Evaluation of Insecticide Toxicity and Field Performance Against Myzus persicae (Hemiptera: Aphididae) in Laboratory and Greenhouse Conditions

Abstract

Myzus persicae Sulzer (Hemiptera: Aphididae) is a pivotal pest affecting various vegetables, fruits, crops, and ornamentals. The primary M. persicae management strategy involves insecticide use. In this study, the toxicity and efficacy of bifenthrin, fenitrothion, fosthiazate, acetamiprid, spirotetramat, afidopyropen, and flonicamid against M. persicae were evaluated under laboratory and greenhouse conditions using the leaf dip method. Laboratory bioassay results revealed that M. persicae exhibited susceptibility to moderate resistance levels for bifenthrin [resistance ratio (RR): 3.00–21.50], fenitrothion (3.13–25.31), fosthiazate (3.00–20.00), and acetamiprid (2.00–14.50), as well as susceptibility to low resistance levels for spirotetramat (0.75 to 6.63). Additionally, M. persicae was susceptible to flonicamid (0.31–1.72) and afidopyropen (0.67–2.00). Furthermore, laboratory bioassays revealed that the Al-Dhabia M. persicae field population showed the highest resistance levels to all tested insecticides compared with other tested field populations, which guided the examination of insecticide field performance under greenhouse conditions. In the greenhouse, most insecticides demonstrated high efficacy (>90%) against M. persicae with enduring effects, except for bifenthrin, which began to lose effectiveness 10 days post-application. In conclusion, M. persicae displayed overall susceptibility to most tested chemical classes, and the prolonged efficacy of these insecticides in the field reinforces their effectiveness in controlling this pest species. To maintain this control level, the registration of novel insecticides such as flonicamid and afidopyropen in Saudi Arabia is imperative, expanding the repertoire of effective chemical tools for M. persicae control. Additionally, a rotational approach to using all effective chemical classes is crucial to preventing or delaying resistance development in M. persicae.

1. Introduction

Myzus persicae, a widespread and highly detrimental pest, poses a worldwide threat to greenhouse and open-field cultivation [1]. With hosts spanning over 40 plant families, including various crops [2], M. persicae thrives under optimal conditions, exhibiting high reproductive rates and multiple generations within short periods [3]. The importance of M. persicae as a pest lies in its ability to severely weaken and damage crops through heavy sap consumption, contaminate yield with sooty mold from its honeydew, and crucially transmit more than 100 destructive plant viruses [4,5,6].

For example, M. persicae has caused crop yield losses of 46% in Australia, 38–42% in Spain, 39% in Chile, and 19% in India, primarily due to virus transmission [7]. These economic losses necessitate effective control measures, mainly achieved through insecticide use across different chemical classes. However, excessive and indiscriminate insecticide application has led to resistance development in M. persicae. For example, resistance of M. persicae to various insecticides has been previously reported in many countries [8,9,10,11,12,13,14]. The Arthropod Pesticide Resistance Database has documented 483 cases of resistance in M. persicae worldwide to 84 different insecticides [15].

Globally, numerous insecticides are registered to control M. persicae, including organophosphates, carbamates, pyrethroids, neonicotinoids, tetramic acid derivatives, pyropenes, flonicamid, sulfoximines, diamides, pyridine azomethine derivatives, butenolides, and mitochondrial complex I electron transport inhibitors (METIs) [16,17]. However, in Saudi Arabia, only a few insecticides are registered for M. persicae control owing to stringent regulations, with many organophosphates, carbamates, pyrethroids, and other small chemical group members having been phased out. Nevertheless, the registered list includes some pyrethroids (pyrethrin, fenpropathrin, fenvalerate, bifenthrin, alpha-cypermethrin, deltamethrin, cypermethrin, lambda-cyhalothrin, and esfenvalerate), organophosphates (fenitrothion), neonicotinoids (acetamiprid, imidacloprid, thiamethoxam, and dinotefuran), METI acaricides and insecticides (tolfenpyrad), tetramic acids (spirotetramat), pyriproxyfen (pyriproxyfen), butenolides (flupyradifurone), sulfoximines (sulfoxaflor), and pyridine azomethine derivatives (pyrifluquinazon) [18]. Regarding sucking pest control, the restricted insecticide selection, coupled with the absence of registered highly effective insecticides (featuring new chemistry and novel modes of action), has elevated the risk of resistance issues in Saudi crop production. Examples of novel, effective insecticides include flonicamid and afidopyropen, which exhibit high efficacy against various aphid species, including M. persicae, with no reported instances of field-evolved resistance.

The fast-acting and broad-spectrum contact neurotoxin organophosphate fenitrothion is used against sucking and chewing insects on various crops, including vegetables, orchard fruits, rice, cereals, forest plants, and cotton [19]. Fosthiazate, a systemic organophosphate primarily employed as a nematicide, is occasionally used as an insecticide for controlling sucking and underground chewing insects [20]. Acetamiprid, a broad-spectrum contact and ingestion neurotoxin neonicotinoid, is effective against major sucking pests, including aphids, whiteflies, leafhoppers, and thrips, across various crops [21]. Spirotetramat, a systemic insecticide, is used effectively to manage a broad spectrum of sucking pests across various crops [22]. It exhibits outstanding systemic and translaminar efficacy, ensuring full mobility throughout the entire plant [22,23]. Notably, spirotetramat’s foliar application effectively protects roots from sucking insect pests, with the insecticide demonstrating unique two-way systemicity among insecticides developed in the last two decades [23,24]. Afidopyropen, a novel systemic insecticide, is highly effective against sucking pests, including M. persicae [25]. Afidopyropen easily penetrates leaves; thus, it shows excellent effectiveness in M. persicae control, lasting more than 30 days. This effectiveness is attributed to its robust translaminar efficacy, systemic activity, and a combination of lethal and sublethal effects, particularly antifeedant effects [26,27]. Flonicamid, a systemic and translaminar insecticide, exhibits highly effective insecticidal activity against major sucking insect pests across diverse crops by rapidly disrupting the feeding behavior of aphids [28].

Our previous study, Sabra et al. [29], was the first to monitor resistance and the corresponding mechanisms in M. persicae to registered and unregistered technical-grade insecticides in Saudi Arabia. Because insecticide technical grades cannot be directly applied in the field because they are unstable, have a shorter shelf life, lower biological performance, and are insoluble in water. Thus, we used formulated insecticide products with the goal of mimicking field situations [30], and the current resistance monitoring of M. persicae against formulated insecticides is a crucial addition to the scientific basis of Saudi crop production. Additionally, formulated insecticide efficacy was determined under greenhouse (semi-field) conditions. And prior to this field evaluation, any doubt about the role of the other (inert) ingredients of each tested insecticide in overcoming this highest level of resistance at the field scale was removed by conducting a baseline toxicity study of the formulation of all tested insecticides vs. testing the active ingredients of these insecticides in our previous study [29]. When addressing the escalating resistance risk, periodic evaluation of registered insecticides’ efficacy against M. persicae becomes imperative to avoid control failure at the field scale. Additionally, assessing the efficacy of new, unregistered insecticides is crucial to broaden the available chemical tools and mitigate resistance development by reducing selection pressure within a particular chemical class. Therefore, this study aimed to evaluate the toxicity and efficacy of registered and unregistered insecticides in controlling M. persicae under laboratory and greenhouse conditions.

2. Materials and Methods

2.1. Myzus persicae

Seven M. persicae populations were collected from different locations in Riyadh Province, Saudi Arabia (

Table 1. Location and collection history of Myzus persicae populations.

Population	Site of Collection	Coordinates	Date of Collection	Host Plants
DHB	Al-Dhabia	24.104548° N, 47.157080° E	30 February 2022	Cabbage
RAF	Al-Rafaa	24.322324° N, 47.113050° E	30 February 2022	Pepper
DWS	Wadi Al-Dawasir	20.392839° N, 44.828053° E	6 February 2022	Mallow
DRH	Diriyah	24.744491° N, 46.573820° E	24 February 2022	Spinach
HAY	Al-Hayer	24.395558° N, 46.757219° E	27 February 2022	Mallow
WSH	Al-Washlah	24.397439° N, 46.665039° E	10 March 2022	Eggplant
NKL	Al-Nakhil	24.724732° N, 46.616787° E	25 March 2022	Eggplant

). The susceptible strain (SS) was obtained from the University of Cattolica del Sacro Core, Italy, where it was established in 1995 and has been continuously reared on pea seedlings without pesticide exposure. Following the method of Sabra, Abbas, and Hafez [29], all populations were consistently maintained under controlled conditions of 60–70% relative humidity, 20 °C ± 2 °C, and a 16:8 h light/dark photoperiod.

2.2. Chemicals

Detailed information regarding the tested insecticides is presented in

Table 2. The list of tested insecticides in the current study.

Common Name	Trade Name (%F)	Field Rate	Manufacturer	IRAC Group	IRAC Mode of Action
Fosthiazate	Thiafos (50EC)	200 mL/100 L	Astrachem, Dammam, Saudi Arabia	1B, Organophosphates	Acetylcholinesterase (ACHE) inhibitors
Fenitrothion	Fentrol (50EC)	100 mL/100 L	Pioneers Chemicals Factory Co., Riyadh, Saudi Arabia	1B, Organophosphates	Acetylcholinesterase (ACHE) inhibitors
Bifenthrin	Bytop (10EC)	50 mL/100 L	Montajat Veterinary pharmaceutical Co., Dammam, Saudi Arabia	3A, Pyrethroids	Sodium channel modulators
Spirotetramat	Movento (10SC)	60 mL/100 L	Bayer Crop Sciences, Leverkusen, Germany	23, Tetramic Acids	Inhibitors of acetyl COA carboxylase
Afidopyropen	Sefina (4.89DC)	22.2 mL/100 L	BASF Corporation, Florham Park, NJ, USA	9D, Pyropenes	Chordotonal organ TRPV channel modulators
Flonicamid	Flonicamid (50WG)	30 g/100 L	ISK Biosciences, Painesville, OH, USA	29, Flonicamids	Chordotonal organ nicotinamidase inhibitors
Acetamiprid	Acetaplan (20SL)	50 mL/100 L	Astrachem, Dammam, Saudi Arabia	4A, Neonicotinoids	Nicotinic acetylcholine receptor (NACHR) competitive modulators

.

2.3. Toxicity Bioassays

Insecticide toxicity against field populations and the SS strain of M. persicae was assessed using 3–4-day-old nymphs following the IRAC leaf dip 019 method [31] with slight modifications. Six serial concentrations of each insecticide were prepared in deionized water. Five mallow plant leaf disks (50 mm) were dipped for 10 s in each concentration and air-dried on a paper towel at room temperature for 2 h. Each treated leaf disk was then placed on an agar bed (10 g/L) in a 60 mL plastic cup. For the control treatment, leaf disks were dipped in deionized water. Ten M. persicae nymphs were placed on each treated leaf disk, and an aerated plastic lid was placed on the cup. For each concentration, 5 replicates (treated leaf disk/plastic cup) were used, with 10 nymphs per replicate, 50 per concentration, and 300 for each bioassay. The control treatment included 50 nymphs (10 per replicate). Mortalities were recorded after 72 h for fosthiazate, bifenthrin, fenitrothion, and acetamiprid and after 120 h for spirotetramat, afidopyropen, and flonicamid. Mortality was determined by the absence of coordinated forward movement when gently pricked with a soft camel-hair brush. All toxicity bioassays were conducted under the aforementioned conditions.

2.4. Greenhouse Insecticide Efficacy Trials

In 2022, greenhouse trials were performed in a 5 × 11 m² greenhouse at the College of Food and Agriculture Sciences, King Saud University, Riyadh, Saudi Arabia. Sweet pepper seeds, Capsicum annuum L., were germinated in a plastic Petri dish (9 × 1.5 cm) lined on the bottom with wet filter paper for three days and then transplanted in plastic seedling trays (50 × 30 × 5.5 cm) containing soil/peatmoss (Goode Green Agricultural Company, Al-Muzahimiyah, Riyadh, Saudi Arabia). After 20 days of growth in greenhouse conditions, seedlings in the primary leaf stage were individually planted in 15 cm plastic pots containing soil/peatmoss (50/50, v/v). Employing a Randomized Complete Block Design, 12 sweet pepper plants (replicates) at the sixth true leaf vegetative growth stage were prepared for each treatment. Then, the plastic pots were individually placed in ventilated plastic containers (30 × 30 × 60 cm³). Fifty adult apterous from a synchronized colony of the highest resistant field population (Al-Dhabia “DHB”) were placed on the upper portion of each sweet pepper plant and allowed to feed and reproduce for seven days. Subsequently, 12 infested plants (replicates) were treated with the recommended labeled field rate of each insecticide. All tested insecticides were applied via foliar application using a 2 L hand-held hydraulic sprayer (each plant was sprayed with 40 mL of each insecticide solution until run-off). In addition to the foliar application, fosthiazate was applied via irrigation application (its recommended application) where each plant was irrigated with 80 mL of fosthiazate solution. In the control treatments, 12 infested plants were treated with water only. Survival numbers were recorded at 0, 1, 3, 7, 10, 14, and 21 days post-application. All greenhouse trials were conducted under consistent conditions of 79–84% relative humidity and 23 °C ± 3 °C.

2.5. Data Analyses

Median lethal concentration values (LC₅₀), 95% fiducial limits (FLs), chi-square (χ²) test results, and slopes with standard errors were calculated through probit analysis [32] using POLO Plus software v2.0 [33]. Resistance ratios (RRs) and 95% confidence intervals (CIs) were calculated as described by Robertson et al. [34]. RRs, considered significantly different if CIs did not include 1, were calculated as follows:

$${\displaystyle \frac{{\mathrm{L}\mathrm{C}}_{50} \mathrm{o}\mathrm{f} \mathrm{i}\mathrm{n}\mathrm{s}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{e} \mathrm{i}\mathrm{n} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{f}\mathrm{i}\mathrm{e}\mathrm{l}\mathrm{d} \mathrm{p}\mathrm{o}\mathrm{p}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}}{{\mathrm{L}\mathrm{C}}_{50} \mathrm{o}\mathrm{f} \mathrm{i}\mathrm{n}\mathrm{s}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{d}\mathrm{e} \mathrm{i}\mathrm{n} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{S}\mathrm{S} \mathrm{s}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{i}\mathrm{n}}}$$

Resistance levels were classified following the scale reported by Li et al. [35]: 1–4-fold RR, susceptibility; 5–10-fold RR, low resistance level; 11–30-fold RR, moderate resistance level; 31–100-fold RR, high resistance level; and >100-fold RR, very high resistance level.

For greenhouse trials, in One-Way ANOVAs, the differences between treatment means were tested using the least significant difference (LSD) test at a 5% significance level through Statistix 8.1v software [36]. Data were presented in two ways: (1) change in percent population density and (2) efficacy percentage relative to untreated plots. The change in population density (CPD) was calculated as follows:

CPD% = [(X_i − X₀)/X₀] × 100,

where X₀ and X_i are the mean number of individuals before spraying and at the ith assessment after spraying, respectively. Positive CPD% values imply a population increase.

The percent efficacy of insecticides (EF) was calculated using the Henderson–Tilton formula [37]:

Ef (%) = [1 − (X_iT/X_iC) (X_0C/X_0T)] × 100,

where X_0C and X_0T are the mean number of individuals in untreated and treated plots before treatment, respectively, and X_iC and X_iT are the mean number of motile forms in untreated and treated plots at the ith assessment after treatment, respectively.

3. Results

3.1. Insecticide Toxicity Against M. persicae

The LC₅₀ values of bifenthrin, fenitrothion, fosthiazate, acetamiprid, spirotetramat, afidopyropen, and flonicamid against the reference strain SS were 0.04, 0.02, 0.01, 0.02, 0.08, 0.03, and 0.16 mg/L, respectively (

Table 3. Susceptibility of seven M. persicae field populations to bifenthrin, fenitrothion, and fosthiazate.

Insecticide	Population	N	Conc. (mg/L)	LC50 (95% FL) (mg/L)	Fit of Probit Line				RR (95% CL)
					Slope ± SE	χ2	df	p
Bifenthrin	SS	350	0.03–1	0.04 (0.03–0.06)	1.54 ± 0.22	2.40	4	0.66	1.00
	DHB	350	0.25–8	0.86 (0.54–1.25) *	1.62 ± 0.18	5.42	4	0.25	21.50 (13.00–32.43) ǂ
	RAF	350	0.25–8	0.60 (0.38–0.86) *	1.56 ± 0.19	4.04	4	0.40	15.00 (9.03–23.37) ǂ
	DWS	350	0.25–8	0.82 (0.49–1.22) *	1.74 ± 0.21	4.98	4	0.29	20.50 (12.25–31.62) ǂ
	DRH	350	0.13–4	0.41 (0.19–0.69) *	1.65 ± 0.20	8.51	4	0.07	10.25 (6.12–15.80) ǂ
	HAY	350	0.06–2	0.20 (0.15–0.25) *	1.68 ± 0.19	3.72	4	0.45	5.00 (2.99–7.46) ǂ
	WSH	350	0.03–1	0.12 (0.08–0.16) *	1.83 ± 0.24	3.80	4	0.43	3.00 (1.73–4.64) ǂ
	NKL	350	0.06–2	0.20 (0.11–0.31) *	1.67 ± 0.22	4.16	4	0.38	5.00 (2.94–8.06) ǂ
Fenitrothion	SS	350	0.01–0.31	0.024 (0.02–0.03)	1.82± 0.21	3.06	4	0.55	1.00
	DHB	350	0.16–5	0.60 (0.11–0.33) *	1.70 ± 0.19	3.26	4	0.52	25.31 (17.87–35.85) ǂ
	RAF	350	0.16–5	0.41 (0.30–0.53) *	2.03 ± 0.25	3.09	4	0.54	17.38 (12.04–25.10) ǂ
	DWS	350	0.08–2.50	0.27 (0.11–0.47) *	1.79 ± 0.23	8.23	4	0.08	11.63 (7.94–17.03) ǂ
	DRH	350	0.04–1.25	0.10 (0.06–0.15) *	1.56 ± 0.18	4.69	4	0.32	4.34 (3.03–6.21) ǂ
	HAY	350	0.08–2.50	0.16 (0.11–0.22) *	1.62 ± 0.21	3.55	4	0.47	6.92 (4.64–10.32) ǂ
	WSH	350	0.04–1.25	0.13 (0.10–0.16) *	2.01 ± 0.22	2.92	4	0.57	5.39 (3.87–7.50) ǂ
	NKL	350	0.04–1.25	0.07 (0.05–0.10) *	1.62 ± 0.22	2.66	4	0.62	3.13 (2.05–4.79) ǂ
Fosthiazate	SS	350	0.01–0.125	0.01 (0.006–0.012)	1.75 ± 0.27	3.33	4	0.50	1.00
	DHB	350	0.06–2	0.20 (0.11–0.33) *	1.59 ± 0.18	8.00	4	0.09	20.00 (14.26–31.62) ǂ
	RAF	350	0.06–2	0.15 (0.09–0.21) *	1.66 ± 0.20	4.86	4	0.30	15.00 (10.20–22.97) ǂ
	DWS	350	0.06–2	0.17 (0.07–0.30) *	1.42 ± 0.18	9.77	4	0.04	17.00 (11.57–26.97) ǂ
	DRH	350	0.03–1	0.07 (0.04–0.11) *	1.43 ± 0.18	5.27	4	0.26	7.00 (4.67–11.16) ǂ
	HAY	350	0.06–2	0.14 (0.08–0.21) *	1.77 ± 0.20	5.86	4	0.21	14.00 (9.49–21.54) ǂ
	WSH	350	0.01–0.25	0.03 (0.02–0.05) *	1.92 ± 0.23	5.66	4	0.23	3.00 (2.16–4.74) ǂ
	NKL	350	0.01–0.25	0.03 (0.02–0.04) *	1.75 ± 0.21	4.40	4	0.35	3.00 (1.80–3.90) ǂ

N = number of tested individuals; Conc. = concentrations used in bioassay; LC₅₀ = median lethal concentration; FL = fiducial limit; χ² = chi-square; RR = resistance ratio (LC₅₀ of insecticide in field population/LC₅₀ of insecticide in susceptible strain). * The field population was significantly more resistant than the susceptible strain. ^ǂ Significantly different from SS based on 95% CIs of RRs that did not include 1 [34].

and

Table 4. Susceptibility of seven M. persicae field populations to acetamiprid, spirotetramat, afidopyropen, and flonicamid.

Insecticide	Population	N	Conc. (mg/L)	LC50 (95% FL) (mg/L)	Fit of Probit Line				RR (95% CL)
					Slope ± SE	χ2	df	p
Acetamiprid	SS	350	0.01–0.10	0.02 (0.01–0.02)	2.38 ± 0.29	5.14	4	0.27	1.00
	DHB	350	0.09–3	0.29 (0.16–0.46) *	1.46 ± 0.18	6.15	4	0.19	14.50 (14.23–6.78) ǂ
	RAF	350	0.09–3	0.20 (0.08–0.34) *	1.38 ± 0.18	7.34	4	0.12	10.00 (9.51–19.50) ǂ
	DWS	350	0.09–3	0.18 (0.09–0.29) *	1.43 ± 0.18	5.68	4	0.22	9.00 (8.61–17.74) ǂ
	DRH	350	0.05–1.5	0.11 (0.07–0.16) *	1.68 ± 0.20	4.29	4	0.37	5.50 (5.72–10.49) ǂ
	HAY	350	0.05–1.5	0.13 (0.09–0.19) *	1.63 ± 0.19	4.61	4	0.33	6.50 (6.71–12.23) ǂ
	WSH	350	0.01–0.38	0.04 (0.03–0.05) *	1.80 ± 0.21	3.22	4	0.52	2.00 (1.82–3.47) ǂ
	NKL	350	0.02–0.75	0.07 (0.05–0.09) *	1.84 ± 0.21	3.17	4	0.53	3.50 (3.57–6.58) ǂ
Spirotetramat	SS	350	0.04–1.25	0.08 (0.06–0.11)	1.46 ± 0.19	3.86	4	0.43	1.00
	DHB	350	0.16–5	0.53 (0.23–0.95) *	1.42 ± 0.17	9.93	4	0.04	6.63 (4.20–9.53) ǂ
	RAF	350	0.16–5	0.43 (0.33–0.54) *	1.66 ± 0.19	3.46	4	0.48	5.38 (3.48–7.69) ǂ
	DWS	350	0.16–5	0.36 (0.15–0.60) *	1.34 ± 0.18	7.13	4	0.13	4.50 (2.77–6.74) ǂ
	DRH	350	0.04–1.25	0.10 (0.06–0.16)	1.47 ± 0.19	4.99	4	0.29	1.25 (0.80–1.91)
	HAY	350	0.04–1.25	0.12 (0.07–0.18)	1.61 ± 0.19	6.14	4	0.19	1.50 (0.95–2.10)
	WSH	350	0.02–0.63	0.06 (0.03–0.10)	1.49 ± 0.20	5.75	4	0.22	0.75 (0.46–1.16)
	NKL	350	0.04–1.25	0.11 (0.07–0.15)	1.62 ± 0.19	4.79	4	0.31	1.38 (0.85–1.90)
Afidopyropen	SS	350	0.01–0.25	0.03 (0.02–0.04)	1.66 ± 0.20	4.92	4	0.30	1.00
	DHB	350	0.02–0.50	0.06 (0.04–0.08)	1.89 ± 0.20	6.65	4	0.16	2.00 (1.59–3.01) ǂ
	RAF	350	0.02–0.50	0.05 (0.03–0.08)	1.79 ± 0.22	6.18	4	0.19	1.67 (1.36–2.84)
	DWS	350	0.02–0.50	0.05 (0.03–0.07)	1.72 ± 0.20	5.36	4	0.25	1.67 (1.33–2.60)
	DRH	350	0.01–0.25	0.02 (0.01–0.04)	1.37 ± 0.19	6.86	4	0.14	0.67 (0.57–1.24)
	HAY	350	0.02–0.50	0.06 (0.04–0.08)	1.96 ± 0.21	5.51	4	0.24	2.00 (1.61–3.11) ǂ
	WSH	350	0.01–0.25	0.02 (0.01–0.03)	1.48 ± 0.20	4.81	4	0.31	0.67 (0.53–1.12)
	NKL	350	0.01–0.25	0.03 (0.02–0.04)	1.71 ± 0.20	4.96	4	0.29	1.00 (0.74–1.45)
Flonicamid	SS	350	0.05–1.50	0.16 (0.07–0.27)	1.48 ± 0.19	7.41	4	0.12	1.00
	DHB	350	0.09–3	0.27 (0.16–0.40)	1.49 ± 0.18	5.14	4	0.27	1.72 (1.13–2.62)
	RAF	350	0.09–3	0.22 (0.13–0.31)	1.66 ± 0.19	4.73	4	0.32	1.40 (0.92–2.12)
	DWS	350	0.09–3	0.17 (0.11–0.22)	1.61 ± 0.21	3.49	4	0.48	1.06 (0.67–1.70)
	DRH	350	0.05–1.50	0.24 (0.15–0.29)	2.15 ± 0.28	4.82	4	0.31	1.54 (1.01–2.33)
	HAY	350	0.05–1.50	0.18 (0.10–0.27)	1.60 ± 0.20	4.73	4	0.32	1.13 (0.73–1.74)
	WSH	350	0.02–0.75	0.06(0.03–0.08)	1.33 ± 0.17	6.18	4	0.19	0.38 (0.22–0.55)
	NKL	350	0.02–0.75	0.05 (0.03–0.06)	1.52 ± 0.18	3.14	4	0.53	0.31 (0.20–0.48)

N = number of tested individuals; Conc. = concentrations used in bioassay; LC₅₀ = median lethal concentration; FL = fiducial limit; χ² = chi-square; RR = resistance ratio (LC₅₀ of insecticide in field population/LC₅₀ of insecticide in susceptible strain). * The field population was significantly more resistant than the susceptible strain. ^ǂ Significantly different from SS based on 95% CIs of RRs that did not include 1 [34].

).

Bifenthrin exhibited LC₅₀ values of 0.12–0.86 mg/L against M. persicae, with RRs of 3.00–21.50 among field populations. The Al-Dhabia (DHB), Al-Rafaa (RAF), and Wadi Al-Dawasir (DWS) populations showed moderate resistance, the Diriyah (DRH), Al-Hayer (HAY), and Al-Nakhil (NKL) populations exhibited low resistance, and the Al-Washlah (WSH) population displayed susceptibility to bifenthrin compared with the SS strain (Table 3). Regarding fenitrothion, LC₅₀ values were 04.07–0.60 mg/L against M. persicae, with RRs of 3.13–25.31 across field populations. The DHB, RAF, and DWS populations showed moderate resistance, the HAY and WSH populations exhibited low resistance, and the DRH and NKL populations displayed susceptibility to fenitrothion compared with the SS strain (Table 3). Fosthiazate showed LC₅₀ values of 0.03–0.20 mg/L against M. persicae, with RRs of 3.00–20.00 among field populations. The DHB, RAF, DWS, and HAY populations showed moderate resistance, the DRH population exhibited low resistance, and the WSH and NKL populations displayed susceptibility to fosthiazate compared with the SS strain (Table 3).

Regarding acetamiprid, LC₅₀ values were 0.04–0.29 mg/L against M. persicae, with RRs of 2.00–14.50 across field populations. The DHB population showed moderate resistance, the RAF, DWS, and DRH populations exhibited low resistance, and the WSH and NKL populations displayed susceptibility to acetamiprid compared with the SS strain (Table 4). For spirotetramat, LC₅₀ values were 0.06–0.53 mg/L against M. persicae, with RRs of 0.75–6.63 among the field populations. The DHB and RAF populations showed low resistance, and the DWS, DRH, HAY, WSH, and NKL populations exhibited susceptibility to spirotetramat compared with the SS strain (Table 4).

Considering afidopyropen, LC₅₀ values were 0.02–0.06 mg/L against M. persicae, with RRs of 0.67–2.00 across field populations. For flonicamid, LC₅₀ values were 0.05–0.27 mg/L against M. persicae, whereas RRs were 0.31–1.72 across field populations. All field populations of M. persicae exhibited susceptibility to afidopyropen and flonicamid compared with the SS strain (Table 4).

3.2. Greenhouse Insecticide Efficacy

Pre-application, the changes in the population density (CPD) and percent efficacy (EF) values of insecticides against the most resistant M. persicae field population (DHB) were not significantly different among treatments except acetamiprid and fenitrothion. Conversely, post-application, significant differences emerged among treatments (

Table 5. Population densities of M. persicae on sweet pepper and effectiveness of insecticides.

Treatments	Pre-Application	1 Day Post-Application			3 Days Post-Application			7 Days Post-Application
		N	CPD %	EF %	N	CPD %	EF %	N	CPD %	EF %
Control	283.75 a	328.25 a	15.68	0	364.17 a	28.34	0	448 a	57.89	0
Spirotetramat	267.83 abc	265.08 b	−1.03	14.44	47.50 d	−82.27	86.18	12 cd	−95.52	97.16
Flonicamid	274.33 ab	287.33 b	4.74	9.46	67.67 c	−75.33	80.78	60.83 b	−77.83	85.96
Afidopyropen	263.67 abc	286.42 b	8.63	6.10	43.33 d	−83.57	87.19	0 d	−100	100
Acetamiprid	246.25 bc	181 d	−26.50	36.46	1.42 f	−99.42	99.55	0 d	−100	100
Bifenthrin	260.67 abc	214.92 c	−17.55	28.73	21.67 e	−91.69	93.52	20.08 c	−92.30	95.12
Fenitrothion	242.25 c	115.58 e	−52.29	58.76	3.67 f	−98.49	98.82	0 d	−100	100
Fosthiazate (IA)	283.58 a	256.92 b	−9.40	21.69	95.92 b	−66.18	73.65	0 d	−100	100
Fosthiazate (FA)	286.08 a	174.17 d	−39.19	47.37	1.67 f	−99.42	99.55	0 d	−100	100
Significance Level	F = 2.53; df = 8; p = 0.015	F = 34.27; df = 8; p ≤ 0.00001			F = 333.35; df = 8; p ≤ 0.00001			F = 611.47; df = 8; p ≤ 0.00001
Treatments	Pre-Application	10 days post-application			14 days post-application			21 days post-application
Control	283.75 a	517.92 a	82.53	0	821.08 a	189.37	0	1148.42 a	304.73	0
Spirotetramat	267.83 abc	1.67 c	−99.38	99.66	0 c	−100	100	0 c	−100	100
Flonicamid	274.33 ab	3 c	−98.91	99.40	0 c	−100	100	0 c	−100	100
Afidopyropen	263.67 abc	0 c	−100	100	0 c	−100	100	0 c	−100	100
Acetamiprid	246.25 bc	0 c	−100	100	0 c	−100	100	0 c	−100	100
Bifenthrin	260.67 abc	47.92 b	−81.62	89.93	87.75 b	−66.34	88.37	143 b	−45.14	86.45
Fenitrothion	242.25 c	0 c	−100	100	0 c	−100	100	0 c	−100	100
Fosthiazate (IA)	283.58 a	0 c	−100	100	0 c	−100	100	0 c	−100	100
Fosthiazate (FA)	286.08 a	0 c	−100	100	0 c	−100	100	0 c	−100	100
Significance Level	F = 2.53; df = 8; p = 0.015	F = 1403.38; df = 8; p ≤ 0.00001			F = 751.94; df = 8; p ≤ 0.00001			F = 714.71; df = 8; p ≤ 0.00001

N: the mean aphid number per plant. Within the same letter column, the means pursued by the same letter are not significantly different (LSD test, α = 0.05). CPD%: change in population density; EF%: percent efficacy calculated according to the Henderson and Tilton [37] formula.

) as follows. One day post-application, fenitrothion showed the maximum reduction (−52.29%) in DHB population size, with an efficacy of 58.76%, followed by fosthiazate “foliar-application, (FA)” (−39.19%) with an efficacy of 47.37%, acetamiprid (−26.50%) with an efficacy of 36.46%, bifenthrin (−17.55%) with an efficacy of 28.73%, fosthiazate “irrigation-application (IA)” (−9.40%) with an efficacy of 21.69%, spirotetramat (−1.03%) with an efficacy of 14.45%, flonicamid (4.74%) with an efficacy of 9.46%, and afidopyropen (8.63%) with an efficacy of 6.10%. Three days post-application, fosthiazate (FA) and acetamiprid exhibited the highest reduction in DHB population size (−99.42% for both) with an efficacy of 99.55%, followed by fenitrothion (−98.49%) with an efficacy of 98.82%, bifenthrin (−91.69%) with an efficacy of 93.52%, afidopyropen (−83.57%) with an efficacy of 87.19%, spirotetramat (−82.27%) with an efficacy of 86.18%, flonicamid (−75.33%) with an efficacy of 80.78%, and fosthiazate (IA) (−66.18%) with an efficacy of 73.65% (Table 5).

Seven days post-application, fenitrothion, acetamiprid, fosthiazate (FA), fosthiazate (IA), and afidopyropen displayed the highest reduction in DHB population size (–100% for each treatment), all with an efficacy of 100%, followed by spirotetramat (−95.52%) with an efficacy of 97.16%, bifenthrin (−92.30%) with an efficacy of 95.12%, and flonicamid (−77.83%) with an efficacy of 85.96%. Ten days post-application, fenitrothion, acetamiprid, fosthiazate (FA), fosthiazate (IA), and afidopyropen maintained the highest possible reduction in DHB population size (−100%), all with 100% efficacy, followed by spirotetramat (−99.38%) with an efficacy of 99.66%, flonicamid (−98.91%) with an efficacy of 99.40%, and bifenthrin (−81.62%) with an efficacy of 89.93%. Notably, bifenthrin began to lose its effectiveness at this 10-day stage (Table 5).

At 14 and 21 days post-application, all insecticides achieved full control of the DHB population, with 100% population reduction and efficacy, except for bifenthrin, which continued to lose efficacy at 14 days (−66.34% population size and 88.37% efficacy) and 21 days (−45.14% population size and 86.45% efficacy) post-application (Table 5).

4. Discussion

Monitoring insecticide susceptibility under laboratory and field conditions is critical for evaluating the efficacy of insecticides, whether registered or showing promise for registration, and subsequently selecting potent chemicals for managing insect pests [29,38]. This study aimed to assess the efficacy of different registered and nonregistered insecticides in Saudi Arabia against M. persicae under laboratory and greenhouse conditions.

4.1. Insecticide Toxicity Against M. persicae

The toxicity results of tested registered and unregistered insecticides in Saudi Arabia revealed susceptibility to moderate resistance, consistent with our previous study [29]. This aligns with the assessment of technical-grade active ingredients of the same insecticides against identical M. persicae populations. The observed association between the highest resistance cases and old chemical classes (organophosphates and pyrethroids), as well as the DHB, RAF, and DWS field populations collected from major crop production governorates (Al-Kharj and Wadi Al-Dawasir), was anticipated. Notably, there was an absence of high resistance cases (>30-fold) among these old insecticides and field populations, possibly attributed to stringent Saudi regulations on insecticide use, including the phasing out of several organophosphates, carbamates, and pyrethroids over the last two decades. This is expected to alleviate selection pressure on M. persicae field populations and reduce the development of resistance against these old chemical classes. Previously, resistance against fenitrothion at various levels has been documented in Aphis gossypii [39] and Aphis craccivora [40]. Similarly, resistance has been reported against bifenthrin in M. persicae [14], A. gossypii [41], and Brevicoryne brassicae [42].

The second-highest cases of resistance were associated with acetamiprid, which was expected given its classification as a neonicotinoid which is the most widely used chemical class for controlling M. persicae in recent years including in Saudi Arabia [29]. The notable cases of low and moderate resistance should raise an alert regarding the potential rapid buildup of resistance against this chemical class. It is advisable to use neonicotinoids in a rotational manner with other chemical classes, periodically monitoring resistance levels to prevent future control failures at the field scale. Previously, high levels of resistance have been reported against acetamiprid in various insect pests, including M. persicae [8], A. gossypii [43], and Amrasca devastans [38]. Spirotetramat, despite showing excellent toxicity against M. persicae field populations, exhibited two cases of low resistance in the most resistant field populations, DHB and RAF. This slight shift in resistance levels should serve as a warning to periodically monitor resistance against this new insecticide to prevent the loss of effectiveness. This concern is justified by the documented worldwide high levels of resistance against spirotetramat in various insect pests, including M. persicae [9], B. tabaci [44], A. devastans [45], and A. gossypii [46]. The most probable mechanisms involved in the development of resistance to different insecticides in M. persicae are detoxifying enzymes and target site mutations [9,12,13,47,48,49].

The outstanding toxicity of the two unregistered insecticides, afidopyropen and flonicamid, against M. persicae field populations, including the most resistant populations, DHB and RAF, is notable in the context of Saudi crop production. Indeed, this finding supports the registration of these two compounds to control M. persicae. Globally, these two insecticides are currently used to control homopterous pests, such as aphids, jassids, and whiteflies [50]. Previous studies have documented cases of resistance in a few pests against afidopyropen, including A. gossypii [51] and B. tabaci [52], and against flonicamid in Rhopalosiphum padi [53] and Oxycarenus hyalinipennis [54].

4.2. Greenhouse Insecticide Efficacy

All insecticides demonstrated high efficacy (>90%) against the DHB M. persicae field population, exhibiting the highest resistance levels in baseline assessments. All tested insecticides, except bifenthrin, displayed prolonged effectiveness, with bifenthrin losing efficacy after 10 days post-application. The insecticides tested in this study can be categorized based on their post-application speed in fully controlling the DHB M. persicae field population: (1) requiring 3 days: acetamiprid, fenitrothion, and fosthiazate (FA); (2) requiring 7 days: afidopyropen and fosthiazate (IA); (3) requiring 10 days: spirotetramat and flonicamid; and (4) failing to achieve full control at all post-application intervals (with a maximum EF value of 95.12% at 7 days post-application of bifenthrin). This study established a robust baseline for crop growers to design effective M. persicae control programs covering the entire growing season by employing insecticides with varying speeds of action. Despite high efficacy at the labeled field rate, except for bifenthrin, the apparent masking of various levels of laboratory-documented resistance raises concerns about “practical resistance.” This highlights the critical need for periodic monitoring of M. persicae resistance to the used insecticides to prevent sudden control failures at the field scale.

The non-systemic and broad-spectrum insecticide and acaricide bifenthrin, rated as “somewhat effective” for aphid control in the 2022–23 Vegetable Production Guide for Commercial Growers [55], emerged as the least effective insecticide in the current study. Although achieving a maximum M. persicae control level of 95.12% within 7 days post-application, its effects gradually decline to 86.45% after 21 days post-application. The suboptimal performance of bifenthrin can be attributed to its non-systemic nature, where systemic characteristics are critical for effectively controlling sucking pests, including aphids. Previous field studies have consistently reported comparable or inferior field performance of pyrethroids. For example, bifenthrin demonstrated analogous field performance, concerning control efficacy and longevity, against Trialeurodes vaporariorum in tomato greenhouse cultivation [56].

In the present study, fenitrothion exhibited excellent field performance in fully controlling M. persicae within only 3 days post-application (98.82%), maintaining 100% control of M. persicae after 7 days post-application. Similar field performance has been reported for other organophosphates. For example, methamidophos and acephate achieved estimated control levels of 95% and 100% in M. persicae, respectively, within five days during sweet pepper open-field cultivation [57]. Chlorpyrifos demonstrated fast action by reducing the Diuraphis noxia population by 33% within the first 2 h after application in a wheat open-field cultivation; at 4 days post-application, full D. noxia population control was achieved [58]. Additionally, chlorpyrifos effectively controlled the D. noxia population within 7 days post-application during wheat greenhouse cultivation [59]. Fosthiazate’s excellent systemic characteristics facilitated rapid control of M. persicae within 3 days post-application when administered foliarly and within 7 days post-application when applied via irrigation. The swifter control observed for foliar application compared with irrigation application may be attributed to direct contact between fosthiazate and M. persicae individuals, along with the plant shoot system, resulting in faster effects and penetration, respectively, thereby reaching lethal concentrations more rapidly. The absence of prior studies about fosthiazate’s field performance against M. persicae was expected owing to its global registration as a nematicide. Notably, fosthiazate was tested in the current study for research purposes, serving as the only registered systemic organophosphate in Saudi Arabia. Additionally, testing it as a foliar application, contrary to its recommended irrigation application, was performed for scientific comparison purposes. Consequently, any recommendations beyond its current application cannot be made without further investigations.

Acetamiprid exhibited excellent field performance in fully controlling M. persicae within only 3 days post-application in this study. Similar to the current study, previous research has found high field performance for acetamiprid. For example, in pepper open-field cultivation, acetamiprid induced 98.9% and 100% mortality in M. persicae at 2 and 7 days post-application, respectively [60]. Similarly, in a canola greenhouse cultivation, it effectively controlled B. brassicae, with 100% efficacy at 120 h post-application [61]. However, in cotton open-field cultivation, acetamiprid required 14 days post-application to control A. gossypii and cotton whitefly populations by 93.0% and 95.7%, respectively [62,63]. Spirotetramat exhibited excellent efficacy (99.66%) against M. persicae within 10 days post-application, maintaining complete control until the study’s conclusion (21 days post-application). In agreement with the current study, the documented efficacy of spirotetramat under greenhouse and open-field conditions includes effective control of M. persicae within 7 and 6 days post-application during cabbage greenhouse and open-field cultivations, respectively [64]. Similarly, spirotetramat achieved effective control of M. persicae over 28 days post-application during peach open-field cultivation [65]. However, during cotton open-field cultivation, spirotetramat showed slightly lower efficacies (although remaining robust) of 95.0% and 89.7% within 14 days post-application in A. gossypii and B. tabaci control assessments, respectively [62,63]. This slight variation in spirotetramat efficacy in these different studies is attributed to variations in trial conditions, targeted populations and species, and resistance levels.

In the present finding, afidopyropen exhibited rapid and prolonged insecticidal activity against the M. persicae field population. It effectively controlled M. persicae within 3 days post-application, reaching complete control by 7 days post-application, and sustaining this efficacy until the end of the study (21 days post-application). Consistent with the current study, afidopyropen has shown excellent insecticidal activity against various common aphid species, e.g., M. persicae, A. gossypii, A. craccivora, and Aphis glycines [26,27,50]. Flonicamid showed excellent efficacy (99.40%) at the 10-day post-application stage, maintaining 100% control of M. persicae until the study’s conclusion (21 days post-application). Similar to our findings, flonicamid completely controlled M. persicae and A. solani within 7 days post-application during head lettuce open-field cultivation [66]. During cotton open-field cultivation, flonicamid showed a slightly lower efficacy of 93% in A. gossypii control within 7 days post-application [67].

5. Conclusions

The tested M. persicae field populations were found to be susceptible to moderate resistance to the registered and unregistered insecticide formulations (in Saudi Arabia) tested in this study. Overall, all tested insecticides displayed high effectiveness and prolonged effects against M. persicae under greenhouse conditions except bifenthrin which lost its efficacy after 10 days post-application. However, their post-application duration varies in fully controlling M. persicae leading by acetamiprid, fenitrothion, and fosthiazate “FO” (required 3 days); followed by afidopyropen and fosthiazate “RG” (required 7 days); and spirotetramat and flonicamid (required 10 days). Specifically, the registered insecticides (acetamiprid, spirotetramat, fenitrothion, and fosthiazate) and unregistered insecticides (flonicamid and afidopyropen) were the most effective insecticides against M. persicae. Consequently, sustaining this effective control necessitates the registration of flonicamid and afidopyropen in Saudi Arabia, expanding the array of available chemical tools for M. persicae control. Employing these effective insecticides in a rotational manner is crucial to preventing or delaying resistance development in M. persicae. Additionally, the use of these effective insecticides should be integrated into a comprehensive management program, including insecticide resistance management, to minimize overreliance on chemicals, prolong insecticide efficacy, and ensure environmental and human safety. This requires periodically monitoring their efficacy and studying their environmental fate and potential negative effects on the non-targeted organisms, especially the newly introduced insecticides.