Toxicity and Repellency Potential of Silver Nano-Formulated Phytoextracts Against Rice Weevil Sitophilus oryzae (L.) (Coleoptera: Curculionidae)

Muhammad Salman Saeed1, Umar Farooq1, Muhammad Zeeshan Majeed1*, Lamya Ahmed Alkeridis2, Samy Sayed3, Abu Bakar Muhammad Raza1 and Asma Akram1

1Department of Entomology, College of Agriculture, University of Sargodha, Sargodha 40100, Pakistan

2Department of Biology, College of Science, Princess Nourah Bint Abdulrahman University, P.O. Box 84428, 11671 Riyadh, Saudi Arabia

3Department of Economic Entomology and Pesticides, Faculty of Agriculture, Cairo University, Giza 12613, Egypt

ABSTRACT

Rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae), is one of the most widely distributed destructive pests of stored rice and wheat grains. Synthetic insecticides and fumigants are predominately used to control this pest. However, wide use of these chemicals manifest pesticidal residues and pest resistance necessitating to look for some other environmental-friendly alternatives such as nano-formulated botanical extracts. This study evaluated nano-formulated leaf extracts of some promising plant species against S. oryzae adults. Initial screening of the acetone extracts of neem (Azadirachta indica A. Juss.), guava (Psidium guajava L.), dhraik (Melia azedarach L.), tobacco (Nicotiana tabacum L.) and eucalyptus (Eucalyptus camaldulensis Dehn.) using grain treatment bioassay method showed maximum and significant weevil mortality by the extracts of A. indica and P. guajava i.e. 98 and 94%, respectively at 120 h post-exposure. These extracts were further nano-formulated using silver nitrate (AgNO3). Toxicity and repellency of different concentrations (i.e. 10, 5, 2.5 and 1.25%) of these nano-formulated extracts were determined against S. oryzae weevils. Results showed significant mortality of the exposed weevil individuals by all treatments and this mortality response was directly proportional to the extract concentration and exposure time. Nano-formulated A. indica extract showed highest mortality (98.7%), followed by M. azedarach (97%), N. tabacum (96%) and P. guajava (84%) at 96 h post-exposure. Probit analysis exhibited minimum LC50 for A. indica (0.62%), followed by P. guajava (0.66%), N. tabacum (0.71%) and M. azedarach (0.94%) at 96 h post-exposure. In case of repellency, 10% concentration of the nano-formulated N. tabacum extract exhibited highest repellency (54%), followed by M. azedarach (48%) and P. guajava (28%) after 24 h of exposure. Overall study results demonstrated the effectiveness of these nano-formulated phytoextracts against S. oryzae advocating their integration in eco-friendly management of stored grain insect pests.

Article Information

Received 08 October 2024

Revised 15 October 2024

Accepted 27 November 2024

Available online 15 May 2025

(early access)

Published 13 February 2026

Authors’ Contribution

MZM and ABMR conceived the idea and designed the study. MSS and UF performed experimentation and recorded data. AA and LAA analyzed the data and prepared results. MSS, UF and AA wrote the initial draft of manuscript. SS and ABMR technically proofread the manuscript. MZM and ABMR supervised the research work. LAA and SS provided technical and financial assistance for the study. All authors have read and approved the final version of the manuscript.

Key words

Stored grain pests, Rice weevil, Silver nanoparticles, Nano-formulation, Phytoextracts, Repellency bioassay, In-vitro toxicity

DOI: https://dx.doi.org/10.17582/journal.pjz/20241008140626

* Corresponding author: [email protected]

0030-9923/2026/0002-0847 $ 9.00/0

Copyright 2026 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).


Introduction

Rice (Oryzae sativa L. Poaceae) is one of the most imperative and essential staple crops, particularly in Asian countries (Chandio et al., 2016). Rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae), is one of the most destructive pests of stored grains worldwide including rice. This pest causes substantial damage to stored rice in tropical and sub-tropical regions with losses reaching up to 40% (Rani et al., 2019; Covele et al., 2020). Synthetic insecticides and fumigants such as methyl bromide, magnesium and aluminum phosphides are the prime source to control S. oryzae infestations both at domestic and commercial levels (Nayak et al., 2020; Wakil et al., 2021). However, their use and effectiveness in stored grain sector have been limited due to their residual toxicity, health hazards, environmental contamination and problems of pest resistance and resurgence (Chayengia et al., 2010; Hubert et al., 2018).

These ecological consequences of extensive and continuous use of synthetic fumigants and other chemicals in stored grain sector necessitate looking for some biorational and environmental-friendly pest control options such as plant-based extracts (Isman, 2020). Phytoextracts can be effective alternatives to synthetic insecticides against stored grain insect pests (Dubey et al., 2008). Due to their plant origin, they exhibit low persistency, low mammalian toxicity, high volatility and effective toxicity against stored grain insect pests (Regnault, 1997; Suteu et al., 2020).

Moreover, nanotechnology is also being perceived as a promising field of research with a significant potential for future pest management (Muraisi et al., 2022). Nano-formulations, particularly silver nanoparticles (AgNPs)-based formulations, have significant impaclications in various agricultural disciplines due to their high surface area to volume ratio, physical and chemical stability, low toxicity and simple surface chemistry (Scrinis and Lyons, 2007; Ale et al., 2023). AgNPs synthesized through green methods can be effective carriers to transport agrochemicals to the targeted sites (Sankar and Abideen, 2015; Abbas et al., 2024). They have proven to be highly effective against insect pests demonstrating considerable insecticidal, antifungal and antiviral properties (Chandran et al., 2006; Srinivasan et al., 2023). Therefore, insecticidal phytoextracts formulated with AgNPs could be an efficient strategy against stored grain insect pests including S. oryzae weevils.

The present research work aimed to evaluate and compare the nano-formulated phytoextracts against S. oryzae weevils under laboratory conditions. Firstly, available promising local phytoextracts were assessed for their effectiveness against S. oryzae adults. Secondly, the most effective phytoextracts were nano-formulated using AgNPs and were evaluated against S. oryzae adults to determine their LC50 and LT50 values. Lastly, the repellency of these nano-formulated phytoextracts was also assessed against S. oryzae adults.

Materials and Methods

Rearing of S. oryzae

S. oryzae population was taken from the local grain market of Sargodha, Punjab, Pakistan from stored rice and was maintained in the Laboratory of Entomology, College of Agriculture, University of Sargodha. Culture was feed on clean and disinfected white fine rice (super basmati, unpolished). Fifty adult weevils were released in glass containers (20×30 cm) and the containers were closed with a fine-mesh muslin cloth. These containers were kept in a growth chamber at 28±2°C temperature and 65±5% relative humidity. Weevil culture was reared up to F3 generation until its use in downstream bioassays.

Preparation of phytoextracts

Fresh leaves of neem (Azadirachta indica A. Juss.), guava (Psidium guajava L.), eucalyptus (Eucalyptus camaldulensis Dehn.), dharaik (Melia azedarach L.) and tobacco (Nicotiana tabacum L.) were collected from the plants in the vicinity of College of Agriculture, University of Sargodha during spring 2023. These leaves were washed with tap-water and were air-dried at room temperature (27°C) for two weeks. Shade dried leaves were powdered using an electric bleeder and were put in hermetic plastic bags. Using pure acetone as extraction solvent, the extraction of plant samples was carried out through Soxhlet apparatus (DH.WHM-12393, Daihan Scientific, South Korea) following the protocol described by Majeed et al. (2020) and Tayyab et al. (2022). Prepared phytoextracts were stored in dark-colored hermetic glass vials in refrigerator at 4 °C until their use in experimentation.

Synthesis and characterization of AgNPs-based nano-formulated phytoextracts

Silver nanoparticles of effective phytoextracts were synthesized using a chemical reduction method. First of all, a solution was prepared by dissolving 0.017g of silver nitrate (AgNO3) in 100 mL of deionized water. The solution was then heated to 80 °C, while continuously stirring. Subsequently, the solution was transferred to a reflux condenser. To this solution, 10 mL of phytoextracts was added using a graduated pipette. The entire solution was heated in an oil bath at 100 °C for duration of 30 min, while maintaining constant stirring. Finally, the solution was allowed to cool to room temperature (27 °C). The formation of silver nanoparticles of both phytoextracts was confirmed and characterized using UV-visible spectrophotometer (UV-Vis, Perkin Elmer, Lambda 35), Raman Spectrometer (Peak Seeker Pro-785; Agiltron, USA) and Scanning Electron Microscope (SEM) (FEI Quanta 250, Hillsboro, OR, USA) following the protocols described by Jafir et al. (2021).

Toxicity bioassay against S. oryzae weevils

Treated grain bioassay method was used to determine the toxicity of phytoextracts against S. oryzae adults. First of all, 10% extracts were screened and then the most effective phytoextracts were formulated with silver (AgNO3) nanoparticles. Experimental design was completely randomized with five replications for each of the four concentrations (i.e. 10, 5, 2.5 and 1.25%) of nano-formulated phytoextracts and a control (acetone) treatment. Five grams of raw white fine rice (Super Basmati, unpolished) were treated with one milliliter of each treatment in 60 mm glass Petri plates and 10 individuals of S. oryzae (containing both male and female ones) were released in each Petri plate and these plates were incubated in dark in a growth chamber at 28 ± 2°C temperature and 65 ± 5% relative humidity. Mortality of the exposed insect individuals was recorded at 12, 24, 48, 72, 96 and 120 h post-exposure and was corrected using Abbott formula (Abbott, 1925).

Repellency bioassay

Half filter-paper disc method was utilized to assess the repellency potential of nano-formulated phytoextracts. In brief, Whatman filter paper (No.1) discs were cut into two halves according to glass Petri plate (60 cm). One half of each paper was treated with 1 ml of either 10 or 5% concentration of each treatment (phytoextracts) and other half was treated with acetone alone (control). After treating, both halves were placed at room temperature (27°C) for drying up to 10 min and were rejoined in 60 cm glass Petri plate. Ten adults of S. oryzae were released in each Petri plate and were covered with lid and were incubated 28±2 ºC temperature and 65±5 % relative humidity. Data regarding the repellency of exposed insect individuals were recorded at 3, 18 and 24 h post-exposure (Elbrense et al., 2022).

Statistical analysis

The percent mortality and repellency of tested S. oryzae adults are presented graphically. Apart from this graphical representation, factorial analysis of variance (ANOVA) was run to analyze the mortality and repellency data by keeping exposure time and treatment concentration as factors, followed by Honestly significant different (HSD) to compare treatment means at 95% probability level (P<0.05). Median lethal concentration (LC50) and median lethal time (LT50) values were calculated by Probit analysis using POLO® statistical regression software. Prior to Probit analyses, data were corrected using Abbott’s formula (Abbott, 1925). Percent repellency was determined by using a previously described equation (Elbrense et al., 2022). Statistical interpretation of data was done using Statistix 8.1® (Abbott, 1925).

Where PR is percent repellency, C is numbers of insects on control and T is numbers of insects on treatment.

Results

Characteristics of nano-formulated phytoextracts

The most effective phytoextracts i.e. A. indica, M. azedarach, P. guajava and N. tabacum were nano-formulated using silver (AgNO3) particles and then the most effective two nano-formulations i.e. A. indica and P. guajava were further characterized using Raman spectroscopy, ultraviolet-visible (UV-vis) spectroscopy and scanning electron microscopy (SEM) techniques. All these determinations confirmed the formation of silver nanoparticles (AgNPs). The absorption spectra of nano-formulated A. indica and P. guajava extracts were determined by UV-visible spectroscopy in the wavelength range of 300–800 nm by taking deionized water as blank. Due to surface plasmon resonance, the color of solution was transformed from yellow to dark brown, which is thought to be the most important indicator of synthesis of AgNPs. The maximum absorbance spectrum of synthesized A. indica and P. guajava AgNPs was observed at 417 and 430 nm, respectively (Figs. 1, 2). Furthermore, the spectral measurement of AgNPs of nano-formulations of both extracts in liquid form was performed with Raman spectrometer (Peak Seeker Pro-785; Agiltron, USA) by a 785 nm laser as a source of excitation transferring a laser power of 50 mW with the help of a 40X objective lens.

The size and morphology of AgNPs of both nano-formulated extracts were determined by scanning electron microscope (SEM) at 100,000X magnification. The average size of both A. indica and P. guajava synthesized AgNPs was approximately 19 and 18 nm, respectively (Figs. 1, 2). Energy dispersive X-ray spectroscopy (EXD) related to SEM corroborated the presence of elemental silver by indicating the conversion of silver ions to silver nanoparticles. Furthermore, peaks of carbon, silver and oxygen appeared in case of aqueous extract of A. indica (Fig. 1), while the peaks of oxygen, potassium, chlorine, silicon and sodium appeared from the aqueous extract of P. guajava extract (Fig. 2).

Toxicity bioassays against S. oryzae adults

In preliminary screening, acetone extracts of five indigenous plant species were tested for their insecticidal potential against S. oryzae adults. Results of this bioassay in which weevils were exposed to 10% extracts of the plants showed that all extracts exhibited significant mortality (F4,20 = 6.68; P = 0.0014) of exposed weevils as compared to the control treatment (Supplementary Table I). Maximum mortality of S. oryzae adults was observed in case of extracts of A. indica (98%) and P. guajava (89%), followed by M. azedarach (87%) and N. tabacum (84%). Minimum weevil mortality (74%) was recorded for the extract of E. camaldulensis (Fig. 3A) Weevil mortality remained less than 5% in control treatment.

 

 

 

In detailed toxicity bioassay, four phytoextracts (i.e. A. indica, P. guajava, M. azedarach and N. tabacum) which showed maximum and significant mortality of S. oryzae weevils were further formulated with silver nanoparticles (AgNPs) and were tested against S. oryzae adults using same protocol as done for previous bioassay. In this bioassay, different concentrations of the nano-formulated phytoextracts were used. Results of this bioassay clearly demonstrated a significant effect on the mortality of S. oryzae adults by the treatments (F3, 64 = 7.53, P < 0.001), their concentrations (F3, 64 = 39.70, P < 0.001) and by their interaction (F9, 64 = 2.32, P= 0.0248) (Supplementary Table II). On overall basis, nano-formulated extracts of A. indica and P. guajava exhibited highest weevil mortality (i.e. 98%) at 96 h post-exposure and were statistically different from other two extracts (Fig. 3B).

Probit analysis of the weevil mortality data demonstrated almost similar trend of toxicity of nano-formulated phytoextracts against S. oryzae and this trend was directly proportional to concentration of the extract and exposure time (Tables I and II). According to probit analysis, A. indica, P. guajava and N. tabacum appeared to be the most toxic extracts exhibiting minimum LC50 and LT50 values i.e. 0.60, 0.66 and 0.71% at 96 h of exposure, and 4.97, 7.57 and 13.7 h for 10% concentration of extracts, respectively (Tables I and II). While, minimum LC50 and LT50 values were recorded for the extract of M. azedarach.

 

Table I. Median lethal concentration (LC50) values of nano-formulated acetone leaf extracts of indigenous plant species evaluated against adults of rice weevil S. oryzae under laboratory conditions.

Botanical

extracts

Observation time (h)

LC50 (%)

95% upper and lower fiducial limits

X2­­-value

P value*

A. indica (Neem)

12

13.96

7.29-158.81

83.27

< 0.05

24

4.32

6.64 -7.65

109.18

< 0.05

48

1.16

0.64-1.63

94.17

< 0.05

72

0.72

0.28-1.13

93.22

< 0.05

96

0.62

0.38-1.03

72.85

< 0.05

M. azedarach (Dhraik)

12

3.78

2.83-5.06

117.32

< 0.05

24

2.11

1.33- 2.86

161.44

< 0.05

48

1.41

0.83 -1.91

148.40

< 0.05

72

1.30

0.66 -1.84

211.82

< 0.05

96

0.94

0.26 -1.49

242.40

< 0.05

N. tabacum (Tobacco)

12

3.49

1.99-6.06

122.91

< 0.05

24

1.81

0.72- 2.77

146.35

< 0.05

48

1.33

0.57-1.97

168.11

< 0.05

72

0.93

0.29-1.49

196.05

< 0.05

96

0.71

0.29-1.27

324.11

< 0.05

P. guajava (Guava)

12

5.83

3.43 -7.63

148.54

< 0.05

24

4.11

2.53-7.65

147.20

< 0.05

48

1.40

0.13-2.53

76.57

< 0.05

72

0.79

0.29-1.27

74.44

< 0.05

96

0.66

0.12- 1.14

117.10

< 0.05

 

*Since the significance level is less than 0.05, a heterogeneity factor is used in the calculation of confidence limits α denotes water used as solvent.

 

Table II. Median lethal time (LT50) values of nano-formulated acetone leaf extracts of indigenous plant species evaluated against adults of rice weevil S. oryzae under laboratory conditions.

Botanical

extracts

Concentration (%)

LT50 (h)

95% upper and lower fiducial limits

X2­­-value

P value*

A. indica (Neem)

10

4.97

42.61-66.31

83.27

< 0.05

5

19.74

15.73-37.47

109.18

< 0.05

2.5

26.69

12.01-26.67

94.17

< 0.05

1.25

52.15

7.3-19.61

93.22

< 0.05

M. azedarach (Dhraik)

10

14.85

0.82-9.39

270.41

< 0.05

5

12.90

2.69-23.61

300.85

< 0.05

2.5

14.10

3.48-19.78

197.07

< 0.05

1.25

36.85

27.69-48.13

94.19

< 0.05

N. tabacum (Tobacco)

10

13.75

3.79-11.02

168.77

< 0.05

5

12.15

2.79-23.94

306.13

< 0.05

2.5

14.10

3.47-19.78

197.07

< 0.05

1.25

36.85

27.68-48.13

94.19

< 0.05

P. guajava (Guava)

10

7.57

7.34-19.61

180.56

< 0.05

5

21.44

12.01-26.67

178.74

< 0.05

2.5

34.69

15.73-37.47

162.15

< 0.05

1.25

55.15

42.61-66.31

87.250

< 0.05

 

*Since the significance level is less than 0.05, a heterogeneity factor is used in the calculation of confidence limits α denotes water used as solvent.

 

Repellency bioassay against S. oryzae weevils

Analysis of variance (ANOVA) performed on the results of repellency bioassay revealed that the treatments (phytoextracts) (F3,32 = 3.50; P= 0.0509) and exposure time (F1, 32 = 3.21; P= 0.0225) exhibited a significant effect on weevils repellency, while the interaction of both these factors exerted a non-significant effect (F3,32= 1.90; P= 0.4521) on the repellence property of S. oryzae adults (Supplementary Table III). High concentration (10%) of nano-formulated N. tabacum extract exhibited the highest repellency effect (52%), followed by M. azedarach (48%), P. guajava (24%) and A. indica (12%). While, about 29, 28, 20 and 12% repellency was exhibited by the 5% extracts of N. tabacum, M. azedarach, P. guajava and A. indica, respectively (Fig. 3C).

Discussion

Rice weevil S. oryzae has been a challenging stored products’ pest being primarily controlled by the extensive applications of synthetic fumigants and other insecticides. Pest resistance, environmental contamination and health hazards are being manifested by this approach necessitating to look for other alternate pest control methods which would be safe and environment-friendly such as using pesticidal phytoextracts (Isman, 2020; Wakil et al., 2021).

This study therefore aimed to determine the insecticidal and repellency potential of some selected phytoextracts against S. oryzae adults. As nano-formulations of pesticidal compounds is being focused as a novel and effective approach (Fraceto et al., 2016), the effective phytoextracts were further nano-formulated using silver nanoparticles. Results of preliminary bioassay with 10% of acetone phytoextracts demonstrated a significant mortality of S. oryzae adults by the extracts of A. indica, P. guajava, N. tabacum and M. azedarach causing considerable weevil mortality as compared to control. These results are in accordance with the findings of Chayengia et al. (2010), Khani et al. (2011) and Hashim et al. (2017) who reported considerable mortality of S. oryzae weevils by these phytoextracts. Particularly, neem (A. indica) and guava (P. guajava) appeared to be the most toxic to S. oryzae in our bioassays. A. indica is a well-known plant species with insecticidal properties (Chaudhary et al., 2017; Ferreira and Alves, 2021).

Later on, AgNPs-based formulations of these phytoextracts were synthesized by green-route chemistry which is an eco-friendly and promising approach for stored grains insect pest management (Rehman et al., 2021; Jasrotia et al., 2022). The results of bioassay with nano-formulated phytoextracts against S. oryzae adults revealed a significant increase in percent mortality with the increase of concentration and exposure time. Our findings are consistent with many previous research reports (Debnath et al., 2011; Stadler et al., 2012; Zahir et al., 2012; Rouhani et al., 2013; Carbone et al., 2020; Srinivasan et al., 2023; Abdel-Megeed et al., 2024). Stadler et al. (2012) evaluated nano-structured alumina (NSA) against S. oryzae and Rhyzopertha dominica and reported high mortality of S. oryzae adults 3 days post-treatment to NSA-treated wheat. Goswami et al. (2010) reported the toxicity of different surface functionalized hydrophilic nanoparticles i.e. silver (AgNPs), silica (SNPs), aluminum oxide (ANPs), zinc oxide (ZNPs) and titanium dioxide (TNPs) nanoparticles against S. oryzae. Insect mortality due to AgNPs treatment was about 85%, obtained at dose rates of almost 10 ml/kg of grain. Similarly, Rani et al. (2019) and Adnan et al. (2022) reported a significantly high mortality of adults of S. oryzae and other sorted product insect pests with green-synthesized AgNPs mediated with the leaf extracts of Moringa oleifera (Moringaceae) and Withania somnifera, respectively.

The results of repellency bioassay revealed that all botanicals exhibited significant repelling effect to S. oryzae adults. In the present study, all nano-formulated phytoextracts showed moderate repellency at minimum exposure time of 3 h, while high repellency as recorded at 24 h post-exposure. These results are consistent with the findings of previous studies (Nerio et al., 2009; Jayakumar et al., 2017; Yeguerman et al., 2022; Arafah et al., 2023). The order of the repellency of phytoextracts at 10% was N. tabacum > M. azedarach > P. guajava > A. indica after 24 h of exposure time. Our results are in line with those of Kanmani et al. (2021) who reported 100% repellent effects of nano-formulated N. tabacum to S. oryzae and C. maculatus at 100 ppm of concentration. Zia-ul-Haq et al. (2014) reported that the repellent effect of N. tabacum and M. azedarach may be due to metabolic activities and sensitivity of insects to these botanicals. Similarly, Reeba and Helen (2021) and Shahzadi et al. (2019) reported highest repellency against S. oryzae and T. castaneum by plant mediated AgNPs prepared by aqueous extract of grapefruit (Citrus paradisi) and acetone essential oil of clove (Syzygium aromaticum), respectively.

Conclusion

On the basis of overall study results, it is concluded that silver nitrate (AgNO3) based nano-formulated extracts of all tested plant species, particularly of neem (A. indica) and guava (P. guajava), exhibited a significant mortality of adults of rice weevil S. oryzae, while nano-formulated extracts of dhraik (M. azedarach) and tobacco (N. tabacum) caused considerable repellency of S. oryzae weevils as compared to control. These findings suggest that the commercial formulations of green nanoparticles could be developed to manage S. oryzae and other coleopterous stored grain pests. Moreover, the characterization of active components of phytoextracts and the validation of these phyto-formulations at large scale constitute the future perspectives of the study.

Declarations

Acknowledgments

Authors are grateful to Dr. Muhammad Irfan Majeed (Department of Chemistry, University of Agriculture, Faisalabad, Pakistan) for his valuable assistance in the fabrication and characterization of nano-formulations of phytoextracts. The authors are also grateful to Princess Nourah Bint Abdulrahman University, Riyadh, Saudi Arabia for supporting this study.

Funding

The authors are thankful to the Princess Nourah Bint Abdulrahman University Researchers Supporting Project number (PNURSP2025R82).

Data availability

All data taken and analyzed in this study are available in the published manuscript.

Supplementary material

There is supplementary material associated with this article. Access the material online at: https://dx.doi.org/10.17582/journal.pjz/20241008140626

Statemen of conflict of interest

The authors have declared no conflict of interest.

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