Fumigant effect of essential oils against pulse beetle (Callosobruchus chinensis L.) and rice weevil (Sitophilus oryzae L.) in stored products

Objectives: To develop a method to replace the usage of synthetic fumigants in stored product pest management. Methodology: For this purpose, we employed four plant based essential oils namely, Lantana camara (Lantana) oil, Citronella nardus (Citronella) oil, Cinnamomum zeylanicum (Cinnamon) oil and Trachyspermmum copicum (Ajwain) oil and evaluated against pulse beetle, Callosobruchus chinensis L., and rice weevil, Sitophilus oryzae L., for their fumigant toxicity and the weight loss caused by them. The oils of Lantana and Citronella were extracted from leaves, Cinnamon and Ajwain from barks and seeds respectively. Findings: Among the essential oils tested for fumigant toxicity, C. zeylanicum performed better with Lethal Concentration (LC50) of 23.16 and 21.91μ l in minimal period of exposure i.e. at 12 h of treatment and found effective against adults of test insects viz., C. chinensis, and S. oryzae respectively. During the maximum period of exposure i.e. at 72 h. the fumigation efficacy of T. copicum was noticed to be the highest (3.43 μ l) against adults of C. chinensis whereas C. zeylanicum was found better against S. oryzae (6.19 μ l). The minimum loss of pulse grains was observed in C. zeylanicum treated at 60 % of 24 h LC50 , with 7.41 % weight loss. The minimum weight loss was noticed in C. zeylanicum about 60 % of 24 h LC50 with 9.68 per cent against S. oryzae. The fumigant toxicity bioassay revealed that as the exposure period increases, mortality of test insects also found increased i.e. exposure period directly proportional to mortality of insects.


Introduction
The food requirement of an increasing human population remains a major global issue. More than one third of food is lost or wasted during post harvest operations. Reducing the post harvest losses, a concrete solution will be to increase the availability of https://www.indjst.org/ food, reduce damage on natural resources, eliminate hunger and improve farmers' livelihoods. Cereal grains are the staple food in most of the developing nations and it account for maximum post harvest losses. Owing to lack of technical knowledge, 50 -60 per cent of cereal grains are lost during storage [1]. As per the report of Food Corporation of India (FCI), about 62,000 tonnes of food grains have been damaged in the storage during the last six years, in 2016-17 (up to 1 st March), a damage of 8,679 tonnes of food grains was noticed. Pest attacks, leakages, poor quality stocks, exposure to rains, floods and negligence are the cause for grain damage in godowns [2]. Storage losses have been estimated as 14 million tonnes of food grain and worth of |7,000 crore every year in India, out of which insects alone account for nearly |1,300 crores. The loss of food grains in the farmer holdings in Tamil Nadu was estimated as 12.9 % in paddy, 16.0 % in sorghum, 14.0 % in bajra and 12.7 % in maize. Out of all the post harvest losses, storage alone 6.58 %, in this insect alone account for 2.0 to 4.2 % followed by rodents (2.50 %), Birds (0.85 %) and moisture (0.68 %) [3].
Among the storage pests, the cowpea beetle, C. chinensis L. and the four spotted bean weevil, Callosobruchus maculatus F. (Bruchidae: Coleoptera) are the notorious pests of common legumes and pulses grown in Asia, Africa, Central and South America. It lays eggs on the seed coat, the grub hollows out the grain and cause huge yield loss [4]. The rice weevil, S. oryzae L. (Coleoptera: Curculionidae) is one of the big menaces and causes huge loss of stored grain both quantitatively and qualitatively worldwide [5,6]. The most effective methods for the protection of stored products from insect pests are fumigation. Usage of phosphine leads to development of resistance in insects and may cause control failures. Although chemical insecticides are effective, their continuous use has led to several problems viz., residue, environmental pollution, and effect on humans. Many insecticides have either been banned or restricted in their use due to the problems mentioned above. This led to the need for biodegradable pesticides with greater selectivity. Plants may act as potential alternatives to the currently used insect control agents as they constitute a rich source of bioactive molecules [7].
Hence, an attention has been given to utilize the plant products against stored grain pests [8]. Further, botanical insecticides are best for organic food production during post harvest protection of food grains [9]. Botanical insecticides composed of essential oils may be an alternative to the synthetic pesticides [10]. Essential oils are volatile oil(s) comprising strong aromatic components and give distinct odour, flavour or scent to a plant and the by-products of plant metabolism. They are obtained from various plant parts namely, leaves, stems, bark, flowers, roots and/or fruits. The aromatic characteristics of essential oils may provide various functions include attracting or repelling insects, utilizing chemical constituents in the oil as defence materials [11]. Plant essential oils and their constituents have been studied against stored product pests as alternatives to classical fumigants [12]. In developing countries, aromatic plants are widely used for stored-product insects in traditional agricultural systems. Essential Oils are produced by steam distillation of plant material and contain many volatile, low-molecular-weight terpenes and phenolics [13]. Essential oils show a broad spectrum of activity against insect pests as insecticidal, antifeedant, repellent, oviposition deterrent and growth regulatory activities. The use of these oils in the control of stored product pests is safer for human and animal health [14]. Hence, essential oils are promising alternatives to chemical insecticides [15]. Many formulations are being used against stored product insects and their selection or synthesis of formulation is important to control target insects [16]. Considering the need for environment friendly, safe, economically feasible and also effective insecticide to manage the insect pests of stored products, the present study was under taken to find out the fumigant toxicity of selected essential oils against the adult stages of two coleopteran pests namely, C. chinensis and S. oryzae inflicting damage to stored produce.

Test Insect culture
The test insects such as pulse beetle, C. chinensis, and rice weevil, S. oryzae were reared at the Department of Entomology, Annamalai University. They were mass cultured in 1kg capacity glass jar of size 15x10 cm containing respective food materials such as green gram for C. chinensis and rice grains for S. oryzae each 500 g as a nutritional source at 60-70 per cent relative humidity and temperature ranged from 30-35 • C. Then glass jars were covered with a fine muslin cloth and secured with rubber band to facilitate aeration. Maximum of seven days were allowed for mating and oviposition in separate rearing containers every day. Then the parent stocks were removed and food media containing eggs were incubated in the temperature/humidity as mentioned above in darkness to obtain same aged insects. In every generation, half of the completely infested grains were replaced with the same quantity of uninfested materials [17]. Thus, a continuous culture was maintained throughout the study period and subsequent progenies of the insects were used for the experiments. https://www.indjst.org/

Procurement of essential oils
Based on the fumigant action of essential oils of plant species from literatures surveyed, the essential oils namely, L. camara (Lantana) oil, C. nardus (Citronella) oil, C. zeylanicum (Cinnamon) oil and T. copicum (Ajwain) oil were selected. The oils of Lantana and Citronella were extracted from leaves, Cinnamon and Ajwain from barks and seeds respectively. The essential oils used for this study were purchased from the Allins Exports Private Limited, Noida, Uttar Pradesh and Surajbala Exports (P) Ltd., New Delhi.

Fumigant toxicity of essential oils against test insects
Glass vials of 10 cm long and 3 cm diameter with polystyrene cap were used for testing the fumigant toxicity of test insects like C. chinensis and S. oryzae. For fumigation, filter paper strip (1 cm 2 ) treated with solutions of five different concentrations (5, 10, 15, 20 and 25 µl for C. chinensis and 10, 20, 30, 40 and 50 µl for S. oryzae) of essential oils prepared in acetone was placed on the inner surface of each screw caps of the glass vials. The treated filter paper strips were air dried for 15 minutes for evaporating the solvents at room temperature. Then ten grams of respective food materials were taken, and ten adults were released in each vial. The open end of the vials was closed by the cap so that the oil treated filter paper remains inside the vial. For each treatment, five different concentrations and for each concentration three replicates were used. The vials were kept at 30 + 2 • C, 75 + 5% RH and a photoperiod of 10:14 (L: D) hours. Mortality of adults was recorded after every 12 h of treatment up to 72 h. Two controls were set one is standard check (i.e.) filter paper strip treated only with acetone and another one was untreated [18].

Estimation of weight loss
Estimation of loss in weight of food sources which was fumigated with two different concentrations of essential oils at 30 % and 60 % of the 24 h LC 50 was done. Ten pairs of freshly emerged adults of the test insects were selected and allowed into the jars containing 100 gm of respective food material. The experiment was replicated thrice. A control was set up without any treatment. The grains were maintained for completion of one generation of test insects and weighed using weighing balance and the per cent loss in weight were determined [19].

Per cent weight loss
Where, W1 = weight of baseline sample, W2 = subsequent sample weight at different storage intervals.

Statistical analysis
The LC 50 values for selected essential oils were calculated by using POLO software program. The data obtained from the assessment of weight loss experiment was analysed statistically by using Completely Randomised Block Design (CRBD). Based on the Analysis of Variance (ANOVA) and Least Significant Difference test (LSD), treatment effects were compared and ranked [20,21].

Toxicity of selected essential oils against test insects due to fumigation
The fumigant effect of essential oils at different exposure periods were tested against the adult beetles of C. chinensis and Lethal Concentration (LC 50 ) along with Upper and Lower Confidence Limit (UCL and LCL 95 %) were obtained and the results presented in Table 1  The results of the essential oils of T. copicum and L. camara were found closer with each other during 48 h of exposure period recorded as 12.36 and 12.28 µl respectively while T. copicum and C. zeylanicum were also caused nearly similar effects during 60 h of exposure period and found with 9.38 and 9.11 µl respectively. At 72 h, it was observed that T. copicum manifests its effectiveness with a LC 50 of 7.24 µl, followed by L. camara and C. nardus with 8.72 µl and 10.63 µl respectively. This might be due to the presence of phytochemicals i.e. secondary plant metabolites in these essential oils had the capability to emit the strong pungent fumigant odour that acts against the target insects. Due to this insecticidal action, the insect became susceptible and showed promising lethal effects. Many plant-derived materials such as monoterpenoids have fumigant action against a variety of insect pests attributed to their high volatility. Monoterpenoids (limonene, linalool, terpineol, carvacrol and myrcene) are the main insecticidal constituents of many essential oils effective against stored product insects [22,23]. The findings of the present study is in accordance with the investigation by [24], two monoterpenes cinnamaldehyde and linalool were selected from different constituents of cinnamon essential oil and the results revealed higher toxicity to C. maculatus than adults of S. oryzae. Further the results are supported by [25] who reported that cinnamon oil provided the highest toxicity to adults and 10, 14 and 18 days old larvae, with LD 50 values of 0.03, 0.05, 0.088 and 0.09 mg cm-3 respectively in fumigation bioassay. Similar findings were also obtained by [26] that the insecticidal efficacy of C. zeylanicum and its two major constituents (Linalool and Cinnamaldehyde) against C. maculatus and S. oryzae. They unveiled that these two components of C. zeylanicum exhibited contact and fumigant toxicity against the adults of both the insect species. The toxic effects of cinnamon essential oil were attributed to its major constituent monoterpenes which are highly volatile and possess high fumigant toxicity. On the same note, [27] reported that the presence of toxic compounds such as 1, 2 naphthalenedione ethanone and borneol in cinnamon where cinnamaldehyde is the principle one. The results of the present investigation supported by previous findings [28]

Effect of fumigation of essential oils on the damage caused by test insects
The minimum loss of pulse grains was observed in C. zeylanicum treated at 60 % of 24 h LC 50 closely followed by L. camara 60 % of 24 h LC 50 and T. copicum 60 % of 24 Table 3). The weight loss of seeds/grains was found ranging from 7.41 % to 37.15% https://www.indjst.org/ in almost all the treatments and also against all the target insects. When comparing all the treatments, C. zeylanicum 60% of 24 h LC 50 provided minimum weight loss per cent and C. nardus 30% of 24 h LC 50 manifested poor results with maximum percent weight loss against target insects. The reduction in weight loss might be due to the fumigant effects of the essential oils tested, the volatile substances are mainly responsible for positive decrease in feeding of the respective food materials by the test insects.

Conclusion
The essential oils obtained from naturally available plant species exhibited their potential fumigant effect against the target pests and also caused minimal damage to grains by them. From the results, it was noticed that as the exposure period to fumigation increases, mortality of test insect pests of grains also increased i.e. exposure period directly proportional to mortality of pests. However, further studies are required on the safety issues of essential oils against non-target organisms and to explore the mechanism of action against target pests. Furthermore, isolation and characterisation of the essential oil will also provide complete details of the compounds responsible for pesticidal activity and helpful in the preparation of easily usable formulations against stored produce pests. Hence, the use of essential oils of plant species may also be incorporated in the stored product pest management programme. It is used as a natural protectant in small scale storage so as to avoid the usage of synthetic chemicals. In India especially Tamil Nadu, it will be ideal if such organic way of protecting the grains in stores and supply of food grains through fair price shops is implemented on experimental basis as a public welfare measure. https://www.indjst.org/

Acknowledgement
The authors are thankful to the authorities of Annamalai University for their permission to carry out the research work.