Effect of Gold Nanoparticles Against Tetranychus urticae and Phytoseiulus persimilis in Tomato

Abstract

The two-spotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae) is a major pest of horticultural, ornamental, fruit, and strawberry crops worldwide. Currently, various management tools have been explored for this pest, with nanoparticles being one of them, which stand out for their characteristics and multiple effects. This study evaluated the effects of green-synthesized gold nanoparticles (AuNPs) on the mortality and repellency of T. urticae and its natural predator Phytoseiulus persimilis under laboratory conditions, as well as their efficacy in greenhouse tomatoes against T. urticae. In the laboratory, a biological window for AuNPs (50–100 mg L⁻¹) on the pest and predator was established using a residual film method and a free-choice assay. In the greenhouse, four concentrations (300, 500, 750, and 1000 mg L⁻¹) were evaluated via foliar application at 10-day intervals. The results showed susceptibility to AuNPs in all stages of T. urticae and the adult P. persimilis. The death times from AuNPs were similar in both species. Furthermore, the AuNPs were selective for the pest rather than the natural enemy. In greenhouses, AuNPs affected T. urticae populations in tomato plants, and significant differences were observed on some continuous and final agronomic variables (associated with fruits). This study showed that T. urticae and P. persimilis were susceptible to green-synthesized AuNPs. AuNPs can be a management tool, although studies on other non-target species and estimating agronomic effects on other crops are recommended.

1. Introduction

The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is widely distributed across most continents [1]. Its small size, high fecundity, short life cycle, and marked ability to develop resistance to acaricides [2,3,4] make it one of the most important agricultural pests worldwide [5]. T. urticae infests >1300 plant species in 77 families, including economically important fruit trees, vegetables, berries, field crops, and ornamentals [1,6]. Managing this mite has become increasingly complex owing to rising global temperatures, intensified agricultural production, management practices, heavy reliance on chemical inputs, and the evolution of resistance to multiple active ingredients [5,7,8]. Such resistance has been driven mainly by repeated applications of synthetic pesticides [9,10]. Consequently, alternative strategies for T. urticae management have been explored within Integrated Pest Management (IPM) [11,12,13]. IPM reduces environmental impact, delays resistance, and lessens risks to non-target organisms such as predators, parasitoids, and pollinators [14,15] by minimizing chemical pesticide use and promoting sustainable, selective options [16,17,18,19].

Nanotechnology is increasingly applied in agriculture [20,21], particularly for pest management [22,23]. Nanoinsecticides and nanoacaricides of diverse compositions and synthesis routes provide controlled release, improved delivery, and reduced loss of active ingredients [24,25,26,27,28]. For T. urticae, metallic (Au, Ag, Zn, Fe, Ti), non-metallic (C, Si), green-synthesized (botanical), and polysaccharide-based nanoparticles (NPs) have been tested [29]. Conventional synthesis routes—hydrothermal, thermal, anodization, deposition–precipitation, wet oxidation, electrodeposition, and sonication—can be costly, energy intensive, and use hazardous reagents [30,31]. Green synthesis, using plants, algae, and microorganisms, offers a sustainable alternative [32], facilitates scale-up [33], lowers costs, and avoids toxic chemicals [34,35].

AuNPs have been explored in the search for alternatives for the control of various urban and agricultural pests [36,37,38,39,40,41]. Given the challenges faced in pest management, such as the emergence of resistance and contamination [5], the use of NPs can be a tool for the control and management of agricultural pests. AuNPs differ in their mode of action due to their unique characteristics [26,31]. In addition to the pest species, they are known to affect multiple processes in insects or arthropods, from interaction with the cuticle, generation of reactive oxygen species (ROS), interference with gene expression, enzyme inhibition, alteration of feeding and reproductive behavior [39], and even affecting plants.

Studies of AuNPs in agricultural pests are limited, and their effects on non-target species such as pollinators, predators, and parasitoids are unknown, even affecting agricultural crops or model plants [42,43].

This study evaluated green, synthetic gold nanoparticles (AuNPs) on the mortality and repellency of T. urticae and their selectivity toward P. persimilis under laboratory conditions. Furthermore, the efficacy of AuNPs on T. urticae and their effects on tomato agronomic variables were evaluated under greenhouse conditions.

2. Materials and Methods

2.1. Green Synthesis and Characterization of Gold Nanoparticles (AuNPs)

Green pecan shells Carya illinoinensis (Wangenh) K. Koch (Juglandaceae) were collected from the experimental field “El Bajío” (25.35599743085867, −101.03874520637001) at the Universidad Autónoma Agraria Antonio Narro (UAAAN) in Saltillo, Coahuila, Mexico. The seeds were cut into small pieces and subsequently dried in an oven (Ecoshel™) at 35 °C for ten days. Finally, the dried material was ground into a fine powder using a Crabby™ ball mill (Retsch, Pulverisette 6, Idar-Oberstein, Germany).

The extract was prepared at the Laboratorio de Nanotecnología of the Departamento de Ciencias Basicas (DCB) of the UAAAN, following the methodology described by Neira et al. [44]. The powdered material obtained after milling was sieved using a 250 μm Tyler™ mesh. For the preparation, 2 g of the sieved powder were suspended in 200 mL of sterile distilled water (pH 6.5) in a round-bottom flask. The mixture was heated to 80 °C for 2 h under constant magnetic stirring. It was then filtered through Whatman No. 4 filter paper, resulting in an aqueous extract of pecan shell. Finally, the extract was stored at 2 °C under refrigeration until use.

The green synthesis of gold nanoparticles (AuNPs) was carried out via direct reduction using the aqueous pecan shell extract and tetrachloroauric acid (HAuCl₄) (Sigma-Aldrich™) as the precursor, following the protocol described by [44] with slight modifications. In a round-bottom flask equipped with a condenser, 985 mL of sterile distilled water and 15 mL of the extract were combined and maintained at 60 °C in a water bath for 10 min. Subsequently, 1.5 g of HAuCl₄ was added under continuous magnetic stirring at 80 °C for 2 h. Upon completion, the resulting solution was stored in amber glass bottles and refrigerated at 2 °C until further analysis and use.

The shape and size of the AuNPs were characterized by energy-dispersive X-ray spectroscopy (EDX) and transmission electron microscopy (TEM) using a FEI-TITAN 80–300 kV microscope (Fisher Scientific™) at the Centro de Investigación en Química Aplicada (CIQA) in Saltillo, Coahuila, Mexico.

2.2. Mites Rearing

Susceptibility assays with AuNPs were conducted at the Laboratorio de Entomología Molecular y Alternativas del Control de Plagas (LEMACP) of the Departamento de Parasitología (DP) of the UAAAN. A colony of T. urticae was established on bean Phaseolus vulgaris L. (Pinto Saltillo variety) plants and maintained in a bioclimatic chamber (Biometer Mark III™, Lab Line) under controlled conditions of 25 ± 2 °C, 60–70% relative humidity, and a 13:11 h (light/dark) photoperiod.

2.3. In Vitro Evaluation of AuNPs on T. urticae and P. persimilis

To evaluate the effect of AuNPs on T. urticae, a biological assay range (50–1000 mg L⁻¹) was established across the egg, larval, protonymph, deutonymph, and adult stages. Population homogenization was performed using the leaf arena technique described by [45], in which two-day-old females were transferred to 35 mm bean leaf discs and allowed to oviposit for 24 h; the resulting population was used in the assays. For P. persimilis, biological material was commercially sourced (Spidex Vital™, Koppert Mexico), selecting only adult individuals.

The susceptibility of the mobile instars of both species was evaluated using method 004 of the Insecticide Resistance Action Committee [46], with slight modifications. Individuals were transferred using a fine brush onto bean leaf discs previously treated with AuNP solutions. The discs were placed adaxial side up on trays containing water-saturated cotton pads to prevent mite escape. AuNP solutions were prepared in sterile distilled water with 0.01% sodium dioctyl sulfosuccinate (DSS) as a dispersant. The leaf discs were immersed in the solution for 10 s; to remove excess and avoid interference with the locomotion of the mites [47], the disc was placed on cotton for 5 min at a 45° angle; then, the females were placed. The treated discs were then placed on the trays, and 30 females were transferred to each disc, with 10 replicates per treatment, totaling 300 individuals.

Mortality was assessed at 24, 48, 72, 96, and 120 h post-treatment. Mites were considered dead when they exhibited ataxia (uncoordinated movements) or remained immobile after stimulation with a fine brush [48]. In the case of eggs, for which stimulation is not feasible, mortality assessment was conducted at the point of complete emergence in the untreated control.

Based on the bioassays, the median lethal concentration (LC₅₀) and median lethal time (LT₅₀) of AuNPs were determined for T. urticae across all developmental stages and for adult P. persimilis. AuNPs were compared against two commercial controls, depending on the instar evaluated: a synthetic acaricide for mobile stages (Thimet 15 G™, 15% phorate™, AMVAC^® Chemical Corporation, Zapopan, Jalisco, Mexico), a botanical ovicide (Atzingao™, 60% Euphorbiaceae plant extracts, GreenCorp, Saltillo, Coahuila, Mexico), and a negative control (distilled water). In both bioassays, when mortality in the negative control was 0%, no correction was applied; if control mortality was less than 10%, the data were adjusted using Abbott’s formula [49].

From the LC₅₀ values obtained in the adult bioassays of T. urticae and P. persimilis, the selectivity ratio (SR) was calculated using the formula proposed by [50]:

Selectivity Ratio (SR) = LC₅₀ of the natural enemy/LC₅₀ of the pest

According to [51], an SR value greater than 1 indicates that the insecticide is selective for the natural enemy (i.e., less toxic to the predator), whereas an SR value less than 1 suggests that selectivity favors the pest (i.e., the product is more toxic to the predator).

2.4. Repellency Effects of AuNPs on T. urticae and P. persimilis

The repellency of AuNPs was evaluated following the methodology described by [52], with slight modifications. The experimental unit (EU) consisted of a free-choice arena designed according to [53,54]. The arena was composed of four plastic Petri dishes (5 cm in diameter), three of which were treated with the same concentration of each treatment, and one left untreated as a control. This design was applied to five evaluated treatments: three sublethal concentrations of AuNPs (LC₁₀, LC₁₅, and LC₂₀), a botanical repellent control (eBioluzion Plus VO™, GreenCorp, Saltillo, Coahuila, Mexico) at 150 mg L⁻¹, and a treatment containing only distilled water and DSS (0.01%). The treated Petri dishes were connected to a central untreated dish via four plastic tubes, each 10 cm long and 0.3 cm in diameter. All the treatments were prepared using sterile distilled water with 0.01% DSS.

Bean leaf discs (Pinto Saltillo variety) were treated by immersion, submerging them in each concentration for 10 s. Excess solution was then removed, and the discs were placed abaxial side up in the Petri dishes, which contained a 5 cm diameter filter paper previously moistened with sterile distilled water.

Individuals of T. urticae were homogenized using the leaf arena technique described by [45]. Adult P. persimilis were obtained commercially (Spidex Vital™, Koppert, Mexico). For both species, 30 females were released into the central dish of the free-choice arena, which was connected to the treated leaf discs. Each treatment consisted of 10 replicates (10 free-choice arenas per treatment), randomly distributed. The bioassay was conducted in a bioclimatic chamber (Biometer Mark III™, Lab Line) for 72 h under controlled conditions: temperature at 25 ± 1 °C, relative humidity at 60 ± 10%, and a 12:12 h light/dark photoperiod. Evaluations were carried out at 2, 4, 6, 12, 24, 48, and 72 h, recording the number of females present on each disc within the free-choice arena.

Based on the data obtained, the repellency percentage (RP) was calculated using the following formula:

Repellency (%) = [(Control population − Treatment population)/(Control population + Treatment population)] × 100

Additionally, the repellency index (RI) was calculated according to [52], as adapted by [55]. This index classifies treatments as neutral (RI = 1), attractive (RI > 1), or repellent (RI < 1). The complementary scale of the repellency index is shown in

Table 1. Complementary scale of the repellency index (RI) proposed by [52], adapted by [55].

Repellency Index (RI) Value	Category
≥1.0	No repellency
0.76–0.99	Weak repellency
0.51–0.75	Moderate repellency
0.26–0.50	High repellency
0.0–0.25	Very high repellency

.

The repellency index (RI) was calculated using the following formula:

RI = (2G/(G + P))

where G represents the percentage of insects in the treatment, and P represents the percentage of insects in the control.

2.5. Greenhouse Evaluation of AuNPs Against T. urticae and Their Effects on Tomato

In the greenhouse phase, six treatments were evaluated: four concentrations of AuNPs (300, 500, 750, and 1000 mg L⁻¹); a chemical control (Thimet 15 G™, 15% phorate™) applied at its recommended commercial rate (60 mg L⁻¹ = 0.5 L/ha, considering an application volume of 300 L/ha); and an untreated control. The trial was conducted using determinate tomato plants of the Río Grande variety, grown in 25 kg plastic pots containing a 1:2 substrate mixture of perlite (Multiperl™) and peat moss (Vigoro™). Crop nutrition was managed with a modified Steiner nutrient solution (NO₃ = 10, H₂PO₄ = 1, SO₄ = 7, K = 7, Ca = 8, and Mg = 3 meq L⁻¹). A randomized block design was used with four replicates, each consisting of five plants, totaling 120 plants. This trial was established in the months of March, April, and May 2024.

To evaluate the biological efficacy of AuNPs against T. urticae, a deliberate infestation was carried out 10 days prior to treatment application, following the methodology described by [56] with slight modifications. Ten two-day-old females were placed on each tomato plant (19 April 2024), 20 days after transplanting (DAT) (March 30). Treatments were applied using a manual sprayer (Truper FDO-2™), with 25 mL of each AuNP concentration in a solution containing 0.01% DSS sprayed onto the foliage. The five developmental stages of T. urticae (egg, larva, protonymph, deutonymph, and adult) were assessed per square inch (in ²) using a portable digital microscope (Jeswo™). Three applications were performed at 10-day intervals, the application start date was 26 April 2024. Evaluations were conducted at 0 (April 29), 10 (May 09), 20 (May 19), and 30 (May 29) days after the first application (DA1A). Biological efficacy was calculated using Abbott’s formula [49] for each developmental stage at the specified time points.

To estimate the effect of AuNPs on the crop, two sets of variables were assessed in tomato plants: continuous variables and final variables. Continuous variables included plant height, petiole Brix degrees, and chlorophyll content (expressed in SPAD units) measured at 0, 10, 20, and 30 days after the first application (DA1A), following the arrows mentioned above. Plant height was recorded with a tape measure (Truper™ FH-5ME); Brix degrees were determined with a handheld refractometer (ATC™); and chlorophyll content was measured with a SPAD-502 Plus™ meter (MCL502). These measurements were taken on the same dates as the AuNP efficacy evaluations, at 10-day intervals.

Final variables related to the fruit were measured at the end of the trial, 80 days after transplanting (DAT) (June 18). The following parameters were recorded: fruit number, polar and equatorial diameter, peduncle length, fruit weight, firmness, Brix degrees, total dissolved solids (TDS), pH, and the fresh weight (FW) and dry weight (DW) of the leaf area. To obtain DW, leaf samples were placed in kraft paper bags and dried in an oven (ICB™) at 40 °C for 15 days. Polar diameter, equatorial diameter, and peduncle length were measured with a digital caliper (LEIDSANY™); fruit weight, FW, and DW were determined with a balance (OHAUS Compass™); fruit firmness with a penetrometer (GY3™); Brix degrees with a handheld refractometer (ATC™); TDS with a TDS-3™ meter; and pH with a potentiometer (HANNA™ Hi98131).

2.6. Statistical Analysis

For the laboratory phase, lethal concentrations (LC₅₀ and LC₉₅) and lethal times (LT₅₀ and LT₉₅), together with their confidence intervals, were estimated by Probit analysis using the maximum-likelihood method [57]. For the repellency assay of AuNPs against T. urticae and P. persimilis, the data were first checked for normality and homoscedasticity, followed by analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) test (p < 0.05).

For the greenhouse data, normality and homoscedasticity were likewise verified. When these assumptions were met, ANOVA was performed for each variable, and means were separated with Fisher’s LSD test (p < 0.05) at each evaluation date. All statistical analyses were carried out in RStudio (R version 3.3.0).

3. Results and Discussion

3.1. EDX and TEM Characterization of AuNPs

Energy-dispersive X-ray spectroscopy (EDX) confirmed that the green-synthesized nanoparticles consisted chiefly of elemental gold (Figure 1). Characteristic plasmonic absorption peaks were observed near 2 and 12 keV, consistent with metallic AuNPs. Minor copper (Cu) signals arose from the TEM grid used during analysis.

Transmission electron microscopy (TEM) micrographs revealed a predominantly spherical morphology with a narrow size distribution (Figure 2a). The size-frequency histogram indicated a mean particle diameter of 29.8 nm (Figure 2b), confirming the homogeneity of the synthesized AuNPs.

3.2. In-Vitro Susceptibility of AuNPs to T. urticae

The concentration–response bioassay performed under controlled laboratory conditions (

Table 2. Egg-hatching inhibition and mortality of the five developmental stages of T. urticae 120 h after exposure to AuNPs.

AuNPs Concentration (mg L−1)	Hatching Inhibition (%)	Mortality (%)
	Eggs	Larva	Protonymph	Deutonymph	Adult
75	25.0 ± 1.5 d	55.0 ± 1.7 d	37.0 ± 1.0 d	35.7 ±2.1 d	31.3 ± 1.5 d
100	33.0 ± 2.5 c	73.0 ± 2.0 c	56.0 ± 1.0 c	50.0 ± 1.0 c	47.3 ± 2.5 c
150	40.0 ± 1.5 b	85.3 ± 0.6 b	85.0 ± 1.0 b	80.0 ± 1.0 b	63.0 ± 2.6 b
175	46.3 ± 1.5 a	98.0 ± 1.5 a	96.0 ± 0.6 a	93.3 ± 1.5 a	73.7 ± 3.2 a
gl	3	3	3	3	3
F value	166.6	203.5	1091	975	275.5
Pr(>F)	1.47 × 10−7	5.66 × 10−8	1.53 × 10−11	7.08 × 10−14	1.32 × 10−8
LSD	3.88	6.82	4.41	2.55	6.02
CV	5.96	4.823	3.54	2.16	6.15

Different letters within columns indicate significant differences (LSD, α = 0.05).

) confirmed that all developmental stages of T. urticae are susceptible to green-synthesized AuNPs. Mortality increased steadily from the lowest rate tested (75 mg L⁻¹) to the highest (175 mg L⁻¹). Egg hatching was inhibited by 25% at 75 mg L⁻¹ and rose to 46% at 175 mg L⁻¹ (F₃,₃₆ = 166.6, p < 0.0001). Among mobile stages, larvae were the most vulnerable: mean mortality climbed from 55 ± 1.7% to 98 ± 1.5% (F₃,₃₆ = 203.5, LSD = 6.82). Protonymphs and deutonymphs exhibited similar concentration-dependent responses, reaching 96% and 93% mortality, respectively, at 175 mg L⁻¹. Adults were consistently the least affected stage, yet mortality still exceeded 70% at the highest concentration (F₃,₃₆ = 275.5, CV = 6.15%). Different letters in Table 2 denote statistically distinct means (LSD, α = 0.05).

Stage-specific LC₅₀ and LC₉₅ values are presented in

Table 3. LC₅₀ and LC₉₅ (mg L⁻¹) of AuNPs (120 h) for each developmental stage of T. urticae.

Stage	Treatments	LC50 (mg L−1)	Fiducial Limits	LC95 (mg L−1)	Fiducial Limits	Slope ± SE	χ2	p
Egg	AuNPs	162	143–180	614	521–763	2.85 ± 0.154	26.4	0.0148
	Atzingao®	2801	2550–3012	7300	6520–8555	3.98 ± 0.216	47.1	0.00025
Larva	AuNPs	60.5	52.9–67.0	207	176–260	3.08 ± 0.195	27.2	0.0116
Protonymph	AuNPs	83.8	77.6–89.7	211	189–245	4.10 ± 0.179	51.1	0.00005
Deutonymph	AuNPs	94.5	88.4–100	205	185–235	4.89 ± 0.283	16.0	0.0988
Adult	AuNPs	112	96.2–124	290	248–375	3.97 ± 0.284	27.8	0.00194
	Phorate™	18.8	15.6–22.0	80.1	64.6–108	2.04 ± 0.114	35.1	0.000827

LC = lethal concentration.

. Eggs required the highest concentration to attain 50% inhibition (LC₅₀ = 162 mg L⁻¹; 95% FL = 143–180 mg L⁻¹), whereas larvae were the most susceptible (LC₅₀ = 60.5 mg L⁻¹). LC₅₀ values for protonymphs (83.8 mg L⁻¹) and deutonymphs (94.5 mg L⁻¹) fell between those of larvae and adults (112 mg L⁻¹). The botanical ovicide Atzingao^® was markedly less potent against eggs (LC₅₀ = 2801 mg L⁻¹), while the synthetic acaricide phorate™ exhibited far greater potency against adults (LC₅₀ = 18.8 mg L⁻¹). Slopes of the probit lines ranged from 2.85 to 4.89, indicating moderately steep concentration–mortality relationships.

For the lethal times (LT₅₀ and LT₉₀) at the three AuNPs concentrations (100, 200, and 300 mg L⁻¹) on the motile stages of T. urticae, the results show that the LT depended on the concentration and the developmental stage of the mite. T. urticae larvae showed a high susceptibility to AuNPs, with an LT that decreased significantly with increasing concentration. The lowest concentration (100 mg L⁻¹) showed an LT ₅₀ of 36.8 h; this time decreased to 24.8 h at a concentration of 200 mg L⁻¹ and to 13.4 h at 300 mg L⁻¹. The LT₉₀ also showed a decreasing trend, going from 69.1 to 31.2 h as the concentration increased. Protonymphs showed a similar response to the larval stage, although with longer lethal times (LTs); at 100 mg L⁻¹, the LT₅₀ was 43.5 h, decreasing to 32.7 h at 200 mg L⁻¹ and reaching 24.8 h at 300 mg L⁻¹, while the LT₉₀ also decreased from 136 to 43.6 h with increasing concentration. Deutonymphs showed LTs comparable to protonymphs; at 100 mg L⁻¹, the LT₅₀ was 43.8 h, decreasing to 32.8 h at 200 mg L⁻¹ and finally to 24.7 h at 300 mg L⁻¹, and the LT₉₀ decreased from 141 to 51.7 h as the concentration increased. Adults were the least susceptible stage to AuNPs, with longer LTs compared to the other stages; at 100 mg L⁻¹, the LT₅₀ was 42.4 h, decreasing to 34.1 and 24.9 h at 200 and 300 mg L⁻¹, respectively; in addition, the LT₉₀ also showed a significant reduction, going from 135 to 49.7 h as the concentration increased (

Table 4. Lethal times (h), fiducial limits, and confidence parameters of AuNPs on the mobile stages of T. urticae.

Stage	AuNPs (mg L−1)	LT50 (h)	Fiducial Limits	LT90 (h)	Fiducial Limits	Slope ± SE	χ2	p
Larva	100	36.8	35.1–38.5	69.1	64.9–74.5	4.68 ± 0.187	23.2	0.109
	200	24.8	23.5–26.0	42.9	40.4–46.3	5.40 ± 0.304	27.2	0.0395
	300	13.4	11.4–15.0	31.2	27.3–38.1	3.49 ± 0.187	73.3	2.61 × 10−9
Protonymph	100	43.5	40.2–46.9	136	117–165	2.59 ± 0.133	38.8	0.00701
	200	32.7	29.2–35.9	101	88.6–121.	2.61 ± 0.135	46.6	0.000677
	300	24.8	23.7–25.8	43.6	41.3–46.5	5.24 ± 0.312	6.56	0.766
Deutonymph	100	43.8	39.8–47.9	141	118–180	2.53 ± 0.132	53.4	0.0000698
	200	32.8	28.3–36.8	101	85.5–127	2.63 ± 0.135	75.4	2.33 × 10−8
	300	24.7	23.2–26.0	51.7	48.6–55.6	3.98 ± 0.229	5.90	0.950
Adult	100	42.4	37.8–46.9	135	111–178.	2.56 ± 0.132	70.5	0.0000001
	200	34.1	30.1–37.8	103	88.9 127.	2.66 ± 0.135	61.9	0.00000363
	300	24.9	23.6–26.2	49.7	47.0–53.2	4.27 ± 0.237	8.58	0.804
	Phorate™ (80 mg L−1)	4.31	3.97–4.63	12.0	11.0–13.4	2.87 ± 0.148	10.3	0.508

LT = lethal time (h = hours).

).

3.3. In-Vitro Susceptibility of AuNPs to P. persimilis

Adult P. persimilis were less susceptible to AuNPs than T. urticae larvae but more tolerant than the pest’s adult stage. Probit analysis at 120 h after application yielded an LC₅₀ of 107 mg L⁻¹ (95% FL = 89.2–120 mg L⁻¹) and an LC₉₅ of 215 mg L⁻¹ (192–257 mg L⁻¹) (

Table 5. Lethal concentrations (LC₅₀ and LC₉₅), fiducial limits, and confidence parameters of AuNPs on P. persimilis adults, under laboratory conditions (120 h after exposure).

Treatments	LC50 (mg L−1)	Fiducial Limits	LC95 (mg L−1)	Fiducial Limits	Slope ± SE	χ2	p
AuNPs	107	89.2–120	215	192–257	4.20 ± 0.281	40.2	0.0000336
Phorate™	15.7	12.8–18.3	65.3	51.9–91.2	2.07 ± 0.139	21.9	0.0155

LC = lethal concentration.

). By contrast, phorate™ (80 mg L⁻¹) produced an LC₅₀ of 15.7 mg L⁻¹, confirming its much higher acute toxicity to the predator.

With respect to the lethal times (LT₅₀ and LT₉₀) of AuNPs on adults of P. persimilis at three concentrations, 100, 200, and 300 mg L⁻¹, these decrease as the concentration of NPs increases, suggesting a clear concentration-dependent relationship, similar to what was observed in T. urticae. At the lowest concentration (100 mg L⁻¹), the LT₅₀ was 40.1 h, with a confidence interval of 35.1 to 44.8 h, while the LT₉₀ was 105 h, with a confidence interval of 88.1 to 136 h. At the intermediate concentration of 200 mg L⁻¹, the LT₅₀ was reduced to 36.4 h, with a confidence interval of 33.0 to 39.6 h, and the LT₉₀ to 93.5 h, with a confidence interval of 82.6 to 110 h. At the highest concentration (300 mg L⁻¹), the LT₅₀ was 23.4 h, with a confidence interval of 22.2 to 24.6 h, and the LT₉₀ was 45.7 h, with a confidence interval of 43.6 to 48.2 h. The chemical control showed significantly shorter lethal times due to its mode of action compared to the AuNPs, with an LT₅₀ of 4.06 h and an LT₉₀ of 11.2 h (

Table 6. Lethal times (LT₅₀ and LT₉₅), fiducial limits and confidence parameters of AuNPs on P. persimilis adults under laboratory conditions.

Treatments	LT50 (h)	Fiducial Limits	LT90 (h)	Fiducial Limits	Slope ± SE	χ2	p
AuNPs (100 mg L−1)	40.1	35.1–44.8	105	88.1–136.	3.06 ± 0.138	116	1.68 × 10−15
AuNPs (200 mg L−1)	36.4	33.0–39.6	93.5	82.6–110.	3.13 ± 0.140	61.7	0.00000387
AuNPs (300 mg L−1)	23.4	22.2–24.6	45.7	43.6–48.2	4.42 ± 0.216	5.12	0.999
Phorate™ (80 mg L−1)	4.06	3.74–4.38	11.2	10.3–12.4	2.90 ± 0.151	10.8	0.458

LT = lethal time (h = hours).

).

3.4. Selectivity Ratio (SR) Based on Adult LC₅₀ Values

The median lethal concentrations (LC₅₀) for T. urticae and P. persimilis adults, along with the selectivity ratio (SR) and their interpretation, are presented in

Table 7. Adult LC₅₀ values for AuNPs and phorate™, corresponding selectivity ratios (SRs), and interpretation.

Treatments	T. urticae (LC50 mg L−1)	P. persimilis (LC50 mg L−1)	SR = LC50 Predator/LC50 Pest	Interpretation *
AuNPs	112	107	0.95	Selective for the pest
Phorate™	4.31	15.7	3.64	Selective for the natural enemy

* Following [52]: SR > 1 indicates lower toxicity to the natural enemy, whereas SR < 1 indicates higher toxicity to the natural enemy.

. The LC₅₀ for T. urticae was 112 mg L⁻¹, while for P. persimilis it was 107 mg L⁻¹, resulting in a SR of 0.95. A SR value close to 1 indicates that the AuNPs do not exhibit selectivity toward P. persimilis. Based on the LC₅₀ values, we can determine that the AuNPs were more toxic to P. persimilis compared to T. urticae (according to SR), although the values are very similar. In contrast, the reference chemical witness (phorate™) showed an LC₅₀ of 4.31 mg L⁻¹ for T. urticae and 15.5 mg L⁻¹ for P. persimilis, with a PS of 3.64. This value reflects selectivity toward P. persimilis compared to T. urticae, causing less harm to the predator.

Gold nanoparticles exhibit unique physicochemical and biological properties that arise from their nanoscale size, high surface–area-to-volume ratio, and capacity to cross biological barriers [58,59]. Numerous green-synthesized AuNPs, prepared from plant extracts of Artemisia vulgaris L. (Asteraceae) and Jatropha curcas L. (Euphorbiaceae), have been explored chiefly against Aedes spp. Meigen (Diptera: Culicidae) mosquitoes and German cockroach Blattella germanica (Blattodea: Blattellidae) [60,61,62,63], highlighting the potential of such nanoparticles for controlling medically important vectors.

Our LC₅₀ for T. urticae adults (112 mg L⁻¹) is higher than the range reported for third-instar Aedes aegypti L. (Diptera: Culicidae) larvae exposed to Cymbopogon citratus Stapf (Poaceae)-derived AuNPs (19–41 mg L⁻¹) [64]. Similarly, Jasminum nervosum Lour (Oleaceae) AuNPs showed an LC₅₀ of 82.6 mg L⁻¹ against Culex quinquefasciatus Say (Diptera: Culicidae) third instars, with an LT₅₀ of 4.5 h at 150 mg L⁻¹ [65], substantially faster than the lethal times recorded here for T. urticae and P. persimilis. These disparities likely reflect inter-specific differences in susceptibility, nanoparticle synthesis routes, and exposure pathways.

The prolonged LT values observed in both mite species relative to the neurotoxic reference compound phorate™ further suggest that AuNPs act via slower, multi-target mechanisms. Proposed modes of action include oxidative stress, cuticular disruption, and digestive system impairment [39]. Tissue-level effects such as ovarian degeneration, altered gene expression, and apoptosis have been documented in Trachyderma hispida (Coleoptera: Tenebrionidae) [66], while accumulation and sublethal developmental impacts were reported for B. germanica [61]. AuNPs have also been shown to impair locomotion in Blaberus discoidalis Serville (Blattodea: Blaberidae) [67] and reduce larval weight, fecundity, and survival in Pericallia ricini (F.) (Lepidoptera: Erebidae) [68], underscoring their potential for chronic or behavioral disruption.

Taking together, our data demonstrates that green-synthesized AuNPs possess acaricidal activity against T. urticae, albeit at higher concentrations and longer exposure periods than those reported for certain insect pests. The moderate selectivity ratio (SR ≈ 1) indicates a risk to P. persimilis at doses required for effective spider-mite control. Future optimization of synthesis parameters (e.g., capping agents, particle size) and delivery methods may enhance efficacy while improving predator safety. Additionally, the inherent ability of T. urticae to develop resistance [4] warrants investigation into potential resistance-breaking properties of AuNPs under semi-field and field conditions.

3.5. Repellency of AuNPs on T. urticae

The repellency results for three sublethal concentrations of AuNPs—53.2 mg L⁻¹ (LC₁₀), 68.6 mg L⁻¹ (LC₂₀), and 82.5 mg L⁻¹ (LC₃₀)—against T. urticae under laboratory conditions show that, at 2 h after arena set-up (haa), LC₃₀ produced the highest repellency percentage (52.0%), with a repellency index (RI) of 0.60, classified as “moderate repellency”. LC₁₀ and LC₂₀ yielded lower values (28.0% and 36.0%, respectively), with no significant differences between them. At 4 haa, treatments LC₂₀ and LC₃₀ (68.6 and 82.5 mg L⁻¹) showed the greatest repellency percentages (both 52.0%), whereas LC₁₀ (53.2 mg L⁻¹) gave the lowest value (38.7%). The RI for LC₃₀ was 0.44, classified as “high repellency”, and differed significantly from LC₁₀ and LC₂₀. At 6 haa, LC₃₀ (82.5 mg L⁻¹) again produced the highest repellency (58.6%), with an RI of 0.40, also classed as “high repellency”; this treatment differed statistically from LC₂₀ (54.6%) and LC₁₀ (42.6%). At 12 haa, values ranged from 41.3% (LC₁₀) to 56.0% (LC₃₀). Although LC₃₀ achieved the greatest repellency, no significant differences were detected among treatments. RI was classified as “high” for LC₂₀ and LC₃₀ and “moderate” for LC₁₀. At 24 haa, LC₂₀ (68.6 mg L⁻¹) and LC₃₀ (82.5 mg L⁻¹) displayed similar repellency (41.3% and 46.6%, respectively) with no significant differences; RI was classified as “high” for LC₂₀ and “moderate” for LC₃₀. At 48 haa, LC₃₀ and LC₂₀ maintained comparable repellency levels (42.6% and 34.6%, respectively) without significant differences; both were rated as “moderate repellency”. During this interval, LC₁₀ (53.2 mg L⁻¹) showed a decline, with an RI of 0.80, classified as “weak repellency”. At 72 haa, the AuNP treatments exhibited similar repellency, with no significant differences; values ranged from 21.3% for LC₁₀ to 37.3% for LC₃₀, with RI classified as “moderate” for LC₂₀ and LC₃₀ and “weak” for LC₁₀. The product eBioluzion Plus VO™ produced the highest effect throughout the test, with repellency categories ranging from “high” to “very high”, whereas the control (water + DSS) showed RI values classified as “weak repellency” or “no repellency” (

Table 8. Percent repellency (mean ± SE), repellency index (RI), and repellency classification of gold nanoparticles (AuNPs) against adults of T. urticae under laboratory conditions.

Treatments	2 h/RI 1		4 h/RI		6 h/RI		12 h/RI		24 h/RI		48 h/RI		72 h/RI
LC10 (53.2 mg L−1)	28.0 ± 8.0 c	0.64 M	38.7 ± 6.1 c	0.56 M	42.6 ± 2.3 d	0.56 M	41.3 ± 2.3 c	0.56 M	36.0 ± 4.0 b	0.64 M	24 ± 4.0 d	0.8 W	21.3 ± 2.3 c	0.8 W
LC20 (68.6 mg L−1)	36.0 ± 4.0 bc	0.68 M	52.0 ± 4.0 b	0.52 M	54.6 ± 2.3 c	0.44 M	46.6 ± 8.3 c	0.44 H	41.3 ± 12 b	0.44 H	34.6 ± 2.3 c	0.64 M	30.6 ± 10.0 bc	0.72 M
LC30 (82.5 mg L−1)	52.0 ± 20.7 b	0.60 M	52.0 ± 6.9 b	0.44 H	58.6 ± 2.3 b	0.4 H	56.0 ± 4.0 b	0.4 H	46.6 ± 4.6 b	0.56 M	42.6 ± 2.3 b	0.6 M	37.3 ± 8.3 b	0.6 M
eBioluzion Plus VO™ (150 mg L−1)	74.6 ± 4.6 a	0.28 H	82.7 ± 4.6 a	0.2 VH	86.6 ± 2.3 a	0.16 VH	85.3 ± 2.3 a	0.12 VH	86.9 ± 2.3 a	0.12 VH	80.0 ± 4.0 a	0.24 VH	78.6 ± 15.1 a	0.28 H
Control (water + DSS 2)	6.6 ± 11.5 d	1 NR	6.6 ± 11.5 d	0.98 W	0 ± 0 e	1 W	0 ± 0 d	1 W	1.3 ± 2.3 c	0.96 W	0 ± 0 e	1 NR	0.66 ± 1.1 d	0.97 W
gl	4		4		4		4		4		4		4
F value	14.7		44.2		700.4		147.9		65.2		300.8		91.6
Pr(>F)	0.00034		2.52 × 10−6		3.41 × 10−12		7.52 × 10−9		4.01× 10−7		2.27 × 10−10		7.78 × 10−8
LSD	21.0		13.0		3.7		8.0		11.8		5.3		9.4
CV	29.2		15.4		4.2		9.5		15.4		8.1		15.4

Means with different letters differ statistically. LSD, α ≤ 0.05. Repellency index (RI) (NR = No Repellency; W = Weak; M = Moderate; H = High; VH = Very High). ¹ Repellency index. ² Dioctyl sodium sulfosuccinate.

).

3.6. Repellency of AuNPs on P. persimilis

The repellency of gold nanoparticles (AuNPs) against adult females of P. persimilis was evaluated under laboratory conditions at three sublethal concentrations: 52.8 mg L⁻¹ (LC₁₀), 67.2 mg L⁻¹ (LC₂₀), and 80.0 mg L⁻¹ (LC₃₀) (

Table 9. Percent repellency (mean ± SE), repellency index (RI), and repellency classification of gold nanoparticles (AuNPs) against adult of P. persimilis under laboratory conditions.

Treatments	2 h/RI 1		4 h/RI		6 h/RI		12 h/RI		24 h/RI		48 h/RI		72 h/RI
LC10 (52.8 mg L−1)	34.0 ± 6.1 c	0.64 M	50.6 ± 12.2 b	0.52 M	62.6 ± 6.1 c	0.44 H	70.6 ± 2.3 c	0.28 H	53.3 ± 6.1 b	0.52 M	52.0 ± 4.0 b	0.52 M	37.3 ± 2.3 c	0.64 M
LC20 (67.2 mg L−1)	40.0 ± 4.0 c	0.56 M	56.0 ± 10.6 b	0.36 H	72.0 ± 4.0 b	0.28 H	78.6 ± 2.3 b	0.2 VH	52.0 ± 4.0 c	0.48 H	52.0 ± 4.0 b	0.48 H	40.0 ± 4.0 bc	0.6 M
LC30 (80.0 mg L−1)	54.7 ± 6.1 b	0.52 M	61.3 ± 2.3 b	0.40 H	73.3 ± 6.1 b	0.32 VH	81.3 ± 2.3 b	0.2 VH	61.3 ± 2.30 c	0.4 VH	44.0 ± 4.0 c	0.56 M	44.0 ± 4.0 b	0.52 M
eBioluzion Plus VO™ (150 mg L−1)	84.0 ± 4.0 a	0.2 VH	92.0 ± 4.0 a	0.08 VH	100.0 ± 0.0 a	0 VH	100.0 ± 0.0 a	0 VH	94.6 ± 4.6 a	0 VH	82.6 ± 4.6 a	0.2 VH	82.6 ± 4.6 a	0.2 VH
Control (water + DSS 2)	0 ± 0 d	1 NR	0 ± 0 c	1 NR	0 ± 0 d	0.9 NR	1.3 ± 2.3 d	1.0 NR	1.3 ± 2.3 d	1.0 NR	1.3 ± 2.3 d	1 NR	1.3 ± 2.3 d	0.96 W
gl	4		4		4		4		4		4		4
F value	131.6		58.5		228.1		101 2		196.7		171.5		44.5
Pr(>F)	1.33 × 10−8		6.71 × 10−7		8.9 × 10− 10		5.45 × 10−13		1.85 × 10−9		3.63 × 10−9		2.44 × 10−6
LSD	8.4		13.7		7.7		3.7		7.5		7.0		12.7
CV	10.8		14.4		6.9		3.1		5.9		8.3		17.8

Means with different letters differ statistically. LSD, α ≤ 0.05. Repellency index (RI) (NR = No Repellency; W = Weak; M = Moderate; H = High; VH = Very High). ¹ Repellency index. ² Dioctyl sodium sulfosuccinate.

). At 2 (haa), LC₂₀ and LC₃₀ exhibited the highest repellency values (40.0% and 54.7%, respectively), both classified as “moderate repellency”, with no significant difference between them. At 4 haa, repellency increased for LC₂₀ (56.0%) and LC₃₀ (66.6%), both rated as “high repellency”, whereas LC₁₀ reached 50.6%, remaining in the “moderate” category. Although statistical differences were not observed among the AuNP treatments at this time, LC₁₀ was consistently lower. At 6 haa, LC₃₀ elicited the highest repellency (73.3%), with a repellency index (RI) of 0.32, classified as “very high repellency”. However, no significant difference was observed compared to LC₂₀ (72.0%; RI = 0.28), also classified as “high repellency”. LC₁₀ reached 62.6% (RI = 0.44), maintaining a “high repellency” rating, with no statistical differences among AuNP treatments. Repellency peaked at 12 haa, with LC₃₀ and LC₂₀ reaching 81.3% and 78.6%, respectively (both RI = 0.20, “very high repellency”). LC₁₀ also showed high repellency (70.6%; RI = 0.28), with no significant differences among treatments at this time.

A general decline in repellency was observed at 24 haa. LC₃₀ maintained the highest effect (61.3%; “very high”), followed by LC₁₀ (53.3%) and LC₂₀ (52.0%), both classified as “high” or “moderate repellency”, depending on the RI. At 48 and 72 haa, repellency continued to decline across all the treatments. Values ranged from 44.0% (LC₃₀) to 52.0% (LC₁₀ and LC₂₀) at 48 haa, and from 37.3% (LC₁₀) to 44.0% (LC₃₀) at 72 haa. Repellency indices at these times were classified as “moderate” for all the AuNP treatments, and no significant differences were detected.

The repellent effect of AuNPs was more pronounced on P. persimilis than on T. urticae, particularly at the LC₁₀ and LC₂₀ concentrations, with maximum repellency observed at 12 h. Thereafter, a gradual decline in repellency was recorded for both species, reaching the lowest values at 72 h. Throughout the assay, P. persimilis consistently exhibited higher repellency percentages, suggesting a greater sensitivity to AuNP exposure compared to T. urticae. These findings are consistent with previous studies [10,69,70], which reported that various botanical compounds and nanoparticle-based products can affect not only pest species but also their natural enemies. This differential response may be attributed to the presence of biologically active constituents—such as primary and secondary metabolites—originating from C. illinoinensis extract, used as a reducing agent during the green synthesis of AuNPs. Metabolites in the extract, particularly phenolic compounds, terpenes, and fatty acids, have been shown to influence arthropod behavior [71]. Their presence in the nanoparticles could explain the behavioral changes observed in both mite species. In particular, fatty acids such as oleic, linoleic, and linolenic acids—identified in Carya extracts—have demonstrated biological activity and may contribute to the repellency effect induced by the AuNPs [72,73,74].

3.7. Efficacy of AuNPs Against T. urticae in Tomato Under Greenhouse Conditions

The results of the efficacy of NPs on the average number of development stages of T. urticae in the tomato crop variety Rio Grande on the four evaluation dates during the efficacy study under greenhouse conditions are shown in

Table 10. Effect of gold nanoparticles (AuNPs) against T. urticae on tomato variety Río Grande under greenhouse conditions.

	Treatment (mg L−1)	Pre Sampling Average (in 2)	10 DA1A *		20 DA1A		30 DA1A
			Average (in 2)	Efficacy (%) 2	Average (in 2)	Efficacy (%)	Average (in 2)	Efficacy (%)
Egg	AuNPs 300	0.70 ± 0.53 a 1	0.75 ± 0.50 b	63	1.50 ± 0.58 b	68	1.17 ± 0.35 b	72
	AuNPs 500	0.65 ± 0.46 a	0.75 ± 0.50 b	63	1.25 ± 0.50 b	73	1.05 ± 0.10 b	75
	AuNPs 750	0.67 ± 0.45 a	0.75 ±0.50 b	63	0.75 ± 0.50 b	84	0.85 ± 0.12 b	80
	AuNPs 1000	0.75 ± 0.51 a	0.75 ± 0.50 b	63	0.75 ± 0.50 b	84	0.67 ± 0.46 b	84
	Phorate™ 60	0.64 ± 0.42 a	0.65 ± 0.45 b	68	1.32 ± 1.0 b	71	1.40 ± 0.97 b	66
	Untreated	0.75 ± 0.51 a	2.00 ± 0.0 a	-	4.62 ± 0.49 a	-	4.17 ± 0.39 a	-
	gl	5	5		5		5
	F value	0.019	5.393		21.47		28.25
	Pr(>F)	0.89	0.00333		5.20 × 10−7		6.32 × 10−8
Larva	AuNPs 300	0.72 ± 0.55 a	1.02 ± 0.05 ab	32	0.55 ± 0.10 b	46	0.45 ± 0.06 b	57
	AuNPs 500	0.62 ± 0.43 a	1.00 ± 0.0 ab	33	0.54 ± 0.12 b	48	0.47 ± 0.09 b	55
	AuNPs 750	0.75 ± 0.53 a	0.80 ± 0.54 ab	47	0.35 ± 0.04 bc	65	0.35 ± 0.06 bc	67
	AuNPs 1000	0.65 ± 0.46 a	0.415 ± 0.28 bc	72	0.22 ± 0.15 c	79	0.22 ± 0.15 c	79
	Phorate™ 60	0.82 ± 0.57 a	0.00 ± 0.00 c	100	0.37 ± 0.25 bc	63	0.37 ± 0.25 bc	64
	Untreated	0.70 ± 0.48 a	1.50 ± 1.00 a		1.02 ± 0.2 a		1.05 ± 0.10 a
	gl	5	5		5		5
	F value	0.081	4.78		12.02		17.86
	Pr(>F)	0.994	0.00592		3.19 × 10−5		2.03 × 10−6
Protonymph	AuNPs 300	0.66 ± 0.45 a	0.75 ± 0.5 ab	25	0.52 ± 0.09 b	62	0.50 ± 0.14 b	60
	AuNPs 500	0.64 ± 0.44 a	0.45 ± 0.33 bc	55	0.45 ± 0.33 b	67	0.49 ± 0.06 b	61
	AuNPs 750	0.57 ± 0.38 a	0.41 ± 0.27 bcd	59	0.45 ± 0.31 b	67	0.46 ± 0.05 b	63
	AuNPs 1000	0.67 ± 0.45 a	0.31 ± 0.22 cd	69	0.00 ± 0.00 c	100	0.17 ± 0.21 c	86
	Phorate™ 60	0.60 ± 0.40 a	0.00 ± 0.00 d	100	0.00 ± 0.00 c	100	0.00 ± 0.00 c	100
	Untreated	0.62 ± 0.42 a	1.00 ± 0.0 a		1.37 ± 0.47 a		1.25 ± 0.29 a
	gl	5	5	-	5	-	5	-
	F value	0.033	5.99	-	13.64	-	28.95	-
	Pr(>F)	0.999	0.00197	-	1.36 × 10−5	-	5.23 × 10−8	-
Deutonymph	AuNPs 300	0.85 ± 0.57 a	1.05 ± 0.10 b	62	0.55 ± 0.04 b	71	0.52 ± 0.05 b	72
	AuNPs 500	0.60 ± 0.41 a	1.02 ± 0.05 b	63	0.55 ± 0.04 b	71	0.52 ± 0.12 b	74
	AuNPs 750	0.75 ± 0.53 a	0.80 ± 0.31 b	71	0.50 ± 0.35 b	74	0.50 ± 0.08 b	79
	AuNPs 1000	0.63 ± 0.42 a	0.00 ± 0.0 c	100	0.00 ± 0.00 c	100	0.17 ± 0.20 c	84
	Phorate™ 60	0.60 ± 0.41 a	0.00 ± 0.0 c	100	0.57 ± 0.05 b	70	0.57 ± 0.05 b	72
	Untreated	0.70 ± 0.47 a	2.75 ± 0.5 a		1.90 ± 0.20 a		2.02 ± 0.05 a
	gl	5	5		5		5
	F value	0.174	67.3		56.39		142
	Pr(>F)	0.969	5.16 × 10−11		2.29 × 10−10		8.21 × 10−14
Adult	AuNPs 300	0.37 ± 0.26 a	0.90 ± 0.20 b	72	1.25 ± 0.50 b	65	0.92 ± 0.09 b	72
	AuNPs 500	0.45 ± 0.31 a	0.75 ± 0.50 b	77	1.00 ± 0.82 b	72	0.85 ± 0.13 b	74
	AuNPs 750	0.38 ± 0.26 a	0.75 ± 0.20 b	77	0.50 ± 0.57 b	86	0.67 ± 0.09 bc	79
	AuNPs 1000	0.27 ± 0.19 a	0.37 ± 0.25 b	88	0.64 ± 0.06 b	82	0.52 ± 0.05 c	84
	Phorate ™ 60	0.30 ± 0.24 a	0.46 ± 0.04 b	87	0.92 ± 0.37 b	74	0.92 ± 0.38 b	72
	Untreated	0.35 ± 0.23 a	3.25 ± 0.95 a		3.55 ± 0.46 a		3.25 ± 0.21 a
	gl	5	5	-	5	-	5	-
	F value	0.246	21.31	-	18.93	-	111.9	-
	Pr(>F)	0.937	5.50 × 10−7	-	1.32 × 10−6	-	6.56 × 10−13	-

* Days after first application ¹ Means with different letters differ statistically. LSD, α ≤ 0.05. ² Abbott efficacy.

.

• Egg Stage

At 10 days after the first application (10 DA1A), all the AuNP treatments—regardless of dose—achieved 63% efficacy, while the chemical standard phorate™ reached 68%. By 20 DA1A, the 750 and 1000 mg L⁻¹ AuNP doses showed the highest efficacy (84%), followed by 500 mg L⁻¹ (73%) and 300 mg L⁻¹ (68%). Phorate™ efficacy decreased slightly to 68%. At 30 DA1A, the 1000 mg L⁻¹ AuNP treatment maintained its efficacy (84%), with 750 mg L⁻¹ at 80%, and the lower doses ranging from 72 to 75%. Phorate™ continued to decline, reaching 66%. Although no significant differences were detected among the AuNP doses, 1000 mg L⁻¹ was consistently the most effective across all sampling dates.

• Larval Stage

At 10 DA1A, the 1000 mg L⁻¹ AuNP treatment provided the highest larval control (72%), while the 300, 500, and 750 mg L⁻¹ doses reached 32%, 33%, and 47%, respectively, with no significant differences among them. Phorate™ achieved 100% efficacy. At both 20 and 30 DA1A, the 1000 mg L⁻¹ dose again outperformed all others (79%), whereas the remaining AuNP treatments showed moderate control (46–65%), and phorate™ decreased to 63–64%.

• Protonymph Stage

Phorate™ yielded complete (100%) control at 10 DA1A, followed by 1000 mg L⁻¹ AuNPs (69%). Lower doses provided reduced efficacy (25–59%). At 20 and 30 DA1A, both 1000 mg L⁻¹ AuNPs and phorate™ achieved 100% control. The remaining AuNP treatments did not differ significantly from each other. No statistical differences were found between 1000 mg L⁻¹ and phorate™ across any evaluation date.

• Deutonymph Stage

At 10 DA1A, both the 1000 mg L⁻¹ AuNP and phorate™ treatments achieved full control (100%). The lower doses (300–750 mg L⁻¹) resulted in efficacy values between 62% and 71%, with no significant differences among them. By 20 DA1A, 1000 mg L⁻¹ maintained 100% efficacy, while phorate™ declined to 70%; the lower AuNP concentrations ranged between 71% and 74%. At 30 DA1A, 1000 mg L⁻¹ remained the most effective (84%), followed by 750 mg L⁻¹ (79%). Phorate™ and 300 mg L⁻¹ both reached 72%. On this date, 1000 mg L⁻¹ AuNPs differed significantly from all other treatments.

• Adult Stage

At 10 DA1A, the 1000 mg L⁻¹ AuNP treatment provided the highest efficacy (88%), slightly surpassing phorate™ (87%). The 300–750 mg L⁻¹ doses showed efficacy ranging from 72% to 77%, without significant differences. At 20 DA1A, 750 mg L⁻¹ AuNPs peaked at 86%, followed by 1000 mg L⁻¹ (82%), while phorate™ was at 74%. The lower doses ranged from 65 to 72%. At 30 DA1A, 1000 mg L⁻¹ AuNPs again achieved the highest efficacy (84%), followed by 750 mg L⁻¹ (79%). Phorate™ dropped to 72%, with the 300 and 500 mg L⁻¹ treatments showing 72% and 74%, respectively. On this final date, the 1000 mg L⁻¹ treatment was significantly more effective than all others.

Across all developmental stages of T. urticae, the 1000 mg L⁻¹ AuNP treatment consistently showed the highest efficacy, achieving up to 100% control in protonymphs and deutonymphs and 79–84% control in eggs, larvae, and adults. Intermediate doses (500–750 mg L⁻¹) provided moderate efficacy, while the lowest concentration (300 mg L⁻¹) was the least effective among the nanoparticle treatments. In contrast, phorate™ exhibited strong initial control but showed a gradual decline in performance over time in several stages. These findings support the concentration-dependent and stage-specific efficacy of AuNPs under greenhouse conditions.

3.8. Effect of AuNPs on Tomato Under Greenhouse Conditions

3.8.1. Continuous Agronomic Variables

The effect of AuNPs on the variables of chlorophyll content, height, and Brix degrees (petiole) during the effectiveness study on T. urticae at each sampling date in the tomato crop is shown in

Table 11. Effect of gold nanoparticles (AuNPs) on chlorophyll, height, and Brix degrees of tomato crops under greenhouse conditions.

Parameter	Treatments (mg L−1)	Pre Sampling	10 DA1A *	20 DA1A	30 DA1A
Chlorophyll (SPAD units)	AuNPs 300	49.27 ± 4.3 a	50.20 ± 3.8 ab	47.83 ± 2.6 ab	36.67 ± 2.9 b
	AuNPs 500	54.87 ± 3.2 a	53.93 ± 1.3 ab	51.27 ± 1.7 ab	49.17 ± 1.10 a
	AuNPs 750	49.77 ± 4.9 a	51.20 ± 5.1 ab	50.60 ± 0.4 ab	49.33 ± 2.0 a
	AuNPs 1000	50.27 ± 3.2 a	55.77 ± 0.9 a	56.30 ± 7.3 a	49.67 ± 2.5 a
	Phorate™ 60	53.83 ± 1.3 a	49.50 ± 4.6 b	43.57 ± 10.4 b	50.50 ± 1.8 a
	Untreated	55.00 ± 4.3 a	50.30 ± 1.5 ab	51.33 ± 1.1 ab	33.33 ± 2.9 b
	gl	5	5	5	5
	F value	1.547	1.652
	Pr(>F)	0.248	0.22
Plant height (cm)	AuNPs 300	28.67 ± 1.1 a	34.33 ± 1.5 b	41.00 ± 1.7 ab	41.0 ± 1.7 ab
	AuNPs 500	30.0 ± 1.0 a	37.00 ± 1.7 ab	38.0 ± 2.0 c	39.0 ± 1.0 b
	AuNPs 750	30.67 ± 1.5 a	36.00 ± 1.7 ab	42.33 ± 2.3 a	42.33 ± 2.3 ab
	AuNPs 1000	28.67 ± 1.1 a	36.00 ± 1.7 ab	39.3 ± 0.5 bc	40.00 ± 1.0 ab
	Phorate™ 60	29.33 ± 1.5 a	36.67 ± 2.3 ab	40.0 ± 1.0 abc	41.0 ± 0.0 ab
	Untreated	30.33 ± 1.1 a	37.67 ± 1.5 a	41.0 ± 1.0 ab	41.3 ± 0.58 ab
	gl
	F value	1.359	1.239	2.805	2.225
	Pr(>F)	0.306	0.35	0.0665	0.119
Degree Brix (°Bx)	AuNPs 300	7.33 ± 0.6 a	6.00 ± 0.0 d	9.0 ± 0.0 d	9.0 ± 0.0 b
	AuNPs 500	7.50 ± 0.9 a	6.50 ± 0.0 c	11.0 ± 0.0 c	10.60 ± 0.6 a
	AuNPs 750	6.67 ± 0.3 a	6.50 ± 0.0 c	10.9 ± 0.2 c	10.3 ± 0.6 a
	AuNPs 1000	7.00 ± 0.0 a	6.90 ± 0.2 b	12.0 ± 0.0 b	10.50 ± 0.0 a
	Phorate™ 60	6.83 ± 0.3 a	7.50 ± 0.0 a	13.0 ± 0.0 a	9.00 ± 0.0 b
	Untreated	7.16 ± 0.8 a	6.66 ± 0.3 bc	9.0 ± 0.0 d	9.00 ± 0.0 b
	gl	5	5	5	5
	F value	0.955	39.65	1537	18.52
	Pr(>F)	0.482	4.7 × 10−7	2 × 10−16	2.86 × 10−5

* Days after first application. Means with different letters differ statistically. LSD, α ≤ 0.05.

.

• Chlorophyll Content

No significant differences in SPAD chlorophyll readings were observed among treatments at 10 and 20 days after the first application (DA1A). However, the highest AuNP concentrations (750 and 1000 mg L⁻¹) tended to maintain slightly elevated values compared to the untreated. At 30 DA1A, significant differences were detected between treatments. Specifically, AuNPs at 500, 750, and 1000 mg L⁻¹ preserved higher SPAD values (ranging from 49.17 to 49.67), while the 300 mg L⁻¹ treatment and the untreated showed the lowest values (36.67 and 33.33, respectively).

• Plant Height

At 10 DA1A, most treatments (AuNPs at 500, 750, and 1000 mg L⁻¹ and phorate™) exhibited similar plant height values, whereas the 300 mg L⁻¹ treatment and the untreated showed significant differences. By 20 DA1A, differences became more evident among treatments, with distinct groupings observed. AuNPs at 750 mg L⁻¹ achieved the highest plant height (42.33 cm), although this value was not statistically different from those obtained with the untreated, phorate™, or 300 mg L⁻¹ AuNPs. The lowest plant height was recorded for 500 mg L⁻¹ (38.0 cm). Most treatments grouped statistically under the “ab” and “abc” categories, indicating intermediate performance. At 30 DA1A, differences were not significant (p = 0.119), although a general trend was observed, with most treatments clustering in the “ab” group. Again, the 750 mg L⁻¹ AuNPs yielded the tallest plants (42.33 cm), maintaining superiority over or equivalence to the untreated at both 20 and 30 DA1A.

• °Brix

At 10 DA1A, phorate™ recorded the highest °Brix value (7.50 °Bx; group “a”), followed by the 1000 mg L⁻¹ AuNP treatment (6.90 °Bx; group “b”). The remaining treatments were statistically grouped into categories “c” and “d”, with 300 mg L⁻¹ showing the lowest value (6.00 °Bx). Highly significant differences were observed at 20 DA1A (p = 2 × 10⁻¹⁶), with phorate™ again yielding the highest °Brix (13.0 °Bx; group “a”), followed by 1000 mg L⁻¹ (12.0 °Bx; group “b”), while 500 and 750 mg L⁻¹ treatments formed group “c”. The lowest values were observed for 300 mg L⁻¹ and the untreated (both at 9.0 °Bx; group “d”). At the final sampling (30 DA1A), significant differences persisted (p = 2.86 × 10⁻⁵). The highest °Brix values were observed in the 500, 750, and 1000 mg L⁻¹ AuNP treatments (10.3 to 10.6 °Bx; group “a”), followed by 300 mg L⁻¹, phorate™, and the untreated (9.0 °Bx; group “b”). Overall, the application of AuNPs positively influenced °Brix content in most treatments when compared to the untreated, with 1000 mg L⁻¹ yielding the highest values among AuNPs treatments, even surpassing phorate™ at the final evaluation date (30 DA1A).

3.8.2. Harvest Agronomic Variables

The effect of AuNPs on fruit-related variables—number, polar and equatorial diameter, stem, weight, Brix, TDS, pH—as well as the fresh and dry weight of the total leaf area of the tomato plant are shown in

Table 12. Effect of gold nanoparticles (AuNPs) on the fruit variables, fresh and dry weight of the leaf area of tomato under greenhouse conditions.

Treatments	Fruit/Plant	Fruit								Fresh Weight of Leaf Area (g)	Dry Weight of Leaf Area (g)
		Polar Diameter (cm)	Equatorial Diameter (cm)	Peduncle (cm)	Fruit Weight (g)	Hardness (kg/cm2)	Degrees Brix (°Bx)	TDS (mg L−1)	pH
AuNPs 300 mg L−1	5.5 ± 0.5 c 1	7.5 ± 1.0 ab	5.8 ± 0.7 bc	0.95 ± 0.1 a	74.9 ± 5.9 b	2.46 ± 0.1 b	6.0 ± 0.0 ab	1373.5 ± 126 c	3.4 ± 0.21 d	389 ± 3.7 ab	94.9 ± 0.9 bc
AuNPs 500 mg L−1	6.3 ± 1.1 bc	8.2 ± 0.3 a	6.9 ± 0.2 a	0.96 ± 0.4 a	80.0 ± 4.1 ab	2.46 ± 0.1 b	5.8 ± 0.3 b	1140.0 ± 141 d	3.6 ± 0.21 d	225 ± 10.2 d	74.6 ± 3.4 e
AuNPs 750 mg L−1	7.6 ± 0.6 b	7.4 ± 0.7 ab	5.5 ± 0.2 c	0.84 ± 0.07 a	55.4 ± 2.0 c	2.83 ± 0.2 a	5.7 ± 0.3 b	1160.0 ± 40 d	4.3 ± 0.29 c	399 ± 7.9 a	88.6 ± 1.8 d
AuNPs 1000 mg L−1	11.6 ± 1.5 a	6.8 ± 0.9 b	5.6 ± 0.9 c	0.93 ± 0.1 a	54.6 ± 2.7 c	2.90 ± 0.1 a	5.2 ± 0.3 c	1079.3 ± 26.1 d	4.7 ± 0.21 bc	407 ± 13.2 a	116.2 ± 3.8 a
Phorate™ (60 mg L−1)	5.6 ± 0.6 c	8.2 ± 0.7 a	6.5 ± 0.3 ab	1.20 ± 0.2 a	82.4 ± 3.3 a	2.30 ± 0.1 b	6.3 ± 0.3 a	2128.0 ± 158 a	5.0 ± 0.33 b	352 ± 16.2 c	90.2 ± 4.1 cd
Untreated	3.3 ± 0.6 d	6.5 ± 0.5 b	5.6 ± 0.4 c	1.16 ± 0.3 a	54.5 ± 2.4 c	2.30 ± 0.1 b	6.3 ± 0.2 a	1588.7 ± 83 b	7.2 ± 0.20 a	372 ± 12.0 b	97.9 ± 3.1 b
gl	5	5	5	5	5	5	5	5	5	5	5
F value	28.98	0.07	3.63	1.02	41.49	11.36	8.48	40.74	92.47	107.1	58.68
Pr(>F)	2.65 × 10−6	2.77	0.031	0.45	3.65 × 10−7	0.001	0.001	4.04 × 10−7	3.72 × 10−9	1.58 × 10−9	5.13 × 10− 8
LSD	1.61	1.2 p	0.91	0.42	6.46	0.23	0.47	192.31	0.44	20.08	5.48
CV	13.52	9.71	8.61	23.49	5.42	5.15	4.47	7.66	5.23	3.16	3.29

Means with different letters differ statistically. LSD, α ≤ 0.05.

.

• Fruits per Plant

The AuNP concentration of 1000 mg L⁻¹ resulted in the highest number of fruits per plant, with an average of 11.6, which was significantly greater than all other treatments. The 750 mg L⁻¹ AuNPs treatment ranked second, producing 7.6 fruits per plant—values significantly higher than both the untreated and phorate™. The 500 mg L⁻¹ AuNPs treatment yielded 6.3 fruits per plant, and phorate™ produced 5.6; no significant difference was found between these two, although both performed better than the untreated. The lowest AuNP dose (300 mg L⁻¹) produced 5.5 fruits per plant, statistically comparable to phorate™ but significantly lower than the 750 and 1000 mg L⁻¹ concentrations. The untreated recorded the lowest fruit count, with an average of 3.3 fruits per plant, which was significantly lower than all other treatments. The 750 and 1000 mg L⁻¹ AuNP concentrations significantly increased fruit production per plant.

• Polar Diameter

Regarding polar fruit diameter, no statistically significant differences were observed among treatments (p > 0.05). Although AuNPs at 500 mg L⁻¹ and phorate™ exhibited the highest mean values (both 8.2 cm), these were not statistically different from the other treatments.

• Equatorial Diameter

The 500 mg L⁻¹ AuNP treatment produced the largest equatorial fruit diameter (6.9 cm), significantly higher than the rest. Phorate™ and 500 mg L⁻¹ AuNPs showed similar values. The 300 mg L⁻¹ concentration (5.8 cm) yielded a slightly smaller diameter compared to phorate™ and 500 mg L⁻¹, though without significant differences. The 1000 mg L⁻¹ treatment (5.6 cm), the 750 mg L⁻¹ (5.5 cm), and the untreated (5.6 cm) exhibited the smallest equatorial diameters, with no significant differences among them. The 500 mg L⁻¹ AuNPs concentration was the only treatment that significantly increased equatorial diameter in tomato fruits.

• Peduncle Length

No statistically significant differences were found among the treatments for peduncle length. All the treatments showed similar values, indicating that none of them significantly affected this trait.

• Fruit Weight

Fruit weight was significantly influenced by phorate™ and AuNPs at 500 mg L⁻¹. Phorate™ produced the heaviest fruits (82.4 g), significantly greater than all other treatments. The 500 mg L⁻¹ AuNP treatment followed closely with 80.0 g. The 300 mg L⁻¹ treatment yielded slightly lower values (74.9 g). The treatments with 750 mg L⁻¹ and 1000 mg L⁻¹ and the untreated produced the lightest fruits, without significant differences among them.

• Fruit Hardness

In terms of fruit firmness, AuNPs at 750 and 1000 mg L⁻¹ resulted in the highest values, reaching 2.83 and 2.90 kg/cm², respectively, and forming a statistically superior group (“a”). These values were significantly higher than those recorded for AuNPs at 300 and 500 mg L⁻¹, phorate™, and the untreated (2.30–2.46 kg/cm²; group “b”).

• Degrees Brix (°Bx)

The highest °Brix values were observed in the phorate™ and untreated treatments (6.3 °Bx), both significantly greater than the other treatments. The 300 mg L⁻¹ AuNP treatment showed similar results (6.0 °Bx), statistically comparable to phorate™ and the control. The 500, 750, and 1000 mg L⁻¹ AuNP treatments showed lower °Brix values (5.8, 5.7, and 5.2 °Bx, respectively), with the 1000 mg L⁻¹ dose being significantly lower than the rest. Phorate™ and the control treatments produced the highest °Brix values in tomato fruit.

• Total Dissolved Solids (TDS)

Phorate™ treatment recorded the highest TDS value (2128 mg L⁻¹), significantly higher than all other treatments. The control ranked second with 1588.7 mg L⁻¹. Among AuNP treatments, the 300 mg L⁻¹ dose had the highest TDS (1373.5 mg L⁻¹), significantly higher than the 500 (1140.0 mg L⁻¹), 750 (1160.0 mg L⁻¹), and 1000 mg L⁻¹ (1079.3 mg L⁻¹) treatments, which did not differ significantly from one another. Phorate™ significantly increased total dissolved solids in tomato fruits.

• pH

The untreated exhibited the highest pH value (7.2), significantly higher than all other treatments, followed by phorate™ at 5.0. Among AuNP treatments, there was a concentration-dependent trend: higher AuNP concentrations led to increased pH values. The 1000 mg L⁻¹ treatment reached 4.7, followed by 750 mg L⁻¹ (4.3), 500 mg L⁻¹ (3.6), and 300 mg L⁻¹ (3.4), though no significant differences were found between the latter two. Overall, AuNPs significantly reduced tomato fruit pH compared to the untreated.

• Fresh and Dry Weight of Leaf Area

The 1000 mg L⁻¹ (407 g) and 750 mg L⁻¹ (399 g) AuNP treatments resulted in the highest fresh weights of leaf area, significantly higher than the remaining treatments. The untreated, with 372 g, showed a similar effect but slightly lower values. The 300 mg L⁻¹ concentration (389 g) did not differ significantly from the 750 and 1000 mg L⁻¹ treatments. Phorate™ (352 g) had a lower value than all the AuNP treatments except for the 500 mg L⁻¹ treatment, which yielded the lowest value (225 g), significantly lower than the rest. Both 750 and 1000 mg L⁻¹ AuNPs significantly increased the fresh weight of tomato leaf area. Regarding dry weight, the 1000 mg L⁻¹ AuNP treatment achieved the highest value (116.2 g), significantly greater than all other treatments. The untreated (97.9 g) and 300 mg L⁻¹ AuNPs (94.9 g) followed, with no significant differences between them. The lowest dry weights were recorded in the phorate™ (90.2 g), 750 mg L⁻¹ (88.6 g), and 500 mg L⁻¹ AuNP (74.6 g) treatments.

Various studies have demonstrated that gold can accumulate in different plant tissues [75]. AuNPs can be absorbed through foliar or root uptake, depending on their size, shape, surface charge, chemical composition, and plant species [76]. While the use of AuNPs in biological systems has been widely studied, their effects on the growth and development of model or crop plants remain poorly understood. Although several investigations have been conducted on different plant species [75], the underlying factors related to AuNP accumulation, translocation, and movement within tissues, as well as their physiological or tolerance-related effects under biotic or abiotic stress conditions, are still not fully elucidated [77,78].

It is known that most plant defenses against herbivores, including phytophagous mites, are induced by the pest itself, beginning with a local response at the site of injury or infestation, followed by systemic responses in undamaged tissues [79]. In the case of T. urticae and its interaction with plants, the induction process is initiated through contact, feeding vibrations, oral secretions, and oviposition fluids [80]. These events trigger changes such as membrane potential alterations (Vm), cytosolic Ca²⁺ influx, and increased levels of reactive oxygen/nitrogen species (ROS/RNS). These processes are further modulated by the salivary recombinant proteins Tet1 and Tet2 in T. urticae, which are known to induce defense responses in host plants [81]. Reactive oxygen species (ROS) can be associated with the application of AuNPs and in turn be associated as defense mechanisms against different pest arthropods.

The role of ROS has been associated not only with T. urticae infestation but also with AuNP exposure. Ref. [82] reported that seedlings of Arabidopsis thaliana (L.) Heynh (Brassicaceae) exposed to AuNPs at concentrations of 10 and 80 μg mL⁻¹ exhibited ROS inhibition levels 3.54 and 2.59 times higher, respectively, suggesting a radical-scavenging effect of AuNPs. Enzymatic activity of ascorbate peroxidase, catalase, glutathione reductase, and superoxide dismutase also significantly increased. In another study, ref. [83] found that AuNP-treated Arabidopsis plants showed upregulation of stress-response genes, including glutathione transferases, cytochrome P450s, glycosyl transferases, and peroxidases. These findings suggest that in our study, the observed reduction in T. urticae populations may have been due not only to the direct toxicity of AuNPs but also to plant-mediated responses, possibly involving changes in ROS levels following foliar AuNP application.

The toxic effects of AuNPs on arthropods have also been documented. Ref. [61] reported effects on B. germanica, specifically on ootheca viability and postembryonic development (nymphal survival and longevity). Gold accumulation was observed in adult cockroaches; while ootheca formation and hatching time were unaffected, viability was reduced by approximately 25%, and the number of nymphs decreased by 32.8%. Similarly, ref. [66] reported ovarian tissue damage in T. hispida caused by sublethal AuNP exposure (0.01 mg/g), accompanied by significant inhibition of antioxidant enzymes—consistent with oxidative damage mechanisms described by [39]. Ref. [84] also reported oxidative stress induced by ZnO-NPs (50–100 µg/mL), affecting hemocyte counts and upregulating genes such as GST, CNDP2, and CE. Ref. [85] described similar toxic effects of Al₂O₃ NPs on Locusta migratoria (L.) (Orthoptera: Acrididae), including antioxidant biomarker disruption, DNA damage, apoptosis, and testicular tissue degeneration, indicating ROS overproduction. Since AuNPs can penetrate biological membranes quickly and generate ROS, they raise concerns about potential toxicity in specific plant and animal tissues [86,87,88,89].

It is also known that AuNPs can affect plant growth. Ref. [90] reported that AuNPs applied to Brassica juncea (L.) Czern (Brassicaceae) altered morphological traits such as plant height, number of stems and leaves, and pod formation [91]. In A. thaliana, ref. [82] found enhanced seed germination, vegetative growth, and ROS scavenging activity after AuNP exposure. Ref. [92] also reported increased root growth, larger rosette diameters, and reduced oxidative stress in A. thaliana treated with AuNPs, findings consistent with [93]. In Vigna unguiculata (L.) Walp (Fabaceae), AuNPs promoted shoot and root growth and induced phenolic compound accumulation, conferring gold tolerance [94]. These phenolic compounds were previously linked to purple coloration in AuNP-exposed plants [95]. Conversely, negative growth effects have also been reported [96], though the mechanisms remain unclear. It is established that AuNPs modulate ROS levels and enzyme systems that mitigate oxidative damage and improve stress tolerance [97,98]. Ref. [99] found that AuNP exposure in B. juncea reduced growth by increasing oxidative stress and antioxidant enzyme activity. These results align with our findings, where plant growth was affected at 20 and 30 DA1A, possibly due to both the AuNPs and damage caused by T. urticae, as ROS are involved in mite defense responses [8].

High AuNP concentrations in chloroplasts may affect photosynthetic sugar storage. Some reducing sugars may interact with AuNPs [77]. Ref. [91] reported increased reducing and total sugars in B. juncea after AuNP exposure. In our study, Brix values in petiole sap increased significantly at 20 and 30 DA1A compared to the untreated. In contrast, fruit Brix values decreased as AuNP concentrations increased. Ref. [100] proposed that sugars may be involved in gold reduction within plant tissues, which may explain the contrasting trends observed between petiole and fruit °Brix values.

In terms of biomass, ref. [91] proposed a relationship between AuNPs and ethylene modulation, suggesting that AuNPs adsorb ethylene, thus delaying senescence and increasing leaf number in B. juncea. Conversely, ref. [101] reported reduced biomass and phytotoxic effects in Hordeum vulgare L. (Poaceae), indicating species-specific responses. Ethylene is a phytohormone involved in ripening and senescence [102,103], which correlates with leaf area and other agronomic traits. In this study, fresh leaf weight was affected by AuNP concentration. Ref. [82] also reported accelerated flowering in A. thaliana (up to 15 days earlier), increasing total seed production in plants treated with 10 μg mL⁻¹ AuNPs. Similarly, in our study, the evaluated tomato variety (determinate type) flowered approximately 10 days earlier under all the AuNP treatments and produced more fruits per plant at higher concentrations.

AuNPs may influence several physiological processes, including growth rate, water exchange, photosynthesis, antioxidant responses, and gene expression under both normal and stress conditions, as shown across various plant taxa [104]. The SPAD values in this study were comparable across treatments, though ROS overproduction may impact chlorophyll content and photosynthetic efficiency. Ref. [97] linked AuNPs with aquaporin disruption, affecting cellular ionic and osmotic balance, potentially altering photosynthesis. Additionally, T. urticae feeding likely impacted SPAD values, especially at the lowest AuNP concentration (300 mg L⁻¹), where mite damage was more pronounced.

This research highlights the potential of AuNPs as tools for integrated pest management. However, further investigation is necessary to evaluate their behavior in agroecosystems, including interactions with soil and rhizosphere, effects on non-target organisms, plant persistence, and implications for food safety and human health. These considerations are crucial to assess their sustainability and viability in modern agricultural systems.

4. Conclusions

In the laboratory, T. urticae showed susceptibility at all the developmental stages evaluated (egg, larva, protonymph, deutonymph, and adult). P. persimilis had a lower LC₅₀ than T. urticae, resulting in a negative selectivity ratio (SR = 0.95), suggesting lower compatibility and greater sensitivity in the natural enemy. Lethal times were influenced by both concentration and developmental stage. AuNPs exerted a moderate to high repellent effect on T. urticae, with even greater repellency observed in P. persimilis.

Under greenhouse conditions, AuNPs showed >80% efficacy against multiple developmental stages of T. urticae. Continuous agronomic parameters were also affected.

Based on the results obtained, the four AuNP concentrations evaluated were effective for controlling T. urticae. However, the concentrations of 750 and 1000 mg L⁻¹ were particularly effective on all three evaluation dates, so their application at 10-day intervals, adjustable according to the mite population density, is suggested. The AuNPs were effective against all biological stages of T. urticae, indicating that they can be applied foliarly with good coverage, preferably as a preventative measure or in situations of low population density. Furthermore, the concentrations evaluated did not show phytotoxicity to plants under the established application frequencies.

For future research, it is recommended to further study the dynamics of nanoparticles in the agroecosystem, especially their interaction with the plant and its edible organs. It is also recommended to include analysis of AuNPs in fruit to establish food safety parameters and contribute to the development of regulatory standards that guarantee their responsible use in agricultural systems, since their impact on the end consumer is unknown. This recommendation is based on the results obtained, which show that AuNPs modify agronomic parameters in tomato crops.

These findings support the potential of AuNPs as a management alternative for T. urticae, in addition to influencing agronomic parameters. However, in IPM programs targeting T. urticae that include P. persimilis as a biological control agent, it is important to consider that the predator is more susceptible to AuNPs than the pest.