Synergistic control of storage pest rice weevil using Hypericum japonicum and deltamethrin combinations: a key to combat pesticide resistance

  • Sreedev Puthur
  • A. N. Anoopkumar
  • Sharrel Rebello
  • Embalil Mathachan AneeshEmail author
Original Article


The augmented rates of pest infestation in stored grains have forced humans to rely on pesticide application in stored food grains even in developed countries. The greater incidence and alarming rate of pesticide resistance and safety concerns about their use in the food industry also contribute to ill-being of grain consumers and environment, thereby alluring constructive attempts to reduce the levels of agrochemical use. The current research elucidated the synergistic role of Hypericum japonicum (a medicinal plant) and pyrethroid deltamethrin in controlling the most common storage pest rice weevil, Sitophilus oryzae even at low concentrations than recommended by FAO. The screening of pesticidal property of methanolic extract of H. japonicum, pyrethroid deltamethrin, and their combinatorial assay was evaluated by standard procedures under laboratory conditions and mortality was gauged after 24 h of exposure. The LC50 and LC90 concentrations of deltamethrin alone (0.725 mg/l and 3.577 mg/l respectively) and in combination with Hypericum methanolic extract (LC50 0.119 mg/l and LC90 1.27 mg/l respectively) were found to be potent. The study revealed that the supplementation of plant extract in the pest controlling formulation substantially reduced the effective individual LC90 concentration of the pesticide required for pest control. The plant extracts showed synergy towards deltamethrin with SF 6.09. This is the foremost report on the synergistic effect of H. japonicum with deltamethrin against rice weevil, which could serve as an effective and more safer storage pest control method against the indiscriminate pesticide use and abuse.


Hypericum japonicum Sitophilus oryzae Deltamethrin Combinatorial bioassay 


The escalating population rates, as well as increased levels of plant raw material utility in industries, has always demanded the need for increased agricultural outputs worldwide (Ribeiro et al. 2003). Grain damage by pests has been a major test to the agricultural sector and the stored grain products being infested from the time of harvest to consumption. Majority of these pests belong to the coleopteran family and the most damaging candidates include Sitophilus Genus members (commonly called weevils) (Huignard et al. 2011; Tennyson et al. 2012). Rice weevil (Sitophilus oryzae) a serious pest of stored grains has a worldwide distribution especially in temperate areas, critically affecting the quality and quantity of the stored grains (Agarwal et al. 1979; Batta 2004). The damages caused by the pest alone accounts to 40% of the worldwide stored grain production (Mishra et al. 2013), as the adult weevils consume the endosperm of the grain and diminish its carbohydrate and protein content (Belloa et al. 2000).

In such a scenario, synthetic pesticides, especially contact insecticides are traditionally used to prevent and eliminate various insect pests (Cengiz et al. 2016). Deltamethrin is one such synthetic pyrethroid-based pesticide widely used in agriculture and veterinary applications for the management of rice weevils (Bekele et al. 2010; Vélez et al. 2017). Regardless of its benefits in pest control, the greatest resultant problem is that the continued application of synthetic pesticides leads to physiological resistance and undesirable ecological effects (Suresh et al. 2017). Moreover, the increasing pyrethroid resistance in grain weevils also demands the use of higher pesticide concentrations to effectively control them (Salerno et al. 2002). Such chaos amidst the adverse effects of chemical control methods and need for quality pest free grains have envisaged the utility of plant-derived materials as alternative pest control agents, individually or in combination with pesticides at very low concentrations (de Andrade Dutra et al. 2016; Herrera et al. 2017). While some plant bioactives have a natural pesticidal role, others serve as repellants not harming the living organisms and most of them are found to be biodegradable (Mavundza et al. 2011; Sreedev 2016).

The present study has been undertaken to evaluate the synergistic property of methanolic extract of H. japonicum with pyrethroid deltamethrin for achieving the highest insecticidal activity with minimal pesticide usage. H. japonicum is a plant widely used as a traditional medicine in China and has great importance because it consists of many active compounds (Hosni et al. 2017; Li et al. 2007). The plant has been used for treatments of hepatitis, gastrointestinal disorders and tumors (Samaga and Rai 2013). Previous studies also proved that the methanolic extract of H. japonicum possesses significant larvicidal and anticancerous activity (Puthur et al. 2018), there by inviting more research interests in the plant. The current research paper primarily targets the effectiveness of deltamethrin and H. japonicum in the control of common rice pest S. oryzae. The study also tries to solve the burden of increased pesticide resistance incidence, by reducing the effective deltamethrin concentrations when used along with phytobased compounds.

Materials and methods

Details of sample

Samples of H. japonicum were collected from Pothumolla, Wayanad district with latitude 11°53′N and latitude 76°0′E, Kerala, India during June 2017 in the vegetative phase. Voucher specimens of the plant were maintained at CDRL Laboratory, Thrissur and authenticated by comparison with the corresponding herbarium documents. The adult rice weevil (S. oryzae) were collected from Chalakudy, Thrissur district Kerala. The pests were collected along with damaged rice grains and were properly stored in a container along with rice grains throughout the study period. Only the adult weevils were used for the subsequent experiment. The analytical grade Deltamethrin standard used for this study was procured from Sigma-Aldrich India.

Preparation of the plant extract

The collected plant material (H. japonicum) were washed, shade dried and powdered. The extraction of the plant was carried out using a Soxhlet apparatus by using methanol as an organic solvent. In reducing the pressure of 20–22 mmHg at 35 °C the solvent undergoes distillation by using a vacuum rotary evaporator. The extract was evaporated to complete aridness at normal room temperature and stored for further analysis (Anoopkumar et al. 2017).


Susceptibility tests were done against the pest using impregnated-paper assays on 80-mm diameter in glass petri dishes (Ali et al. 2012). Whatman No. 1 filter papers were cut into two halves and dipped into the five differently concentrated plant extract solutions for 5 s against distilled water as a control. After drying in a hood for 2 min, each paper was placed at the bottom of the Petri dish and adults of S. oryzae were introduced (into the Petri dish). Both the test and control beakers were placed under similar conditions of 25 ± 2 °C, 12 h light/dark regime, with no food for 24 h, and mortality was monitored. The pest was considered as dead if they were not responsive to gentle prodding with a fine needle. All the tests in the following experiments were done in triplicate using 20 individuals, and values were expressed as mean ± standard deviation. The results were expressed as percent mortality.

Similarly, deltamethrin stock solutions were diluted using distilled water and the susceptibility test was carried out with five varying concentrations using the above-mentioned protocol. Data from all replicates were pooled for analysis. LC50 and LC90 values were calculated from a log dosage-probit mortality regression line using a computer software program (IBM SPPSS version. 17.0). Mortality was measured in percentage, and if the control mortality fluctuated, it was corrected using Abbott’s formula (Abbott 1925).

Combinatorial bioassay

For combinatorial studies, varying concentrations of deltamethrin were prepared to range 1.125–2 mg/l. The LC 50 value of plant extract obtained from the probit analysis was kept as a standard and each concentration of the pesticide formulation was mixed with the plant extract accordingly. Mortality obtained after 24 h were recorded. The resulting data from the observation were subjected to statistical analysis to calculate the LC50 and LC90 values. The synergistic factor (SF) (Kalyanasundaram and Das 1985) and the co-toxicity coefficient (CTC) (Sun and Johnson 1960) are also calculated.


Bioassay of individual components

The pesticide deltamethrin alone showed potent activity with LC50 and LC90 value of 0.725 mg/l and 3.577 mg/l causing the death of 50% and 90% of pest populations respectively in post-treatment. On the other hand, the plant extracts required a higher LC50 and LC90 value of 498.29 mg/l, 2507.79 mg/l respectively, to induce significant lethality of rice weevil. The bioassay results of methanolic extracts of H. japonicum and deltamethrin are summarized in Tables 1 and 2 respectively. The survival of the pest species varied significantly by doses and time of exposure. The LC50 value of plant extract was a bit on the higher side compared to deltamethrin. The survival values were found in good proportion to the concentration applied and ranged from 75 to 15% of low to a high dose of plant extract applied; whereas in deltamethrin, the values ranged from 90 and 10% for low to high doses. However, the survival rate in control was 100% throughout the experiment proving the pesticidal activity of the individual components considered in the above study. Figures 1 and 2 depict the log dose probit mortality responses of plant extract and deltamethrin against rice weevil.
Table 1

Efficacy of methanol extract of H. japonicum against S. oryzae


Concentration (mg/l)

Percent mortality

LC50 (LCL–UCL) (mg/l)

LC90 (LCL–UCL) (mg/l)

Chi square value

P value




Plant extract






498.29 (603.82–725.48)

2507.79 (3567.24–6075.67)










LCL lower confidence limit, UCL upper confidence limit

*P < 0.15 significant difference at 1%

Table 2

Efficacy of deltamethrin against S. oryzae


Concentration (mg/l)

Percent mortality

LC50 (LCL–UCL) (mg/l)

LC90 (LCL–UCL) (mg/l)

Chi square value

P value










0.725 (1.017–1.439)

3.577 (5.960–14.272)



















LCL lower confidence limit, UCL upper confidence limit

*P < 0.50 significant difference at 5%

Fig. 1

Log concentration-percent mortality relationship of S. oryzae to H. japonicum methanolic extract

Fig. 2

Log concentration-percent mortality relationship of S. oryzae to deltamethrin

Combinatorial bioassay

The combinatorial use of pesticide deltamethrin along with the methanolic plant extract yielded better pest control results compared to their individual effects against rice weevil as depicted in Table 3. Moreover, the study also succeeded in lowering the effective LC50 and LC90 values of deltamethrin to 0.119 mg/l and 1.27 mg/l respectively, when supplemented with H. japonicum phytoextract at a concentration of 498.29 mg/l (corresponding to its individual LC90 value) for a 24-h treatment. The combinatorial assay showed synergy with a synergistic factor of 6.09. The log dose probit mortality responses of combinatorial bioassay are shown in Fig. 3.
Table 3

Synergistic effect of H. japonicum methanol extract and deltamethrin against S. oryzae


Concentration (mg/l)

Percent mortality

LC50 (LCL–UCL) (mg/l)

LC90 (LCL–UCL) (mg/l)






Plant extract + deltamethrin

498.2 + 0.125



498.29 + 0.25


0.119 (0.266–0.426)

1.271 (2.602–18.099)




498.29 + 0.5



498.29 + 1



498.29 + 2



LCL lower confidence limit, UCL upper confidence limit, CTC co-toxicity coefficient, SF synergistic factor

Fig. 3

Log concentration-percent mortality relationship S. oryzae to combinatorial bioassay


S. oryzae (rice weevil) occurs as a cosmopolitan pest in rice, sorghum, maize, wheat and almost all grains with infestation caused by both adult and larvae (Srivastava and Subramanian 2016). The damage is characterized by the formation of an intact pericarp shell of grains when infested with rice weevils, leading to high levels of economic damage to stored crops worldwide (Hong et al. 2018). The individual use of deltamethrin was found effective in controlling rice weevils during this study, as high mortality rate was observed but the development of resistance is a major drawback that raises a caution. The growing pesticide resistance, especially against pyrethroids has been reported in a wide variety of pests attributed to knockdown resistance mutations in sodium channels and altered detoxification strategies (Haddi et al. 2018). Apart from pests, even vectors such as mosquitoes are reported to show pesticide resistance (Amelia-Yap et al. 2018), thus demanding alternate strategies to control pests or reducing the unchecked usage of pesticides.

Literature survey revealed the exploration of various plant species for insecticidal properties. The lethal effect of Datura alba seed extract against onion thrips (Malik 2005), Datura stramonium leaf extract against Tribolium castaneum (Pascual-Villalobos and Robledo 1998) and Datura fastuosa extracts against rice weevil (Kamruzzaman et al. 2005) are some examples of herbal extracts against pests. H. japonicum is an annual herb widely distributed through Asia and used as traditional medicine for viral and bacterial disorders in China. This plant produces phloroglucinol flavonoid derivatives with potential antioxidant activity (Peng et al. 2006). References to the insecticidal activity of Hypericum species analyzed using essential oils was displayed in insecticidal action against Brachiacantha dentipes (Liu et al. 2007).

Significant mortality was shown by H. japonicum plant extract (p < 0.15) against S. oryzae, thereby raising its prospects as a herbal insecticide. The methanolic extract of H. japonicum comprises potent compounds which attribute its insecticidal activity (Puthur et al. 2018). There is a direct correlation between efficacy and concentration of phytoextract, the lethal effect was maximum at high concentration (Kuganathan et al. 2008). The present results agree with the studies conducted by Ali et al. (2012) in which D. alba leaf extracts active against S. oryzae and Trogoderma granarium.

Irrespective of its insecticidal properties, the individual use of botanicals face a major drawback of bio compound deterioration that promotes the prospects of synergistic use of plant extracts with insecticides (Nenaah 2011). Synergistic effects are defined as the action of multiple compounds on a pest that is greater than the action of individual compounds (Kumar and Parmar 1996; Scott et al. 2002). Muroi and Kubo (1993) reported the mechanism of synergy, such compounds will destroy the ability of the pest to metabolize toxins and leads to death. Plant extracts in combination with phenthoate and fenthion also showed significant synergism against malarial vector (Kalyanasundaram and Das 1985). Extracts of Rhizophora apiculata, Caulerpa scalpelliformis and Dictyota dichotoma showed synergism with synthetic insecticides against Aedes aegypti (Thangam and Kathiresan 1991).

According to the findings of the present study, methanolic extracts H. japonicum can be used as a natural weapon against stored grains pest S. oryzae and the extracts show synergy in combinational use with deltamethrin. Data verified by the above study thus suggests a combinatorial use of methanolic extracts of the plant at concentrations of 498.29 mg/l, and deltamethrin at 0.119 mg/l concentrations could serve as an effective decoction to reduce the pest population of stored grain by 50%. As per the regulations of FAO, pyrethroids are applied at a rate of 3 mg/l in stored grains to prevent infestation by pests ( This would reduce the current pesticide use from 3 mg/l to lower concentrations gains relevance.

Our study revealed the pesticidal efficacy of H. japonicum plant extract, Deltamethrin and their combinatorial effect against S. oryzae with a synergy frequency of SF 6.09. This indicates that the insecticidal efficacy of the individual components is increased approximately seven times when used in combination. This method has the dual benefit in reducing the amount of synthetic insecticide and making the application more effective, the problems of insecticide resistance of pests can be overcome to an extent by using such control measures (Mohan et al. 2007; Morales-Rodriguez and Peck 2009). The exact mode of synergistic action is still not fully understood, however, previous studies conducted shows that plant secondary metabolites and synthetic pesticide will inhibit the acetylcholine esterase (AChE) activity of S. oryzae (Maazoun et al. 2017; Saad et al. 2018). This is the first report on synergistic and pest control role of H. japonicum. Thus synergistic combinations of H. japonicum methanolic extracts with substantially low concentrations of deltamethrin could be used as an effective solution for commercial grain storage. This investigation has the benefit of pesticide usage reduction maintaining environmental sustainability, and reducing the chance of developing toxicity to non-target organisms.



We thank Principal, St. Joseph’s College for the laboratory facilities provided.

Author contributions

SP and EMA designed experiments and analyzed data. SP conducted experiments. ANA and SR did the statistical analysis. All authors contributed to writing the paper.


We are greatly thankful to the University Grants Commission, New Delhi for providing funds under the UGC Research Award - F30-6/20-16(SA-II).

Compliance with ethical standards

Conflict of interest

The Authors SP, ANA, SR, and EMA declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


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Copyright information

© Society for Environmental Sustainability 2019

Authors and Affiliations

  1. 1.Communicable Disease Research Laboratory, Department of ZoologySt. Joseph’s College, IrinjalakudaThrissurIndia

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