Effect of Biosynthesized Nanoselenium on Controlling Tomato Root-Knot Nematode Meloidogyne incognita

Abstract

Tomato is a globally important fruit crop, which is easily susceptible to infection by plant-parasitic nematodes during growth. In this study, selenium nanoparticles were biosynthesized through the selenite reduction pathway in a wild-type Pseudomonas stutzeri BB19 and were characterized by uniform nanostructured needle-like forms with an average size of 95.2 nm. The nematicidal effect of biosynthesized selenium nanoparticles (BioSeNPs) at different concentrations (2, 6, and 10 ppm) during the pre- or post-infection of tomato root-knot nematode (RKN), Meloidogyne incognita, was assessed under greenhouse conditions. The BioSeNPs were applied as a foliar spray or a soil drench, compared to ethoprophos nematicide (100 mL/pot). The results showed that the ethoprophos nematicide significantly reduced the number of galls, egg mass, and eggs/egg mass of M. incognita by 94.2, 92.8, and 49.8%, respectively. BioSeNPs, as foliar sprays, significantly reduced the number of galls in post-treated infected tomato plants at 10 ppm and in pre-treated infected tomato plants at 2 ppm by 91.9 and 91.4%, respectively. Concerning the egg mass, BioSeNPs significantly reduced it in pre-treated infected tomato plants at 2 ppm as a foliar spray by 90.9%. Moreover, BioSeNPs significantly reduced the eggs/egg mass in pre-treated infected tomato plants at 2 ppm as a soil drench by 43.3%. On the other hand, the BioSeNPs considerably improved tomato growth, chlorophyll a and b, carotenoid content, and enzymes (i.e., catalase and peroxidase) activity compared to untreated infected tomato plants (negative control). Hence, the BioSeNPs show a significant application potential as a cost-effective and environmentally friendly biocontrol agent for RKN management in tomato plants.

1. Introduction

Tomato (Solanum lycopersicum L.) is a globally planted and economically important vegetable fruit that serves to meet the fundamental nutritional essentials of humans [1]. Root-knot nematodes (RKNs) are notorious pests associated with tomato crop, causing damage, ranging from 32–40% and an annual yield loss of approximately 22–30% [2]. One of the most prevalent RKNs, Meloidogyne incognita, causes biochemical and physical damage to tomato plants, such as changes in their energy and water metabolism, production of reactive oxygen species (ROS) [3], severe gall formation, yellowed and stunted leaves, and eventually the loss of whole plant [4]. Therefore, it is necessary to develop strategies for RKN control.

Currently, various methods are applied to reduce the damage of tomato crop caused by RKNs, such as biological control [5], chemical nematicides [6], and the development of resistant crops [7]. Among these, nematicides are commonly used to protect the tomato crop from RKNs because they are more effective than resistant varieties and control the cultivation environment [8]. Some nematicides, however, have also been identified as potentially serious dangers to the environment and human health, as well as higher application costs on farms [9,10]. Furthermore, several previous studies have confirmed that the development of nematode susceptibility to pesticides is additionally related to the occurrence of resistance after exposure to nematicides [11]. Traditional strategies are still meeting major obstructions. Therefore, there is a critical need for novel techniques to achieve the most significant effect with the least economic and environmental impact.

Recently, nanotechnology had excellent potential applications in plant pest and disease control [12]. Many nanomaterials can be used against plant parasitic nematodes and are directly applied either in the soil (soil drench) or on the leaves (foliar spray) [13]. Moreover, nanomaterials can be used as inducers, which can effectively activate the natural immune potential of plants through triggering with a complex integrated system of defense mechanisms [14]. More recently, the biosynthesis of nanomaterials via biological techniques using bacteria has represented a promising alternative to harmful traditional preparation methods [15].

Selenium nanoparticles (SeNPs) possess physicochemical characteristics, such as melting point, electrical/thermal conductivity, and light absorption, which enable them to exhibit excellent performance in numerous fields of science [16,17]. Many microorganisms can be used for biological synthesis to obtain SeNPs, such as Bacillus megaterium, Bacillus subtilis, Bacillus cereus, Aspergillus terreus, Enterococcus faecalis, and countless others [18,19,20]. Microorganisms can produce complex and specific nanoparticles (NPs) using their enzymes that determine unique morphological features of the SeNPs, which are often impossible to achieve through chemical synthesis [21].

In the current study, the biosynthesized SeNPs (BioSeNPs) were carried out using a wild-type Pseudomonas stutzeri strain BB19 through the selenite reduction pathway of the cell. The current study investigated the impact of BioSeNPs on the root-knot nematode M. incognita under greenhouse conditions. The study also investigated the effectiveness of BioSeNPs in controlling root galling and egg hatching in M. incognita, which infected tomato plants (Figure 1).

2. Materials and Methods

2.1. Chemicals, Bacterial and Nematode Strains, and Culture Conditions

Chemicals were supplied by Sigma-Aldrich or Merck and were of analytical grade. The chemical nematicide ethoprophos Smart-N (UPI CropScience Co., Ltd., Tianjin, China) was purchased from the local supplier Starchem Industrial Chemicals, 6th of October City, Egypt. A wild-type P. stutzeri strain BB19 previously isolated and identified from bean plants in the Baloza area, North Sinai Governorate, Egypt [22], and it was utilized to synthesize BioSeNPs in the present study. P. stutzeri cells were routinely grown in Luria-Bertani (LB) broth medium [23] at 28 °C. The culture of M. incognita was isolated from the okra plant grown in a greenhouse at El-Behera Governorate, Egypt. Perineal patterns of adult females and the morphology of second-stage juveniles (J2) were used to identify root-knot nematodes [24]. RKN egg masses were incubated for 48 h at room temperature (25 ± 2 °C) in a sodium hypochlorite solution of double-distilled H₂O (ddH₂O). Newly born second-stage juveniles (J2) were collected daily [25].

2.2. Biosynthesis and Purification of Selenium Nanoparticles Using P. stutzeri BB19

Biosynthesis and purification were performed according to a previously reported protocol with few modifications [26]. Briefly, 1% (v/v) of P. stutzeri inoculations were cultivated for 12 h in 250 mL Erlenmeyer flasks, usually containing 100 mL LB broth, then 500 µL of sodium selenite (1 M) was added, and incubation proceeded for 72 h at 28 °C with shaking at 150 rpm. The cultures with red color, indicating the production of BioSeNPs, were harvested by centrifugation at 8000 rpm for 10 min. After rinsing twice with ddH₂O and scraping the supernatant, the pellets were resuspended in ddH₂O and centrifuged twice for 30 min with 80% (w/v) sucrose to extract biomass. The separated pellets were washed with ddH₂O and again resuspended to remove the impurities. The pellets were later dried and stored at −20 °C until they were used.

2.3. Characterization of Biosynthesized Selenium Nanoparticles

The rapid biosynthesis of SeNPs was characterized by a UV-visible spectrophotometer (Spectro UV-Vis double beam DUV 3500, Shimadzu Corp., Kyoto, Japan) at wavelengths between 250 to 700 nm. The SEM (Hitachi S-4500, Tokyo, Japan) and EDX were employed to investigate the size, morphology, and elemental composition of the BioSeNPs. The phase formation and crystalline nature of BioSeNPs were assayed by XRD (Model D/Max-2500, Kyoto, Japan) using Cu-Kα radiation (λ = 1.5406 Å) at a tube voltage of 40 kV and a tube current of 30 mA. The measurement was recorded over the range of 10–70 at the 2θ scale. The functional groups on the surface of fabricated materials were identified by FTIR (Model Perkin Elmer, Waltham, MA, USA), and the FTIR spectrum was recorded at wavelengths ranging from 400 to 4000 cm⁻¹ at a resolution of 4 cm^–1 in potassium bromide disk. The particle size distribution was measured by a particle analyzer (Zetasizer Nano ZS, Malvern Co., Malvern, UK) in the range of 232 nm under the following conditions: laser wavelength of 632 nm, water refractive index of 1.33, and at room temperature.

2.4. Effect of BioSeNPs on Tomato Plants Infected with M. incognita, Root Galling, and Egg Hatching of M. incognita (Greenhouse Experiments)

The experiment was conducted in the greenhouse of the Faculty of Agriculture, Cairo University, Giza, Egypt. Tomato 30-days-old uniform seedlings (cv. Super Strain B) were transplanted (seedling/pot) in a plastic pot (15 cm diameter) filled with 1 kg mixture of sand and clay (3:1, v/v). The plants were infected with 5000 nematode eggs in each pot by pouring the nematode suspension into holes with a diameter of 2–4 cm in the soil surface around the base of the plants. The commercial ethoprophos nematicide was the positive control with the recommended rate (3 L per hectare ≅ 100 mL/pot). The tomato seedlings inoculated with M. incognita without the nematicide or BioSeNPs treatments served as the negative control. Three concentrations of 2, 6, and 10 ppm of BioSeNPs were applied to tomato plants as a foliar spray, and soil drench treatment was performed individually two times; the first time was 7 days before inoculation with M. incognita (pre-infection treatment experiment), while the second time was 7 days after inoculation (post-infection treatment experiment). All treatments were replicated three times and arranged in a completely randomized design. During the experiment, the greenhouse temperature ranged between 25–30 °C, and irrigation and fertilization were applied as needed.

After 60 days from transplantation, tomato plants were uprooted, and the roots were washed and dried for 15 min in an aqueous solution of Phloxine-Bstain (0.15 g/L ddH₂O) before being gently rinsed in tap water. The roots were carefully washed in tap water, and the red-stained egg masses were counted on infected roots [27]. Then, the number of galls/root system of a plant was counted visually, and egg mass and eggs per egg mass were also counted [28]. At harvest, roots were examined to count galls and egg masses on a 0–5 scale as follows [24]. Gall index: 0 = 0 gall/egg mass/root system; 1 = 1–2 galls/egg mass/root system; 2 = 3–10 gall/egg mass/root system; 3 = 11–30 gall/egg mass/root system; 4 = 31–100 gall/egg mass/root system; and 5 ≥ 100 gall/egg mass/root system.

2.5. Estimation of Growth Parameters and Photosynthetic Pigments

Growth parameters of tomato plants, including plant length, shoot, and root lengths, as well as shoot and root fresh weights, were recorded at the end of the experiment, 60 days after transplantation. Additionally, the photosynthetic pigments, including chlorophyll a, chlorophyll b, and carotenoids (mg/g fresh weight), were determined according to Mackinney [29] using 0.5 g of the fresh plant, homogenized and grounded with 5 mL of 85% of acetone, followed by filtration with wetted filter paper. This technique was repeated several times with acetone, and the filtrate volume was completed to 50 mL by 85% of acetone. Finally, the absorbance was immediately measured using the UV-Vis spectrophotometer at 662, 644, and 440 nm for calculating chlorophyll a, chlorophyll b, and carotenoids, respectively.

2.6. Assay of Antioxidant Enzymes Activity

One gram of tomato leaves was taken and homogenized to investigate the enzymatic activity, in 2 mL of 0.1 M sodium phosphate buffer (SPB), at a pH of 6.5 at 4 °C. The filtrate was centrifuged at 20,000 rpm at 4 °C for 15 min. The supernatant served as the enzyme extract for catalase (CAT) and peroxidase (POD) assays.

Catalase activity was measured by quantifying the decomposition at an absorbance of 240 nm every 10 s for 1 min. The reaction mixture comprised 50 mM of potassium phosphate buffer (pH = 7) and 10 mM H₂O₂, to which 30 µL of protein extract was added. CAT activity was calculated as a micromole of decomposed H₂O₂ per min per mg of protein [30].

Peroxidase activity was assayed colorimetrically by measuring the oxidation of pyrogallol in the presence of H₂O₂. The reaction mixture consisted of 1.5 mL of SPB, 20 μL of enzyme extract, 1 mL of 0.5 M pyrogallol, and 480 μL of 1% H₂O₂ solution (v/v). The increase in the optical density at 460 nm against blank was continuously recorded every minute. POD enzyme activity was expressed as a change in absorbance per 3 min/g fresh tissue [31].

2.7. Statistical Analysis

The obtained data from the greenhouse tests were statistically analyzed by analysis of variance using Infostat v2009 software (Córdoba, Argentina), and the means were compared by Duncan’s Multiple Range Test at a 0.05 significance level [32].

3. Results and Discussion

3.1. Biosynthesis and Characterization of Selenium Nanoparticles Using P. stutzeri BB19

The SeNPs were successfully and rapidly synthesized from P. stutzeri BB19 LB aqueous extract. The color changed from light yellow to red after 72 h of incubation (Figure 2A), which was the first evidence of selenite reduction, was followed by the synthesis of SeNPs. The surface morphology of the BioSeNPs was characterized by SEM, in which the BioSeNPs had uniform nanostructures with needle-like shapes and an average particle size of 95.2 nm (Figure 2B). These results showed a close similarity with previous results of a particle size ranging from 50 to 150 nm [33]. The preliminarily substantiated characterization of BioSeNPs was performed by UV-Visible spectrum scanning, which showed peak absorption values between 200–800 nm (UV-Visible range) and exhibited a high-intensity absorption peak at 380 nm (Figure 2C). The formation of such a peak occurs due to the Surface Plasmon Resonance (SPR) of SeNPs, and the result of a wide SPR peak strongly reveals the polydispersity of the SeNPs [34]. Similar observations were recorded in some recent studies [35,36,37,38]. In addition, the results of particle size analyzer showed that SeNPs had an average size of 50–500 nm with a high polydispersity index (Figure 2D).

The EDX elemental analysis of BioSeNPs in Figure 3 confirms the sample composition through a high-intensity peak. The EDX intensity peaks indicated the presence of selenium, carbon, oxygen, and sodium with 14.2, 49.4, 23.3, and 4.75% by weight, respectively. These results confirm the successful preparation of SeNPs.

The XRD patterns of BioSeNPs are shown in Figure 4, where the diffraction intensity was observed from 2θ angles varying from 10 to 100°. The distinct sharp peaks at 23.5, 29.8, 31.8, 41.3, 43.8, 45.5, 51.7, 61.7, 65.2, and 75.4° indicated the crystalline nature of BioSeNPs, which is in agreement with previous research [39,40,41].

FTIR is used to determine the functional groups of organic and inorganic materials. As shown in Figure 5, the FTIR spectra show several absorption peaks at 3267, 2923, and 2850 cm⁻¹. These peaks indicate stretching vibrations of the O–H or N–H group, asymmetric C-H stretching vibration, and symmetric C-H stretching vibration, respectively. In addition, other peaks were observed at 1627 and 1524 cm^–1, which represent the stretching vibration absorption peak of C=O and bending vibration of N–H. This is most likely due to the presence of an amide group coated on the surface of the BioSeNPs. Moreover, another absorption band was observed at 1109 cm^–1, representing the stretching vibration absorption peak of the C–O group. These functional groups are found in phenols, lipids, proteins, and carbohydrates, which are responsible for BioSeNPs stabilization [42,43].

3.2. Effect of BioSeNPs Treatments on Tomato Plants Infected with M. incognita under Greenhouse Conditions

3.2.1. Effect of BioSeNPs on Growth Parameters of Tomato Plants

Data in

Table 1. The growth parameters of tomato seedlings infected with M. incognita pre- and post-treated with different concentrations of BioSeNPs ¹.

Factor	Plant Length (cm)	Root Length (cm)	Shoot Length (cm)	Root Fresh Weight (g)	Shoot Fresh Weight (g)
Treatment time (TT)	NS	NS	NS	NS	**
Pre-infection (Pre)	70.8 a ± 1.6	25.8 a ± 1.2	44.9 a ± 1.0	5.92 a ± 0.4	11.7 b ± 0.4
Post-infection (Post)	71.7 a ± 2.5	24.7 a ± 0.8	47.0 a ± 1.8	6.31 a ± 0.4	13.7 a ± 0.7
Application method (AM)	NS	*	**	NS	**
Foliar spray (FS)	72.0 a ± 1.7	24.0 b ± 0.9	48.0 a ± 1.3	6.05 a ± 0.4	13.5 a ± 0.7
Soil drench (SD)	70.4 a ± 2.4	26.5 a ± 1.1	43.9 b ± 1.5	6.18 a ± 0.4	11.9 b ± 0.4
BioSeNPs (ppm)	**	**	**	**	**
NC	61.0 d ± 2.3	21.7 b ± 0.9	39.3 d ± 1.4	4.80 c ± 0.1	11.0 c ± 0.2
BioSe2	69.3 c ± 3.5	24.3 ab ± 2.2	44.9 c ± 2.3	4.73 c ± 0.6	12.7 ab ± 1.1
BioSe6	75.3 b ± 2.4	26.8 a ± 2.0	48.5 b ± 1.7	5.64 c ± 0.4	13.7 ab ± 1.1
BioSe10	80.6 a ± 3.7	27.6 a ± 1.4	53.0 a ± 3.0	6.68 b ± 0.5	13.9 a ± 1.3
PC (Ethoprophos Smart-N)	70.0 c ± 1.3	26.0 a ± 0.9	44.0 c ± 0.4	8.73 a ± 0.5	12.2 bc ± 0.6
TT ×AM × BioSeNPs	**	**	**	*	**
NC	61.0 fg ± 5.3	21.7 d–g ± 2.2	39.3 fg ± 3.2	4.80 c–e ± 0.2	11.0 c–e ± 0.6
Pre × FS × BioSe2	68.0 d–f ± 1.2	18.7 fg ± 1.8	49.3 c–e ± 2.9	2.30 f ± 0.8	8.67 e ± 1.5
Pre × FS× BioSe6	78.3 b–d ± 6.2	27.7 b–e ± 6.9	50.7 b–d ± 3.5	5.33 b–e ± 0.1	13.8 bc ± 0.9
Pre × FS × BioSe10	71.3 c–f ± 3.2	20.7 e–g ± 1.8	50.7 b–d ± 3.5	5.13 b–e ± 0.6	12.1 c–e ± 0.6
Pre × SD × BioSe2	81.7 bc ± 1.2	35.7 a ± 0.3	46.0 d–f ± 1.2	6.40 b–d ± 0.6	13.5 bc ± 1.0
Pre × SD × BioSe6	78.0 b–d ± 3.5	31.3 a–c ± 3.2	46.7 d–f ± 0.3	6.03 b–e ± 1.0	13.0 cd ± 1.7
Pre × SD × BioSe10	68.3 d–f ± 0.7	29.0 a–d ± 0.0	39.3 fg ± 0.7	6.96 a–c ± 0.8	9.47 de ± 0.9
Post × FS × BioSe2	75.7 b–e ± 3.5	25.0 b–g ± 0.0	50.7 b–d ± 3.5	6.07 b–e ± 1.1	17.7 a ± 1.2
Post × FS× BioSe6	79.7 b–d ± 1.5	24.7 c–g ± 2.6	55.0 bc ± 1.2	7.30 ab ± 0.4	18.3 a ± 1.9
Post × FS × BioSe10	85.3 b ± 4.4	28.0 b–e ± 0.6	57.3 b ± 3.8	7.27 ab ± 1.4	17.6 a ± 3.4
Post × SD × BioSe2	51.7 g ± 2.0	18.0 g ± 0.0	33.7 g ± 2.0	4.13 d–e ± 0.6	10.9 c–e ± 0.3
Post × SD × BioSe6	65.0 ef ± 2.1	23.3 d–g ± 1.2	41.7 ef ± 0.9	3.90 ef ± 0.3	9.43 de ± 0.5
Post × SD × BioSe10	97.3 a ± 2.9	32.7 ab ± 1.5	64.7 a ± 1.5	7.35 ab ± 0.8	16.3 ab ± 0.3
PC (Ethoprophos Smart-N)	70.0 d–f ± 3.1	26.0 b–f ± 2.1	44.0 d–f ± 1.0	8.73 a ± 1.2	12.2 c–e ± 1.3

¹ Each value represents mean ± standard error. Means in each column for each factor followed by the same letter do not differ significantly according to Duncan’s multiple range test at p ≤ 0.05. (* = p ≤ 0.05, ** = p ≤ 0.01 and NS = non-significant). NC (Negative control) denotes tomato plants inoculated with M. incognita without any treatment. PC (Positive control) denotes tomato plants treated with the chemical nematicide ethoprophos Smart-N. BioSe₂, BioSe₆, and BioSe₁₀ denote 2, 6, and 10 ppm of BioSeNPs, respectively.

show that the growth parameters of infected tomato seedlings significantly increased under BioSeNPs treatments. The results show that the growth parameters increased substantially after the treatment with BioSeNPs compared with infected untreated tomato plants (negative control). The BioSeNPs concentration, treatment time, and application method significantly affected plant growth. There was no significant difference between the two treatment times in plant length, root, shoot lengths, and root fresh weight, except shoot fresh weight, which has significantly increased with the post-infection treatment. Soil drenching significantly increased root length, while foliar spraying significantly increased shoot length and shoot fresh weight. However, there was no significant difference in plant length and root fresh weight between the two application methods. Post-infection treatment with BioSeNPs at 10 ppm as a soil drench resulted in the highest plant and shoot length. Pre-infection treatment with BioSeNPs at 2 ppm as a soil drench resulted in the highest root length. Post-infection treatment at all concentrations of BioSeNPs as a foliar spray resulted in the best shoot fresh weight. Consistent with previous findings, the obtained results indicated a positive role of SeNPs on growth parameters [44,45]. These might be due to the biological activity of SeNPs, where the uptake of SeNPs by the roots is slow. Then, they are oxidized rapidly inside the plant to selenite and become organic forms, improving root growth. Moreover, the interaction of SeNPs with proteins inside the plant cells and other biomolecules containing functionally active groups leads to changes in the responses of plants and improves their growth [46,47].

3.2.2. Effect of BioSeNPs on Photosynthetic Pigments of Leaves of Tomato Plants

The results of photosynthetic pigments are presented in

Table 2. The chlorophyll content of tomato seedlings infected with M. incognita pre- and post-treated with different concentrations of BioSeNPs ¹.

Factors	Chlorophyll a (mg/g FW)	Chlorophyll b (mg/g FW)	Carotenoids (mg/g FW)
Treatment time (TT)	**	**	**
Pre-infection (Pre)	2.90 b ± 0.29	2.04 b ± 0.25	1.21 b ± 0.22
Post-infection (Post)	3.89 a ± 0.31	2.50 a ± 0.39	1.37 a ± 0.17
Application method (AM)	**	*	**
Foliar spray (FS)	3.47 a ± 0.29	2.07b ± 0.24	1.48 a ± 0.22
Soil drench (SD)	3.32 b ± 0.34	2.47 a ± 0.40	1.10 b ± 0.16
BioSeNPs (ppm)	**	**	**
NC	1.98 e ± 0.02	1.58 b ± 0.02	0.51 e ± 0.03
BioSe2	3.66 b ± 0.13	2.57 a ± 0.40	2.11 a ± 0.43
BioSe6	2.51 d ± 0.16	1.78 b ± 0.17	0.61 d ± 0.14
BioSe10	3.45 c ± 0.78	2.87 a ± 1.01	1.87 b ± 0.34
PC (Ethoprophos Smart-N)	5.40 a ± 0.04	2.54 a ± 0.38	1.35 c ± 0.02
TT × AM × BioSeNPs	**	**	**
NC	1.98 g ± 0.04	1.58 c–e ± 0.04	0.51 ij ± 0.06
Pre × FS × BioSe2	4.20 c ± 0.06	4.77 b ± 0.02	4.55 a ± 0.04
Pre × FS× BioSe6	2.90 e ± 0.05	2.54 c ± 0.02	0.75 h ± 0.03
Pre × FS × BioSe10	0.75 j ± 0.03	0.04 f ± 0.01	0.65 hi ± 0.03
Pre × SD × BioSe2	3.05 e ± 0.01	2.25 cd ± 0.03	1.08 f ± 0.11
Pre × SD × BioSe6	2.30 f ± 0.15	2.05 cd ± 0.08	0.42 j ± 0.01
Pre × SD × BioSe10	1.05 i ± 0.01	0.50 ef ± 0.01	0.93 g ± 0.01
Post × FS × BioSe2	3.76 d ± 0.02	1.37 c–e ± 0.02	1.36 de ± 0.08
Post × FS× BioSe6	3.09 e ± 0.05	1.17 de ± 0.02	1.26 e ± 0.02
Post × FS × BioSe10	5.26 b ± 0.03	2.54 c ± 0.03	2.54 c ± 0.03
Post × SD × BioSe2	3.60 d ± 0.08	1.88 cd ± 0.06	1.43 d ± 0.02
Post × SD × BioSe6	1.75 h ± 0.01	1.35 c–e ± 0.03	0.03 k ± 0.01
Post × SD × BioSe10	6.73 a ± 0.02	8.42 a ± 0.02	3.36 b ± 0.03
PC (Ethoprophos Smart-N)	5.40 b ± 0.09	2.54 c ± 0.9	1.35 de ± 0.04

¹ Each value represents mean ± standard error. Means in each column for each factor followed by the same letter do not differ significantly according to Duncan’s multiple range test at p ≤ 0.05. (* = p ≤ 0.05, ** = p ≤ 0.01). NC (Negative control) denotes tomato plants inoculated with M. incognita without any treatment. PC (Positive control) denotes tomato plants treated with the chemical nematicide ethoprophos Smart-N. BioSe₂, BioSe₆, and BioSe₁₀ denote 2, 6, and 10 ppm of BioSeNPs, respectively. FW denotes fresh weight.

and show that chlorophyll a, chlorophyll b, and carotenoids increased significantly after the treatment with BioSeNPs compared with negative control. Regarding the treatment time, post-infection treatment significantly increased the chlorophyll content more than the pre-infection treatment. As for the two application methods, foliar spray recorded the highest increase in chlorophyll a and carotenoids, while soil drench recorded the highest increase in chlorophyll b. In the interaction, compared to the negative control, chlorophyll a and b showed the highest results with BioSeNPs at 10 ppm as a soil drench in post-infected treated tomato plants.

In contrast, carotenoids showed the highest results in pre-infected treated tomato plants with BioSeNPs at 2 ppm as a foliar spray compared to the negative control. The obtained results align with those reported in earlier studies, wherein SeNPs enhanced the growth and photosynthetic pigments in several plants [48,49]. The chlorophyll pigment acts as the light-capturing center and plays an essential role in photosynthesis, and any alterations in chlorophyll are harmful to plant health [50]. The mechanism behind the improved chlorophyll content has been attributed to the role of SeNPs that protected photosynthetic pigments, and this type of effect depends on the concentration of NPs used, where NPs can induce positive responses to specific doses. In contrast, they can induce the opposite effect or do not have effect on others. This behavior, called hormesis, has been reported when NPs have applied as bio-stimulants in crops [51]. The best effect of a low Se dose is reflected in the reconstruction of the lipid and fatty acids of the plastids in the cell membrane, which increase the photosynthetic rate of the plants [52].

3.2.3. Effect of BioSeNPs on Enzyme Activity of Leaves of Tomato Plants

The results presented in

Table 3. The activity of some antioxidant enzymes of tomato seedlings infected with M. incognita pre- and post-treated with different concentrations of BioSeNPs ¹.

Factor	CAT Activity (ΔAbs 240 mg−1 Protein min−1)	POD Activity (ΔAbs 460 mg−1 Protein min−1)
Treatment time (TT)	**	**
Pre-infection (Pre)	81.4 b ± 2.7	21.6 b ± 1.1
Post-infection (Post)	84.1 a ± 3.3	23.8 a ± 2.1
Application method (AM)	**	**
Foliar spray (FS)	86.5 a ± 2.2	25.7 a ± 2.1
Soil drench (SD)	79.0 b ± 3.5	19.6 b ± 0.8
BioSeNPs (ppm)	**	**
NC	89.0 c ± 0.1	19.9 d ± 0.2
BioSe2	54.9 e ± 3.2	21.6 c ± 2.8
BioSe6	82.3 d ± 3.1	17.6 e ± 1.2
BioSe10	91.7 b ± 2.1	29.9 a ± 4.5
PC (Ethoprophos Smart-N)	95.8 a ± 0.1	24.4 b ± 0.1
TT ×AM × BioSeNPs	**	**
NC	89.0 e ± 0.3	19.9 e ± 0.4
Pre × FS × BioSe2	68.7 k ± 0.4	36.7 b ± 0.4
Pre × FS× BioSe6	84.7 g ± 0.4	13.8 h ± 0.1
Pre × FS × BioSe10	82.7 h ± 0.4	20.8 de ± 0.2
Pre × SD × BioSe2	44.5 m ± 0.3	15.2 g ± 0.1
Pre × SD × BioSe6	75.9 i ± 0.2	21.9 d ± 0.2
Pre × SD × BioSe10	87.8 f ± 0.1	18.6 f ± 0.3
Post × FS × BioSe2	61.8 l ± 0.1	20.8 de ± 0.4
Post × FS× BioSe6	97.8 b ±0.3	20.9 de ± 0.5
Post × FS × BioSe10	99.8 a ± 0.03	55.4 a ± 0.2
Post × SD × BioSe2	44.6 m ± 0.2	13.7 h ± 0.4
Post × SD × BioSe6	70.8 j ± 0.4	13.8 h ± 0.5
Post × SD × BioSe10	96.6 c ± 0.4	24.7 c ± 0.5
PC (Ethoprophos Smart-N)	95.8 d ± 0.1	24.4 c ± 0.3

¹ Each value represents mean ± standard error. Means in each column for each factor followed by the same letter do not differ significantly according to Duncan’s multiple range test at p ≤ 0.05 (** = p ≤ 0.01). NC (Negative control) denotes tomato plants inoculated with M. incognita without any treatment. PC (Positive control) denotes tomato plants treated with the chemical nematicide ethoprophos Smart-N. BioSe₂, BioSe₆, and BioSe₁₀ denote 2, 6, and 10 ppm of BioSeNPs, respectively.

show that post-infection treatment significantly enhanced CAT and POD activities more than the pre-infection treatment. Concerning the two application methods, foliar spray achieved the best CAT and POD activities, more than the soil drench. Regarding the interaction, assays of CAT and POD showed their superior activities in post-infected treated tomato plants with BioSeNPs at 10 ppm as a foliar spray by 12.1 and 178.4%, respectively, over negative control. Similarly, the literature data showed the role of SeNPs in enhancing antioxidant enzymes [48,53,54]. Activating the antioxidant enzymes has been regarded as an essential factor in enhancing plant resistance against pathogenic infection [55]. The significant antioxidant activity of SeNPs might be due to the highest level of Se, which plays a vital role in the enhancement of enzymes, such as CAT and POD, that protect plants from the damaging effects of ROS [56].

3.2.4. Effect of BioSeNPs on Number of Galls and Egg Hatching of M. incognita-infected Tomato Plants

The results in

Table 4. The number of M. incognita galls, egg mass, and eggs/egg mass per each g root of tomato seedlings pre- and post-treated with different concentrations of BioSeNPs ¹.

Factor	No. of Galls (g root−1)	Reduction (%)	Egg Mass (g root−1)	Reduction (%)	Eggs/Egg Mass (g root−1)	Reduction (%)
Treatment time (TT)	*		**		NS
Pre-infection (Pre)	846.1 a ± 165	-	232.9 b ± 41	-	345.2 a ± 16	-
Post-infection (Post)	799.0 b ± 163	-	263.5 a ± 39	-	341.4 a ± 14	-
Application method (AM)	**		**		NS
Foliar spray (FS)	716.1 b ± 166	-	225.5 b ± 41	-	349.9 a ± 15	-
Soil drench (SD)	929.0 a ± 160	-	270.9 a ± 38	-	336.6 a ± 14	-
BioSeNPs (ppm)	**		**		**
NC	2484.3 a ± 50	-	651.0 a ± 10	-	456.6 a ± 18	-
BioSe2	491.6 bc ± 94	80.2	160.7 c ± 28	75.3	364.2 a ± 21	6.37
BioSe6	454.3 c ± 59	81.7	189.4 b ± 12	70.9	357.1 a ± 8	8.20
BioSe10	538.4 b ± 134	78.3	192.9 b ± 14	70.3	377.2 a± 23	3.03
PC (Ethoprophos Smart-N)	144.0 d ± 8	94.2	47.0 d ± 1	92.7	229.0 b ± 7	41.1
TT ×AM × BioSeNPs	**		**		**
NC	2484.3 a ± 117	-	651.0 a ± 24	-	456.6 a ± 41	-
Pre × FS × BioSe2	212.3 g ± 8	91.4	59.0 g ± 3	90.9	378.3 c ± 6	17.1
Pre × FS× BioSe6	213.0 g ± 9	91.4	129.7 f ± 3	80.6	383.3 c ± 3	16.0
Pre × FS × BioSe10	443.7 e ± 4	82.1	196.7 de ±4	69.8	446.6 a ± 9	2.19
Pre × SD × BioSe2	290.0 fg ± 13	88.3	132.0 f ± 3	79.7	259.0 ef ± 4	43.3
Pre × SD × BioSe6	755.0 d ± 16	69.6	181.0 e ± 6	72.2	330.0 cd ± 12	27.7
Pre × SD × BioSe10	1290.0 b ± 3	48.0	235.0 c ± 5	63.9	366.7 c ± 21	19.7
Post × FS × BioSe2	451.7 e ± 4	81.8	141.7 f ± 7	78.2	363.3 cd ± 3	20.4
Post × FS× BioSe6	383.3 ef ± 12	84.5	211.7 c–e ± 6	67.5	341.7 cd ± 13	25.2
Post × FS × BioSe10	200.0 g ± 10	91.9	120.0 f ± 6	81.6	289.3 de ± 9	36.6
Post × SD × BioSe2	1012.3 c ± 7	59.2	310.0 b ± 6	52.4	386.3 c ± 4	15.4
Post × SD × BioSe6	466.0 e ± 5	81.2	235.3 c ± 4	63.9	373.3 c ± 4	18.2
Post × SD × BioSe10	220.0 g ± 5	91.1	220.0 cd ± 8	66.2	345.3 cd ± 4	24.4
PC (Ethoprophos Smart-N)	144.0 g ± 19	94.2	47.0 g ± 3	92.8	229.0 f ± 16	49.8

¹ Each value represents mean ± standard error. Means in each column for each factor followed by the same letter do not differ significantly according to Duncan’s multiple range test at p ≤ 0.05. (* = p ≤ 0.05, ** = p ≤ 0.01 and NS = non-significant). NC (Negative control) denotes tomato plants inoculated with M. incognita without any treatment. PC (Positive control) denotes tomato plants treated with the chemical nematicide ethoprophos Smart-N. BioSe₂, BioSe₆, and BioSe₁₀ denote 2, 6, and 10 ppm of BioSeNPs, respectively.

demonstrate that treating inoculated tomato plants with chemical nematicide ethoprophos followed by BioSeNPs significantly reduced the numbers of galls, egg mass, and eggs/egg mass compared to the negative control. Concerning the treatment time, post-infection treatment significantly reduced the number of galls more than the pre-infection treatment; however, the pre-infection treatment significantly reduced the egg mass more than the post-infection treatment, whereas eggs/egg mass was not significantly different. Comparing the two application methods, the foliar spray caused a higher reduction in the number of galls and egg mass than the soil drench, while eggs/egg mass were not significantly different. In the interaction, the highest reduction in the number of root galls appeared in the positive control, which was an amount of 94.2%. In addition, the post-infected treated tomato plants with BioSeNPs at 10 ppm as foliar spray or soil drenching changed by 91.9 and 91.1%, respectively, compared to the negative control. The results of the interaction also showed that the highest reduction in egg mass was performed in infected tomato plants treated with ethoprophos nematicide (positive control) by 92.8% and pre-infected treated tomato plants with BioSeNPs at 2 ppm as a foliar spray by 90.9%, compared with the negative control. The number of eggs/egg mass showed the highest reduction in infected tomato plants treated with ethoprophos nematicide (positive control) by 49.8%, followed by pre-infected treated tomato plants with BioSeNPs at 2 ppm as a soil drench by 43.3% over the negative control. These results support the conclusion drawn from the previous research that the SeNPs showed significant suppressive activity against root-knot nematodes [44,57]. Hence, the treatment of tomato plants with BioSeNPs reduced the infestation of plants by the reduction in the number of galls, egg mass and, eggs per egg mass of M. incognita in the roots. These findings suggest the vital role of Se in plants’ life as a protector against biotic stresses, including pathogens infection by the interaction in plant cells with proteins and other biomolecules containing active groups, which enhances the signal transmission between living cells and modification of receptor proteins, which improved some plant innate mechanisms in relation to M. incognita [44,47].

4. Conclusions

In this study, the SeNPs were biosynthesized using P. stutzeri BB19 and used as a promising effective resistance inductor against tomato root-knot nematode M. incognita for the first time. The treatment of tomato plants infected with M. incognita under greenhouse conditions with BioSeNPs at 2, 6, and 10 ppm as a foliar spray or a soil drench exhibited inhibitory effects on M. incognita. Accordingly, the treatment with BioSeNPs induced tomato systemic resistance to M. incognita by the reduction in root galling, egg mass, and eggs per egg mass. Furthermore, the BioSeNPs treatments stimulated tomato growth parameters, chlorophyll a, chlorophyll b, carotenoid content, and CAT and POD enzymes activity compared with the untreated infected tomato plants. The mechanism of the activity of BioSeNPs is determined by their interaction in tomato plant cells with proteins and other biomolecules containing functionally active groups. Such interaction promotes signal transmission between living cells and modification of receptor proteins, which may lead to changes in the responses of plants to invasion. The known properties of Se as a substance with a wide range of antistress activities contributed to the development of studies of its NPs in relation to using them as biogenic elicitors of plant resistance to parasitic nematodes. Consequently, the BioSeNPs in this study show application potential as cost-effective and eco-friendly resistance inductors for the biocontrol of the tomato RKN M. incognita.