Insecticide resistance in agricultural pests represents a major challenge to sustainable crop protection worldwide. Resistance can arise through multiple mechanisms, including target-site mutations, enhanced metabolic detoxification, reduced cuticular penetration, and behavioral avoidance (Jan et al., 2017; Kim et al., 2025). Among these, metabolic resistance mediated by cytochrome P450 monooxygenases (CYPs) is particularly widespread and has been identified as a dominant factor reducing insecticide efficacy in many pest species.

The beet armyworm (Spodoptera exigua) is a globally distributed and highly polyphagous pest, with resistance to diamide insecticides reported across Asia, the Americas, and other regions (Lai and Su, 2011; Huang et al., 2021). Numerous studies have demonstrated that CYP-mediated metabolic detoxification is a major mechanism underlying diamide resistance in S. exigua. Recent molecular and functional analysis identified CYP9A40 as a key enzyme contributing to chlorantraniliprole resistance through enhanced detoxification capacity (Han et al., 2024; Kim et al., 2025). In Korea, nationwide surveys in 2025 further confirmed the widespread establishment of diamide-resistant S. exigua populations, underscoring the urgency of developing resistance management strategies targeting metabolic mechanisms (Park et al., 2025).

Similarly, the cotton bollworm (Helicoverpa armigera) has evolved resistance to pyrethroid insecticides in numerous countries worldwide and is recognized as one of the most globally problematic lepidopteran pests (Walsh et al., 2018). Metabolic resistance mediated by CYP enzymes is considered the primary mechanism underlying pyrethroid resistance in this species (Joußen et al., 2012). A chimeric CYP gene CYP337B3, has been shown to confer resistance to pyrethroids such as fenvalerate and cypermethrin and is widely distributed in resistant populations across Asia, Africa, Australia, and South America (Walsh et al., 2018). The repeated emergence of multiple CYP337B3 alleles through independent recombination events highlights the strong and persistent selection pressure imposed by pyrethroid use (Joußen et al., 2012; Xu et al., 2016; Walsh et al., 2018).

While the development of new insecticides is often proposed as a solution to resistance, this approach is limited by high costs, long development timelines, and the rapid emergence of resistance to newly introduced compounds (Sparks 2013; Nishimoto 2019). These limitations emphasize the need for alternative and complementary strategies that enhance the efficacy of existing insecticides while reducing further selection pressure.

Azole fungicides, including both imidazoles and triazoles, are well-established inhibitors of CYP enzymes and have attracted increasing attention as potential insecticide synergists. These compounds inhibit monooxygenase activity by coordinating with the heme iron at the catalytic center of CYP enzymes, thereby suppressing detoxification pathways and enhancing insecticide toxicity (Balding et al., 2008). Imidazole fungicides, such as imazalil, have long been recognized for their strong CYP-inhibitory properties; however, their broader toxicity profiles and regulatory restrictions limit their practical applicability in integrated pest management programs (Dalhoff et al., 2016; Gottardi and Cedergreen, 2019).

In contrast, triazole fungicides have been widely adopted in agriculture due to their favorable environmental and toxicological profiles, including low acute toxicity to non-target organisms, reduced mammalian toxicity, and relatively rapid environmental degradation compared with many insecticides (Balding et al., 2008, Lewis et al., 2016). Among triazoles, propiconazole, hexaconazole, and metconazole are extensively used worldwide, possess well-characterized modes of action, and have been reported to inhibit CYP activity in both fungi and insects, making them suitable candidates for evaluating synergistic interactions with insecticides (Johnson et al., 2013, Haas and Nauen, 2021). Importantly, these compounds exhibit negligible insecticidal activity when applied alone, reducing the risk of direct selection pressure on insect populations while offering potential utility as resistance-modifying agents.

Within this context, the present study evaluated the synergistic effects of selected azole fungicides—propiconazole, hexaconazole, and metconazole—in combination with chlorantraniliprole and deltamethrin against S. exigua and H. armigera, respectively. By assessing larval mortality following co-application treatments, we aimed to determine whether CYP inhibition by azole fungicides could be exploited as a practical and environmentally compatible component of insecticide resistance management strategies for controlling diamide- and pyrethroidresistant populations.

Materials and Methods

Insects

Field populations of S. exigua were collected in 2025 from three agricultural regions in Korea (Nonsan, Haenam, and Gimjae), where reduced susceptibility to diamide insecticides had been previously reported (Park et al., 2025). The susceptibility of these populations to chlorantraniliprole was first evaluated using diet-based bioassays, and toxicity parameters (LC₅₀ and LC₉₀ values with 95% confidence intervals) were estimated by probit analysis (Table 1). All tested field populations exhibited markedly reduced susceptibility to chlorantraniliprole compared with the susceptible strain, confirming the presence of a diamide-resistant phenotype.

To establish a representative resistant strain for subsequent synergism experiments, individuals from all field populations were pooled and mass-mated. This pooled population was maintained under laboratory conditions and used as a diamideresistant strain. Bioassays were conducted using the first (F₁) and second (F₂) laboratory generations derived from this strain to minimize field-related variability while preserving resistance characteristics.

In addition, a laboratory-maintained pyrethroid-resistant strain of H. armigera was included in this study and reared under controlled conditions as previously described (Kwon 2025). All insect colonies were maintained at 25 ± 3°C with 50 ± 10% relative humidity under a 16:8 h (light:dark) photoperiod. Larvae were fed an artificial diet, and general rearing procedures followed established protocols (Han et al., 2023).

Bioassay

Two insecticides; chlorantraniliprole (4%, Wettable Powder, Nonghyup Chemical Co., Ltd.), and deltamethrin (1%, Emulsifiable Concentrate, Kyung Nong Co., Ltd.), and three azole fungicides; propiconazole (25%, Emulsifiable Concentrate, Syngenta Korea Co., Ltd.), hexaconazole (2%, Water-dispersible Granule, FarmHannong), and metconazole (20%, Suspension Concentrate, Dongbang Agro Corporation) were evaluated as commercial formulations obtained from the Korean domestic market. Piperonyl butoxide (PBO; technical grade, 90% purity; Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control synergist and dissolved in acetone prior to application.

Bioassays were conducted using a modified artificial diet– based method adapted from IRAC Method 020. Artificial diet blocks were treated using a dip-based surface application procedure, incorporating the dipping and surface-drying steps described in IRAC Method 007 prior to larval exposure, as previously reported (Kim et al., 2021). Third instar larvae were used in all bioassays.

For bioassays, insecticides such as chlorantraniliprole or deltamethrin was tested alone or in combination with azole fungicides, with 2 mM PBO pretreatment (1 ul per larva) followed by insecticide exposure serving as a positive control.

Mortality was assessed at 96 h post-treatment for chlorantraniliprole- based assays and at 72 h post-treatment for deltamethrin- based assays. All treatments were replicated three times using independent biological replicates.

Data analysis

Larval mortality was calculated as the proportion of dead individuals relative to the total number of treated larvae. Larvae were considered dead if they exhibited no feeding activity, no growth, and no response to tactile stimulation. Observed mortality values were used to evaluate synergistic effects among treatments.

Statistical analyses were performed using SAS software. Differences among treatments were analyzed using one-way analysis of variance, followed by Duncan’s multiple range test for post hoc comparisons. When multiple treatments were compared with a single control, Dennett’s test was applied. Statistical significance was determined at P < 0.05. Graphical representations were generated using Sigmaplot version 15.0.

Results

Synergistic effects of azole fungicides in diamideresistant S. exigua

Larval mortality in the diamide-resistant S. exigua strain remained low in both the acetone-only control and the chlorantraniliprole treatment at the recommended concentration (RC; 20 ppm), with no significant difference detected between the two groups (Fig. 1A). In contrast, pretreatment with the CYP inhibitor PBO followed by chlorantraniliprole exposure caused a marked increase in larval mortality, reaching approximately 80–85%, which was significantly higher than that observed in both the acetone control and chlorantraniliprolealone treatments (Duncan’s multiple range test, P < 0.05).

When propiconazole was applied alone, larval mortality remained low at all tested concentrations (83, 830, and 8,300 ppm), with no significant differences among treatments (Fig. 1B), indicating that propiconazole itself exerted little to no insecticidal activity against S. exigua larvae.

By contrast, co-application of chlorantraniliprole (20 ppm) with propiconazole resulted in clear concentration-dependent increases in larval mortality over time (Fig. 1C). At the lowest propiconazole concentration (83 ppm), mortality remained comparable to that observed with chlorantraniliprole alone throughout the observation period. However, co-treatment with the intermediate (830 ppm) and highest (8,300 ppm) propiconazole concentrations led to progressively greater mortality from day 1 through day 4. Notably, the highest concentration of propiconazole (8,300 ppm) significantly enhanced chlorantraniliprole toxicity, resulting in near-complete larval mortality by days 3 and 4. These mortality levels were statistically comparable to those observed in the PBO synergist treatment and were significantly higher than those in all control and lowerconcentration treatments (Duncan’s multiple range test, P < 0.05).

Synergistic effects of azole fungicides in pyrethroidresistant H. armigera

In the pyrethroid-resistant strain of H. armigera, co-application of deltamethrin at the recommended concentration (RC; 10 ppm) with the synergist PBO resulted in 100% larval mortality (Fig. 2A). By contrast, the acetone-only control showed a background mortality of approximately 40%, which was corrected using Abbott’s formula prior to statistical analysis. Deltamethrin applied alone caused only moderate mortality, further supporting the resistant phenotype of the tested population.

When deltamethrin was co-applied with propiconazole, larval mortality did not differ significantly from that observed with deltamethrin alone across the tested concentration range (83–8,300 ppm), suggesting that propiconazole did not exert a synergistic effect with deltamethrin in this strain (Fig. 2B).

In contrast, simultaneous treatment with deltamethrin and metconazole increased mortality to 83% at 6,700 ppm, corresponding to 100 times the recommended concentration. This was approximately twofold higher than the 43% mortality observed at either the recommended concentration (67 ppm) or 10 times the recommended concentration (670 ppm).

Hexaconazole likewise exhibited synergistic activity with deltamethrin. Co-treatment with hexaconazole significantly increased larval mortality at all tested concentrations (10, 100, and 1,000 ppm) relative to deltamethrin alone. Mortality at 100 and 1,000 ppm hexaconazole was comparable, indicating that the synergistic effect reached a plateau at higher concentrations. All statistically significant increases in mortality observed in the azole–deltamethrin co-treatments were confirmed by Dunnett’s test (P < 0.05).

Discussion

Diamide and pyrethroid insecticides remain cornerstone tools for the control of major lepidopteran pests, including S. exigua and H. armigera. However, resistance to both chemical classes has been widely reported and is now recognized as a major threat to sustainable pest management. In Korea, resistance to diamide insecticides such as chlorantraniliprole has been documented in multiple field populations of S. exigua, with resistance levels increasing rapidly in recent years (Cho et al., 2018, Park et al., 2021). Similarly, pyrethroid resistance in H. armigera has been reported globally and is primarily associated with enhanced CYP–mediated detoxification (Walsh et al., 2018). Within this context, the present study evaluated whether azole fungicides could synergistically enhance the toxicity of chlorantraniliprole and deltamethrin in resistant populations of S. exigua and H. armigera, respectively.

In diamide-resistant S. exigua, chlorantraniliprole applied at the RC caused mortality comparable to that of the solvent control, confirming a strong resistance phenotype (Fig. 1A). In contrast, pretreatment with the broad-spectrum CYP inhibitor PBO restored chlorantraniliprole toxicity, resulting in significantly elevated mortality. This result provides strong evidence that metabolic detoxification mediated by CYP enzymes plays a major role in diamide resistance in these populations, consistent with previous molecular and transcriptional studies reporting overexpression of CYP genes, including CYP9 family members, in resistant S. exigua (Han et al., 2024).

Propiconazole alone exhibited negligible insecticidal activity across all tested concentrations (Fig. 1B), indicating that its effects in combination treatments were not attributable to direct toxicity. However, co-application of propiconazole with chlorantraniliprole resulted in clear, concentration-dependent increases in larval mortality over time (Fig. 1C). At higher concentrations, propiconazole restored chlorantraniliprole efficacy to levels comparable to those achieved with PBO, suggesting that propiconazole can partially inhibit CYP-mediated detoxification pathways involved in diamide resistance. The absence of synergistic effects at the lowest concentration indicates that a threshold level of CYP inhibition may be required to overcome resistance, a phenomenon previously reported for azole–insecticide interactions and mixture toxicity scenarios (Johnson et al., 2013).

In contrast to S. exigua, a different pattern of synergistic interaction was observed in the pyrethroid-resistant H. armigera strain. Deltamethrin alone produced moderate mortality, confirming the resistant phenotype, whereas PBO pretreatment resulted in complete larval mortality (Fig. 2A). This finding aligns with extensive evidence demonstrating that pyrethroid resistance in H. armigera is predominantly mediated by CYP enzymes, particularly the chimeric gene CYP337B3, which confers resistance to multiple pyrethroids and is widely distributed across resistant populations worldwide (Walsh et al., 2018).

Among the azole fungicides tested, propiconazole did not significantly enhance deltamethrin toxicity at any concentration (Fig. 2B), despite its clear synergistic effect in S. exigua. This species-specific response suggests that propiconazole does not effectively inhibit the CYP isoforms responsible for pyrethroid detoxification in H. armigera. In contrast, hexaconazole showed strong and consistent synergistic effects with deltamethrin across all tested concentrations, while metconazole exhibited a significant effect only at the highest concentration. These differences highlight the compound-specific nature of azole– CYP interactions and suggest that different azole fungicides vary in their affinity for distinct CYP enzymes.

The species- and compound-specific synergistic patterns observed in this study provide critical mechanistic support for the role of CYP-mediated detoxification in both diamide- and pyrethroid-resistant phenotypes. In both S. exigua and H. armigera, pretreatment with the broad-spectrum CYP inhibitor PBO resulted in a pronounced restoration of insecticide susceptibility (Figs. 1A and 2A), confirming that CYP enzymes constitute a dominant resistance mechanism in these populations. In contrast, azole fungicides displayed differential and selective synergistic effects that varied not only between insect species but also among individual compounds, indicating that they inhibit only a subset of CYP isoforms involved in insecticide metabolism. In S. exigua, propiconazole effectively enhanced chlorantraniliprole toxicity (Fig. 1C), consistent with inhibition of CYPs associated with diamide detoxification, whereas in H. armigera the same compound failed to synergize deltamethrin (Fig. 2B), suggesting limited activity against CYP isoforms such as CYP337B3 that are central to pyrethroid resistance. Conversely, hexaconazole exhibited strong synergistic effects with deltamethrin across all tested concentrations (Fig. 2B), highlighting compound-specific differences in CYP affinity and inhibitory capacity. These findings align with previous reports demonstrating substantial interspecific variation in CYP expression profiles, substrate selectivity, and inhibitor sensitivity, particularly in resistant lepidopteran pests (Feyereisen, 2012, Haas and Nauen, 2021, Haas et al., 2022, Han et al., 2024).

Importantly, while the mechanistic evidence for synergism is robust, the practical relevance of these interactions must be carefully considered. In the present study, significant enhancement of insecticide toxicity was primarily observed at relatively high concentrations of azole fungicides (e.g., 8300 ppm of propiconazole and 6700 ppm of metconazole), which exceed typical field application rates. This suggests that the direct implementation of such high-dose azole–insecticide combinations may have limited feasibility in real-world agricultural systems due to economic, regulatory, and environmental constraints.

However, these findings remain relevant when interpreted in the context of agricultural practices where fungicides and insecticides are frequently co-applied. In intensive cropping systems, repeated fungicide applications, tank mixtures, or environmental persistence may result in cumulative or sublethal exposure levels that could partially inhibit CYP-mediated detoxification. Although such effects may be weaker than those observed under laboratory conditions, they could still contribute to variability in insecticide performance or influence resistance dynamics over time. From an integrated pest management and insecticide resistance management perspective, the present findings suggest that azole fungicides may function as selective modulators of detoxification enzymes rather than direct synergists under field conditions. The strong synergistic effects observed at high concentrations provide proof-of-concept evidence that CYPmediated resistance can be suppressed, thereby offering valuable insight for the development of more potent and selective synergists. Unlike the broad-spectrum inhibitor PBO, azole fungicides exhibit compound-specific interactions with CYP isoforms, indicating potential for the design of targeted inhibitors with improved efficacy at lower, field-relevant concentrations.

At the same time, the use of pesticide mixtures must be approached with caution. Previous studies have highlighted potential risks associated with combined pesticide exposure, including unintended effects on non-target organisms and environmental safety (Haas and Nauen, 2021). Therefore, the application of azole–insecticide combinations should be guided by a mechanistic understanding of their interactions and evaluated under realistic field conditions.

Future studies should focus on identifying the specific CYP isoforms inhibited by individual azole fungicides through protein-level approaches such as heterologous expression and enzyme inhibition assays. In addition, field-based and semifield evaluations will be necessary to determine whether the synergistic effects observed under laboratory conditions translate into practical benefits without increasing ecological risks or accelerating resistance evolution.

Overall, this study provides mechanistic and comparative insights into how azole fungicides interact with diamide and pyrethroid insecticides in resistant S. exigua and H. armigera. While the direct application of high-concentration mixtures may be l imited i n practice, t he r esults o ffer a v aluable foundation for understanding CYP-mediated resistance and for guiding the rational development of improved resistance management strategies.

Conclusion

This study demonstrates that azole fungicides can act as effective synergists of diamide and pyrethroid insecticides in resistant populations of S. exigua and H. armigera, respectively, primarily through interference with CYP–mediated detoxification. In diamide-resistant S. exigua field populations from Korea, co-application of propiconazole significantly enhanced the toxicity of chlorantraniliprole in a concentration-dependent manner, restoring larval mortality to levels comparable with those achieved using the established CYP inhibitor piperonyl butoxide. In pyrethroid-resistant H. armigera, strong synergistic effects were observed with hexaconazole and, to a lesser extent, metconazole in combination with deltamethrin, whereas propiconazole showed no appreciable effect, highlighting pronounced species- and compound-specific interactions.

The contrasting responses observed between insect species and azole fungicides underscore the selective nature of CYP inhibition and reflect differences in detoxification pathways underlying resistance to diamide and pyrethroid insecticides. Importantly, azole fungicides alone exhibited negligible insecticidal activity, indicating that their utility lies not as standalone control agents but as auxiliary synergists capable of enhancing insecticide efficacy under specific resistance scenarios.

Overall, these findings provide mechanistic support for the involvement of CYP-mediated metabolism in resistance to both diamide and pyrethroid insecticides and suggest that carefully selected azole fungicides may contribute to resistance mitigation strategies when used judiciously. Further studies focusing on CYP–azole interactions at the molecular level, as well as evaluations of ecological safety and field performance, will be essential before such combinations can be incorporated into practical insecticide resistance management programs.