Evaluation of Co-toxicity Coefficient for Combined Impact of Botanicals with Spinosad against Pulse Beetle, Callosobruchus maculatus in stored Blackgram

Blackgram holds significant importance as a pulse crop in India, constituting around 10% of the total pulses produced and it is a nutritionally rich source of protein, especially for the vegetarian population in India, there’s a pressing demand to boost pulses production. Adequate storage of pulse seeds is essential to fulfill both household consumption and commercial purposes. However, a critical challenge lies in pest infestation, both in the field and during storage, which poses major hurdles to pulse production and preservation (Sanon et al., 2018).
       
The pulse beetle, Callosobruchus maculatus (Fabricius) (Coleoptera: Bruchidae), is a widely distributed insect pest among various pulse crops. It is considered the primary post-harvest insect pest leading to decreased commercial value and seed germination, rendering the grains unsuitable for human consumption (Atwal and Dhaliwal, 2005). While infestation typically starts in the field, it poses significant threats to seed integrity during storage, causing substantial damage (Kayode and Ileke, 2019).
       
Currently, pest management strategies in storage predominantly rely on synthetic insecticides and fumigants, which offer swift and reliable pest control. However, mixing these chemicals directly with food grains is not recommended. The widespread use of such chemicals in storage facilities has resulted in various issues, including the development of insecticide resistance, the resurgence of pest populations, the presence of toxic residues in food grains and soil (Fishwick, 1998) and environmental pollution. In addition, many local farmers who lack resources find the cost of chemical pest control unaffordable. Considering the challenges associated with synthetic insecticides and fumigants, there’s a global imperative to minimize their usage and adopt safer alternatives for safeguarding stored grains against insect infestations. Numerous plants exhibit efficacy against pests commonly found in stored grains (Musa and Uddin, 2016). The insecticidal properties of many plant powders are attributed to their abundance of phytochemicals like alkaloids, tannins and flavonoids (Afolabi et al., 2018). Studies have shown that incorporating plant products such as leaves, bark, powders, or extracted oils into stored pulses can effectively reduce oviposition rates, inhibit adult emergence of bruchids and minimize seed damage (Soe et al., 2020). Additionally, botanicals are biodegradable and environmentally friendly, posing a minimal risk of pollution. Spinosad, a bacterium-derived insecticide, is approved for use in more than 250 crops across 60 countries (Hagstrum and Subramanyam, 2006). It functions as both a contact and stomach poison against insect pests and exhibits low toxicity to mammals. In the USA, it has been authorized for application on millet and various cereals at a concentration of 1 mg per kilogram.
       
It should be noted that using botanical powders to protect seeds from insect pests has a significant drawback of low persistence. This is because the powders tend to lose their effectiveness due to their short post-application duration (Babarinde et al., 2008). To enhance the efficacy of botanical powders, it would be worth exploring the option of combining them with other bio-rational products like spinosad. The combination of spinosad and botanical powders can offer an opportunity to achieve both cost-effectiveness and prolonged efficacy, thus making it a promising area for empirical exploration. Therefore, this study aims to evaluate the synergistic effects of mixing spinosad with botanical powders on their toxicity against C. maculatus.

The experiment was conducted in the laboratory of the Department of Entomology, Agricultural College, Bapatla during 2020-2021.
 
Materials used in the experiment
 
Sweet flag powder, Neem kernel powder and Spinosad
 
Preparation of botanical powders
 
Powders were prepared by grinding of shade-dried neem kernels and dried rhizomes of sweet flag separately. The obtained powders were sieved through a 0.25 mm sieve, stored in an air-tight labeled container and used in the experiment.
 
Methodology
 
Spinosad was mixed with botanical powders in 1:1, 1:5, 1:10, 1:20, 1:50 and 1:100 ratios. The ratio of 1:10 was found effective for combining botanical powders with spinosad. The mixed formulations were used for the bio-assay with one-day-old adults of C. maculatus by mixing with seed method. The combined doses of botanical powders with spinosad by mixing with seed method were given in Table 1.
 

 
Statistical analysis
 
Data was recorded at 1,2,3,5 and 7 days after the release of the test insect. The mortality percentages were corrected by using Abbott’s formula (Abbott, 1925) and probit analysis was done according to Finney (1971) using SPSS software to calculate LD₅₀ and other parameters for botanicals in combination with spinosad. By using LD₅₀ of botanicals alone and in combination with spinosad, the co-toxicity coefficient with respect to botanicals were calculated by using the following formula (Sun and Johnson, 1960).
         
When the co-toxicity coefficient of a mixture is 100, the effect of this mixture indicates probability of similar action. If the mixture gives a coefficient significantly greater than 100, it indicates a synergistic action. An independent action usually will give a coefficient less than 100, but the toxicity of the mixture will be higher than that of either component (Sun, 1950).

Sweet flag powder when mixed with spinosad in 1:10 ratio has resulted in 13.33 to 93.33 corrected per cent mortality of C. maculatus at the dosages ranging from 0.012 to 0.024 g per 50 g at 7 DAT. As a result, the combined LD₅₀ value was reduced from 24 mg per 50 g to 17 mg per 50 g when used the treatment alone as mentioned in Fig 1. The slope of log dose probit (ldp) line was 8.02 and the co-toxicity co-efficient value was 141.17. Whereas, when neem kernel powder was mixed with spinosad in a 1:10 ratio it resulted in 13.33 to 93.33 corrected per cent mortality of C. maculatus at the dosages ranging from 0.015 to 0.027 g per 50 g at 7 DAT. As a result, the LD₅₀ value was reduced from 26 mg per 50 g to 20 mg per 50 g when used the treatment alone. The slope of log dose probit (ldp) line was 9.48 and the co-toxicity co-efficient value was 130 as mentioned in Table 2.
 

 

       
The highest co-toxicity co-efficient value was observed for sweet flag power compared to neem kernel powder. After 7 days of exposure, both the sweet flag powder and neem kernel powder admixed with spinosad showed similar and higher percentage mortality (93.33%), but the dose (LD₅₀) required to kill 50% of the assayed insects when sweet flag powder (17) was admixed with spinosad was lesser than neem kernel powder (20). The results also indicate that spinosad synergistically improved the toxicity of sweet flag powder at a combined dosage of 0.0024 + 0.024 g per 50 g and neem kernel powder at a combined dosage of 0.0027 + 0.027 g per 50 g against C. maculatus. It implies that as the dosages of spinosad botanical powder mixtures increased, the mortality rate of C. maculatus also increased.
       
In this study, the toxicity of botanical powder and spinosad mixtures was exposure dose-dependent. The co-toxicity co-efficient values for both treatments were greater than 100, indicating good synergism and increased efficacy between spinosad and treatments. The probable cause of this synergism may be due to a combination of different modes of action i.e., physical toxicity due to botanical powders and chemical toxicity due to spinosad. The mode of action of powder formulations that kills the target arthropods involves the abrasion of the cuticle that causes desiccation (Awam et al., 2012) and the blockage of the spiracle by the dust particles. These two mechanisms were also postulated by Babarinde et al., (2018) for the observed toxicity of the botanicals against Tribolium castaneum. Insects can be killed by ingesting or coming into contact with spinosad and botanical powder-spinosad mixtures in lethal amounts. This is due to the way spinosad works, which affects the insect’s nervous system by targeting the gamma-aminobutyric acid and nicotinic acetylcholine receptor sites. The results obtained in the present study were supported by the findings of Babarinde et al., (2018) who stated that the admixture of spinosad with botanical powders like Aframomum melegueta, Eugenia aromatica and Piper guineense evoked a higher percentage mortality of the red flour beetle in melon seeds than the sole application of botanical powders throughout the experimental period. Similarly, Ajiboye et al., (2023) found that after 14 days of exposure, a combination of citrus species and spintor, applied at a rate of 500 mg per kg, was more effective in killing a higher percentage of assayed adults (72.22–90.28%) than using solely spintor (62.10%). This mixture was also found to be significantly more effective than a combination of spintor with Trema orientalis (22.08%) or spintor with Crataeva religiosa (17.92%), both of which were also applied at a rate of 500 mg per kg.

Among the two botanical powders tested, the highest co-toxicity coefficient was recorded for sweet flag powder. The combination of spinosad with sweet flag powder performed better with less LD₅₀ values than the combination of neem kernel powder and is therefore recommended as a biorational approach for the control of C. maculatus. Spinosad improved the toxicity of sweet flag powder against C. maculatus. The results of this study show spinosad as a synergist for the botanical powders and establish its relevance in stored product protection.

All the authors, certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria, educational grants, participation in speakers’ bureaus, membership, employment, consultancies, stock ownership, or other equity interest and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.