Efficacy of Vitex trifolia Leaf Powder as a Sustainable Botanical Insecticide Against the Rice Weevil, Sitophilus oryzae (Coleoptera: Curculionidae)

Rice (Oryza sativa) is the primary staple food for most of the Indonesian population, and its demand continues to increase in line with population growth [1]. This increasing demand poses a threat to national food security [2]. One of the major pests of stored rice is Sitophilus oryzae (Coleoptera: Curculionidae), which causes significant post-harvest damage by boring into rice grains, resulting in breakage, powdering, and overall quality deterioration [3]. Therefore, effective control of S. oryzae is essential to minimize both quantitative and qualitative losses during storage [4].

Currently, the control of Sitophilus oryzae relies primarily on synthetic insecticides, which have adverse effects on human health, non-target organisms, and the environment. In addition, fumigation with phosphine and methyl bromide, commonly used in Indonesian rice storage facilities, may lead to residue accumulation in stored rice and the development of pest resistance [4]. Consequently, there is an urgent need for environmentally friendly alternatives, such as botanical insecticides [5].

Botanical insecticides have attracted increasing attention due to their biodegradability, lower mammalian toxicity, and multiple modes of action [6, 7]. Plant-derived materials contain diverse secondary metabolites, including terpenoids, sesquiterpenes, alkaloids, flavonoids, glycosides, and phenolic compounds, which may exert insecticidal, repellent, antifeedant, and growth-regulating effects [8, 9]. Unlike single-active synthetic insecticides, botanical products may act through multiple physiological pathways, thereby potentially reducing the likelihood of resistance development [10]. Bioactive compounds in V. trifolia (e.g., casticin, artemetin, and vitricin) and their mechanisms of action have been investigated, including reported acetylcholinesterase (AChE) inhibition [6, 8].

Vitex trifolia L., traditionally used by local farmers in Aceh to repel storage pests by sprinkling dried leaves in rice warehouses, has shown potential as a botanical insecticide [11]. The leaves of V. trifolia contain various bioactive compounds, including sesquiterpenes, terpenoids, alkaloids, glycosides, and flavonoids. These compounds are known to inhibit insect growth, disrupt nervous system function, and exhibit repellent activity [11]. Previous studies reported that V. trifolia leaf extract at a concentration of 20% repelled Sitophilus oryzae by 56.7% after 120 h, whereas a 1% concentration resulted in only 3.3% repellency after 24 h [12]. In powder form, 10 g/100 g rice reportedly repelled adults and caused adult mortality within 1 h of application, suggesting interference with the insect’s respiratory and nervous systems.

Given these promising preliminary findings, Vitex trifolia leaf powder represents a viable botanical insecticide candidate for sustainable pest management in stored rice systems. However, while liquid extracts of V. trifolia have shown repellent activity against S. oryzae [12], the efficacy of its powdered leaf form—a simpler, more accessible, and more practical formulation for smallholder farmers—has not been comprehensively evaluated. Specifically, the dose-dependent effects of V. trifolia leaf powder on multiple biological endpoints, including adult mortality, F1 progeny development and emergence, reproductive inhibition, sex ratio dynamics, and grain weight loss, remain largely uncharacterized. Addressing this gap is essential for developing evidence-based recommendations for the use of botanical insecticides as alternatives to synthetic chemicals in rice storage facilities. This study aimed to (1) determine the dose-dependent repellent and lethal effects of V. trifolia leaf powder against adult S. oryzae; (2) assess its impact on F1 progeny development, emergence, and sex ratio; and (3) quantify the resulting protection of rice grains from weight loss.

3.1 Percentage of repellent power of Sitophilus oryzae adults

Figure 2 shows that the mean repellency of adult Sitophilus oryzae following application of Vitex trifolia leaf powder differed significantly among treatments (F = 21.85; P < 0.01). The highest repellency was recorded at the 25 g dose (54.50%), whereas the lowest was observed at the 5 g dose (44.50%).

Figure 2. The insect repellent power of Sitophilus oryzae was observed in 2 days after application (DAA)

The repellent activity of Vitex trifolia leaf powder is associated with the presence of terpenoid compounds, particularly essential oils with fumigant properties. These compounds are reported to interfere with the nervous system of Sitophilus oryzae and emit odors that are unfavorable to insects, resulting in reduced activity and avoidance behavior. Increasing doses of V. trifolia leaf powder correspond to higher concentrations of bioactive compounds, thereby enhancing its effectiveness as a botanical insecticide.

Terpenoids present in V. trifolia leaves have been reported to exhibit toxic and repellent effects against various insect pests [12, 17]. Essential oils primarily exert their repellent action through the respiratory system, whereby inhaled volatile compounds diffuse throughout the insect body and disrupt neural function, potentially leading to mortality [12, 18]. Repellency is attributed to volatile terpenoids (e.g., sesquiterpenes in essential oils) acting as fumigants through olfactory disruption and interference with octopaminergic receptors [19].

3.2 Mortality of Sitophilus oryzae adults

The insecticidal activity of Vitex trifolia leaf powder against S. oryzae adults was dose- and time-dependent (Figure 3). Mortality across all treatment groups increased progressively from day 1 to day 9 after application (DAA). The highest application rate of 25 g exhibited the most pronounced effect, with mortality rising sharply from 27.50% on day 1 to 77.50% by day 9. This trend continued, reaching a peak of 82.50% between 10 and 11 DAA, after which it plateaued until the end of the observation period on day 15. The 20 g dose also showed substantial efficacy, achieving 52.50% mortality by day 9.

In contrast, lower doses resulted in considerably slower and lower mortality. By day 9, the 10 g and 5 g doses had only induced 40.00% and 30.00% mortality, respectively. Mortality at the 5 g dose remained consistently low, stabilizing at approximately 32.50% from day 10 onwards. The 15 g dose showed an intermediate effect, reaching 52.50% mortality by the end of the trial.

This response is likely influenced by the essential oil content of V. trifolia leaf powder, which functions as a repellent through the emission of strong odors that drive Sitophilus oryzae away from food sources. In addition, volatile compounds such as terpenoids and alkaloids act as respiratory toxins that can enter the insect body through the spiracles, disrupt nervous system function, and cause physiological tissue damage, ultimately leading to mortality. This finding is consistent with that of researcher [20], who reported that alkaloids and tannins act as antifeedants by reducing larval feeding activity, thereby decreasing nutrient intake. Such nutritional stress may be further exacerbated by saponins, which interfere with metabolic processes and induce fluid loss.

Figure 3. Mortality of Sitophilus oryzae adults following the application of Vitex trifolia leaf powder was observed at 1–15 days after application (DAA)

According to Ainun et al. [12], higher application doses result in stronger repellent effects on rice weevils, and prolonged exposure to essential oils increases insect migration away from food sources. Similarly, Utomo et al. [4] reported that increasing the dosage of botanical insecticides is directly proportional to their effects on target organisms, as higher amounts of active compounds enter the insect body. Comparable results have been observed with V. trifolia leaf extract, which caused increased mortality of Spodoptera litura larvae at 36 h after application.

It is noteworthy that mortality at the earlier observation points (5, 7, and 9 DAA) did not differ significantly among treatments (p > 0.05), despite clear numerical trends. This lack of statistical significance may be attributed to several factors: (1) the inherent high variability among replications in botanical insecticide bioassays, as the bioactive compound concentrations in plant-derived preparations are less uniform compared to synthetic formulations; (2) the volatile nature of essential oils, whose active compounds may evaporate at different rates across replications depending on micro-environmental conditions within each container; and (3) the relatively small sample size (10 adults per experimental unit), which may limit the statistical power to detect moderate treatment effects. However, the cumulative mortality data at days 10-15 revealed more pronounced dose-dependent differences, suggesting that the insecticidal effect of V. trifolia leaf powder requires sustained exposure for full expression. This time-dependent pattern is consistent with the mode of action of essential oil-based insecticides, which act primarily through chronic respiratory exposure and gradual disruption of neural and metabolic functions rather than acute contact toxicity. Furthermore, adult mortality is linked to alkaloids (e.g., vitricin) and saponins, which cause acetylcholinesterase (AChE) inhibition and cell membrane disruption, respectively [21].

3.3 Time to first F1 adult emergence of Sitophilus oryzae

As shown in Figure 4, the mean F1 adult emergence time of Sitophilus oryzae differed significantly among Vitex trifolia leaf powder treatments. Increasing application rates significantly delayed development to the adult stage. Developmental delay in F1 is associated with flavonoid glycosides (artemetin, casticin) that interfere with juvenile hormone and ecdysone-mediated developmental pathways [22]. The longest emergence time was recorded at the 25 g dose (42.32 days), whereas the shortest was observed at the 5 g dose (29.75 days).

Figure 4. The emergence time of F1 adults Sitophilus oryzae at 60 days after application (DAA) observations

Differences in adult emergence time indicate that increasing doses of Vitex trifolia leaf powder prolong the life cycle of Sitophilus oryzae. This effect may be associated with the activity of bioactive compounds, such as essential oils, flavonoids, and alkaloids, which are known to inhibit insect development within rice grains. Previous studies have reported that delayed adult emergence of Sitophilus zeamais is influenced by essential oil compounds and alkaloids, including dillapiole, myristicin, Z-carpacin, piperamidine, and constituents of clove flower essential oil (eugenol, caryophyllene, α-caryophyllene, and eugenol acetate), which disrupt insect reproductive systems [23]. Prolongation of the egg, larval, and pupal stages consequently increases the time required for adult emergence. This finding is consistent with Hikal et al. [10], who reported that essential oils exhibit repellent, growth-inhibitory, ovicidal, and oviposition-suppressing effects, thereby reducing the population growth rate of Sitophilus spp.

3.4 Number of F1 adults emerged

Based on Figure 5, the application of V. trifolia leaf powder resulted in a highly significant difference in the number of F1 adults S. oryzae emerging among treatments (F = 10.11; p < 0.01). The lowest number of F1 adults was recorded at a dose of 25 g, i.e., 36.67 individuals, while the highest number occurred at a dose of 5 g with an average of 60.50 individuals.

Figure 5. Number of F1 adults of S. oryzae appearing at 60 days after application (DAA) observation

The variation in the number of F1 adults s emerging between treatments is thought to be related to the activity of chemical compounds in the powdered leaves of the V. trifolia. The aroma of these compounds affects the physiological activity of S. oryzae, including nervous system disruption, decreased feeding activity, and reduced copulation frequency.

Ripoll-Aristizábal et al. [24] reported that the application of soursop leaf flour disrupted oviposition and reduced the hatchability of Callosobruchus analis beetle eggs on mung bean seeds. Azadirachtin, a terpenoid derivative with a pungent aroma, is known to have repellent and antioviposition activities, reduce fecundity, and inhibit insect growth and development [25]. also stated that antifeedant compounds can inhibit the copulation of female S. oryzae adults and reduce feeding activity, so that increasing the dose has implications for reducing the number of F1 adults that emerge. In addition, flavonoids and tannins have been reported to interfere with feed digestion by reducing the activity of protease and amylase enzymes, which in turn inhibits insect growth and development.

3.5 Percentage inhibition of F1 adult emergence

As shown in Figure 6, Vitex trifolia leaf powder significantly affected the inhibition rate (IR) of F1 adult emergence of Sitophilus oryzae (F = 12.15; p < 0.01). The highest reproductive inhibition was recorded at the 25 g dose (79.58%), whereas the lowest inhibition occurred at the 15 g dose (55.65%).

Figure 6. Average percentage inhibition of F1 adult emergence

This variation is likely associated with differences in the concentration of bioactive compounds present in V. trifolia leaf powder, particularly terpenoids, saponins, and alkaloids, which have been reported to suppress insect reproductive activity. The inhibition of reproduction is likely attributed to the combined action of terpenoids and phenolic compounds, which may cause oviposition deterrence and ovicidal effects [10].

This finding is consistent with the researchers [26, 27], who reported that low F1 adult emergence is influenced by the mortality of infesting adults. The applied materials are known to contain volatile compounds, including phenols, flavonoids, and saponins, which exhibit toxic effects against insect pests. According to researchers [28], inhibition of adult emergence may result from volatile compounds derived from botanical insecticides that induce mortality in the egg and larval stages of Sitophilus oryzae.

Essential oils contribute to the repellent activity against rice weevils; moreover, Abdellaoui et al. [29] reported that essential oils possess both repellent and toxic properties. The insecticidal activity of essential oils is dose-dependent, whereby sublethal concentrations primarily induce repellent responses, while higher doses reduce egg viability and oviposition success. In addition, flavonoids exhibit a wide range of biological activities; although commonly recognized for their antioxidant and anti-inflammatory properties [30], flavonoids have also been reported to exert toxic effects on insects [31]. Phenolic compounds within flavonoids contribute less to repellent activity due to their low volatility; however, they may negatively affect insect fitness by reducing fertility and lifespan.

3.6 Percentage grain weight loss

Based on Figure 7, the average percentage of rice weight loss caused by Sitophilus oryzae infestation did not differ significantly among treatments, although numerical variations were observed. The highest rice weight loss occurred at the 10 g dose (5.40%), followed by the 15 g dose (4.35%). The lowest rice weight loss was recorded at the 20 g dose (3.05%), closely followed by the 5 g dose (3.40%) and the 25 g dose (3.53%). The non-linear pattern across treatment doses, where the 5 g dose showed lower weight loss than higher doses such as 10 g and 15 g, may be attributable to inherent variability in individual insect feeding behavior and oviposition patterns among replications.

Figure 7. Average percentage of damage due to weight loss in rice

Rice weight loss was positively correlated with the population density of S. oryzae adults in each treatment. Higher adult populations resulted in greater levels of grain damage and weight loss, while treatments with lower adult populations exhibited reduced rice weight loss. This relationship indicates that the extent of rice damage is strongly influenced by the abundance of S. oryzae adults present during the storage period.

Sitophilus spp. utilize rice grains both as a food source and as an oviposition site by boring into the kernels to deposit eggs. Elimam et al. [17] reported that a reduction in pest population density is associated with a corresponding decrease in the percentage of damaged grains, which directly leads to lower rice weight loss. Reduced grain damage results in minimized quantitative losses because fewer kernels are physically destroyed during infestation.

Hidayati et al. [32] reported that rice grains with high nutrient content, relatively high moisture levels, and a softer texture provide favorable conditions for the growth and development of Sitophilus oryzae. Such characteristics create an optimal environment for feeding activity, oviposition, and successful completion of the insect’s life cycle within the grain. Consequently, food substrate suitability plays a crucial role in shaping population dynamics, as S. oryzae preferentially selects media with adequate nutritional quality to support survival and reproduction. Although S. oryzae requires nutrients throughout its life cycle, rice with high amylose content alone, in the absence of sufficient nutritional support, is insufficient to sustain long-term population growth.

3.7 Sex ratio of F1 adults of Sitophilus oryzae

The results of observations on the sex ratio due to the application of the V. trifolia leaf powder treatment can be seen in Table 2 below.

Based on Table 2, the aroma emitted by Vitex trifolia leaf powder appeared to influence the sex ratio of adult Sitophilus oryzae. At a dose of 10 g, the numbers of male and female adults were 231 and 41, respectively. In contrast, application of 25 g resulted in a markedly lower adult emergence, with 84 males and 23 females recorded. This effect is likely associated with the activity of bioactive compounds present in V. trifolia leaf powder, which interfere with the reproductive capacity of S. oryzae. These compounds may reduce nutrient intake and disrupt physiological processes during development, leading to impaired regeneration and an imbalance in the F1 sex ratio, characterized by a higher proportion of males than females.

Table 2. Sex ratio of S. oryzae adults due to the application of V. trifolia leaf powder

Hendrival and Melinda [33] reported that limited food resources can influence the sex ratio of Sitophilus zeamais adults, with suboptimal nutritional conditions tending to produce a higher proportion of males than females. Conversely, under conditions of optimal nutrient availability, the male-to-female ratio tends to be more balanced [34]. This pattern is further supported by researcher [35], who demonstrated that increased food availability is positively correlated with a higher number of female adults, whereas nutritional constraints result in male-biased populations.

From an ecological and pest management perspective, the observed male-biased sex ratio at higher doses of V. trifolia leaf powder has important implications for long-term population suppression of S. oryzae in storage. A reduction in the proportion of females directly limits the reproductive potential of the population, as fewer females are available for oviposition in subsequent generations. Combined with the observed reduction in total F1 progeny numbers and delayed adult emergence, these effects suggest that V. trifolia leaf powder exerts multi-level suppressive pressure on S. oryzae populations not only through direct adult mortality, but also through disruption of reproductive output and population sex structure. In practical stored-grain management, this means that even sublethal doses that do not achieve high acute mortality may still provide meaningful long-term protection by progressively reducing the reproductive capacity and growth rate of pest populations across successive storage cycles.

3.8 Comparative efficacy with other botanical powders

Although LC₅₀ and LC₉₀ values were not calculated in the present study due to the design using fixed application rates in a rice matrix, the mortality level achieved at 25 g/100 g rice (82.50% at day 10–15) indicates relatively strong insecticidal activity compared with several plant powders previously reported against S. oryzae.

For example, Annona squamosa (Annona) leaf powder has been reported to cause mortality levels of weevils of about 98.3% after the 8^th week [36]. Another study shows that the extract of Cymbopogon citratus root and leaves at 200 ppm cause 100% mortality of weevils after 24 h exposure [37]. The mortality levels of weevils observed in this study (82.50% after 11 days) are also comparatively high that typically reported as moderate suppression of F₁ emergence [38].

Importantly, the present study used crude leaf powder rather than solvent extracts or purified essential oils. Considering that crude botanical materials generally exhibit lower bioavailability than concentrated extracts, the mortality and reproductive suppression levels observed here suggest promising relative efficacy. Future studies incorporating probit analysis to determine LC₅₀ and LC₉₀ values would further strengthen comparative evaluation and allow standardized toxicity assessment.

The authors express their sincere gratitude and high appreciation to all parties who contributed to this research, particularly the Department of Plant Protection and the Plant Protection Laboratory, Faculty of Agriculture, Universitas Syiah Kuala, Banda Aceh.