Laboratory Evaluation of the Combine Usage Possibilities of Entomopathogenic Nematodes with Insecticides against Mediterranean Corn Borer Sesamia nonagrioides (Lefebvre)

Abstract

The Mediterranean Corn Borer (MCB), Sesamia nonagrioides, poses a significant threat to maize crops, necessitating effective pest management strategies. This study investigates the compatibility of two entomopathogenic nematode (EPN) isolates, Steinernema feltiae KV6 and Heterorhabditis bacteriophora EO7, with four registered insecticides for MCB control: deltamethrin, flubendiamide, spinetoram, and betacyfluthrin. The impact of these insecticides on EPN mortality, infectivity, and reproduction was assessed. Results indicate that deltamethrin exhibits the lowest toxicity to EPNs, with mortality rates of 1.3% for S. feltiae and 0.63% for H. bacteriophora at field dose (FD) after 24 h and 4.63% and 1.96%, respectively, after 48 h. In contrast, betacyfluthrin showed higher toxicity, with mortality rates of 38.04% and 14.17% for S. feltiae at 2FD and FD after 48 h. The infectivity assays demonstrated that deltamethrin-treated EPNs caused up to 100% mortality in MCB larvae, while the reproduction capacity varied significantly between the EPN species and insecticides. H. bacteriophora exhibited higher progeny production, especially in the presence of deltamethrin (87,900 IJs/larva). The findings suggest that integrating EPNs with selective insecticides like deltamethrin can enhance pest control efficacy and support sustainable agricultural practices. This study provides valuable insights for developing integrated pest management (IPM) strategies aimed at mitigating MCB infestations in maize while minimizing environmental impacts.

1. Introduction

Corn (Zea mays L.) is one of the most important cereal crops globally, yielding higher productivity per unit area compared to other cereals like wheat and barley [1]. In the Mediterranean region, corn ranks as the second most significant agricultural product; however, its production is threatened by the Mediterranean Corn Borer (MCB), Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae) [2,3,4,5]. This pest exhibits a polyphagous nature, infesting a variety of host plants, including corn, sorghum, millet, rice, sugar cane, grasses, melon, asparagus, palms, and bananas [6,7]. Originating from the Mediterranean region, the MCB has spread extensively across Europe, North Africa, and the Middle East due to its adaptability to different climates and broad host range [2,8].

Studies have shown that MCB infestations can cause significant yield losses, particularly in late and second crop maize productions, with potential losses reaching up to 100% if not adequately controlled [4]. Chemical insecticides are often recommended and applied multiple times throughout the growing season to manage this pest; however, even with intensive insecticide use, yield losses can still exceed 30% during severe outbreaks [9]. Additionally, MCB infestations lead to secondary issues such as fungal infections at feeding sites, further compromising crop quality and safety [10]. Despite the implementation of various control methods, including the frequent use of chemical insecticides, damage from MCB remains pervasive. However, chemical insecticides, while effective in reducing pest populations, pose significant threats to the environment and human health.

Entomopathogenic nematodes (EPNs) are soil-inhabiting organisms known for their capability to parasitize and eliminate insect pests [11,12,13], making them valuable biocontrol agents against MCB. These nematodes, particularly those from the genera Steinernema and Heterorhabditis, display efficient host-seeking behavior through their infective juvenile stage, which actively locates and invades insect hosts [14,15]. Upon entering the host, EPNs release symbiotic bacteria (e.g., Xenorhabdus spp. for Steinernema and Photorhabdus spp. for Heterorhabditis), which proliferate, produce toxins, and ultimately cause insect death by septicemia within 24–48 h [16]. EPNs’ effectiveness in targeting soil-dwelling stages of pests, such as the larvae of the MCB, has been well documented [17,18,19,20]. Their use is particularly advantageous in integrated pest management (IPM) programs due to their minimal impact on non-target organisms, including beneficial arthropods and soil microbiota, and their compatibility with organic farming practices [21,22]. Additionally, EPNs’ resilience to various environmental conditions enhances their potential as a sustainable and reliable pest control method [23].

Recent studies have also highlighted the synergistic potential of combining EPNs with other biocontrol agents or compatible chemical insecticides, which can lead to improved pest management outcomes while reducing the reliance on conventional pesticides [24,25]. Such integrated approaches not only improve the efficacy of pest control strategies but also align with the increasing demand for environmentally sustainable agricultural practices [26,27]. The integration of entomopathogenic nematodes (EPNs) with chemical insecticides offers a potentially synergistic approach to enhancing pest control efficacy while reducing reliance on conventional pesticides. Numerous studies have investigated the compatibility of EPNs with various chemical agents, including pesticides [12,13,28,29,30,31,32,33,34,35,36,37,38,39], fertilizers, and microbial control agents.

The findings from these studies have been mixed, with some indicating negative effects of various pesticides on nematode infectivity and survival, while others have reported synergistic behavior that enhances pest control [12,40]. The susceptibility of infective juveniles (IJs) of EPNs to these chemical agents can vary widely depending on several factors, including the species and strain of the nematode, the application method and dose of the pesticide, the timing of application, and environmental conditions [13]. For instance, it has been demonstrated that certain chemical insecticides could be combined with EPNs without compromising their effectiveness [24], while some other studies have highlighted the adverse impacts of some pesticides on EPN survival and performance [13,28,38]. These varying outcomes underscore the importance of understanding specific interactions between EPNs and pesticides to optimize their combined use.

Incorporating EPNs into integrated pest management (IPM) programs allows for more sustainable and environmentally friendly pest control strategies. This combined approach can reduce the application rates of chemical insecticides, thereby mitigating environmental contamination risks and minimizing the development of insecticide resistance [12,34]. Furthermore, the use of EPNs supports the principles of IPM by promoting biological control methods and reducing the ecological footprint of pest management practices [26,27]. By leveraging the synergistic potential of EPNs and chemical insecticides, farmers can achieve effective pest control while supporting sustainable agricultural practices.

This study aims to investigate the compatibility of two EPN species, Steinernema feltiae and Heterorhabditis bacteriophora, with four commonly used insecticides: deltamethrin, flubendiamide, spinetoram, and betacyfluthrin. By assessing the impact of these insecticides on EPN mortality, infectivity, and reproduction, this research seeks to identify combinations that maximize pest control efficacy while minimizing adverse effects on non-target organisms and the environment. The findings will contribute to the development of more effective and sustainable IPM strategies for managing MCB infestations in maize crops.

2. Materials and Methods

2.1. Entomopathogenic Nematodes

Steinernema feltiae KV6 and Heterorhabditis bacteriophora EO7 isolates were used in the study (

Table 1. Entomopathogenic nematodes were used in the study.

Isolate	Accession Number	Reference
Steinernema feltiae KV6	MN853593	[13]
Heterorhabditis bacteriophora EO7	MN853594	[38]

).

Nematodes were cultivated in the final instar larvae of the greater wax moth, G. mellonella (L.) (Lepidoptera: Pyralidae), at 25 °C, following the method described by [41]. The collected infective juveniles (IJs) from modified White’s traps were kept in Ringer solution in culture flasks at 10–12 °C for up to one week, with a density of 3000 IJ/mL. Prior to the experiments, the viability of EPNs was verified, and only stocks with over 95% viability were utilized [12].

2.2. Collection and Identification of MCB Larvae

MCB larvae were collected from infested corn fields (37°18′9125″ N, 39°73′0863″ W; 37°19′7926″ N, 39°65′9425″ W; 37°22′9385″ N, 39°70′3475″ W; and 37°24′7937″ N, 39°71′0127″ W) in Şanlıurfa, Türkiye. The stems were dissected to remove larvae, which were placed in perforated plastic boxes (HS-020, Hi-Paş Plastik Eşya Tic. Ve San. Ltd. Şti, Istanbul, Türkiye) for airflow and provided with fresh corn stems as food. These boxes were then transported to the laboratory. Healthy larvae were selected after two days of feeding under a 12:12 photoperiod at room temperature for bioassays.

To extract genomic DNA, the GeneMATRIX tissue and bacterial DNA purification kit (EURx Ltd., Gdańsk, Poland) was employed. The extracted DNA was stored at −20 °C until required for amplification. The PCR primers used were LCO1490 (5-GGTCAACAAATCATAAAGATATTGG-3) and HCO2198 (5-AAACTTCAGGGTGACCAAAAAATCA-3) [42]. The PCR amplifcation protocol consisted of an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of denaturation at 94 °C for 1 min, annealing at 60 °C for 1 min, extension at 72 °C for 1 min, and a final extension step at 72 °C for 5 min. PCR products were verified by electrophoresis and sequenced by Macrogen, Inc. (Republic of Korea), with sequences deposited in GenBank (Accession numbers: PP504953 and PP504954).

2.3. Pesticides Used in Compatibility Bioassays

The insecticides used in this study are listed in

Table 2. Insecticides used in this study.

Active İngredient	Commercial Name	Formulation *	Field Dose (mL/da)	Chemical Group	Mode of Action
25% Spinetoram	Delegate®	WG	20	Spinosyns	Nicotinic acetylcholine receptor (nAChR) allosteric modulators
25 g/L Deltamethrin	Decis®	EC	50	Pyrethroids	Sodium channel modulators
222 g/L Flubendiamide	Tunga®	SC	30	Diamides	Ryanodine receptor modulators
25 g/L Betacyfluthrin	Betagard®	EC	75	Pyrethroids	Sodium channel modulators

* Stock solutions for all pesticides were prepared using their commercial formulations, following the recommended field dose (FD), as well as double the recommended dose (2FD) and half the recommended dose (FD/2) according to the product labels.

. These insecticides were selected based on their common usage in cornfields across Turkey, as confirmed through personal communications with representatives from both the Ministry of Agriculture and Forestry of Turkey and private-sector companies.

2.4. Effect of Pesticides on the Survival of EPNs

Insecticide solutions were prepared by diluting them with distilled water in 50 mL beakers (ISOLAB, ISOLAB Laborgerate GmbH, Germany). For each pesticide, 2 mL of the solution was dispensed into 35 mm diameter Petri dishes (ISOLAB, ISOLAB Laborgerate GmbH, Germany), achieving final concentrations that matched the field dose (FD), half the field dose (FD/2), and double the field dose (2FD). Each Petri dish received 100 infective juveniles (IJs) of each nematode species, suspended in 10 µL of distilled water. Control groups were treated with distilled water only. The pesticide-treated IJs were then incubated in a shaking incubator (Biosan ES-20/60, SIA Biosan, Riga, Latvia) set to 150 rpm in a dark environment at 25 ± 2 °C for durations of 24 and 48 h. After incubation, the nematodes were examined under a stereomicroscope (Olympus SZ61, Olympus Corporation, Tokyo, Japan) to count the live and dead IJs. Nematodes were classified as dead if they did not respond to mechanical probing [13]. This experiment was conducted with ten biological replicates and two technical replicates for each species of nematode.

2.5. Effect of Pesticides on the Infectivity of EPNs

The infectivity of infective juveniles (IJs) treated with pesticides was assessed using last-instar MCB larvae as hosts. Pesticide solutions were prepared at field dose concentrations in 10 mL volumes within 50 mL flasks (ISOLAB, ISOLAB Laborgerate GmbH, Germany). Following the introduction of EPNs, the flasks were placed in an orbital shaker set to 26 °C [34]. After a 48 h exposure period, the nematodes were filtered using a 20-micron sieve and then transferred to Falcon tubes (ISOLAB, ISOLAB Laborgerate GmbH, Germany) containing distilled water. The supernatant was removed after 30 min, and this rinsing process was repeated three times to ensure thorough removal of pesticides. After the final rinse, 150 IJs were suspended in 100 µL of distilled water and applied to 35 mm diameter Petri dishes (ISOLAB, ISOLAB Laborgerate GmbH, Germany) lined with filter paper. Each Petri dish then received a single last instar MCB larva and was incubated at 25 ± 1 °C. After 24 and 48 h, the larvae were examined for mortality, and the cadavers were dissected under a stereomicroscope (Olympus SZ61, Olympus Corporation, Tokyo, Japan) to confirm the presence of nematodes. This experiment included twelve biological replicates and three technical replicates.

2.6. Effect of Pesticides on the Reproduction Capacity of EPNs

The reproductive capacity of nematodes for each species was determined following the method used for assessing the impact of pesticides on infectivity. Upon observing the death of MCB larvae, they were promptly transferred to White’s traps and incubated at 25 ± 1 °C in dark conditions for a duration of 10 days. After this incubation period, the total number of infective juveniles (IJs) that emerged from each larva was counted [13]. This experiment was carried out with six biological replicates and two technical replicates for each nematode species.

2.7. Statistical Analysis

The data were initially converted to percent mortality and then transformed using the arcsine (n′ = arcsin√n) method before being subjected to ANOVA. Variance analysis was conducted on the transformed data, and treatment differences were evaluated using Tukey’s multiple comparison tests (p < 0.05). All statistical analyses were performed using the MINITAB^® Release 21 software package. The survival assay data were analyzed with one-way ANOVAs, with mean separation achieved through Tukey’s procedure at α = 0.05. For the infectivity assay, a two-way ANOVA was utilized, while the reproduction capacity assay was analyzed using a one-way ANOVA. The mean separations for these assays were also conducted using Tukey’s procedure with α = 0.05.

3. Results

3.1. Effect of Insecticides on Survival

The ANOVA table shows significant effects of the main factors (insecticide, EPN, time, and dose) and their interactions on nematode mortality rates (p < 0.01). This indicates that the type of insecticide, EPN, the duration of exposure, and the dose all significantly influence the mortality rates of the nematodes (

Table 3. ANOVA analysis of mortality rates for entomopathogenic nematodes (EPNs).

Source	DF	Adj SS	Adj MS	F-Value	p-Value
Insecticide (I)	3	4918.3	1639.43	354.93	<0.01 *
EPN (N)	1	2136.9	2136.95	462.64	<0.01 *
Time (T)	1	3905.1	3905.07	845.44	<0.01 *
Dose (D)	2	2080.1	1040.03	225.16	<0.01 *
IXN	3	3020.8	1006.95	218.00	<0.01 *
IXT	3	4765.0	1588.34	343.87	<0.01 *
IXD	6	2353.5	392.24	84.92	<0.01 *
NXT	1	1660.3	1660.32	359.45	<0.01 *
NXD	2	725.3	362.63	78.51	<0.01 *
TXD	2	1135.4	567.71	122.91	<0.01 *
IXNXT	3	3303.4	1101.15	238.40	<0.01 *
IXNXD	6	1871.1	311.85	67.51	<0.01 *
IXTX*D	6	2155.2	359.20	77.77	<0.01 *
NXTXD	2	716.8	358.38	77.59	<0.01 *
IX NXTXD	6	2032.1	338.69	73.32	<0.01 *
Error	1104	5099.4	4.62
Total	1151	41,878.7

*: significant at 0.01.

).

Table 4. Mortalities of EPNs (100 IJs) following insecticide treatment at 24 and 48 h post-exposure.

EPN Strain	Dose	Deltamethrin		Flubendiamide		Spinetoram		Betacyfluthrin
		24 h	48 h	24 h	48 h	24 h	48 h	24 h	48 h
H.b EO7	FD/2 1	0.17 ± 0.38 Ca 2	0.42 ± 0.58 Ca	0.13 ± 0.34 Bb	0.67 ± 0.64 Ba	0.58 ± 0.65 Aa	0.58 ± 0.72 Ba	0.38 ± 0.50 Bb	2.17 ± 1.24 Ca
	FD	0.63 ± 0.71 Ba	0.95 ± 1.08 Ba	0.38 ± 0.78 ABb	1.83 ± 0.92 Aa	0.63 ± 0.71 Ab	2.25 ± 1.07 Aa	0.67 ± 0.64 Bb	3.29 ± 1.23 Ba
	2FD	1.29 ± 1.04 Ab	1.96 ± 1.20 Aa	0.83 ± 0.92 Ab	2.5 ± 1.32 Aa	0.96 ± 0.86 Ab	2.67 ± 1.71 Aa	1.63 ± 1.25 Ab	4.83 ± 1.83 Aa
S.f KV6	FD/2	0.88 ± 1.40 Ab	2.33 ± 1.34 Ba	0.50 ± 0.66 Bb	1.33 ± 1.00 Ba	0.50 ± 0.59 Bb	1.50 ± 0.98 Ba	0.50 ± 0.66 Bb	7.25 ± 2.21 Ca
	FD	1.30 ± 1.12 Ab	4.63 ± 2.93 Aa	0.83 ± 0.81 ABb	2.33 ± 1.20 Aa	1.04 ± 1.00 Bb	2.20 ± 1.56 ABa	0.88 ± 1.00 ABb	14.17 ± 6.00 Ba
	2FD	1.70 ± 1.33 Ab	6.41 ± 3.59 Aa	1.08 ± 0.83 Ab	2.67 ± 1.50 Aa	1.75 ± 1.26 Ab	2.50 ± 1.29 Aa	1.42 ± 1.39 Ab	38.04 ± 10.51 Aa

¹ FD = Field dose. ² Capital letters among doses and lowercase letters among time in each insecticide are indicated as statistically different from each other (p < 0.05, Tukey test).

presents the mortality rates of EPNs treated with various pesticides 24 and 48 h after exposure. The data includes field dose (FD), half field dose (FD/2), and double field dose (2FD) of pesticides. Among the insecticides, the highest rate of nematode mortality for S. feltiae was observed in betacyfluthrin at 2FD at 48 h. A statistically significant difference in mortalities is observed between the doses at 24 h, with the highest mortality at 2FD for S. feltiae in deltamethrin. At 48 h, the mortality rate significantly increases for all doses of deltamethrin, indicating time-dependent toxicity. At 48 h after exposure, the mortality rates were still higher (p < 0.01) in deltamethrin. However, for H. bacteriophora, the lowest nematode mortality was observed in deltamethrin at 48 h for all application doses. Flubendiamide and spinetoram exhibited higher mortality rates, particularly at 48 h and higher doses, suggesting greater toxicity. At 24 h, the mortality rates caused by flubendiamide were relatively low across all doses, but at 48 h, there was a significant increase. This indicates a delayed toxicity effect of flubendiamide on EPNs. Spinetoram also showed significantly higher mortality rates at 48 h for both EPN isolates. Betacyfluthrin exhibits lower toxicity at 24 h but shows increased mortality at 48 h, for both nematode isolates.

3.2. Effect of Insecticides on Infectivity

Table 5. ANOVA results for the mortality rates of MCB infected with insecticide-treated EPNs.

Source	DF	Adj SS	Adj MS	F-Value	p-Value
Time (T)	1	21.852	21.8522	90.23	<0.01
Insecticide (I)	4	48.639	12.1598	50.21	<0.01
EPN (N)	1	0.164	0.1636	0.68	0.412
TXI	4	4.580	1.1449	4.73	<0.01
TXN	1	0.500	0.5000	2.06	0.152
IXN	4	10.182	2.5456	10.51	<0.01
TXIXN	4	1.428	0.3571	1.47	0.211
Error	220	53.279	0.2422

presents the data analysis of the combined results. Time and insecticide treatment significantly influence the mortality rates of Mediterranean Corn Borer (MCB) larvae infected with pesticide-treated EPNs.

The infectivity assays demonstrated that the infection rates for both species increased over time.

Deltamethrin showed the highest compatibility with both EPN species. At 24 h, H. bacteriophora treated with deltamethrin was slightly lower but not significantly different from the untreated control (

Table 6. Mortality rates (%) of MCB larvae with pesticide-treated EPN strains at 24 and 48 h after exposure (n = 100 IJs).

EPN Strain	Time	Deltamethrin		Flubendiamide		Spinetoram		Betacyfluthrin
		Treated	Untreated	Treated	Untreated	Treated	Untreated	Treated	Untreated
H.b EO7	24 h	90.00 ± 4.92 Ba *	95.83 ± 6.69 Aa	85.83 ± 5.43 Ba	91.67 ± 9.38 Aa	64.17 ± 9.96 Bb	95.83 ± 6.69 Aa	71.67 ± 11.15 Bb	95.83 ± 6.69 Aa
	48 h	96.67 ± 9.53 Aa	99.17 ± 2.89 Aa	99.17 ± 2.89 Aa	95.83 ± 6.69 Aa	77.50 ± 9.65 Ab	99.17 ± 2.89 Aa	87.50 ± 7.54 Ab	99.17 ± 2.89 Aa
S.f KV6	24 h	94.17 ± 5.15 Ba	80.00 ± 9.53 Bb	82.50 ± 12.88 Ba	80.00 ± 9.53 Ba	61.67 ± 11.15 Bb	80.00 ± 9.53 Ba	85.00 ± 5.22 Ba	80.00 ± 9.53 Ba
	48 h	100.00 ± 0.00 Aa	90.83 ± 9.00 Ab	94.17 ± 9.96 Aa	90.83 ± 9.00 Aa	84.17 ± 9.00 Aa	90.83 ± 9.00 Aa	96.67 ± 4.92 Aa	90.83 ± 9.00 Aa

* Capital letters among time and lowercase letters among insecticides in each time interval are indicated as statistically different from each other (p < 0.05, Tukey test).

). By 48 h, the mortality rate had increased, closely matching the untreated control. S. feltiae treated with deltamethrin exhibited a mortality rate at 24 h, significantly higher than the untreated control. At 48 h, the treated S. feltiae reached a 100% mortality rate, significantly higher than the untreated group. Flubendiamide also supported the good infectivity of EPNs. For H. bacteriophora, the flubendiamide-treated group showed a slightly lower mortality rate than the untreated control (p < 0.05). At 48 h, the treated group reached 99.17%, similar to the untreated group at 95.83%. The mortality rate of S. feltiae treated with flubendiamide was not significantly different from the untreated control at 24 h. At 48 h, the treated group showed a comparable mortality rate to the untreated group (p > 0.05). In contrast, spinetoram significantly reduced the infectivity of both EPN species. For H. bacteriophora, the treated group showed a significantly lower mortality rate at 24 h compared to the untreated control. At 48 h, the mortality rate for the treated group was still markedly lower than the untreated control. The mortality rate of S. feltiae treated with spinetoram was significantly lower than the untreated control at 24 h. At 48 h, the mortality rate of the spinetoram-treated group was still lower than that of the untreated control. Betacyfluthrin exhibited a similar negative effect on EPN infectivity. For H. bacteriophora, the mortality rate of the treated group was significantly lower than the untreated control at 24 h. At 48 h, the mortality rate for the treated group was again lower than the untreated control. S. feltiae treated with betacyfluthrin showed a mortality rate that was not significantly different from the untreated control at 24 h. However, at 48 h, the mortality rate of betacyfluthrin-treated S. feltiae was slightly higher than the untreated control at 90.83% (p > 0.05).

3.3. Effect of Pesticides on Reproduction Capacity

The progeny production capacity of H. bacteriophora and S. feltiae varies significantly depending on pesticide exposure (

Table 7. Progeny production capacity (Mean ± SE) of Heterorhabditis bacteriophora and Steinernema feltiae after 10 days of exposure to a field dose of insecticides.

	H.b EO7	S.f KV6
Deltamethrin	87,900 ± 21,655 Aa *	53,317 ± 34,032 Ba
Flubendiamide	60,292 ± 26,772 Aa	73,500 ± 54,173 Aa
Spinetoram	30,500 ± 27,649 Ab	53,500 ± 43,847 Aa
Betacyfluthrin	31,875 ± 19,649 Ab	43,583 ± 32,636 Aa
Control	74,862 ± 45,688 Aa	37,965 ± 29,245 Ba

* Capital letters among EPNs in each insecticide and lowercase letters among insecticides in each EPN’s isolates are indicated as statistically different from each other (p < 0.05, Tukey test).

). Deltamethrin and flubendiamide demonstrated the highest compatibility with H. bacteriophora when compared to spinetoram and betacyfluthrin (p < 0.05). In contrast, S. feltiae treated with deltamethrin is significantly lower than H. bacteriophora. S. feltiae treated with flubendiamide has the highest progeny production for this species among all treatments. But there are no statistically significant differences among the treatments. Spinetoram and betacyfluthrin had a significant negative impact on the reproductive capacity of H. bacteriophora (p < 0.05). However, S. feltiae treated with spinetoram produced 53,500 IJs per larva, indicating that spinetoram’s impact on S. feltiae is less detrimental compared to H. bacteriophora. This suggests species-specific effects, with S. feltiae being more resilient to spinetoram than H. bacteriophora. In the control (untreated) group, H. bacteriophora produced 74,862 IJs per larva, indicating high reproductive capacity in the absence of insecticides but still lower than the deltamethrin-treated group. S. feltiae in the control group produced 37,965 IJs per larva, lower than the insecticide-treated groups, indicating that untreated S. feltiae have a lower reproductive output compared to treated groups (p > 0.05).

4. Discussion

This study provides valuable insights into the interactions between entomopathogenic nematodes (EPNs) and various chemical insecticides in managing the MCB. The findings suggest that EPNs can be as effective as chemical insecticides under optimal conditions [43,44], and their combined use in integrated pest management (IPM) programs can be efficient in terms of time, effort, and cost [45]. The results indicate the potential for incorporating EPNs with insecticides in IPM strategies to optimize pest control and reduce environmental impacts.

This study showed that the type of insecticide used significantly influences EPN mortality rates. Among the tested insecticides, deltamethrin showed high compatibility with both EPN species, maintaining high infectivity and reproductive capacity. This observation is in line with recent studies that highlight the lower toxicity of pyrethroids, such as deltamethrin, on EPNs due to their specific action on insect nervous systems [30,34,46]. Deltamethrin, a pyrethroid, acts on sodium channels in nerve cells, leading to pest paralysis and death. Several authors have reported the non-toxic nature of various pyrethroids to EPNs [24,30,47,48]. Although Head et al. (2000) [49] noted that pyrethroids strongly influence EPN infectivity but not viability, Mráček (2010) [50] found minimal impact of pyrethroids on both mortality and infectivity of Steinernema feltiae, S. arenarium, and S. kraussei. Specifically, deltamethrin did not reduce EPN survival [34,46]. Conversely, flubendiamide and spinetoram exhibited higher toxicity, particularly after 48 h and at higher doses. The delayed toxicity effect of flubendiamide may be related to EPN resilience to immediate calcium disruption caused by ryanodine receptor modulators, but adverse effects were noted over prolonged exposure. El Roby et al. (2023) [51] evaluated the compatibility of EPNs Heterorhabditis bacteriophora (HP88) and Steinernema carpocapsae (AT4) with lambda-cyhalothrin and flubendiamide against Spodoptera frugiperda larvae, finding synergistic effects. Spinetoram, a spinosyn targeting nicotinic acetylcholine receptors, causing continuous activation and eventual paralysis, exhibited significantly higher mortality rates at 48 h, corroborating findings by De Nardo and Grewal (2003) [40] on the adverse impacts of certain insecticides on EPN performance. Betacyfluthrin, another pyrethroid acting on sodium channels, showed variability in toxicity, suggesting different EPN species have varying resistance levels. De Nardo and Grewal (2003) [40] and Özdemir et al. (2020) [13] highlighted the synergistic use of imidacloprid with EPNs to enhance pest control efficacy without significantly affecting nematode viability.

The infectivity assays demonstrated that deltamethrin had a minimal impact on nematode infectivity, preserving their ability to infect the host. This aligns with recent studies suggesting pyrethroids do not significantly hinder EPN infectivity [13,47,48,52]. Flubendiamide and spinetoram significantly reduced infectivity, likely due to their action on nematode physiology and behavior. Conversely, some chlorantraniliprole formulations were reported to have no adverse effects on EPN survival or infectivity [53]. In [54], synergistic or additive interactions were found when combining H. bacteriophora with chlorantraniliprole against Holotrichia oblita (Faldermann) (Coleoptera: Scarabaeidae) larvae, leading to faster larval mortality than EPNs or insecticides alone. It was observed that there were additive effects when combining flubendiamide with various EPNs for controlling Helicoverpa armigera [55]. Özdemir et al. (2021) [38] reported no adverse effects of chlorantraniliprole on the survival and infectivity of S. feltiae KV6 Turkish isolate used against Leptinotarsa decemlineata larvae. However, it was noted that there was high larval mortality when combining flubendiamide with H. indica [56]. Incompatibility of some EPNs with certain insecticides was also reported in various studies, suggesting careful selection is needed [55,56,57,58].

The progeny production capacity varied significantly between H. bacteriophora and S. Feltiae, depending on insecticide exposure. H. bacteriophora showed higher progeny production in the presence of deltamethrin, flubendiamide, and the control group, whereas S. feltiae exhibited higher progeny production when exposed to spinetoram and flubendiamide. This species-specific response underscores the importance of selecting appropriate EPN species for use with particular insecticides to maximize biocontrol efficacy. Rovesti and Deseö (1990) [32] and Özdemir et al. (2020) [13] reported varying effects of insecticides on EPN reproductive capacity, consistent with our results. It was reported that long-term exposure to sub-lethal doses of insecticides could adversely affect EPN reproductive capabilities, aligning with our findings that betacyfluthrin and spinetoram significantly influenced progeny production [29]. A study showed that sublethal effects on EPN reproduction vary widely depending on nematode species and the specific chemicals used, supporting the need for tailored pest management strategies [13].

The integration of EPNs with chemical insecticides offers a promising approach to reducing reliance on chemical controls alone, mitigating environmental contamination, and minimizing insecticide resistance development. Previous studies have highlighted the compatibility of EPNs with various agricultural practices and their minimal impact on non-target organisms. The findings of this study support the potential for EPNs to enhance the efficacy of chemical insecticides to control MCB, particularly when used strategically.

The varying responses of EPNs to different insecticides emphasize the need for careful selection and optimization of pest control strategies. For instance, the lower toxicity of deltamethrin and flubendiamide to EPNs suggests they could be effectively combined with EPNs without significantly impacting their survival and reproductive capabilities. Conversely, the higher toxicity of betacyfluthrin necessitates more cautious application to avoid detrimental effects on EPN populations.

5. Conclusions

This study provides valuable insights into the interactions between EPNs and chemical insecticides, highlighting the potential for their integrated use in IPM programs. The findings indicate that deltamethrin and flubendiamide are the most compatible insecticides, not adversely affecting the survival, infectivity, and reproduction of EPNs. However, the most important bottlenecks of this study are the variability in nematode susceptibility to different insecticides and the environmental conditions influencing the results. The complex interactions between EPNs and insecticides require further research to optimize these combinations and evaluate their long-term effectiveness and environmental impact under diverse field conditions. Future research should explore the field application of these findings and investigate the physiological mechanisms underlying EPNs’ resistance to certain insecticides. By leveraging the synergistic potential of EPNs and insecticides, sustainable and effective pest management solutions can be developed, contributing to the long-term health and productivity of agricultural systems.