Insecticidal, Oviposition Deterrent and Antifeedant Property of Certain Plant Extracts against Pulse Beetle, Callosobruchus chinensis Linn. (Coleoptera: Bruchidae)

Pulses have a prominent place in daily diet and they are rich source of vegetable proteins, minerals and vitamins. They are of special significance of the people in developing countries like India, who can hardly afford animal protein in adequate quantities. India has achieved a record of pulse production of 17.82 million ton in 2016-2017. Post-harvest losses are often more significant than crop losses which occur in the field. As in field crops, a wide range of insect pests also attack stored products. In pulses, over 200 species of insects have been recorded in India (Saranya et al., 2019). Among the insect pests attacking stored products, the Pulse beetle Callosobruchus chinensis L. is reported to be the major pest infesting all types of pulses both in the field and in storage condition in India, Bangladesh and other tropical and subtropical countries (Mummigatti and Krishnaiah, 2007). C. chinensis is also known to attack cotton seed, sorghum and maize (Ahmed et al., 2003) The beetles breed rapidly in the storage condition in the tropical and subtropical environment. Their larvae can easily penetrate the grain and feed the endosperms, (Ahad et al., 2015) while feeding scoop out the contents of grains. The pulse beetles assume serious proportions usually during July-August in the stores (Varma and Anandhi, 2010). The infested grains become unsuitable for human consumption, deteriorate nutritional value and loss germination potential (Deeba et al., 2006). Ratnasekera and Nayanathara (2010) reported that the moisture content in pulse grains to less than 10% could significantly reduce the beetle infestation. Fumigation with synthetic chemicals like methyl bromide and phosphine is an effective method being used only in the warehouses. This method is expensive and unbearable to rural farmers and impractical in the primitive nature of storage in many of the villages. On the contrary, injudicious application of the synthetic fumigants creates serious health hazards and environment pollution (Kim et al., 2003). These problems directed the need for biodegradable pesticides in the management of stored grain pests (Daglish, 2008). Control of the pest using botanical pesticides, pressurized carbon dioxide and temperature management techniques are becoming popular (Yuya et al., 2009). The plant-derived materials are selective in action and their compounds are readily biodegradable and less likely to contaminate the environment. A number of authors have reported the toxicity, repellency, antifeedant, growth and progeny inhibition activity of plant materials against field and stored grains. Moreover, the farmers and small-scale industrialists can easily produce and store the crude or partially purified extracts from leaves, stems, fruits or roots of the plants. Recently, attention has been given to the possible use of plant products or plant derived compounds as promising alternative to synthetic insecticides in controlling insect pests of stored products (Ratnasekera and Rajapakse, 2009). The present study aims at investigating the insecticidal property, ovipositional and antifeedant effect of different plant extracts against C. chinensis.

Mass culturing of pulse beetle
 
Freshly harvested pulse seeds were obtained from farmers of Thuraiyur region. Seeds of the following crops like black gram, green gram and bengal gram and local varieties VBN 8, VBN (Gg) 3 were used in the storage experiments. Mass culturing of pulse beetle, C. chinensis was done at the Department of Agricultural Entomology, Imayam Institute of Agriculture and Technology, Thuraiyur during 2019-20. The adult pulse beetle was collected from Black gram and Bengal gram seed samples of the Department of Seed Science and Technology and from Department of Plant Breeding and Genetics and utilized for mass culturing. These beetles were reared on fresh black gram and green gram seeds following the method developed by Credland and Wright (1989). Black gram and green gram seeds @ 500 g each were placed in 600 ml plastic jars, into which approximately 30 pairs of freshly emerged adult beetles were introduced. The plastic jars were covered with muslin cloth and placed in dark to facilitate maximum oviposition, maintained at a room temperature of 30±5°C and 70±5% RH throughout the period of study. After 25 to 30 days, adults that emerged from the culture were utilized for maintenance of sub cultures following the same procedure as described above. Sub culturing of this beetle was done at weekly intervals so as to get continuous supply of insects for experiments. Freshly emerged adults were used for conducting the experiments.
 
Collection and aqueous extraction of plants
 
Locally available plants viz., Notchi (Vitex negundo Linn.), Senna (Cassia angustifolia Vahl), Calotropis (Calotropis gigantea Linn.) and Pungam (Pongamia glabra Linn.) were collected from the farm of IIAT, Thuraiyur. Leaves of above mentioned plants were shade dried for one week and ground into fine powder (Vanmathi et al., 2010). Plant powders were sieved through 0.25 mm pore size mesh sieve to obtain uniform fine dust particles (Jembere et al., 2005). The resulting powders were kept separately in plastic containers with tight cap and stored at room temperature in dark prior to use. Ten gram of each powder was soaked in 100 ml of distilled water and left for 24 hrs. Thereafter, the extracts were decanted and filtered using muslin cloth. Later the resultant solutions were used for laboratory study.
 
Insecticidal action of plant extracts
 
Seeds of green gram, black gram and bengal gram were taken @ 10gram in each plastic container. Plant extracts were added to the pulse seeds in each container individually at concentration of 10% and shaken thoroughly. Ten newly emerged adults of C.chinensis were released in each plastic container with treated seeds and were covered firmly and kept at the room temperature. Untreated pulse seeds were maintained as control and Azadirachtin was used as a standard check. Mortality (lack of locomotion and/or response to repeated probing) was recorded at 24 h interval for three days. The experiments were conducted in complete randomized block design (CRBD) with six treatments and four replications.

               
               

 
Ovipositional deterrent
 
In order to study the ovipositional deterrent effect of plant extract, seeds viz.,Green gram, Black gram and Bengal gram were taken @ 20 grams in each plastic container and mixed with plant extracts at the concentration of 10%. Five pairs of newly emerged adults of pulse beetle, C. chinensis were released in each plastic container and covered firmly and were kept under the laboratory conditions. Four replications were maintained for each treatment. The number of eggs laid was recorded after 48 h and oviposition deterrence was calculated with the following formula (Pascual-Villalobos and Robledo, 1998).

 
Oviposition deterrence = 100 × (1 - NE_t/NE_c)

 
Where
NE_t is the number of eggs in treatment and NE_c is the number of eggs in control.
 
Antifeedant effect
 
After 24 hours of treatment, grains were taken from the treated seeds of each treatment. Total weight loss in each replication was measured using a weighing balance. After data collection, grains were kept in plastic container of the  respective treatments. The extent of damage caused by pulse beetle on black gram, bengal gram and green gram seeds was determined on the basis of the weight loss from the total weight was calculated. The data was recorded for each replication. Effect on infestation was calculated by computing infested/unhealthy grain after 48 and 72 hours after release (HAR).
 
Statistical analysis
 
The data collected under different laboratory experiments were analyzed using analysis of variance (ANOVA) using AGRES 3.01 and AGDATA software. Data in the form of percentages were transformed to arcsine values and those in numbers were transformed to and analyzed. The mean values of the treatments were compared using LSD at 5 per cent level of significance.

Insecticidal effect of plant extracts on C. chinensis in different hosts
 
The results of insecticidal activity of different plant extracts viz., Vitex negundo, Cassia angustifolia, Calotropis gigantea, Pongamia glabra on pulse beetle, C. chinensis in green gram, black gram and bengal gram are furnished in Table 1. On 24 HAT, the highest mortality was recorded in Azadirachtin with 100 per cent followed by V. negundo (72.5%), P. glabra (57.5%), C. angustifolia (52.50%) and lowest mortality was recorded in C. gigantea (7.50%) and control(12.5.%). On 72 HAT, the highest mortality was recorded in Azadirachtin with 100 per cent, V. negundo (97.5%), C. angustifolia (87.5%), P. glabra (85.0%) and lowest mortality was recorded in C. gigantea (27.5%) and control (20%). The insecticidal activity of different plant extracts on pulse beetle in black gram, on 24HAT the highest mortality was recorded in Azadirachtin (100%) followed by V. negundo (72.5%), C. angustifolia (57.5%), P. glabra (55%) and lowest mortality was recorded in C. gigantea (12.5%) on par with control. On 72 HAT, the highest mortality was recorded in Azadirachtin (100%) followed by V. negundo (97.5 %), C. angustifolia (92.5%), P. glabra (90%) and lowest mortality was recorded in C. gigantea (40%). The results of insecticidal activity of different plant extracts in On 72 HAT, the highest mortality was recorded in Azadirachtin (100%) followed by V. negundo (97.5%), C. angustifolia (90%), P. glabra (77.5%) and lowest mortality were recorded in C. gigantea (27%). The results are in line with the findings of Fabiola et al., (2016) have studied the larvicidal effect of V. negundo extract (50 ppm) on Aedes aegypti and reported 77.80 per cent mortality in the 1^st instar larvae at 24 HAT. Insecticidal property of 10% V. negundo extracts were reported earlier by Sahayaraj (1998). Similar effects were observed by Gautham et al., (2003) in which they reported 51.7% mortality of Spodoptera litura at 6% aqueous extract of V. negundo leaves. Sharma et al., (2018) reported that mortality range (84-100%) against pulse beetle was observed in neem treated pigeonpea grains.
 

 
Oviposition deterrent effect of plant extracts on C. chinensis in different hosts
       
The results of the oviposition deterrent activity study showed that egg laying of C. chinensis was lowest in Azadirachtin with oviposition deterrent 91.70% followed by Cassia angustifolia (68.56%) and Pongamia (57.01%) treated green gram. In black gram, highest oviposition deterrent effect (92.18%) was observed in Azadirachtin treated seeds. Calotropis showing least oviposition deterrent effect (40.30%) in black gram. In the case of bengal gram, Vitex negundo treated seed showed 91.86%. oviposition deterrent (Table 2). Highest oviposition deterrent indicates minimum egg laying capacity by C. chinensis on pulses. Current findings were supported by the results of Vanmathi et al., (2010) who reported the oviposition deterrent effect of 1, 3 and 5 per cent aqueous extracts of C. gigantea and V. negundo on C. maculates in black gram seeds. It appears that these plant extracts might possess repellent or oviposition deterrent principles. Olaifa and Erhun (1988), who observed a complete suppression of oviposition by C. maculatus when treated with 42% powder of P. guineense. Elhag (2000) studied the oviposition deterrence of nine plant materials on C. maculatus and found seed treatment with 0.1% crude extract resulted in significant reduction in egg-laying by the bruchid. Oviposition deterrence may be due to the changes induced in physiology and behaviour in the adult of C. chinensis as reflected by their egg laying capacity.
 

 
Antifeedant effect of plant extracts on C. chinensisin different hosts
 
Any substance that reduces food consumption by an insect can be considered as an antifeedant or feeding deterrent (Isman, 2002). In green gram, the mean food consumption was minimum (0.05g) in V. negundo treated seeds. In Black gram and Bengal gram seeds, the mean food consumption was 0.03g and 0.10g, respectively (Table 3). Haridasan et al. (2017) exhibited the feeding deterrence activity of methanol and petroleum ether extracts of V. negundo on Tribolium castaneum. The results of the present study is in concordance with the findings of Arivoli and Samuel (2013) who recorded maximum antifeedant activity (86.41%) of V. negundo extract on S. litura. This indicated that the active principles present in the V. negundo inhibit feeding behaviour or make the food unpalatable or the substances directly act on the chemo sensilla of the beetle resulting in feeding deterrence.
 

The present investigation has brought out the efficacy of different plant extracts on C. chinensis. Especially, Notchi (Vitex negundo) leaf extract at 10 per cent concentration has highest insecticidal, antifeedant and oviposition deterrent activity on pulse beetle. Preparation of these aqueous extracts and application on the seeds are comparatively easy and cost effective. Vitex negundo is traditionally used by farmers as a storage insecticide and a common insecticide.  Hence, the plant extracts of V. negundo can be used as one of the component in Integrated Pest Management especially in small godowns or shop retailer for short term storage.