Evaluation of Botanical Powders for the Management of Rice Weevil (Sitophilus oryzae L. Coleoptera: Curculionidae) in Rupandehi, Nepal

An experiment to manage rice weevil (Sitophilus oryzae L. Coleoptera: Curculionidae) in wheat (Triticum aestivum L. Gramineae) was carried out at Institute of Agriculture and Animal Science (IAAS), Paklihawa Campus, Rupandehi, Nepal.(e experiment was conducted under completely randomized design (CRD) with seven treatments viz. neem leaf dust (Azadirachta indica A. Juss) 15 g/kg, tobacco leaf dust (Nicotiana tabacum L.) 10 g/kg, ginger rhizome powder (Zingiber officinale Roscoe) 20 g/kg, garlic cloves powder (Allium sativum L.) 20 g/kg, Sichuan pepper seed powder (Zanthoxylum armatum Roxb.) 10 g/kg, sweet flag rhizome dust (Acorus calamus L.) 5 g/kg, and control with three replication. Result revealed that the highest mortality of weevils was observed in the wheat seed treated with A. calamus (98.33%), followed by N. tabacum (85.67%), A. sativum (73.34%), A. indica (70.67%), Z. armatum (70.34%), and Z. officinale (58.34%). Similarly, the lowest percent weight loss (3.32%) and damage of seed (4.0%) were observed in wheat treated with A. calamus. Moreover, the highest germination (89%) was observed in seeds treated with A. calamus rhizome powder when tested at 90 days after treatment application. Based on weevil mortality and the germination test, it is found that sweet flag rhizome powder is the best treatment against rice weevil followed by tobacco leaf dust and garlic clove powder. (erefore, these botanicals could be one of the effective alternatives for the management of weevil especially to the farmers who do not use chemical insecticides in the rural areas of Nepal.


Introduction
In Nepal, wheat (Triticum aestivum L.) is the third ranking cereal crop after rice (Oryza sativa L.) and maize (Zea mays L.) in terms of area of production and productivity. Wheat occupies about 22.58% of the total area in the cereal production and contributes 20.13% of the total cereal production [1]. is crop is grown in most of the agroclimatic regions from Terai (tropical plain area) to foothills of mountains in Nepal [1]. Wheat is believed to be originated in southwestern Asia [2]. Wheat is considered nutritionally better than other cereals as it contains more protein, and it has a relatively high amount of amino acids [3]. Insects such as Sitophilus spp, Tribolium spp, Rhizopertha spp, and Sitotroga cerealella are major stored grain pests in Nepal. It is reported that the loss due to different insect pests and postharvest handling is around 15-20% of total grain production in Nepal [4]. e estimated postharvest loss of food grains due to insect pests in Nepal in traditional storage conditions is 5.5% [5]. Among these insects, rice weevil Sitophilus oryzae L. is the most ubiquitous, and it severely damages a range of cereals such as wheat, maize, and rice [6]. When wheat is stored for long time for human consumption or seed, it is prone to get infested by S. oryzae that eventually leads to severe economic loss as the quantity and quality of stored wheat are largely compromised. Both larva and adult of the weevil are capable of damaging the wheat by boring and feeding on the grains. Such damage is often accelerated along with high grain moisture content (>12%), high relative humidity (>70%), and high temperature (>27%) by favoring the growth and development of the weevils. e study of the efficacy of some botanicals against various insects has been carried out by many researchers [7,8]. In Nepal, Bhusal and Sharma used different combination and concentrations of botanicals of which some are effective against this pest [8,9]. e botanicals used for these studies are traditionally popular for their culinary as well as medicinal purpose in Nepal. Although the mechanism of these botanicals in controlling the insects and pests are not very clear, efforts have been made to estimate their efficacy in controlling the insects in various experiments in Nepal. As these plants have antifeedant, repellent, and pesticidal properties against insects, various local products made from such plants are found to be very effective in controlling stored grain pests especially in small scale. While some of these plants have growth disrupting properties as well [10].
Among the common botanicals used as pesticides, the Zanthoxylum armatum DC, Timur in Nepalese language, is grown extensively in the hills of Nepal and is also a common plant in southeast Asia. Its seed is popular for its strong volatile smell; so, it is used for culinary purposes and is known to be traditionally used as a repellent against different insect pests. Similarly, Acorus calamus L. is grown in marshy land from the Terai (topical plain area) to the hills of Nepal. It is popularly used as medicine for sore throat and cold traditionally. Similarly, A. calamus powder is used to control stored grain pests on various cereals in Nepal. Paneru et al. reviewed about properties of A. calamus and stated that β-asarone found in stolons has an insecticidal property, whereas dried rhizome possesses antifeedant property [11,12]. Garlic (Allium sativum L) and Zinger (Zingiber officinale Roscoe) are popular herbs grown from plains to high hills of Nepal. Garlic and zinger own an important place in the Nepalese kitchen, and these are also traditionally used as medicine for abdominal pain, several digestive health issues, and common cold and in some cases used for pest management. Likewise, neem (Azadirachta indica A. Juss) is another popular plant of subtropical and tropical regions of Nepal. e leaves of this plant are popular for insect control since a long period of time. Moreover, the nicotine, extract from the tobacco (Nicotiana tabacum L.), is being used as an insecticide against many pest since a long. e nicotine is very effective against insects causing the uncontrolled nerve firing and masking acetylcholine [13]. Complete reliance on a chemical has led to insecticide resistance within the insects, and insects have become more and more adapted to these chemicals [14]. On the other hand, the issues related to the persistence of chemical on the treated grains and also their toxicity to natural enemies of these insects are equally dangerous in many ways. Similarly, other problems caused by chemical pesticides are insect resurgence, secondary pest outbreak, health hazard to human beings, and the environment.
is led us to refocus the way we think and encouraged us to conduct research for a pest management program that is safer, economical, and scientifically justifiable. In rural Nepal, farmers lack proper knowledge on techniques of grain protection in storage, and they often practice other risky methods of protection of stored grains. ey use synthetic pesticides such as malathion and aluminium phosphide to protect their stored grains which can cause health hazards. Hence, the plant products which have insecticidal properties present within them are a very good alternative to protect stored grains instead of these hazardous synthetic pesticides [15]. e major objective of our study is to devise an effective and ecofriendly alternative of chemical insecticides to control rice weevil in stored grains in Nepal.

Materials and Methods
e research was conducted at the laboratory of the Department of Entomology, Paklihawa Campus, Rupandehi, Nepal, in 2015. e laboratory is located at the geographical coordinates of 27°30′ 0″ North, 83°27′ 0″ East. e average temperature and relative humidity during the experiment period was 32°C (ranging from 29°C to 35°C) and 70 ± 5%, respectively. e room was well ventilated to allow enough air circulation throughout the room.

Materials.
Plastic made insect rearing box (one liter capacity), with a perforated lid was used to store 250 g of wheat. e plastic container was covered with a muslin cloth to ensure air circulation and to prevent the escape of inoculated weevil from the box. An aspirator was used to collect insects into the vial. maximum and minimum digital thermometer was used to detect the temperature of the laboratory during the study period. A digital RH meter was used to measure humidity in the lab. An electronic weight scale (Changzhoo Accurate Weight Co., Ltd.) of measuring capacity up to 350 g and 3 digits more behind decimal point was used to weight the test seeds.

Sampling of Wheat
Grains. Gautam (BL 1887) variety of wheat (released by Nepal Agriculture Research Council in 2002) was used in the entire test. Gautam is a high yielding variety recommended for the Terai, tars, and lower valleys of Nepal to grow in irrigated land as a timely or late sown variety. Using the results from a preliminary survey, this variety was found as most popular among farmers in Rupandehi because of its bold grain, taste, flour quality, and milling quality for making "chapatti" (local bread). us, the seed was collected from local farmers for this research. e sample wheat was disinfected by hot air treatment, and infestation free grains were used in the experiment.

Preparation of Botanicals.
All the botanicals used in the experiment viz. leaf of neem and tobacco, rhizomes of sweet flag and zinger, garlic cloves, and Sichuan pepper seed were collected from farmers field, dried in the shade, and powdered using a grinder. ose botanicals were applied in the following dosage: Nicotiana tabacum L. 10 g/kg, Zingiber officinale Roscoe 20 g/kg, Allium sativum L. 20 g/kg, Azadirachta indica A. Juss 15 g/kg, Zanthoxylum armatum DC. 10 g/kg, Acorus calamus L. 5 g/kg, and control (not treated with any materials). e digital balance was used in weighing purpose.

Collection, Mass Rearing, and Inoculation of S. oryzae.
e initial stock of adult weevils was collected from local farmers of Bhairahawa. e weevils were later identified as the S. oryzae L. in the laboratory. Mass rearing of weevils was performed to achieve uniform aged weevil stock of the second generation under controlled lab environment at 27°C, 85% RH, and 14 : 10 day night ratio in the Entomology lab of Paklihawa Campus for three weeks. Two hundred and fifty grams of the sample wheat were treated with botanical powder at 5-20 g/kg of grain, taken in a small plastic container (capacity 500 g), and 50 pairs/treatment of S. oryzae belonging to the second generation were released in the wheat, as the new stock of weevils would be more active, vigorous, and prolific, and they would be more liable to damage more wheat than the old stock.

Germination Test.
e germination of seeds was made using a random sample of hundred grains of each treatment and replication before and after treatment application. e seeds were cleaned properly and distributed homogeneously in well-soaked blotting paper in Petri dishes (8.8 cm diameter). All Petri dishes were then placed in a germination chamber at a normal room temperature of 27°C. Blotting paper was used as a germination medium because it has a lower risk of getting mold infestation, it is easier to handle than sand and soil, but it still permits penetration of the radical that provides a better evaluation of abnormal germination. Moreover, moisture also does not stratify within the paper medium as it might do so if sand or soil was used. Finally, the germinated seeds were counted, and the germination percentage was calculated using the following equation: germination percentage � (number of germinated seeds) (total number of seeds) × 100. (1) Additionally, counts were also taken for physically damaged grains and intact grains for 100 randomly sampled seeds, and weight loss comparison was also made for the seeds used in the experiment both before and after treating with botanicals in these seeds.

Statistical Analysis.
Data were subjected to analysis of variance (ANOVA) and significant differences existed; treatment means were compared at 0.05 significant level using the DMRT test. Arcsine data transformation was conducted whenever required to satisfy the assumptions of ANOVA [16]. Data were analyzed using MS-EXCEL 2007 and R-Stat 3.5.0 (R core team 2013).
Even in previous experiments, it was evident that the powder of A. calamus was very effective to maize weevil at 2 g, 1 g, and 0.5 g/100 g of maize grain in which 100% mortality was seen at 8 th and 10 th days after treatment application [17]. Similar results were obtained in another experiment where 100% mortality of S. oryzae L. was obtained when weevil was treated with A. calamus at 3 g/kg of wheat grain [18]. Similarly, in another test conducted against Angoumois grain moth, (Sitotroga cerealella O.), it was found that 100% mortality was resulted when rhizome dust of A. calamus was used at 10 g/kg of rice grain [19]. A. calamus dust was also found to have excellent efficacy in controlling Callosobruchus chinensis L. [20]. In another experiment, A. calamus was even found more effective than malathion at 1 g/kg in controlling pulses beetle C. chinensis for up to two generations [21]. is indicates that A. calamus could be used as a safer alternative to commonly used commercial pesticides.

Effect of Botanicals on Percent Germination, Seed Damage, Weight Loss, and Live Weevil Population at 90 DAT.
e mean germination of wheat was 95% before the seeds were treated in the experiment. When the germination test was performed at 90 DAT, the germination percent was significantly higher in A. calamus (89%) treated wheat than in control (23.66%) ( Table 2). Apart from germination, the physical damage of wheat seed was quantified by counting the weevil bored seeds and intact seeds in a randomly sampled 100 grains from all the treated wheat stock. e physical damage percentage was lowest in wheat treated with A. calamus (4%) which was followed by N. tabacum (14.33%) treated wheat seed. Furthermore, the percentage of seed damage was recorded highest in the control, i.e., 83% ( Table 2). On quantifying the percent weight loss of wheat, the highest percentage of weight loss was recorded in control (50.95) and lowest was recorded in A. calamus (3.32%) treated wheat seed ( Table 2).
Most of the findings of this study lie closely in accordance with the finding of some previously conducted experiments. For example, extracts of N. tabacum were found effective in controlling the various stages of maize weevil (Sitophilus zeamays M.) [22]. A. sativum demonstrated to have great potential at reducing the population of S. oryzae in some cereals [23]. Similarly, the progeny emergence of maize weevil and cowpea weevil (Callosobruchus maculates F.) was low in A. sativum treated grains as compared to the Advances in Agriculture grains without any protectant used [24]. Garlic also performed better in reducing the number of holes per seed, number of damaged seeds, percentage damaged seeds, and weight loss of beans infected with Phaseolus vulgaris L. tested against bean bruchid (Zabrotes subfasciatus Boh) compared to no pesticide application treatment [25]. Botanicals are easily available herbal plants, which have almost no adverse effects on human health and the environment. We agree that some traditionally used botanicals can be an excellent alternative to chemical pesticides in stored grains if used in the correct dose and method. e ease in availability and simplicity in these botanicals are most likely to be accepted and adopted by the farmers in Nepal [26]. Apart from sweet flag, most other herbs used in this experiment have been found to be very effective in controlling various other stored insect pests and which has been mentioned in many reports.

Conclusion
Among botanical materials tested, it is found that three safe botanical materials such as A. calamus rhizome dust, A. sativum clove powder, and N. tabacum leaf dust can be explored as excellent alternatives over the poisonous pesticides for the management of rice weevil in wheat grain storage. ese ecofriendly treatments could be accessible and economically viable alternatives to hazardous chemical pesticides in integrated insect pest management in rural Nepal. However, testing over three generations' weevils in laboratory research is slightly inadequate in drawing conclusions about the effectiveness of these botanical powders. So, they must be evaluated under different climatic conditions and different ecological zones as per need to expand their uses in a wide variety of stored seeds.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.