The pulse beetle, Callosobruchus chinensis (Chrysomelidae: Coleoptera) is notorious, global pest of stored legume grains (Seni and Mishra 2022; Singh and Boopathi 2022). Five species of the genus Callosobruchus are problematic in stored legume grains in South-Asia (Revanasidda et al., 2022). C. chinensis is the most abundant and destructive pest of legume grains, with the potential to cause 30% to 40% post-harvest losses, which can reach up to 100%, in grain legumes alone (Jaiswal et al., 2025). These beetles are known for their destructive feeding habits on the germ of the whole kernel, leaving only the pericarp (Mukherjee et al., 2024). Pulse beetles cause 50% to 92% germination loss, 55% to 69% seed weight loss and 45.6% to 66.3% protein content loss (Banga et al., 2020). Adoption of recommended storage techniques can reduce the losses to as little as 1 to 2% (Kumar and Kalita, 2017). Poor storage structures in developing nations have aggravated storage losses resulting in unfulfilled demand for legume grains (Paudel et al., 2020).
       
Grain legumes are major component of the traditional Nepalese farming system and are important in human and animal nutrition and soil fertility management (Gautam et al., 2022; Darai et al., 2023). Despite Nepal’s high export and production potential, storage pests are causing significant post-harvest damage, resulting in economic losses for both farmers and the country (Manandhar et al., 2018; Kandel et al., 2021). Conventionally, chemical fumigants such as phosphine, carbonyl sulfide and sulfuryl fluoride used against storage pests, their hazardous impact on human health, environment and food safety concerns (Mohapatra et al., 2015). However increased awareness raised the demand of organic products (Nile et al., 2019).
       
Approximately 372 plant species are known for their essential oils, which have been tested against variety of insect pests (Kaur et al., 2023). Plant essential oils are highly concentrated volatile compounds produced in oil sacs/glands in aromatic that serve as a plant defense mechanism (Sharma et al., 2022). Terpenoids present in these PEOs act as neurotoxins, interfering key metabolic and energy-producing pathways, culminating in insect mortality. The selective toxicity of these compounds is due to their different modes of action on insect physiological targets, rendering them non-toxic to mammals (Kavallieratos et al., 2021; Wojtunik-Kulesza, 2022).
       
Despite Nepal’s position as a global biodiversity hotspot-ranking 49^th with 1.3% of the world’s total biodiversity and exports of PEOs valued at $3.3 million in 2017, which make it the 62^nd country globally in export, we lack a clear understanding of the specific uses, purposes and  researches surrounding PEOs (Chhetri et al., 2021; Phuyal et al., 2019). This research aims to evaluate the potential of plant essential oils against the pulse beetle, C. chinensis.

The study was conducted from February to August 2023 in temperature-controlled and humidity-fluctuating conditions at the NARC laboratory. At the time of the toxicity assay, the temperature was set to 29.81±0.83°C with a RH of 58.23± 10.23%. Lentil seeds (cv. Simal) were obtained from the Grain Legume Research Program (GLRP) Khajura, Banke (MoALD, 2023). Using a diaphanoscope, only whole, clean, healthy, non-infested seeds that were free from foreign particles were selected. The selected seed grains then placed in a hot air oven at 70°C for one hour to ensure they were free from infestation (Aguilera and Steinsapir, 1985). Plant essential oils extracted from mint, citronella, sweet flag, zanthoxylum, prickly ash, artemisia were sourced from specialized essential oil processing industries in their 100% pure form and diluted with ethanol by 5% v/v, followed by centrifuging at 3000 rpm for 10 minutes. Adult pulse beetles were confirmed via stereomicroscope using standard keys (Southgate, 1958).  A total of 110 pairs (1:1 sex ratio) were placed in a aerated 5-liter plastic container with lentil grains. Conditions were maintained at 27.23±0.84°C, 44.42±4.36% RH and a photoperiod of 10:14 light and dark hours (Chaubey, 2008). Daily screening was done to clean the container from dead adults. Newly emerged adults, up to two days old, were chilled for 10 minutes for better handling.
       
The research was laid out in a Completely Randomized Design (CRD) to study fumigant toxicity and ovicidal action  of PEOs. The treatments were tested at two levels: 100 μl and 200 μl for fumigant toxicity and 10 μl and 20 μl for the ovicidal action. Each  treatment was replicated four times thus comprising a total of 52 experimental units. The experimental unit consisted of a 420 ml plastic jar containing with 200 gm of lentils and 10 pairs of adults (1:1 sex ratio). Whatman’s No. 1 filter paper with a 4 cm diameter was used for essential oil application and was placed in between the muslin cloth and the cap to prevent direct contact. An untreated jar served as the control and data were recorded at 24-hour intervals post-inoculation. An ovicidal assay consisted of 8.9cm diameter petri dishes each having 20 grains with freshly laid eggs (< 24 hours). The PEOs were applied to 2 sq. cm filter paper within the dishes and each treatment replicated four times. After 14 days, the hatching rate was assessed using a dissecting needle to observe larval presence.
       
Beetles were confirmed dead when no antennal or leg movement was detected or no coordinated movement observed within 15 seconds of gentle prodding with an entomological pin (Durmusoglu et al., 2015). The grains with adult emergence holes were considered damaged. The initial grain count in each unit was estimated (6360±0.13) using the thousand-grain weight (TGW) from four composite samples. The moisture content of grains before fumigation (11.5%) and after adult emergence was recorded using a digital hand-held moisture meter (Wile 55 device). Seed germination was tested before and after the treatment using three hundred seeds from each treatment combination on water-saturated germination paper and incubated at 18°C for 5 days (Santosh et al., 2020). After the incubation period, normal seedlings were counted to calculate the germination percentage. A paired t-test was conducted to detect the impact of essential oils on germination.
       
The raw data were initially entered, organized and tabulated in Microsoft Excel 2016 software. Prior to analysis, normality of data and homogeneity of variance were assessed by Shapiro-Wilk and gvlma tests. For statistical procedures, ‘doebioresearch’ package within RStudio, 2024/12/1+536, was used. To assess the impact of essential oils and exposure times on the beetle mortality and hatching ability of eggs, a Two-way Analysis of Variance (ANOVA) was conducted to separate the mean mortalities across the six essential oils and control group. After a significant (p<0.05) ANOVA result, treatment means were compared using both Tukey’s Honest Significant Difference (HSD) and Duncan’s multiple range test (DMRT) to identify statistically significant differences among the essential oils. All the statistical outputs including ANOVA, residual plots and post-hoc comparisons were visualized using the ggplot2 package ensuring clear interpretation of findings.

Time-dependent mortality of adult C. chinensis following fumigation
 
Adult mortality (Table 1) in citronella treated unit was significantly the highest, 25.6±4.9 %, (p<0.001) on the first day following fumigation. Mint EO had showed the significantly lowest adult mortality rate at 7.5%, followed by artemisia (21.3±1.8 %) and prickly ash (20±2.9%). By the second day, artemisia resulted significantly (p<0.001) the highest mortality rate (59.4±4.9%), closely followed by citronella (54.4±7.1%), prickly ash (55.6±6.9 %) and lowest in mint treated units (26.9±4.9%). Citronella, artemisia and prickly ash all attributed to mortality rates exceeding 79% by the third day, continuing the trend of significant mortality from these three EOs. Prickly ash and citronella (94.4±2.2%) were the most effective by the fourth day, resulting in over 93% mortality. In contrast, control unit did not exhibit any mortality during the course of the nighty six hours. These observations align with the findings of Manju et al., (2018), who reported that citronella EO caused 93.75% mortality of adult beetles on first day of fumigation at 100% concentration. The monoterpenoid components of citronella oil; citronellal, citronellol and geraniol have potent neurotoxic and insect-repelling effects that accounts for the oil’s strong fumigant toxicity (Das et al., 2021; Abdelgaleil et al., 2021; Subedi et al., 2020). Similarly, cyclic ketonic monoterpenes thujone and cineole, which caused acute poisioning by disrupting insect metabolism, are associated with the effect of artemisia essential oil (Abd-Elhady, 2012; Di Lorenzo et al., 2018). Variations in the actions of plant essential oils depend on the species and their geographic origin, chemotypes, species, metabolic pathways and synergism of major and minor constitutients (Titouhi et al., 2017; Abbassy et al., 2009; Abd-Elhady, 2012). Amzouar et al., (2016) reported that the effectiveness of the essential oils depends upon the timing of collection during distillation, plant parts selected, insect pest tasted, extraction methods, species of the plants and major constituents of specific essential oils. Al-Hayali et al. (2025) also reported that increasing artemisia oil dose and exposure time resulted higher adult mortality.


 
Percentage adult mortality of C. chinensis across PEOs and doses after fumigation
 
After the first day of fumigation, citronella EO at 200 µl significantly resulted the highest adult mortality at 37.5% (p<0.05). Prickly ash (26.3%) and artemisia (25.0%) at the same dose were also significantly effective. Overall, the 200 µl dose consistently outperformed the 100 µl dose for all the plant essential oils, resulting the lowest adult mortality at 100 µl in sweet flag and tea tree EOs (Table 2). By the second day, citronella, prickly ash and artemisia EOs at 200 µl all resulted in significantly the higher mortality rates of 72.5%, 72.5% and 71.3%, respectively (P<0.05). Mint at 100 µl showed significantly (p<0.05) the lowest mortality among the tested essential oils with a mortality rate of 15%. The result demonstrated indeed a dose-dependent response, with greater doses being associated with higher fatality rates (p<0.001) aligns with the findings of Jayakumar et al., (2017) and Saranya et al., (2019), who found that increased dose raised the mortality percentage. Monoterpenes present in citronella caused strong neurotoxicity and repellent action (Das et al., 2021; Abdelgaleil et al., 2021; Subedi et al., 2020). However, mint essential oil shows more repellency and a less toxic effect due to its alcoholic monoterpene menthol, resulting in a lower mortality effect on pulse beetles (Saeidi and Mirfakhraie, 2017). The trend of resulting higher mortality from the top three essential oils continued (Table 2). On the third day, citronella and prickly ash at 200 µl had the highest mortality rates, at 97.5% and 95.0% respectively (p<0.05). These were significantly more effective than all other treatments. Artemisia at 200 µl also showed high efficacy, with a mortality rate of 91.3%. Tea tree and sweet flag at 100 µl both had mortality rates of 58.75%, whereas mint at 100 µl resulted the lowest mortality rate (51.25%), while there was not adult mortality recorded in the control group. No significant difference (P<0.05) in adult mortality was observed among the essential oils after four days of fumigation.


       
The significant (p<0.05) interaction between dose and essential oil type found in the early hours of fumigation time suggested a clear dose-dependent response, aligned with Hamza et al., (2016). The cumulative effect over time, sublethal effects, dose-dependent metabolic reactions, synergistic effect and mode of actions are attributed to the interaction effects and complex interactions phenomenon between oil type and doses (Hamza et al., 2016; Amzouar et al., 2016).
 
Ovicidal potentials of essential oils against pulse beetle, C. chinensis
 
Among the six PEOs tested, citronella EO resulted the significantly (p<0.05) highest mean ovicidal potential of 96.25±2.06%, followed by prickly ash, sweet flag, mint and tea tree essential oil (Table 3). Artemisia oil showed the lowest ovicidal rate with 16.25±2.45%, while all the eggs were hatched in the control group after fourteen days. Higher dose significantly increased ovicidal potential, with the mean ovicidal rate at the 20 µl dose being 68.13 ± 6.10% as compared to 37.68 ±5.59% at the 10 µl dose. Interaction between oil type and doses (Fig 1) on ovicidal actions found significant (p<0.001). This finding aligns with Raja and William (2008), who reported that citronella EO effectively restricted egg development by blocking the micropyle region of the chorion. Citronellal, linalool and β-caryophyllene attributed to the ovicidal potential of citronella (Gharsan et al., 2022). They also reported that nano-emulsified volatile oils have more impact than pure oils.




 
Ovicidal potential of six essential oils against C. chinensis across doses
 
The impact of six essential oils on the hatching rate of C. chinensis eggs across the doses was statistically differ (p<0.001) after fumigation (Fig 1). Citronella oil at 20 µl exhibited the highest average ovicidal potential, achieving 100%. This was followed by prickly ash oil (20 µl) citronella oil (10 µl), sweet flag (20 µl), mint oil at (20 µl), tea tree oil (10 µl), mint oil (10 µl) and sweet flag oil (10 µl). In contrast, the lowest ovicidal rates were recorded for tea tree oil at 10 µl, artemisia oil at both doses with average ovicidal rates of 18.75%, 16.25% and 16.25%, respectively. All eggs were found hatched in the control group. Increased volatile oil dosages raised the ovicidal rates, supporting a dose-dependent response that was documented by Jayakumar et al., (2017). Variation in ovicidal potential is due to chemical compositions, modes of action and different levels of toxicity to embryonic development. Volatile oils show a dose-dependent response as they have different synergistic effects (Hamza et al., 2016; Amzouar et al., 2016). This resulted some oils exhibited higher efficacy at specific doses. This differential response highlighted the complex interaction between essential oil type and doses used. In contrast, artemisia essential oil’s least effectiveness also reported by Gao et al., (2020) due to its strong repellency trait rather than toxicity. A study by Papachristos and Stamopoulos (2004) reported that these volatile oils have a toxic latent effect on the egg and progeny with significant impact of exposure length.




 
Grain damage percentage and grain moisture content
 
After 1^st generation of adult emergence until their lifespan, there was significance difference (p<0.001) in the grain damage and grain moisture percentage in between the essential oils and the control group (Table 4 and 5). Among PEOs, citronella-treated units exhibited the lowest grain damage and grain moisture percentage (0.86±0.19% and 12.4±0.2%), followed by artemisia, prickly ash, tea tree, mint and the highest in sweet flag-treated units (5.17±0.35% and 14.5±0.3%) treated units. The control group showed a significantly the higher average grain damage and grain moisture percentage (13.37±1.63% and 16.6±0.2%) as compared to all treatments. At the higher dose (200 µl), the average grain damage was 2.39±0.82%, while at the lower dose (100 µl), it was 5.33±0.39%. These findings are similar with the findings of Nyamador et al., (2017). They reported that fumigating PEOs reduce oviposition, impart toxic effect on insects, rapid onset of negative feedback of the nervous system and repellent properties lead to lesser grain damage. Grain damage percentage was reduced on increasing the dose of essential oils which is consistent with the Jayakumar et al., (2017). The higher grain moisture in untreated and less effective PEOs is due to the higher biological (respiratory) activity of beetles (Srivastava and Mishra, 2021). Variation on effect is due to interaction effect of oil types and doses (Hamza et al., 2016).
 
Germination of fumigated grains
 
Regardless of doses, the six PEOs maintained a high germination rate (>96%) and exhibited no toxicity to the treated grains’ germination rate after a month. The germination percentage for the control group was 99.3%. The paired t-test revealed no statistically significant difference (p<0.05) in the germination rates between the 100 µl and 200 µl doses of plant essential oils, with the computed t-values (2.12 and 2.21) being smaller than the critical t-values (2.47 and 2.57) for 6 and 5 degrees of freedom. Nayanathara and Ratnasekera (2010) also reported no significant impact of PEOs on the germination of the stored legume grains. Furthermore, Keita et al., (2001) and Biswas and Biswas (2005) all reported that the higher germination rates after essential oil treatment, confirming that essential oils had no negative impact on seed germination.

Among the six EOs, citronella, prickly ash and artemisia essential oils resulted remarkable fumigant and ovicidal efficacy and safety for seed viability, suggesting their potential as natural alternatives to chemical insecticides for the management of C. chinensis in stored legume grains. These compounds can be used in sealed storage structure against C. chinensis in stored legume grains maintaining grain quality parameters.

We sincerely thankful to the United States Agency for International Development (USAID) funding support. We are appreciative of the Nepal Agricultural Research Council’s (NARC) and everyone who helped by offering technical support and insightful advice.
 
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The views and conclusions expressed in this article are solely those of the authors. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.