Synergized Toxicity of Promising Plant Extracts and Synthetic Chemicals against Fall Armyworm Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae) in Pakistan

Abstract

Fall armyworm, Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae), is a destructive pest of a wide array of agricultural and horticultural crops worldwide. This in vitro research assessed the combined effect of methanolic extracts of indigenous flora of Soone Valley (Khushab, Pakistan) and nine commonly used synthetic insecticides against 3rd instar larvae of S. frugiperda using the leaf-dip bioassay method. Toxicity bioassays with twelve plant extracts revealed that the extracts of Withania somnifera (L.) Dunal, Sophora mollis (Royle) Baker and Rhazya stricta Decne. were the most effective, exhibiting minimum LC₅₀ and LT₅₀ values. Bioassays with synthetic insecticides revealed a significantly higher mortality of S. frugiperda larvae by emamectin benzoate (45%), chlorpyrifos (40%) and chlorantraniliprole (38%). Further bioassays with 10 binary combinations of these most effective botanical and synthetic insecticides showed that seven pesticidal combinations exhibited synergistic toxicity, and three combinations comprising emamectin benzoate exhibited an additive effect on the mortality of S. frugiperda larvae. GC–MS analyses of methanolic extracts of W. somnifera, S. mollis and R. stricta revealed 1,2,4-trimethyl-benzene and 3,5-dimethyl-octane, 1-ethyl-2-methyl-benzene, and 1-monolinoleoylglycerol trimethylsilyl ether, decane, and lupeol as major bioconstituents, respectively. Our results demonstrated that combining botanicals with synthetic insecticides can synergize their toxicity against S. frugiperda larvae, suggesting their potential incorporation into future IPM programs against S. frugiperda and other lepidopterous pests.

1. Introduction

The fall armyworm Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae) is a polyphagous pest of many agricultural and horticultural crops. It is native to the western tropical hemisphere and was recognized as a severe threat to farmers in West and Sub-Saharan Africa in 2016 [1,2]. Later, it was reported that these armyworms were infesting maize crops in China and India in May 2018 [3,4]. In March 2019, this exotic species was reported from various localities in Sindh Province, Pakistan, where they were damaging maize crops [5]. Spodoptera frugiperda is a polyphagous pest that infests a wide array of host plants, comprising approximately of 350 plant species from 76 families, including maize, sorghum, millet, wheat, sugarcane and vegetable crops. Maize and cabbage are the most vulnerable crops to S. frugiperda infestation worldwide [6]. Loss of these crops causes an economic loss of approximately 9.4 billion US dollars annually in Africa alone [7].

Synthetic insecticides have been prime and inevitable control options for combating S. frugiperda infestations worldwide. In Pakistan, growers rely exclusively upon synthetic pesticides to control lepidopterous pests, including Spodoptera species [8,9]. Unfortunately, farmers have reported that the available pesticides do not effectively control S. frugiperda in the field. As a result, they arbitrarily increase the labeled dose to eradicate this insect pest, which will lead to the development of insecticide resistance in S. frugiperda in the future. The overuse of synthetic insecticides to eradicate this pest is manifested as environmental contamination and insecticidal resistance in S. frugiperda. Approximately 46 and 60% of farmers in Ethiopia and Kenya, respectively, claimed the ineffectiveness of synthetic insecticides against S. frugiperda [10]. Indeed, repeated applications of insecticides with the same mode of action have resulted in resistance to S. frugiperda in Africa [2]. Furthermore, Zhang et al. [11] monitored the resistance in S. frugiperda against commonly used insecticides and revealed the evolution of resistance in S. frugiperda against chlorpyrifos, spinosad, lambda-cyhalothrin, malathion, fenvalerate, deltamethrin, emamectin benzoate and chlorantraniliprole. Therefore, there is a need to develop an integrated management approach to effectively control this invasive pest.

In response to the global spread of this pest, especially in Pakistan, many studies have recently focused on developing biopesticides with the integration of various control strategies as a component of integrated pest management (IPM) against S. frugiperda [12,13]. However, plant-based insecticides have long been recognized as promising alternatives to synthetic insecticides for insect pest management [14,15]. Botanical pesticides are usually environmentally friendly, cost-effective and exhibit relatively low toxicity to on-target organisms [16,17]. Many native plants having the ethnomedicinal value of certain biogeographic regions may also exhibit insecticidal potential. For instance, Soone Valley and its surrounding salt range (Khushab, Punjab, Pakistan) are enriched with flora of ethnomedicinal value and insecticidal potential [18,19,20]. The comparative toxicity of the extracts of forty plant species, including herbs, shrubs and trees, from this area determined against Spodoptera litura by Majeed et al. [21] provides a basis for further research on evaluating the combined insecticidal effect of promising botanicals along with the synthetic insecticide against S. frugiperda. Many previous studies have demonstrated the potential of plant-derived compounds to enhance the toxicity and to reduce the inhibitory concentration of different synthetic insecticides [22]. Therefore, this laboratory research aimed to assess the combined toxicity of promising local plant extracts and commonly used synthetic insecticides against the 3rd instar larvae of S. frugiperda. In brief, binary and/or tertiary combinations of LC₃₃ and LC₅₀ of the selected botanical extracts were bioassayed along with half of the label-recommended dose rates of selected synthetic insecticides.

2. Materials and Methods

2.1. Insect Culture

For the rearing of S. frugiperda, mature larvae were collected from the maize field (32°13′35″ N, 72°68′67″ E) and were brought to the laboratory of Entomology, College of Agriculture, University of Sargodha (Punjab, Pakistan). These larvae were reared in glass Petri plates (diameter 9 cm) lined with a corn-based artificial diet [23] under controlled conditions of 25 ± 2 °C, 60 ± 5% RH and 16 h:8 h (L:D) photoperiod. Only few larvae (5–8 larvae per Petri plate) were maintained in order to avoid cannibalism. The larval diet was changed regularly until pupation. Pupae were maintained on moist Whatman No. 1 filter paper (diameter 9 cm) in glass Petri plates. After emergence, adult moths were provided a 10% honey solution and were housed separately in rearing plastic cages (30 × 30 × 30 cm; Bugdorm-I, Taiwan) with hanging muslin cloth strips for oviposition. The egg masses of S. frugiperda were collected from the cages, maintained on Petri plates lined with a thin layer of artificial diet and reared to obtain subsequent generations. Healthy and active 3rd instar larvae of the laboratory-reared F₃ generation of S. frugiperda were utilized in all bioassays.

2.2. Collection and Extraction of Plant Materials

Samples of promising local plant species, as detailed in

Table 1. Description of plant samples collected from the selected sites of Soone Valley (Khushab) and the surrounding salt range of Pakistan.

Plant Species	Vernacular Name	Family	Locality	Part(s) Used
Buxus papillosa C. K. Schneid.	Shamshad	Buxaceae	Kufri	Leaves
Maerua arenaria Hook. f. & Thomson	Hemkand	Capparaceae	Uchhali	leaves
Monotheca buxifolia Falc. A. DC.	Kohair	Sapotaceae	Khura	Leaves
Olea ferruginea Wall. ex Aitch.	Kao	Oleaceae	Uchhali	Leaves
Peganum harmala L.	Harmal	Nitrariaceae	Anga	Leaves
Periploca aphylla Decne.	Jangli bata	Apocynaceae	Anga	Leaves
Rhazya stricta Decne.	Akri	Apocynaceae	Uchhali	Leaves and flowers
Salvia moorcroftiana Wall. ex Benth.	Khalatra	Lamiaceae	Kufri	Leaves
Solanum incanum L.	Mahori	Solanaceae	Kufri	Fruits
Solanum nigrum L.	Black nightshade	Solanaceae	Khabeki	Leaves and flowers
Sophora mollis (Royle) Baker	Kohni	Leguminosae	Khabeki	Leaves and flowers
Withania somnifera (L.) Dunal	Aksan	Solanaceae	Khura	Roots

, were collected from six distinct locations (Figure 1) of the Soon Valley and adjacent salt range of district Khushab (Punjab, Pakistan) (

Table 2. Geographical coordinates of the study sites of Soone Valley (Khushab) and the surrounding salt range of Pakistan.

Localities	Latitude N	Longitude E	Elevation (m)
Anga	32.35° N	72.05° E	821
Khabbeki	32.35° N	72.12° E	774
Khura	32.23° N	72.11° E	866
Uchhali	32.56° N	72.02° E	794
Kufri	32.56° N	72.02° E	723

). Collected plants were identified up to the species level with the help of an online identification portal (http://www.theplantlist.org/1.1, accessed on 20 April 2022) generated by the botanical community in response to the Global Strategy for Plant Conservation (GSPC) and by the local experts of the Department of Botany, University of Sargodha, Pakistan. These plant samples were prepared and extracted by a Soxhlet apparatus (DH. WHM-12393, Daihan Scientific, Seoul, Korea) using methanol as the extraction solvent in a 1:10 ratio as described previously [21]. The extraction time for most of the samples was 4–6 h. Further purification of extracted plant samples was performed using a rotary evaporator (WEV-1001 L, Daihan Scientific, South Korea) fitted with a vacuum pump and chiller. Extracted plant materials were stored at 4 °C in 50 mL hermetic dark glass vials until their use in the toxicity bioassays.

2.3. Bioassays with Plant Extracts

In the first bioassay, 20% methanolic plant extracts were screened against S. frugiperda larvae using the standard leaf-dip method. In brief, fresh cauliflower (Brassica oleracea L. botrytis) leaves were collected, washed with tap water and allowed to air dry for 3 min at room temperature (27 °C). Leaf discs (9 cm) were made and dipped for 30 s in 20% methanolic extracts of plants and were placed on filter paper sheets to drain out the excess solution. After drying, the treated leaf discs were placed in glass Petri plates (diameter 9 cm) lined with 2.0% agar solution to keep them fresh, and 10 pre-starved (4 h) larvae of S. frugiperda were released in each Petri plate. These plates were incubated in an environment chamber (Sanyo MLR-350H, Sanyo, Kyoto, Japan) under controlled conditions of 25 ± 2 °C, 60 ± 5% RH and 16 h:8 h (L:D) photoperiod. Five replicates were maintained for each treatment, and methanol-soaked leaves were used as a control. Larval mortality was examined at 12, 24, 48 and 72 h posttreatment. Furthermore, four different concentrations (i.e., 5, 10, 20 and 40%) of the three most effective botanical extracts were prepared with methanol and were bioassayed against 3rd instar larvae of S. frugiperda to determine their median lethal concentration (LC₅₀) and lethal time (LT₅₀) values. The bioassay protocol for this second botanical bioassay was the same as that described above.

2.4. Bioassay with Synthetic Insecticides

The comparative toxicity of synthetic insecticides against the immature S. frugiperda was assessed using the standard lead-dip method as described previously [24]. For this purpose, nine synthetic insecticides were purchased from authenticated pesticide dealers from the local grain market of Sargodha (Punjab, Pakistan) and were tested according to their label-recommended dose rates (

Table 3. Description of synthetic insecticides bioassayed against 3rd instar larvae of the fall armyworm Spodoptera frugiperda.

Insecticide	Label dose (mL/Acre)	IRAC Group *	Manufacturer
abamectin	400	Avermectins	FMC, Lahore, Pakistan®
chlorantraniliprole	50	Diamides	Orange, Karachi, Pakistan®
chlorpyrifos	1000	Organophosphate	Orange, Karachi, Pakistan®
deltamethrin	80	Pyrethroid	Bayer, Karachi, Pakistan®
emamectin benzoate	200	Avermectins	Syngenta, Karachi, Pakistan®
fipronil	480	Phenylpyrazole	Orange, Karachi, Pakistan®
lambda cyhalothrin	250	Pyrethroid	FMC, Lahore, Pakistan®
lufenuron	200	Benzoylurea	Syngenta, Karachi, Pakistan®
profenofos	250	Organophosphate	Syngenta, Karachi, Pakistan®

* Insecticide Resistance Action Committee (v10.2_23March22).

). These insecticidal solutions were prepared in laboratory according to recommended dose per 80 L water as recommended for one acre coverage out in the field. Freshly prepared discs of B. oleracea leaves were dipped into aqueous solutions of insecticides, and after draining and drying on filter paper sheets, these discs were placed in glass Petri plates (9 cm). Ten 4 h pre-starved 3rd larvae of S. frugiperda were exposed to these treated leaves. Each treatment was replicated five times with water-soaked leaves acting as a control. All procedures were conducted under controlled conditions (at 25 ± 2 °C, 60 ± 5% RH and 16 h:8 h (L:D) photoperiod). Larval mortality was recorded at 12, 24, 48 and 72 h post-treatment.

2.5. Efficacy of Binary/Tertiary Mixtures

The toxicity of binary and/or tertiary mixtures of the most effective botanical and synthetic insecticide treatments was further determined using the LC₃₃ and LC₅₀ of botanicals and half of the label-recommended doses of synthetic insecticides. The LC₅₀ values at 72 h were considered for these combination treatments. The calculation of all treatment solutions was based on previous experiments and is mentioned in

Table 4. Selected effective treatments and their combinations bioassayed against 3rd instar larvae of the fall armyworm Spodoptera frugiperda.

Sr. No.	Botanicals or Synthetic Treatments	Concentration/Dose Used
T1	LC33 (Withania somnifera)	20%
T2	LC33 (Sophora mollis)	28%
T3	LC33 (Rhazya stricta)	29%
T4	LC50 (W. somnifera)	30.21%
T5	LC50 (S. mollis)	33.32%
T6	LC50 (R. stricta)	36.41%
T7	emamectin benzoate (1/2 of LD) *	100 mL/acre
T8	chlorpyrifos (1/2 of LD) *	500 mL/acre
T9	chlorantraniliprole (1/2 of LD) *	25 mL/acre
T10	LC33 (W. somnifera + S. mollis + R. stricta)	20% + 28% + 29%
T11	LC50 (W. somnifera) + emamectin benzoate (1/2 of LD) *	30.21% + 125 mL/acre
T12	LC50 (W. somnifera) + chlorpyrifos (1/2 of LD) *	30.21% + 500 mL/acre
T13	LC50 (W. somnifera) + chlorantraniliprole (1/2 of LD) *	30.21% + 25 mL/acre
T14	LC50 (S. mollis) + emamectin benzoate (1/2 of LD) *	33.32% + 125 mL/acre
T15	LC50 (S. mollis) + chlorpyrifos (1/2 of LD) *	33.32% + 500 mL/acre
T16	LC50 (S. mollis) + chlorantraniliprole (1/2 of LD) *	33.32% + 25 mL/acre
T17	LC50 (R. stricta) + emamectin benzoate (1/2 of LD) *	36.41% + 125 mL/acre
T18	LC50 (R. stricta) + chlorpyrifos (1/2 of LD) *	36.41% + 500 mL/acre
T19	LC50 (R. stricta) + chlorantraniliprole (1/2 of LD) *	36.41% + 25 mL/acre
T20	Control (water only)	0.00%

* LD = labeled dose.

. Twenty treatments, including the control, were assessed against S. frugiperda larvae. Here, LC₃₃ concentration of each plant extract was compared alone and in tertiary combination, while LC₅₀ concentrations were evaluated alone and in combination with half of the label-recommended dose rates of synthetic insecticides. All bioassay protocols were the same as those detailed above. Actual larval mortalities were compared to the expected mortalities based on the formula derived after Trisyono and Whalon [25] as follows:

For tertiary combination:

$$E=O_a+O_b \left(1-O_a\right)+O_c \left(1-O_b\right)$$

For binary combinations:

$$E=O_a+O_b \left(1-O_a\right)$$

where E is the expected mortality for the combination and O_a, O_b and O_c are the observed mortalities of W. somnifera, S. mollis and R. stricta alone at a given concentration. The effect of mixtures was designated antagonistic, additive or synergistic based on χ² comparisons as follows:

$${\chi }^2 = \frac{ {\left(O_m-E\right)}^2}{E}$$

where O_m is the observed mortality for the binary mixture and E is the expected mortality; χ² with α = 0.05 was 3.84. A pair with χ² values > 3.84 and having greater than the expected mortality was considered to be synergistic, with χ² values < 3.84 representing additive effects.

2.6. GC–MS Characterization of Effective Plant Extracts

A GC–MS-DSQ II (Thermo Scientific, San Jose, CA, USA) with a gas chromatograph interfaced to a mass spectrometer apparatus was used to analyze the crude methanolic extracts of W. somnifera, S. mollis and R. stricta. The following conditions were employed: a TR-5MS fused silica capillary column (30 × 250 × 0.25 m, composed of 5% phenyl/95% dimethylpolysiloxane) operating in electron impact mode at 70 eV; helium (99.99%) was used as the carrier gas at a constant flow rate of 1 mL min⁻¹, and an injection volume of 1 μL was used (ay split ratio of 10:1); injector temperature was 240 °C, and the ion-source temperature was 200 °C. Initially, the oven temperature was adjusted to 70 °C (isothermal for 2 min) and then rose to 240 °C at a rate of 10 °C min⁻¹, followed by a 9 min isothermal at 280 °C. The mass spectra were acquired at 70 eV with a scan interval of 0.5 s with fragments ranging in size from 40–440 Da. The total time spent running the GC was 40 min [26]. The compounds were identified by comparing the GC–MS mass spectra to those in the Wiley/NIST databases [27].

2.7. Statistical Analysis

Data regarding S. frugiperda larval mortality were interpreted statistically using the program Statistix 8.1^® (Analytical Software, Tallahassee, FL, USA). Factorial analysis of variance (ANOVA) was used to examine the mortality data, and the treatment means were compared using an honestly significant difference (HSD) post hoc test at the 95% probability level (p ≤ 0.05). Lethal concentration 33 percent (LC₃₃), median lethal concentration (LC₅₀) and time (LT₅₀) values were calculated by probit analysis [28] using regression software IBM SPSS^® (Version 20.0). Prior to probit analysis, mortality data were corrected using Abbott’s formula [29] and were normalized by arcsine square root transformation (arsin (sqrt(x))) [30].

3. Results

3.1. Screening of Plant Extracts for Insecticidal Potential

Factorial analysis revealed a significant effect of both botanical treatments (F_11,144 = 86.55; p < 0.001) and the time factor (F_2,64 = 164.24; p < 0.001) and of their interactions (F_22,144 = 5.34; p< 0.001) on the mortality of S. frugiperda larvae. The 20% methanolic extracts of S. mollis and W. somnifera showed the highest mean corrected mortality (~37%) of the 3rd instar larvae of S. frugiperda, whereas R. stricta and O. ferruginea extracts caused 32 and 17% mortality, respectively. The lowest mortality of S. frugiperda larvae was observed in the case of B. papillosa and P. aphylla (7%), followed by M. arenaria (8%), S. moorcroftiana (8%), S. incanum (8%) and S. nigrum (8%) (Figure 2). The toxicity of each botanical extract against S. frugiperda concerning the exposure time indicated that S. mollis and W. somnifera caused the highest corrected mortality (51%), followed by R. stricta (49%) at 72 h post-treatment. Although M. arenaria, P. aphylla, S. moorcroftiana, S. incanum and S. nigrum showed the least mortality (~12% each) (Supplementary Figure S1), no mortality of S. frugiperda was observed by P. aphylla at 24 h posttreatment, whereas B. papillosa, M. arenaria, S. moorcroftiana, S. incanum and S. nigrum caused only 2% mortality at 24 h post-treatment (Supplementary Figure S1).

For the second bioassay using different serial concentrations of botanical extracts, probit analysis revealed that the most effective botanical extract was W. somnifera (LC₅₀ = 40.42 and 30.21% at 48 and 72 h posttreatment, respectively), followed by S. mollis (LC₅₀ = 44.09 and 33.32%) and R. stricta (LC₅₀ = 75.10 and 36.41%) at 48 and 72 h of application, respectively (Supplementary Table S1). In comparison, the extract of O. ferruginea resulted in the lowest toxicity to the 3rd instar larvae of S. frugiperda, with an LC₅₀ value of 245.79% at 72 h post-treatment. In the case of the medial lethal time (LT₅₀) values, the 40 and 20% extracts of W. somnifera exhibited minimum LT₅₀ values (i.e., 48.59 and 51.82 h), followed by S. mollis (49.06 and 52.31 h) and R. stricta (54.89 and 55.02 h) (Supplementary Table S2).

3.2. Toxicity of Synthetic Insecticides

The toxicity bioassay with nine synthetic insecticides against the 3rd instar larvae of S. frugiperda revealed a significant effect of both insecticidal treatments (F_8,324 = 141.81; p < 0.001) and the time factor (F_3,324 = 710.51; p < 0.001), and revealed their interactions (F_24,324 = 4.84; p < 0.001) on the mortality of S. frugiperda larvae, where the label-recommended dose of emamectin benzoate showed the highest mean corrected mortality (~45%), followed by chlorpyrifos (40%) and chlorantraniliprole (38%). In comparison, lufenuron caused minimum larval mortality (~16%), followed by fipronil (17%) and lambda-cyhalothrin (18%). There was no significant difference among the larval mortalities of S. frugiperda caused by profenofos, abamectin and deltamethrin (i.e., 28, 28 and 27%, respectively) (Figure 3).

3.3. Efficacy of Binary/Tertiary Combinations of Effective Synthetic and Botanical Treatments

Ten binary/tertiary combinations of the most effective botanical and synthetic insecticidal treatments were tested in the third bioassay. Among these combinations, seven pesticide combinations exhibited synergistic toxicity, while three combinations showed an additive effect on the mortality of 3rd instar larvae of S. frugiperda (

Table 5. Combined effect of binary/tertiary pesticidal mixtures against 3rd instar larvae of the fall armyworm Spodoptera frugiperda.

Pesticides (Dose)			Larval Mortality (%)
			Pesticides			Binary/Tertiary Pesticidal Mixtures		χ2	Effect
A	B	C	Observed A	Observed B	Observed C	Expected *	Observed
LC33 (Withania somnifera 1)	LC33 (Sophora mollis)	LC33 (Rhazya stricta)	24.0	24.0	22.7	59.5	29.3	15.3	Synergistic
LC50 (Withania somnifera)	1/2 of LD emamectin benzoate	-	32.0	44.0	-	61.9	49.3	2.6	Additive
LC50 (Withania somnifera)	1/2 of LD chlorpyrifos	-	32.0	38.7	-	58.3	40.0	5.7	Synergistic
LC50 (Withania somnifera)	1/2 of LD chlorantraniliprole	-	32.0	36.0	-	56.5	36.0	7.4	Synergistic
LC50 (Sophora mollis)	1/2 of LD emamectin benzoate	-	33.3	44.0	-	62.7	49.3	2.8	Additive
LC50 (Sophora mollis)	1/2 of LD chlorpyrifos	-	33.3	38.7	-	59.1	40.0	6.2	Synergistic
LC50 (Sophora mollis)	1/2 of LD chlorantraniliprole	-	33.3	36.0	-	57.3	36.0	7.9	Synergistic
LC50 (Rhazya stricta)	1/2 of LD emamectin benzoate	-	33.3	44.0	-	62.7	49.3	2.8	Additive
LC50 (Rhazya stricta)	1/2 of LD chlorpyrifos	-	33.3	38.7	-	59.1	40.0	6.2	Synergistic
LC50 (Rhazya stricta)	1/2 of LD chlorantraniliprole	-	33.3	36.0	-	57.3	36.0	7.9	Synergistic

* Indicates the expected larval mortality derived from the formula of Trisyono and Whalon (1999). χ² = 3.84 (at α = 0.05). A combination with χ² value > 3.84 was considered to be synergistic, while with χ² value < 3.84 indicates an additive effect.

). The tertiary combination of LC₃₃ values of W. somnifera, S. mollis and R. stricta exhibited 29.3% mortality compared to an expected mortality of 65.2% and demonstrated synergistic toxicity. In all treatments, the application of individual insecticidal treatments caused lower larval mortality than their combined application. The combination of chlorpyrifos (at its half label-recommended dose) with LC_50s of W. somnifera, S. mollis and R. stricta demonstrated an observed mortality of 40.0% for each combination compared to the expected mortality of 58.3, 59.1, 59.1%, respectively. Similarly, all binary combinations of emamectin benzoate with all three botanical extracts of W. somnifera, S. mollis and R. stricta showed an additive effect on larval mortality (Table 5).

3.4. Biochemical Composition of Plant Extracts

GC–MS analysis was used to determine the presence of biologically active components in methanolic extracts of W. somnifera, S. mollis, and R. stricta. The major bioconstituents, their molecular weight (g mol⁻¹, M.W.), molecular formula (M.F.), retention time (s, R.T.), and peak area (%) are given in

Table 6. Chemical composition of the methanolic extract of Withania somnifera roots.

Peak No.	R.T.	Compounds	Area (%)	M.F.	M.W.
1	3.53	Benzene, 1,2,4-trimethyl-	9.40	C9H12	120
2	4	Octane, 3,5-dimethyl-	7.34	C10H22	142
3	4.49	Tumerone	2.09	C15H22O	218
4	6.06	Limonen-6-ol, pivalate	1.56	C15H24O2	236
5	6.50	2-Oxazolamine, 4,5-dihydro-5-(phenoxymethyl)-N-[(phe nylamino)carbonyl]-	1.41	C17H17N3O3	311
6	6.83	12,15-Octadecadiynoic acid, methyl Ester	1.40	C19H30O2	290
7	7.40	Dodecane	3.05	C12H26	170
8	7.81	(2-Aminocyclohexyl)-phenyl-methanol	0.62	C13H19NO	205
9	8.81	Pyridine, 2-(1H-tetrazol-5-yl)-	2.21	C6H5N5	147
10	9.97	2-Vinyl-9-[3-deoxy-á-d-ribofuranosyl]hypoxanthine	0.47	C12H14N4O4	278
11	12.33	Bicyclo [4.4.0]dec-2-ene-4-ol, 2-methyl-9-(prop-1-en-3-ol-2-yl)-	0.73	C15H24O2	236
12	14.25	2-Oxazolamine, 4,5-dihydro-5-(phenoxymethyl)-N-[(phenylamino)carbonyl]-	0.44	C17H17N3O3,	311
13	16.02	Cholestan-3-ol, 2-methylene-, (3á,5à)-	0.28	C28H48O	400
14	18.67	Cystathionine, bis(triemthylsilyl) ester	2.05	C13H30N2O4SSi2	366
15	20.66	Dihydroxanthin	1.37	C17H24O5	308
16	22.41	9,12,15-Octadecatrienoic acid, 2,3-bis[(trimethylsilyl)oxy]propyl ester, (Z,Z,Z)-	1.16	C27H52O4Si2	496
17	24.33	1-(2-Acetoxyethyl)-3,6-diazahomoadamantan-9-one oxime	2.03	C13H21N3O3	267
18	26.94	Cyclotrisiloxane, hexamethyl-	3.75	C6H18O3Si3	222

R.T., Area (%), M.F., and M.W., indicates the retention time, peak area, molecular formula and molecular weight.

,

Table 7. Chemical composition of the methanolic extract of Sophora mollis leaves and flowers.

Peak No.	R.T.	Compounds	Area (%)	M.F.	M.W.
1	3.59	Benzene, 1-ethyl-2-methyl-	6.49	C9H12	120
2	5.20	1-Hexadecanol, 2-methyl-	1.20	C17H36O	256
3	6.93	E-9-Tetradecenoic acid	1.37	C14H26O2	226
4	8.09	Pregnane-3,11,20,21-tetrol, cyclic 20,21-(butyl boronate), (3à,5á,11á,20R)-	1.28	C25H43BO4	418
5	9.93	Oxirane, hexadecyl-	0.57	C18H36O	268
6	11.82	Naphthalene, 1,1′-(1,10-decanediyl)bis-	0.43	C30H34	394
7	13.80	Tetraethylrhodamine	0.95	C28H31N2O3	443
8	15.26	1-Oxaspiro [4.4]non8-ene-4,7-dione, 9-hydroxy-6-(3-methyl-2-butenyl)-2-(1-methylethyl)-8-(3-methyl-1-oxobutyl)-	0.26	C21H30O5	362
9	16.61	10-Hydroxy-5,7-dimethoxy-2,3-dimethyl-1,4-anthracenedione	0.38	C18H16O5	312
10	18.58	7,8,12-Tri-O-acetylingol	0.74	C26H36O9	492
11	19.40	Digitoxin	0.27	C41H64O13	764
12	20.74	Z-10-Methyl-11-tetradecen-1-ol Propionate	0.75	C18H34O2	282
13	23.09	Isoproturon	0.51	C12H18N2O	206
14	24.02	1-Monolinoleoylglycerol trimethylsilyl Ether	0.22	C27H54O4Si2	498
15	27.30	Silane, 1,4-phenylenebis-trimethyl-	0.52	C12H22Si2	2221

R.T., Area (%), M.F., and M.W., indicates the retention time, peak area, molecular formula and molecular weight.

and

Table 8. Chemical composition of the methanolic extract of Rhazya stricta leaves and flowers.

Peak No.	R.T.	Compounds	Area (%)	M.F.	M.W.
1	4.04	Decane	5.08	C10H22	142
2	5.73	Octane, 5-ethyl-2-methyl-	1.18	C11H24	156
3	6.14	Palmitic acid, (2-phenyl-1,3-dioxolan-4-yl)methyl Ester	0.29	C26H42O4	418
4	9.56	2-Propenoic acid, tridecyl ester Acetic acid	0.65	C16H30O2	254
5	10.48	17-(1-acetoxy-ethyl)-10,13-dimethyl-3-oxo-2,3,8,9,10,11,12,13,14,15,16, 17-dodecahydro-1H-cyclopenta[a]phenanthren-11-yl (ester)	1.77	C25H34O5	414
6	11.62	Stearic acid, 3-(octadecyloxy)propyl ester	0.54	C39H78O3	594
7	12.31	Methyl abietate isomer	0.61	C21H32O2	316
8	14.31	2,5-Octadecadiynoic acid, methyl ester	1.19	C19H30O2	290
9	16.69	10-Heptadecen-8-ynoic acid, methyl ester, (E)-	0.54	C18H30O2	278
10	17.24	Hexadecanoic acid, 1-(hydroxymethyl)-1,2-ethanediyl ester	1.01	C35H68O5	568
11	19.01	Akuammilan-16-carboxylic acid, 17-(acetyloxy)-, methyl ester, (16R)-	0.64	C23H26N2O4	394
12	20.46	à-N-Normethadol	1.75	C20H27NO	297
13	22.19	9,12,15-Octadecatrienoic acid, 2,3-bis[(trimethylsilyl)oxy]propyl ester, (Z,Z,Z)-	1.06	C27H52O4Si2	496
14	24.90	1-Monolinoleoylglycerol trimethylsilyl ether	8.73	C27H54O4Si2	498
15	26.98	Lupeol	4.24	C30H50O	426

R.T., Area (%), M.F., and M.W., indicates the retention time, peak area, molecular formula and molecular weight.

, respectively. The crude extract of W. somnifera roots primarily comprised eighteen compounds. 1,2,4-trimethyl-benzene and 3,5-dimethyl-octane were the most abundant compounds, with areas of 9.40 and 7.34%, respectively. In comparison, the other minor compounds were present in low quantities, with relative peak areas ranging from 0.28–3.75% (Table 6). The GC–MS profile of the S. mollis extract revealed the presence of fifteen compounds. Among these compounds, 1-ethyl-2-methyl-benzene was the major compound with a 6.49% relative peak area, whereas the other fourteen identified compounds were recognized as minor compounds with relative peak areas ranging from 0.22–1.37% (Table 7). Chemical profiling of R. stricta indicated the presence of fifteen substances in its extract. The principal compounds were 1-monolinoleoylglycerol trimethylsilyl ether, decane, and lupeol with 8.73, 5.08 and 4.24% relative peak areas, respectively, while the other twelve minor compounds had relative peak areas ranging from 0.29–1.77% (Table 8).

4. Discussion

This research work revealed the synergistic effect of some promising indigenous plant extracts and commonly used synthetic insecticides on the 3rd instar larvae of S. frugiperda. An initial screening bioassay performed with 20% methanolic extracts of twelve indigenous plant species demonstrated that the extracts of S. mollis, W. somnifera and R. stricta were the most effective botanicals exhibiting the highest mortality of S. frugiperda larvae. Our results corroborate the findings of some recent studies that have demonstrated the toxicity of acetone extracts of indigenous plant species of the same study area against the termite (Odontotermes obesus), mosquito (Culex quinquefasciatus), psyllid (Diaphorina citri) and armyworm (S. litura) [20,21,31]. Similarly, Phambala et al. [32] demonstrated the insecticidal activity of 10% methanolic extracts of some indigenous ethnomedicinal plants of Mitundu (Malawi) against S. frugiperda larvae. They showed that extracts of Nicotiana tabacum and Lippia javanica caused significantly higher mortality (62–66%) of S. frugiperda larvae. Moreover, Rioba and Stevenson [33] reviewed a number of previous studies documenting significant larvicidal and ovicidal activity of local plant extracts against S. frugiperda.

Our second feeding bioassay conducted using different concentrations of these plant extracts revealed W. somnifera, S. mollis and R. stricta as the most effective botanical treatments, with minimum LC₅₀ values of 30.21, 33.32 and 36.41% at 72 h of application, respectively, and minimum LT₅₀ values of 48.59, 49.06 and 54.89 h by 40% extracts, respectively. Although these three plant species are not well studied regarding their insecticidal potential, many phytoextracts, including plant extracts and essential oils, have been demonstrated to show larvicidal [21,33,34,35,36,37], ovicidal and other anti-insect activities [32,36] against S. frugiperda and other Spodoptera species. Our results are in line with Gupta and Srivastava [38], who showed significant mortality (63.33%) of Callosobruchus chinensis adults by 10% ethanolic extracts of W. somnifera roots. Similarly, extracts of S. mollis (and other plants from the genus Sophora) and R. stricta have been known to contain many plants secondary metabolites with allelopathic, antibiotic, nematicidal and insecticidal potential [39,40].

The results of the third bioassay with commonly used synthetic insecticides revealed emamectin benzoate, chlorpyrifos and chlorantraniliprole as the most effective insecticides against S. frugiperda larvae. Many previous studies based on socioeconomic surveys and laboratory and field bioassays have demonstrated the effectiveness of emamectin benzoate, chlorpyrifos and chlorantraniliprole against different species of Spodoptera [41,42]. Emamectin benzoate is an effective insecticide that is effectively used alone or in combination with other insecticides in East Africa and South Asia. For instance, 92% and 88% of farmers from Rwanda and Uganda utilized a combination of emamectin benzoate and cypermethrin against fall armyworm, respectively [43]. Shallot growers in Java (Indonesia) use chlorpyrifos for effective control of S. exigua [44], whereas some studies have indicated the effectiveness of chlorantraniliprole against the 3rd instar larvae of S. litura [45,46]. A recent study was reported on the efficacy of abamectin and broflanilide belong to the avermectin and diamides group cause significant mortality of 87.3 and 91.3% against second instar S. frugiperda larvae at 72 h post-treatment in China [47].

In the fourth bioassay, combinations of LC₃₃ and LC₅₀ of the most effective botanicals (R. stricta, S. mollis and W. somnifera) and half of the labeled dose of synthetic insecticides (emamectin benzoate, chlorpyrifos and chlorantraniliprole) were assessed against 3rd instar larvae of S. frugiperda. Among the ten combinations, seven exhibited synergy, and three produced additive toxicity against S. frugiperda. Our findings regarding the synergistic and additive effects of different botanical and synthetic insecticides are consistent with those of many previous studies. For instance, our results are consistent with those of Fazolin et al. [48], who revealed a synergistic toxicity of beta-cypermethrin and fenpropathrin against S. frugiperda by combining with Piper aduncum essential oil. Similarly, binary combinations of different phyto-constituents (α-thujone, (+)-camphor, 1,8-cineole, and α-caryophyllene) from Salvia hispanica exhibited synergistic toxicity against S. exigua [49]. The insecticidal activity of garlic and thymol oils is enhanced against S. littoralis when combined with cypermethrin and chlorpyrifos [50]. Similarly, Rao and Dhingr [51], and Ruttanaphan et al. [52] revealed synergistic and additive activity of different vegetable and plant essential oils along with cypermethrin against larvae of S. litura. Similarly, Silva et al. [53] reported significantly higher mortality of 3rd instar larvae of S. frugiperda by the binary combinations of LD₅₀ doses of pyrethroid deltamethrin and Ocimum basilicum-derived linalool oil.

5. Conclusions

Based on the overall study results, it is concluded that the methanolic extracts of R. stricta, S. mollis, and W. somnifera exhibited significant toxicity potential against fall armyworm larvae. The combination of LC_33s and LC_50s of these plant extracts along with half of the label-recommended doses of chlorpyrifos, chlorantraniliprole and emamectin benzoate synergized the toxicity against 3rd instar larvae of S. frugiperda, suggesting their potential incorporation into future integrated pest management of S. frugiperda. Nevertheless, field evaluation of these botanical and synthetic insecticidal combinations regarding their effect on S. frugiperda and its natural enemies (insect predators and parasitoids) constitute future perspectives of this study.