A plant-based extract mixture for controlling Spodoptera litura (Lepidoptera: Noctuidae)

Spodoptera litura (Lepidoptera: Noctuidae) is a polyphagous insect pest that feeds on at least 87 plant species in over 40 plant families, including many vegetables, fruit, cotton, groundnut, chili, tobacco, castor, lady finger, cauliflower, and pulses, in many Asian countries, such as Thailand, China, Japan, India [1–3]. It has gradually become a significant insect pest in recent years [4]. The spread of this pest has led to the continuous usage of insecticides on crops. Pesticide residues impact the environment and people’s health [5]. These results of insecticide usage have encouraged scientists to seek less hazardous chemicals and identify an alternative method for integrated pest management (IPM).

Botanical extract products have become more prominent in assessments of current and future pest control alternatives. They are biodegradable and ecologically safe and an important component of IPM programs [6]. For instance, numerous studies have focused on neem (Azadirachta indica A. Juss), and a number of studies demonstrate efficacy against a variety of pests [7, 8]. Its compound has numerous activities against insects, such as antifeedant, growth inhibition, growth regulation, reduced fecundity and sterility, and inhibition of protein synthesis, as well as toxic effects in a wide variety of insect taxa, including Lepidoptera [7, 8]. Plants comprise a source of novel chemical compounds that are used in medicine and other applications. Each plant contains many active compounds, such as terpenes, terpenoids alkaloids, steroids, phenols, and flavonoids [9], which are found in specific parts, i.e., leaves, flowers, stems, fruits, seeds, roots. Plant defense compounds typically result from a combination of these plant products and not as individual compounds.

A variety of documents suggest that complex mixtures would be more efficient, and synergistic effects have been reported [10, 11]. Singh et al. [12] demonstrated that thymol and α-terpineol synergized the impacts of both linalool and 1,8-cineole, but linalool with 1,8-cineole exhibited only an additive effect against Chilo partellus. trans-Anethole acted synergistically with thymol, citronellal and α-terpineol [13]. Consequently, the mixtures of plant compounds are also likely to be more durable against insects evolving resistance and developing behavioral desensitization.

This study was focused on the effect of six plant extracts, including Acorus calamus (Acorus calamus: Acorus calamus), Alpinia galanga (Zingiberaceae: Zingiberales), Curcuma longa (Zingiberaceae: Zingiberales), Piper nigrum (Piperaceae: Piperales), Piper retrofractum (Piperaceae: Piperales), and Sphagneticola trilobata (Asteraceae: Asterales), on S. litura control. These plant species are well known for their natural properties in the Thai traditional system of medicine or growth as a weed and have been reported to possess numerous types of biological activities.

Numerous anti-insect properties of the six plants involve toxic effects against many insects. Lee et al. [14] demonstrated the insecticidal effect of the methanol extract of C. longa rhizome on Plutella xylostella larvae. The hexane and ethanol extracts of rhizomes of A. galanga exhibit insecticidal activity against the fruit fly Bactrocera dorsalis when applied using a direct spray technique [15]. The essential rhizome oil of A. calamus showed a sterilizing effect against the eggs of Sitophilus granarius, Sitophilus oryzae, and Culiosobruchus chinensis [16] and larvicidal activity against Culex quinquefasciatus [17]. Upadhyay and Jaiswal found that 0.2 μl of P. nigrum oil significantly repelled Tribolium castaneum [18]. The dichloromethane extract of P. nigrum has pesticidal activity against Callosobruchus maculates and Sitophilus zeamais [19]. In addition to P. nigrum, P. retrofractum, which is in the same genus, also showed larvicidal activity against mosquito larvae C. quinquefasciatus [20]. Moreover, S. trilobata crude extracts have a larvicidal effect on S. litura, S. exigua, and P. xylostella larvae after topical application [21].

However, there is no research on the extract efficiency of the binary mixture in S. litura. The literature suggests that complex mixtures would be more effective than pure or only one crude extract [12, 13]. Thus, this research has the main goal of producing a plant-based mixture for control of S. litura with the possibility of increasing the control efficiency. Additionally, detoxification enzyme activities on treated S. litura were analyzed to search for the possibility of controlling this pest and examining the trends of resistance to this plant-based product in the future.

Plant extracts as sources of bioactive and therapeutic purposes have been used for thousands of years [27]. Plant extracts and their derivatives have been evaluated for different pest control properties, e.g., their toxicity, repellent, antifeedant, fumigation, and effect on oviposition activities [28].

In this study, six plant extract compounds were extracted by ethanol, and the goal is that the farmer can use this knowledge to develop insecticides to control S. litura by themselves, potentially reducing the cost for pest control. All crude extracts evaluated via topical application against second instar S. litura larvae revealed that the ethanolic extract of P. retrofractum had the highest control efficiency, followed by A. calamus (Table 2). P. retrofractum and A. calamus are used as alternative medicines and supplements in primary health care worldwide [29, 30]. They are less toxic to nontarget organisms and mammals. A previous study from Wiwattanawanichakun et al. showed that P. retrofractum was moderately toxic for guppy fish compared with the known data available for synthetic pesticides [31]. Moreover, the LD₅₀ values for oral doses of A. calamus extracts were greater than 5000 mg/kg body weight in Wistar rats [32] and 5070.59 mg/kg in mice [33].

Among the six plant extracts, P. retrofractum had the most potent extract with an LD₅₀ value of 5.575 µg/larva. Similar results were reported by Chansang et al. [20]. Among aqueous extracts of nine medicinal plants, P. retrofractum showed the highest level of activity against Cx. quinquefasciatus and Aedes aegypti (L.) larvae with LD₅₀ values of 135 and 79 µg/g, respectively. P. retrofractum exhibits insecticidal activity against many insect pests. It caused 100% mortality at a concentration of 0.5% against second instar Crocidolomia pavonana larvae [34]. This extract also exhibited a high knockdown effect on several test insects, including P. xylostella, C. pavonana, Cx. quinquefasciatus, Ae. aegypti, and Coptotermes gestroi [20, 35].

Another Piperaceae plant in this research is P. nigrum, which is also among the top three for S. litura control. Kumar et al. [36] revealed that the ethanolic extracts of black and white P. nigrum were 30–40% less toxic than the extracts of Piper longum against A. aegypti larvae. Additionally, a hexane extract showed a toxic effect against second instar larvae of S. litura with an LD₅₀ of 1824 µg/g insect [37].

Some reports described that the active compound extracted from the fruit part of P. retrofractum and P. nigrum is piperine [38, 39]. This compound also showed insecticidal activity against larvae of plant insects and antimicrobial activity [38]. However, in our results, piperine does not provide efficient control of S. litura. Our study assessed another new active ingredient from this piperaceae plant crude extract called (2E,4E,14Z)-N-isobutylicosa-2,4,14-trienamide.

The other crude extract that showed the highest S. litura control in our results is A. calamus. Many papers have been published on the biological activities of A. calamus. A total of 0.4% of ethanolic extracts yielded 63.3% mortality against third instar larvae of P. xylostella based on the leaf-dipping method [40]. Phongpaichit et al. [41] revealed that the crude methanol extract exhibited high antimicrobial activity on various microorganisms and fungi. The larvicidal activity of A. calamus is due to the presence of the primary chemical compound beta-asarone [17].

Schmidt and Streloke [42] investigated the chemical composition of A. calamus rhizome using beta-asarone as the major compound for control Prostephanus truncatus (Horn). Other chemicals investigated in A. calamus include ethyl isoeugenol, 3.9-decadien-ol-1,3-methyl-6-(1-methylethenyl), 4-pentyl-1-(4propylcyclohexyl)1cyclohexene, and alpha-asarone, which also has toxicity to insects, such as S. litura and Liposcelis bostrychophila, and other bioactivities [43–45].

Furthermore, synergistic effects of complex mixtures are thought to be important in plant defenses against herbivory. Plants usually present defenses based on a group of compounds and not individual compounds [13]. Although the highest mortality was noted with the P. retrofractum extract compared with other extracts at the same dose in our study, the combination of each extract in a binary assay produced stronger toxicity.

In this study, P. retrofractum synergized the toxicity of A. calamus at all doses. Among all combinations, the mixture of P. retrofractum + A. calamus (LD₃₀:LD₁₀) could be the chosen mixture for controlling this insect. Both combinations showed a synergistic effect and also exhibited higher antifeedant activity at 82.43% compared with others (Tables 4, 5).

Dadang et al. [46] reported the robust efficacy of a mixture of P. retrofractum with Annona squamosa and Aglaia odorata, which produced 100% and 94% mortality, respectively, in Crocidolomia pavonana after a 48-h treatment with a 0.05% extract mixture. The extract mixture of A. odorata and A. squamosa yielded a synergistic combination with multiple actions, such as feeding inhibition and insecticidal activity [46].

Insects have well-developed defense mechanisms against insecticides and natural pathogens that involve various enzyme systems. It is well known that herbivorous insects use detoxification enzymes, including EST, GST, and cytochrome P450 monooxygenases, to metabolize toxic chemicals and secondary plant metabolites [47]. However, these enzymes are also induced by xenobiotics as one of the mechanisms responsible for the development of resistance in insects [48]. The enzyme activities from in vivo treatment are presented in Table 5. GST activities in treated insects that survived after 24 h of exposure revealed a significant increase in GST in the P. retrofractum + A. calamus mixture at all combinations compared with the control group (Table 6). Conversely, EST activity showed no significant difference between groups (p ≥ 0.05, df = 7) (Table 6). Kaur et al. [49] reported that the induction of detoxification enzyme activities depended on both the duration of treatment and concentrations [50]. Specifically, a prolonged treatment and high dose showed a higher increase in enzyme activity. Therefore, the ratio and concentration of the mixed compounds are essential given that a suitable concentration could possibly affect detoxification enzymes.

An induction of detoxification enzyme activity has been reported by Zhou et al. [51], demonstrating that extracts from Illicium verum fruit induced EST activity in Myzus persicae and also increased EST activity in the gypsy moth Lymantria dispar after feeding on a diet of aspen leaves supplemented with phenolic glycosides [52]. A similar result was found in S. litura after they fed on Melia toosendan extract, demonstrating that midgut esterase activities were significantly increased after 24 and 48 h of feeding and decreased after 72 h [53].

The results demonstrated that ethanolic crude extracts of A. calamus L., A. galangal, C. longa L., P. nigrum, P. retrofractum, and S. trilobata caused toxicity in S. litura. Additionally, binary mixtures of P. retrofractum and A. calamus showed synergistic effects, suggesting that these mixtures could serve as an acute toxicant or antifeedant. In particular, the mixture of P. retrofractum + A. calamus (LD₃₀:LD₁₀) at this combination exhibited the highest antifeedant activity at 82.43%, showing synergistic contact toxicity effects and no significant differences in both EST and GST activities compared with controls. Therefore, the combination of these two compounds may constitute a useful alternative approach for the development of a binary mixture formulation to control lepidopteran pests for use in an IPM system.