is clearly evident that the acceptability of neem by O. surinamensis for the purpose of feeding decreased as the protein-rich pea fraction concentration in the seeds increased. This suggests that the protein-rich pea fraction has the potential of repelling insects from the seeds which would lead to the non-preference of the protein-rich pea fraction treated seeds by the insects. At low concentrations, the repellent response was not very high, whereas at 1% concentration, there was a significant increase in the repellency. The reduction in the per cent population of insects in the treated seeds could be caused by chemosensory effects of the protein-rich pea fraction, either olfactory or gustatory. Progeny The average emergence of adult O. surinamensis from 1% whole-pea flour and protein-rich fraction treated neem seeds were 33.0 and 7.8, respectively, during a period of 7 weeks (Table 2). Progeny build-up of O. surinamensis was markedly suppressed in neem seeds admixed with protein-rich pea fraction. A significant decline in the number of adults offspring was noticed in 1% concentration compared to other tested concentrations. All these reductions were dose-dependent. The reproduction process was normal for O. surinamensis adults in the untreated seeds. Disruption of feeding by protein-rich pea fraction treated neem seeds would depress oviposition resulting in progeny reduction as feeding is vital for egg laying. Probe trap Studies revealed that O. surinamensis adults trapped in the probe traps placed in untreated and 1% whole-pea flour treated seeds were similar. No significant difference was observed in the trap catches of both untreated and whole-pea flour treated neem seeds. A greater percentage of insects got trapped in the probe traps placed in protein-rich pea fraction treated seeds indicating its good repellent property. Probe traps were known to trap stored product insects in large numbers (Mohan, 1993). Adults of O. surinamensis caught in probe traps placed in protein-rich pea fraction treated neem seeds were 60.5, 76.0 and 92.5% as against 15.0, 28.5 and 42.0% in untreated seeds in a time period of 12, 18 and 24 h, respectively (Table 3).

4 70 Sky. J. Agric. Res. Table 3. Adults of Oryzaephilus surinamensis caught in probe traps placed in untreated and 1% whole-pea flour or protein-rich pea fraction treated neem seeds. Duration (after trap placement) % insects trapped (mean±s.e.) Untreated Control Whole-pea flour Protein-rich pea fraction 0.5 h 0 Ad 0 Ad 1.5±1.0 Ae 1 h 0 Ad 0 Ad 2.0±1.6 Ae 6 h 3.5±1.0 Bd 3.0±1.1 Bd 39.5±2.5 Ad 12 h 15.0±2.5 Bc 15.5±1.9 Bc 60.5±1.9 Ac 18 h 28.5±1.9 Bb 29.0±2.5 Bb 76.0±2.3 Ab 24 h 42.0±1.6 Ba 41.5±1.0 Ba 92.5±2.5 Aa In a column, means followed by the same letter (lower case) and in a row, means followed by the same letter (upper case) are not significantly different by Duncan s multiple range test (P=0.05). Table 4. Mortality of Oryzaephilus surinamensis exposed to neem seeds treated with pea extracts at 1% concentration. Test product % mortality of insects (mean±s.e.) 1 week 2 weeks 3 weeks 4 weeks Protein-rich fraction 31.3±2.5 Ca 73.8±2.5 Ba 100.0±0.0 Aa 100.0±0.0 Aa Whole-pea flour 11.3±2.5 Db 17.5±2.8 Cb 28.8±2.5 Bb 36.3±2.5 Ab Untreated control 0 Bc 0 Bc 5.0±0.0 Ac 6.3±2.5 Ac In a column, means followed by the same letter (lower case) and in a row, means followed by the same letter (upper case) are not significantly different by Duncan s multiple range test (P=0.05). Protein-rich pea fraction would have increased the movement of insects in the seeds ending in greater emigration and trapping of insects in probe traps, hence, reducing insect populations. Repellent activity of proteinrich pea fraction enhanced the trapping efficiency of probe traps. Toxicity The study revealed that the toxic action of whole-pea flour or protein-rich pea fraction varied with test duration. Among the pea extracts evaluated, protein-rich pea fraction was found to be highly efficient in its toxic property than the whole-pea flour. Among the concentrations tested, 1% concentration exhibited greater insecticidal activity against O. surinamensis. Whole-pea flour admixed with neem seeds at 1% concentration caused 17.5% and 36.3% mortality of O. surinamensis adults after 2 and 4 weeks exposure, respectively (Table 4). At the same concentration, protein-rich pea fraction treated neem seeds resulted in 31.3% and 100% mortality after an exposure period of 1 and 3 weeks, respectively. Based on the toxicity studies, the protein-rich fraction of Bonneville pea significantly increased the mortality of O. surinamensis in a dose-dependent manner. Protein-rich pea fraction treated neem seeds at 1% concentration offered good control of O. surinamensis causing mortality of the insects due to its toxicity. The antifeedant property of protein-rich pea fractions, which may constitute a complex of chemicals, was reflected by the increased mortality rate. Neem seeds treated with protein-rich fraction of Bonneville pea variety offered greater protection against O. surinamensis compared to the seeds treated with whole pea flour of the same pea variety. Nonacceptability of protein-rich pea fraction treated neem seeds by O. surinamensis suggests that the protein-rich pea fraction could be a useful protectant for stored seeds from insect attack. Acknowledgements We would like to thank the Science and Engineering Research Board (SERB), Government of India, for providing financial assistance to carry out this research work. REFERENCES Bell EA (1978). Toxins in seeds. In JB Harborne, ed, Biochemical aspects of plant and animal co evolution. Academic Press, New York pp Bodnaryk R, Fields PG, Xie Y, Fulcher K (1999). Insecticidal factors from field pea. United States of America Patent 5, 955, 082. Brahmachari G (2004). Neem - an omnipotent plant: a retrospection. Chembiochem., 5(4): Girish K, Bhat SS (2008). Neem - A green treasure. Electronic Journal of Biology, 4(3): Gomez KA, Gomez AA (1984). Statistical Procedures for Agricultural Research. John Wiley and Sons, New York, 680 p.

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