Fumigant toxicity, oviposition deterrence and repellency of three plant essential oils against Callosobruchus maculatus on five bean types

Research Article

Fumigant toxicity, oviposition deterrence and repellency of three plant essential oils against Callosobruchus maculatus on five bean types

Marziyeh Namdari¹, Masumeh Ziaee¹, Seyed Ali Hemmati^1* and Fatemeh Nasernakhaei²

1. Department of Plant Protection, Faculty of Agriculture, Shahid Chamran University of Ahvaz, Ahvaz, Iran.

2. Department of Genetic and Plant Production, Faculty of Agriculture, Shahid Chamran University of Ahvaz, Ahvaz, Iran.

Abstract: One of the most destructive pests affecting stored legumes is the bruchid beetle, Callosobruchus maculatus (F.). This study investigated the fumigant toxicity, oviposition deterrence, and repellency of three essential oils (EOs), including Trachyspermum ammi (L.) Sprague, Cuminum cyminum (L.), and Melaleuca viminalis (Sol. ex Gaertn.) against C. maculatus adults. The lethal concentrations (LC₅₀) for the EOs were determined as 1.727 µL/L air for T. ammi, 1.219 µL/L air for C. cyminum, and 1.622 µL/L air for M. viminalis, with a dose-dependent increase in adult mortality with rising EO concentrations. Oviposition deterrence was assessed on various bean types, including white kidney beans, common beans, cowpeas, navy beans, and red kidney beans. Notably, T. ammi oil at 5 µL/g of seeds completely inhibited oviposition, whereas 2 µL/g resulted in 88-94% inhibition. In contrast, M. viminalis oil showed the least deterrence at the same concentration, ranging from 60% to 77%. The control group exhibited significant variation in egg-laying across bean types, with cowpeas showing the highest egg count, followed by white kidney and common beans. All EOs effectively reduced both egg numbers and hatch rates. The repellent efficacy was highest in treated cowpeas, with C. cyminum oil exhibiting a notable repellent activity of 80%, compared to 46.66% for T. ammi and 33.33% for M. viminalis oils. Our findings underscore the potential of these oils as natural alternatives for managing C. maculatus infestations in stored legumes.

Keywords: Beans, mortality, essential oil, oviposition, repellent

Introduction[1][2]

The protection of stored products, particularly grains and pulses, is a critical issue in agricultural practices worldwide. Insect pests pose a significant threat to these commodities, leading to both qualitative and quantitative losses that can have severe economic repercussions. Among the various culprits, storage beetles are notorious for causing extensive postharvest losses, spoilage, and diminished market demand for affected products (Ahmad et al., 2021). Pulses, such as beans, lentils, and peas, are particularly vulnerable to damage from weevils and beetles during both field cultivation and storage (Stathas et al., 2023). One of the most prominent pests affecting stored legumes is the bruchid beetle, Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae). This insect plays a destructive role not only in agricultural fields but also during storage, particularly in warm, Mediterranean regions where it thrives. The beetles can inflict considerable damage on stored legumes of the genus Phaseolus, leading to significant losses in seed weight, germination rate, and overall market value (Hill, 2002; Rees, 2007).

To combat the pest, synthetic chemical pesticides have traditionally been employed. However, the widespread use of these chemicals has led to numerous problems. These include insect resistance development, environmental hazards, risks to human health, and the presence of pesticide residues that can affect end consumers (Ahmad et al., 2024). Protecting stored products with plant-based products, while considering environmental concerns, has highlighted the need for a new generation of pesticides (Pathak et al., 2022). As a result, there is an increasing need to explore alternative pest control methods that are more environmentally friendly and sustainable. One promising approach is using plant-based products as natural pesticides. The search for new-generation pesticides has highlighted the potential of plant compounds that exhibit a wide range of insecticidal activities. Many of these compounds have toxic properties, as well as repellent effects and deterrent characteristics that can inhibit oviposition and feeding behaviors (Enan, 2001). Essential oils (EOs) derived from various plants have attracted particular attention due to their low toxicity to humans and mammals, as well as their medicinal and nutritional benefits. Moreover, these EOs tend to decompose rapidly in nature, reducing their environmental impacts (Ivanova et al., 2025; Sharifi-Rad et al., 2017). The effectiveness of these natural products has led to growing interest among pesticide companies in developing plant-based insecticides as viable alternatives to synthetic chemicals (Isman, 2000; Isman et al., 2010). The exploration of native plants for their insecticidal properties represents a crucial first step in optimizing the production of plant-derived pesticides. By assessing the insecticidal potential of local flora, researchers can identify species with optimal efficacy against specific pests. This approach not only supports sustainable agricultural practices but also aligns with global efforts to reduce reliance on harmful chemical pesticides. In light of these considerations, this study aimed to evaluate the fumigant toxicity, oviposition deterrence, and repellency of three selected plant essential oils against C. maculatus. The findings could lead to the development of effective and environmentally friendly pest control strategies that protect stored pulses while minimizing risks to human health and the environment.

Materials and Methods

Bean preparation

Various types of beans, including white kidney beans, common beans, cowpeas, navy beans, and red kidney beans, were purchased from local markets for experimental purposes. To ensure consistency in moisture content, the bean seeds were maintained under controlled conditions of 25 ± 1 ºC and 60 ± 5% relative humidity (R.H.) for one week, allowing them to acclimate to the environmental R.H.

Insect rearing

Callosobruchus maculatus was collected from infested cowpeas stored in a warehouse. The C. maculatus rearing method was similar to that of Zandi Sohani and Ramazani (2012). A stock culture of C. maculatus was established by rearing the insects on the beans above under the same controlled conditions in an incubator (Grouc Co, Tehran, Iran) set at 25 ± 1 ºC, 60 ± 5% R.H., and continuous darkness. The colony was maintained for five generations without any exposure to insecticides. Adults aged 1-3 days old were used in the experiments.

Plant materials and extraction of EOs

The seeds of Trachyspermum ammi (L.) Sprague (Apiaceae) and Cuminum cyminum (L.) (Apiaceae) were acquired from local markets in Mashhad. Meanwhile, leaves from Melaleuca viminalis (Sol. ex Gaertn.) (Myrtaceae) were collected from trees at the Faculty of Agriculture, Shahid Chamran University of Ahvaz, Ahvaz, Iran. The EOs from these plants were extracted through hydrodistillation using a Clevenger-type apparatus. For the extraction process, approximately 50 grams of T. ammi and C. cyminum seeds, or 100 grams of M. viminalis leaves, were milled using a National dry mill (MJ-176NR, Osaka, Japan) and placed in a round-bottom flask over water, with the temperature maintained at approximately 100 ºC. After a four-hour distillation period, the oils that accumulated in the reservoir were collected. To ensure purity, anhydrous sodium sulfate was used to remove residual water after extraction. The extracted oils were then stored in a refrigerator at 4 ºC to preserve their integrity for subsequent experiments.

Fumigant toxicity of EOs on Callosobruchus maculatus adults

To evaluate the fumigant toxicity of the essential oils, 10 adults of C. maculatus (1-3 days old) were placed in each vial with six replications per treatment. Filter papers (Whatman No. 1) were placed beneath the caps of the glass vials (140 mL) to facilitate the exposure to the oils. A preliminary concentration-setting experiment was performed to identify effective concentrations that would result in 20-80% mortality after 24 h of exposure. Employing a symmetric five-dose design, as outlined by Robertson et al. (1984), specific concentrations were selected: T. ammi at 1, 1.3, 1.7, 2.3, and 2.7 µL/L air; C. cyminum at 1.1, 1.5, 1.9, 2.4, and 3.2 µL/L air; and M. viminalis at 0.5, 0.8, 1.1, 1.6, and 2.4 µL/L air were impregnated onto the filter papers. The vials were tightly sealed and incubated at 25 ± 1 ºC, 60 ± 5% R.H. in continuous darkness. An untreated filter paper served as a control group. Mortality was assessed after 24 h. The lethal concentration (LC₅₀) was subsequently calculated to evaluate the efficacy of each EO.

The oviposition deterrence effects of EOs on Callosobruchus maculatus adults

To evaluate the oviposition-deterrent effects of the essential oils on C. maculatus, an experiment was conducted using 10 grams of each bean seed type placed in glass vials (140 mL). Each vial received 0.02 or 0.05 µL/g of the respective EOs, which were thoroughly mixed with the seeds. A pair of adult beetles (one male and one female) was introduced into each vial. Ten replicates were prepared for each oil concentration. The vials were maintained for five days at 25 ± 1 ºC, 60 ± 5% R.H., and in continuous darkness. After this exposure period, the number of eggs laid on the seeds was counted. The percentage of oviposition deterrence was calculated using the following formula (Elhag, 2000):

Oviposition deterrence = (NC-NT)/NC × 100, where NC represents the number of eggs in the control group and NT represents the number of eggs in the treatment group.

The repellent effects of EOs on C. maculatus adults

An olfactometer was used to investigate the repellent effects of the essential oils (Nikooei and Moharramipour, 2011). Fifteen minutes before the experiment began, the device was turned on to allow air to enter the olfactometer after passing through activated charcoal. Each EO was applied at 2000 mg/kg and sprayed onto 10 g of bean seeds. Untreated beans served as the control, placed in one arm, and treated beans with each oil in the other arm of the olfactometer. The tested adults were kept without food for 24 h. An adult was released individually at the entrance of the main arm, and the experiment was replicated 30 times for each essential oil. To eliminate the effects of arm side, the treated beans were placed in the right arm for 15 replications and in the left arm for the other 15 replications. After each EO test was completed, the device's arms were washed with acetone to eliminate the odor and the effects of the previous oil.

The repellence percentages of the EOs were calculated according to the following formula:

;

Where R is the percentage of repellent effect, C is the number of adults in the control arm, E is the number of adults in the essential oil treatment arm, and T is the total number of adults tested.

Data analysis

The normality of the data was examined by the Shapiro-Wilk test. For the fumigant toxicity test, the LC₅₀ values and 95% confidence limits were estimated by probit analysis. The mortality and repellent data were subjected to one-way analysis of variance (ANOVA). For each dose rate, data on oviposition deterrence, egg number, and hatch rate were analyzed using one-way ANOVA to determine differences among bean types or essential oils. Tukey-Kramer honestly significant difference (HSD) test was used to determine significant differences. All analyses were performed using SPSS 16.0 software (IBM Corp., 2007).

Results

The lethal concentrations (LC₅₀) of T. ammi, C. cyminum, and M. viminalis EOs are presented in Table 1. Based on LC₅₀ values and their confidence limits, the beetles were more susceptible to the C. cyminum oil (Table 1).

For the fumigant toxicity, there were significant differences among five different concentrations of T. ammi (F_4,25 = 69.286, P < 0.001), C. cyminum (F_4,25 = 105.50, P < 0.001), and M. viminalis oil (F_4,25 = 68.315, P < 0.001). The highest mortality percentage was observed when adults were exposed to 2.7 µL/L air of T. ammi oil. For C. cyminum oil, there were no significant differences between 2.4 and 3.2 µL/L air concentrations, which caused 88.33 and 95% mortality, respectively. For M. viminalis oil, the highest mortality was observed at 1.6 and 2.4 µL/L air (Fig. 1).

The results of the oviposition deterrence of C. maculatus adults when exposed to essential oils on different beans are summarized in Figure 2. Significant differences were observed among the various beans exposed to 0.02 µL/g of seeds (F4,26 = 3.078, P = 0.034). The T. ammi oil at 0.05 µL/g of seeds achieved 100% deterrence. For 0.02 µL of C. cyminum oil/g seeds, the highest oviposition deterrence was recorded when adults fed on cowpeas (88.57%) (F_4,26= 34.264, P < 0.001), while for 0.05 µL/g the highest deterrence value was found in cowpeas, navy beans, and red kidney beans (F_4,26= 5.918, P < 0.001). For M. viminalis oil, the highest oviposition deterrence was observed when adults were exposed to treated cowpeas at both 0.02 µL/g of seeds (F_4,26 = 20.018, P < 0.001) and 0.05 µL/g of seeds (F_4,26 = 5.188, P < 0.001) (Fig. 2).

At the rate of 0.02 µL/g of seeds, the highest oviposition deterrence was observed in all bean seeds including white kidney beans (F_2,15 = 144.368, P < 0.001), common beans (F_2,15 = 36.935, P < 0.001), cowpeas (F_2,15 = 100.849, P < 0.001), navy beans (F_2,15 = 172.193, P < 0.001), and red kidney beans (F_2,15 = 104.597, P < 0.001) treated with T. ammi oil in comparison with C. cyminum and M. viminalis oils. For 0.05 µL/g of seeds, T. ammi oil caused the highest oviposition deterrence in white kidney beans (F_2,15 = 36.60, P < 0.001), common beans (F_2,15 = 47.50, P < 0.001), cowpeas (F_2,15 = 15.0, P < 0.001), navy beans (F_2,15 = 47.50, P < 0.001), and red kidney beans (F_2,15 = 14.231, P < 0.001) (Fig. 3).

Table 1 Lethal concentrations (LC₅₀) of three essential oils against Callosobruchus maculatus adults 24 h after exposure.

Figure 1 Mean (± SE) mortality of Callosobruchus maculatus adults due to fumigant toxicity of Trachyspermum ammi (A), Cuminum cyminum (B), and Melaleuca viminalis (C) essential oils 24 h after exposure. Means followed by the same letter are not significantly different using Tukey-Kramer (HSD) test at P < 0. 5.

Figure 2 Mean (± SE) oviposition deterrence of Callosobruchus maculatus when exposed to Trachyspermum ammi (A), Cuminum cyminum (B), and Melaleuca viminalis (C) essential oils on five different beans. Means followed by the same lowercase letter for 0.02 µL/g and same uppercase letter for 0.05 µL/g of seeds are not significantly different using Tukey-Kramer (HSD) test at P < 0.05.

Figure 3 Mean (± SE) oviposition deterrence of Callosobruchus maculatus when exposed to three essential oils on white kidney beans (A), common beans (B), cowpeas (C), navy beans (D), and red kidney beans (E). Means followed by the same lowercase letter for 0.02 µL/g and same uppercase letter for 0.05 µL/g of seeds are not significantly different using Tukey-Kramer (HSD) test at P < 0.05.

The number of eggs laid by C. maculatus differed significantly among the bean types in the control (F_4,25 = 7.462; P < 0.001), and the highest number of eggs was found on cowpeas, followed by white kidney and common beans. Also, significant differences were observed when the seeds were treated with T. ammi oil at 0.02 µL/g (F_4,25 = 3.978; P < 0.001). No eggs were laid on T. ammi oil-treated seeds at 0.05 µL/g of seeds; for C. cyminum oil, the highest eggs were observed when adults were exposed to white kidney beans treated with 0.02 µL/g of seeds (F_4,25 = 40.733; P < 0.001), followed by treated common beans. There were no significant differences among bean seeds when exposed to the C. cyminum oil at the rate of 0.05 µL/g of seeds. For M. viminalis oil, the highest number of eggs was laid in common beans treated with 0.02 µL/g. However, there were no significant differences among bean seeds when exposed to 0.05 µL/g of seeds (Fig. 4).

The results of hatch rates of C. maculatus eggs are presented in Figure 5. In the control group, the highest hatch rate was recorded on cowpeas, while there were no significant differences among other seed types (F_4,25 = 6.864; P < 0.001). No eggs hatched across all bean seed types treated with both T. ammi oil concentration rates. A small percentage of eggs were hatched in white kidney and common beans treated with 0.02 µL/g of seeds of C. cyminum oil, which did not exceed 6% (F_4,25 = 3.750; P = 0.016). For C. cyminum and M. viminalis oils, no eggs were hatched at the rate of 0.05 µL/g of seeds. For M. viminalis oil, there were no significant differences among bean seeds when exposed to 0.02 µL/g of seeds (F_4,25 = 1.243; P = 0.318) (Fig. 5).

The repellent effects of the essential oils are presented in Figure 6. For T. ammi oil, no significant differences were observed among bean seeds (F_4,10 = 3.071; P = 0.068). However, the repellent activity was high in treated cowpeas, which was 46.66%. In the case of C. cyminum oil, the highest repellent activity was found in cowpea (80%), followed by navy beans (66.66%). However, the lowest repellent activity was observed in white kidney beans (F_4,10 = 11.50; P < 0.001). For M. viminalis oil, no significant differences among bean seeds were recorded, and the repellent activity did not exceed 33.33% in the case of treated cowpea (F_4,10 = 3.125; P = 0.066) (Fig. 6).

Discussion

The increasing prevalence of C. maculatus, a significant pest of stored legumes, highlights the urgent need for eco-friendly pest control methods. This study investigates the efficacy of various EOs, particularly T. ammi, C. cyminum, and M. viminalis, in managing C. maculatus populations. The findings align with previous studies indicating that EOs can serve as effective bio-pesticides, exhibiting varying levels of fumigant toxicity and deterrence against C. maculatus. For example, the fumigant toxicity of essential oils from four Ocimum species was assessed against C. maculatus adults. Notably, O. canum oil resulted in the highest mortality rate (94%) in adults. Additionally, minimal oviposition was observed in the treatments with O. canum and O. basilicum L. oils, with only 0.6 and 0.8 eggs, respectively, demonstrating significant differences compared to other treatments and the control group (Keita et al., 2000). In another study, at the highest dose of 25 mL of oil per 8 mL vial, O. basilicum achieved an 80% mortality rate, while O. gratissimum L. recorded a 70% mortality. The LC₅₀ values of the oils were 0.66 mL/mL for O. basilicum and 1.06 mL/mL for O. gratissimum (Keita et al., 2001).

Our results indicated that C. cyminum oil had the lowest LC₅₀ value, suggesting that C. maculatus was particularly susceptible to it compared with other oils. Raja et al. (2001) found that the highest adult mortality of C. maculatus occurred with Mentha spicata L. (100% mortality), followed by Mentha piperita L. (70%) and Mentha arvensis L. (55%) oils, assessed one month after treatment. In their study, cowpea seed protection lasted up to 2 months post-treatment, due to reduced oviposition, low adult emergence, and high adult mortality during this period. However, pest damage increased significantly by four months post-treatment.

Figure 4 Mean (± SE) number of Callosobruchus maculatus eggs when exposed to Trachyspermum ammi (A), Cuminum cyminum (B), and Melaleuca viminalis (C) essential oils. Means followed by the same lowercase letter for control and same uppercase letter for 0.02 µL/g are not significantly different using Tukey-Kramer (HSD) test at P < 0.05.

Figure 5 Mean (± SE) hatch rates of Callosobruchus maculatus eggs when exposed to Trachyspermum ammi (A), Cuminum cyminum (B), and Melaleuca viminalis (C) essential oils. Means followed by the same lowercase letter for control are not significantly different using Tukey-Kramer (HSD) test at P < 0.05.

Figure 6 Mean (± SE) repellent effects of Trachyspermum ammi (A), Cuminum cyminum (B), and Melaleuca viminalis (C) essential oils against Callosobruchus maculatus. Means followed by different letters are significantly different using Tukey-Kramer (HSD) test at P < 0.05.

A further study reported LC₅₀ values for the essential oils against adults of Tribolium castaneum Herbst as follows: C. cyminum (16.26 µL), Piper nigrum L. (15.26 µL), and Foeniculum vulgare Mill. (18.55 µL) (Chaubey, 2007). Sahaf and Moharramipour (2008) found that the LC₅₀ for different life stages of C. maculatus exposed to T. ammi oil were 1.01 µL/L air for eggs, 2.50 µL/L air for larvae, and 0.90 µL/L air for adults, indicating that T. ammi was more toxic across all growth stages than the other EOs used in their study (Vitex pseudo-negundo Hand I. MZT.). Our study confirms that all three tested oils, particularly T. ammi, were toxic to adult C. maculatus. Other plant EOs also demonstrated fumigant activity against various stored product pests; for instance, O. gratissimum oil at 1 µL/L air caused 98%, 99%, and 100% mortality in Rhyzopertha dominica F., Oryzaephilus surinamensis (L.), and Callosobruchus chinensis L., respectively, within 24 hours post-treatment (Ogendo et al., 2008).​ Ilboudo et al. (2010) reported that Ocimum americanum L. oil was particularly toxic to adults of C. maculatus, with an LC50 of 0.23 µL/L, and was more effective than other tested oils, such as Hyptis suaveolens (L.) Poit., Hyptis spicigera Lam., and Lippia multiflora Moldenke. The comparative analysis of essential oils suggests prioritizing specific oils in pest management strategies. Evaluating the activity of ten Apiaceae species indicated that oils from Anethum graveolens (Dill), Carum carvi L., and C. cyminum exhibited strong fumigant toxicity against the adults of Sitophilus oryzae L. The LC₅₀ values for these oils were reported as 2.45 mg/L air for C. carvi, 3.29 mg/L air for A. graveolens, and 4.75 mg/L air for C. cyminum (Kim et al., 2013). Additionally, recent findings showed LC₅₀ values of 0.421 and 0.271 mL/mL for M. piperita, and 0.135 and 0.136 mL/mL for C. cyminum oil against T. castaneum and S. oryzae, respectively (Lashgari et al., 2014).

The susceptibility of insect species to EOs varies. Adults of O. surinamensis (LC₅₀ = 1.69 µL/L air) were significantly more susceptible to T. ammi oil than R. dominica (LC₅₀ = 19.01 µL/L air) and Tribolium confusum Jacquelin du Val. (LC₅₀ = 58.70 µL/L air) at 24 h length of exposure to this oil (Shokri Habashi et al., 2011). Ziaee et al. (2014) documented high fumigant toxicity of T. ammi oil and also essential oil-loaded nanogel against R. dominica and T. confusum adults. Our results indicated that mortality increased with increasing oil concentration. The findings from Kedia et al. (2015) further emphasized the importance of concentration in determining the effectiveness of C. cyminum oil. Moreover, the higher sensitivity of C. chinensis compared to S. oryzae in their studies suggests that optimal concentrations must be identified for each species to enhance EOs' activity.

In our study, significant oviposition deterrence was observed with T. ammi and C. cyminum oils, suggesting that these oils could disrupt the reproductive cycle of C. maculatus, thereby reducing its population growth. This finding is consistent with earlier studies, such as the one by Boeke et al. (2004), who mentioned that C. maculatus adults on beans treated with Capsicum frutescens L., Tagetes minuta L., and Tephrosia vogelii Hook f. laid fewer eggs in the first 24-h period than the ones on untreated beans. Additionally, the work by Nyamador et al. (2010) indicated that the eggs and adults of C. maculatus were susceptible to EOs, whereas Callosobruchus subinnotatus (Pic.) showed greater tolerance, suggesting that pest management strategies may need to be tailored to species-specific responses to EOs. This differential sensitivity is crucial for developing targeted interventions that maximize efficacy against specific pests. Based on their investigations, Cymbopogon giganteus Chiov. oil was toxic to adults of both species, while Cymbopogon nardus (L.) oil showed better ovicidal activity.

The findings from this study highlight the significant potential of C. cyminum oil as a natural repellent against C. maculatus. The observed high repellent activity aligns with previous research indicating that various EOs can effectively deter insect pests. For instance, Boeke et al. (2004) investigated 33 African plant oils. They reported that Clausena anisata (Willd.) Hook, Dracaena arborea (Willd.) Link, T. vogelii, Momordica charantia L., and Blumea aurita (L.) showed repellent properties against C. maculatus. This finding suggests that traditional knowledge regarding plant-based repellents can be leveraged to enhance modern pest management practices. Moreover, the comprehensive evaluation by Upadhyay et al. (2007) into 14 essential oils, including C. cyminum, underscores the multifaceted role of plant oils in managing pest populations. The reported repellent and antifeedant effects indicated that these oils not only had the potential to repel pests but also to inhibit their feeding behavior, thus potentially reducing damage to stored products. The findings from Khodadoust et al. (2011) further confirmed the effectiveness of C. cyminum and T. ammi oils against other pests, showcasing their broad-spectrum efficacy. As reported in their study, 0.7 µL of C. cyminum and T. ammi oil had 100% and 73.33% repellency against T. confusum, and 100% and 80% repellency against Ephestia kuehniella Zeller, respectively.

In conclusion, the results underscore the potential of specific essential oils as eco-friendly alternatives for managing C. maculatus populations, thereby contributing to sustainable agricultural practices in stored legume pest control. This is particularly crucial in light of growing concerns about pesticide resistance and the environmental impacts of chemical use in agriculture. Future research should focus on field trials and the formulation of these EOs to develop practical applications in agricultural settings, ensuring a balance between pest management and environmental sustainability. Overall, this study contributes valuable insights into the potential of essential oils as natural pest control agents, paving the way for innovative strategies in stored legume pest management.

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.

Author contributions

Masumeh Ziaee and Seyed Ali Hemmati conceived and designed the experiments; Marziyeh Namdari performed the experiments; Masumeh Ziaee analyzed the data and wrote the original draft of the paper; Seyed Ali Hemmati reviewed and edited the paper. Fatemeh Nasernakhaei was involved in the methodology. All authors have read and approved the final manuscript.

Acknowledgements

This research was financially supported by Shahid Chamran University of Ahvaz, Ahvaz, Iran (Grant No. SCU.AP1402.39134), which is greatly appreciated.

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