Fumigant and contact toxicities of individual and additive combinations of biorational-essential oils for control of rice weevil (Sitophilus oryzae)

Abstract Fumigant and contact activity of individual and combination of EOs (cumin seed and black pepper oil) were conducted against rice weevil (Sitophilus oryzae). Cumin seed and black pepper oil individually exhibited 100% insecticidal activity (Contact and Fumigant) against S. oryzae at 24 and 168 h of exposure in the absence and presence of food, respectively. Overall, differential insect mortality rates were observed with individual EOs; however, cumin seed EO showed higher insecticidal activity in both fumigant and contact bioassays than black pepper EO. Binary mixtures of cumin seed and black pepper oil at 60:40 and 80:20 ratios showed higher toxicity than individual EOs against S. oryzae and further, additive interaction of binary combinations of EOs resulted in complete insecticidal activity of S. oryzae. Thus, we conclude binary mixtures of cumin seed and black pepper EOs might be a promising prophylactic treatment solution for control of S. oryzae to prevent stored grain infestation. Graphical abstract


Introduction
Sustainable methods of grain storage and prevention of insect infestation in food commodities are growing global concern for both developing and developed countries. Presently, the control methods of insects in the storage units of various grains are heavily dependent on the usage of gases like phosphine or synthetic insecticides that increase the risk for human population and environment (Zongo et al. 2021). Generally, Sitophilus oryzae (L.) (Coleoptera: Curculionidae) is a weak beetle but it prefers to infest on many cereal grains and its feeding activity enhances with increase in temperature and relative humidity. Fumigation of food grains with phosphine is the preferred choice in India; however, indiscriminate dosage and prolonged usage leads to development of heritable resistance in S. oryzae. Hence to address to these emerging issues of resistance development in stored grain insect pests and safe storage methods for food grain management, an alternative approach needs to be explored.
Plant-based biorational products such as extracts, essential oils and phytochemical compounds etc. have been looked upon as an adequate alternative to control pests of stored durable products (Haddi et al. 2020;Isman 2020;Jumbo et al. 2022) because of their less hazardous nature towards the non-target organisms. Black pepper (Piper nigrum L.) is widely cultivated as a perennial spice crop in Malabar coast and tropical regions of India. Essential oil of black pepper contains terpenes, such as caryophyllene, limonene, pinene (alpha and beta) and sabinene as major aromatic compounds. Cumin (Cuminum cyminum) widely cultivated herbaceous and traditional spice plant of India contains cuminaldehyde, gamma-terpinene, para-cymene, beta-pinene as major components of its essential oil.
Essential oils (EO) have gained greater attention from the scientific community attributing to its highly diverse number of chemical constituents present in the EOs and its combined mixtures (de Oliveira et al. 2017). Among the various interactions observed in mixtures of EOs, the synergistic mixtures (Trisyono and Whalon 1999) allow the application of lower doses preferably because of relatively increased insecticidal activity. Thus, ensuring reduced management costs, environmental risks and resistance development as compared to the conventional insecticides (Hummelbrunner and Isman 2001;Tak and Isman 2015;de Oliveira et al. 2017).
The current report would shed light on the individual and binary combination effect (Synergistic, Antagonistic or additive) of cumin seed and black pepper essential oil with its insecticidal activity mainly through fumigation and contact assay method against S. oryzae (Rice weevil) which is a major pest of cereal grains and its products.

Insecticidal activity of cumin seed oil
In the fumigant assays of cumin seed oil without food ( Figure S2 (a)), from a treatment dose of 20 mL of cumin seed EO, a mortality of 100% is observed for an exposure period of 12 hours. The fumigant toxicity of cumin seed oil treated-grain ( Figure S2 (b)) assays showed mortality caused due to the fumigant achieves 100% mortality at 144 h (Day 6) of exposure using a dose of 25 mL. In the contact treated assays of Cumin seed oil, a rapid response (100%) is noted against S. oryzae in without food conditions ( Figure S2 (c)) for a treatment dose of 10 mL and an exposure period of 12 h. Whereas in contact treated-grain assays ( Figure S2(d)) of cumin seed oil, a notable mortality is observed in a treatment dose of 25 mL where it reaches 100% within a period of 24 h for a dose of 25 mL. In contrast to our results, Kedia et al. (2015) showed poor fumigant toxicity by cumin seed EO (LC 50 ¼ 104.07 mL/L air) against S. oryzae using a treatment dose of 60 mL/L air concentration for 24 h.

Insecticidal activity of black pepper oil
In the fumigant assays of black pepper ( Figure S3 (a)) exhibits 100% mortality within 24 h from a treatment of 20 mL. The fumigant treated-grain assays ( Figure S3(b)) of black pepper oil show complete mortality (100%) from a period of 96 h of exposure using a dose of 25 mL. In the contact treated assays without grains ( Figure S3 (c)) of black pepper oil, it shows complete mortality (100%) for an exposure period of 24 h using a treatment dose of 25 mL. The contact treated-grain assays ( Figure S3 (d)) of black pepper oil, a treatment dose of 20-25 mL a notable mortality (100%) is observed from an exposure period of 72-168 h (Day 3-7). According to a report by Khani et al. (2011), the rice weevil was tested for toxicity by Piper nigrumpetroleum and chloroform extracts of seeds having major chemical compounds as caryophyllene (18.53), piperine (74.34%) and cinnamic acid (5.20%) which showed high mortality rate with LC 50 values of 1.61 ml/g and 1.7 ml/g, respectively.

Insecticidal activity of binary combination
In the fumigant assays of all the binary combinations in without food condition ( Figure S4 (a) and Table S3 (a)) exhibited a highest activity (100% mortality) within an exposure period of 24 h. In fumigant treated-grain assays ( Figure S4 (b) and Table  S3 (b)), all combinations showed mean mortality more than 50% from an exposure period of 48 h (Day 2) onwards. The 60:40 ratio shows better toxicity exhibiting 100% mortality within 72 h (Day 3) of exposure. In the contact assays of all the combinations in without grain ( Figure S5 (a) and Table S4 (a)) condition, the mixtures tested were significantly (F ¼ 75.93, p < 0.05) more toxic than the fumigant method. The binary mixtures of ratio 60:40 and 80:20 achieves a significant mortality of 100% within a time period of 8 h. And in contact treated-grain assays ( Figure S5 (b) and Table S4 (b)) all combinations showed mean mortality more than 50% for an exposure period of 48 h (Day 2) onwards. Among all the combinations, the 60:40 ratio exhibited complete mortality (100%) within 72 h (Day 3) of exposure. The fumigant and contact toxicity caused by binary mixtures of cumin seed and black pepper oil (Combination ratios of 40:60, 60:40, 80:20 etc.) in with and without food conditions for an exposure period of 24 and 72 h of exposure, respectively, were elucidated to be a cause of additive effect from the individual compounds in the mixture. In a study by Santana et al. (2022), combinations of essential oils (EOs) as a potential strategy for management of stored grain insect pests and they had reported, fumigant toxicity and repellent effects of six essential oils on Sitophilus zeamais.

Lethal concentrations of cumin seed and black pepper oil
The lethal concentrations of the essential oils (Table S5) of cumin seed and black pepper oil against S. oryzae show contact and fumigant activities in without and with food conditions. For the fumigant, the LC 50 values of these treatments using cumin seed oil showed highest toxicity with 0.022 mL/mL air in without food condition for an exposure period of 24 h and 0.799 mL/mL air in with food condition for a period of 48 h. Whereas the LC 50 values of the treatments using Black pepper oil showed 0.099 mL/mL air for an exposure period of 24 h and 0.71 mL/mL air for a period of 48 h.

Conclusion
Potentiality of binary combination of biorational-essential oil for control and prophylactic treatment of Sitophilus oryzae might be a promising area of stored grain insect pest management. Minimising the usage of phosphine and suggesting an alternate chemical for control of economically deleterious stored grain insect pest still remains as potential area of research. Hence our present study is attempted to understand the individual and binary combination effect of black pepper essential oil and cumin seed oil for effective control of S. oryzae. Both the oils exhibit a profound insecticidal activity (Mortality100%) in with and without food conditions against S. oryzae during the fumigant and contact treatments. The binary combinations (60:40 and 80:20) resulted in complete mortality due to additive effect. Binary mixtures of phytogenic EOs might be a future alternative for currently used prophylactic treatments for control of S. oryzae and it is also a step towards organic method of food grain storage.

Acknowledgement
Authors, acknowledge the support of Director, CSIR-Central Food Technological Research Institute Mysore (CFTRI), 570020, Karnataka for providing the necessary facilities. Authors also thank Ms. M Asha (Sr. Technical officer), CIFS, CSIR-CFTRI for assistance in GC analysis and Mr. Bhavani Eswaran M (Sr. Technical Officer) CIFS, CSIR-CFTRI for GC-MS analysis.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
VR acknowledges the financial support of UGC NET-JRF Fellowship by University Grant Commissions (UGC), Delhi, India. CSV greatly acknowledges the support of MLP 297 In-house project for the financial assistance to carry out the work.

Authors contributions
AVR and CSV conceptualised the topic; AVR -performed the experiments and wrote the manuscript; CSV -Analysed the data and corrected the manuscript. All the authors read and approved the final manuscript. ORCID C.S Vivek Babu http://orcid.org/0000-0002-3946-4123