Green fabrication of titanium dioxide nanoparticles via Syzygium cumini leaves extract: characterizations, photocatalytic activity and nematicidal evaluation

ABSTRACT

The most significant plant parasitic nematode in terms of economic impact is root-knot nematode (Meloidogyne incognita), which severely harms essential crops used for agriculture. This study investigated green fabrication of titanium dioxide nanoparticles (TiO₂ NPs) utilizing Syzygium cumini (SC) leaf extract and evaluated its nematotoxic activity against M. incognita in vitro and in pots. During experiments, second-stage juveniles (J2s) and egg masses of M. incognita were treated with different concentrations (125, 250, 375 and 500 ppm) of SC-TiO₂ NPs. After 48 h and five days of exposure, respectively, lowest J2s mortality and per cent inhibition in egg hatching was discovered at 125 ppm, and highest J2s mortality and maximum per cent inhibition in egg hatching determined at 500 ppm. The treatment of SC TiO₂ NPs considerably reduced M. incognita infection and enhanced carrot plants' growth and physiological characteristics. The characterization of green synthesized SC-TiO₂ NPs made by X-ray diffraction, Photoluminescence, FTIR, SEM, UV-Visible Diffuse Reflectance Spectrophotometer, TEM, and photocatalytic activity also examined. In conclusion, it can be said that SC-TiO₂ NPs possess nematode-toxic potential against M. incognita and are used as substitutes for high-risk chemical nematicides without causing any phytotoxicity.

GRAPHICAL ABSTRACT

KEYWORDS:

• Nano-materials
• botanicals
• green synthesis
• sustainable management
• plant parasitic nematodes

1. Introduction

In the global context, plant diseases cause significant agricultural production losses, which are believed to be a severe consequence for food safety (1, 2). Agriculture must increase to produce enough food for the growing global population. This might be accomplished by using nanotechnology, which has enormous promise in agriculture, to manage pests, pathogens, weeds, and diseases, protect plants, maintain soil and water quality, monitor pollution, enhance crop quality, and generate nano-sensors (3, 4). Moreover, in both conventional and organic agricultural systems, plant parasitic nematodes (PPNs) are among the most destructive and damaging parasites that target a variety of plants (5, 6). The root-knot nematode (RKN) of the genus Meloidogyne, could infect nearly all crops worldwide (7). Interestingly, M. incognita is the most damage-causing species in this genus among the other known species. Mainly, M. incognita is bio-trophic parasite that, over several weeks, drains nourishment from the roots of hosts, resulting in the host's early demise and decreased agricultural output (8).

It has been estimated that losses to the crops produced by the PPNs have been measured to be 80 billion yearly in open cultivation (9); but it may have crossed 30-60% in protected cultivation (10). Due to the growing demand for food production via intensive agriculture, RKN's effects on agriculture have typically been controlled by chemical methods. Moreover, at the high economic expense of nematicides, this has long-term adverse effects on the environment and public health. As a result, hunting for alternate RKN control strategies became vital by examining how RKN interacts with the soil's biological composition (11). The appliance of phyto-nanotechnology in agriculture is now seen as an environmentally beneficial practice with the potential to increase crop yield. Furthermore, bio-safety and eco-toxicological research on interactions between nanoparticles and biological elements of soil should be included in the production of nanoparticles for agricultural usage (12).

Nano agriculture utilizes nanoparticles as fertilizers and pesticides to enhance soil quality and offer environmentally friendly ways to boost plant health. Due to their large surface area and tiny size, nanoparticles are helpful in various industries, including agriculture, medicine, food, catalysis, electronics, and the environment (13, 14). Green synthesis, which employs plant extract, is a time- and money-saving alternative to employing hazardous chemicals (15) and, therefore, found to be more advantageous than other methods used presently. Metal oxides, metalloids, nonmetals, and carbon nano-materials are only a few examples of nanoparticles that are useful in preventing plant diseases and improving plant health (16). Further, silver (Ag) and copper oxide nanoparticles could also be utilized as effective nematicidal, insecticidal, anti-fungal and antibacterial agents (17, 18). The creation of agricultural systems based on nanotechnology currently offers a ground-breaking way to enhance the efficacy of conventional pesticide formulations (19, 20).

Syzygium cumini, a fruit-bearing plant in the Myrtaceae family, is referred to as Jamun locally in Asia. Several plant parts, including the bark, leaves, seeds, and fruit, have been used to treat a variety of illnesses (21). It was discovered that the leaves of Jamun included following, betulinic acid, crategolic acid, n-dotricontanol, n-hentriacontane, n-hepatcosane, mycaminose, myricetin, myricitrin, myricetin 3-O-(4″-acetyl)-α-L-rhamnopyranosides, n-nonacosane, quercetin, β-sitosterol, noctacosanol, n-triacontanol, triterpenoids, tannins, eicosane, octacosane and octadecane (22–24). There have been reports of low to high concentrations of cardiac glycosides, flavonoids, alkaloids, glycosides, anthraquinones, saponins, phenols, steroids, phytosterols, triterpenoids, tannins, terpenoids, proteins, amino acids, volatile oils, carbohydrates, fixed oils, mucilage, and fats in hexane, petroleum ether, chloroform, ethanol, ethyl acetate, methanol, and water extracts of Jamun leaves (25). Conversely, minerals like sodium, potassium, calcium, zinc, iron, magnesium, copper, manganese, lead, and chromium were also found in jamun leaves (26). The present work synthesized TiO₂ NPs using aqueous S. cumini leaf extract solution as a capping and reducing agent. These days, the main use of jamun fruits, seeds, and leaves is in pharmaceutical treatments for a range of metabolic diseases, including obesity, diabetes, hypertension, and hyperlipidemia. Because S. cumini leaf extract is non-toxic, safe for environment, and naturally caps, reduces, and stabilizes synthesized TiO₂ NPs without causing agglomeration. Green-generated nanoparticles (NPs) have been proposed as potential new avenues for advancement of nanotechnology due to their less hazardous nature. In green synthesis, biological agents primarily plant extracts are employed more frequently than hazardous chemical agents. Among the many benefits of green synthesis are low manufacturing costs and a simple one-step process. Different plant extracts have been used to make green synthetic nanoparticles since they are simple to make. The nematicidial activity of plant extracts against the main plant pathogenic nematodes was significantly increased upon conversion to nanoparticles. Such an approach could lead to a new, safe, and successful nematode management program. Its accessibility and ease of use were probably the causes of this. In addition to being a more ecologically friendly formulation, green technique for synthesized nanoparticles has potential to offer practical solutions for a number of issues pertaining to agricultural crops. Before they can be suggested for field application and integrated pest management (IPM) programs against PPNs, develop bio-fabricated green synthetic nanoparticles that are poisonous and kill nematodes while also having biodegradation modes of action. These eco-friendly nanoparticles showed excellent photocatalytic activity and nematicidal assessment. Additionally, we examined the antagonistic potential of greenly synthesized TiO₂ NPs against M. incognita (Mortality, hatching and penetration bioassay) and characterization of TiO₂ NPs was done using X-ray diffraction (XRD), Fourier transform-infrared spectroscopy (FT-IR), Photoluminescence (PL), UV-Visible Diffuse Reflectance Spectrophotometer, Scanning electron microscopy (SEM) and Transmission Electron Microscopy-Selected Area Electron Diffraction (TEM-SAED).

2. Materials and methods

2.1. Materials

The RKN, M. incognita, was chosen as an objective pathogen, and the carrot (Daucus carota) was selected as the test plant. Titanium tetraisopropoxide (TTIP, C12H28O4Ti, 97%) was purchased from Merck, and double distilled water (DDW) was taken from the water purification plant at our lab.

2.2. Green synthesis of tiO₂ NPs

Jamun leaves extract includes alkaloids, coumarins, and flavonoids, taken as reducing, capping, and stabilizing mediators in the green fabrication of TiO₂ NPs. TiO₂ NPs synthesized via jamun leaves extract prevents agglomeration of NPs, which is visible in TEM images. The jamun leaves were plucked from the Naqvi Park of Aligarh, Uttar Pradesh. The extract from jamun leaves was made by combining 50 g of jamun leaves, one hundred millilitres of double-distilled water (DDW), and autoclaving mixture for 30 min at 121°C. The aqueous solution was filtered via Whatman filters paper No. 1 and kept for subsequent research. Add 20 mL of the leaf extract, one drop at a time, to 50 mL of TTIP (1M) in a 100 mL beaker. At room temperature, the solution swirled for three hours. The transformation of the solution's color from pure white to yellowish-grey indicated that TiO₂ NPs had formed. The solution was then filtered and dried for four hours at 80°C (Figure 1). The dried samples were ground to a powder via a motor pestle and then heated in a muffle furnace to 650°C for 6 hr (27, 28).

Figure 1. Flowchart represents the various stages involved in green synthesis of TiO₂ NPs using jamun leaves extract.

2.3. Characterization of tiO₂ nPs

XRD of TiO₂ rutile phase NPs was recorded using a Rigaku Miniflex Diffractometer in the range of 10°−80° using the CuKα as the primary radiation source, corresponding to 0.15,406 nm. FT-IR spectrum was obtained using Perkin Elmerin in the 400–4000 cm−1 range. The surface morphology and visual studies of TiO₂ NPs using SEM-EDX, along with the mapping spectrum, revealed the homogeneity and elemental composition of the sample. SEM and mapping images were recorded using the JEOL Japan model, Model No. JSM-6510LV. Cary 5000 UV-Visible Diffuse Reflectance Spectrophotometer recorded the UV-visible spectrum. The absorption spectrum was observed in the range of 200–800 nm. A fluorescence spectrometer (Perkin Elmer LS 55) was used to document the spectrum in the 325–550 nm range. Furthermore, the shape, size, particle size distribution, and surface morphology of green synthesized TiO₂ NPs were confirmed by analyzing TEM-SAED images and particle size distribution histograms.

2.4. Photocatalytic activity

TiO₂ NPs were used as photo-catalysts against UV and visible light irradiation to ascertain photo-catalytic MB dye degradation. The light source is crucial in modifying the pace at which organic pollutants decompose. A 100 mg photo-catalyst was inoculated into a 100 mL dye solution at 10 ppm in MB. The combined solution was then maintained in a dark environment to reach the adsorption–desorption equilibrium plane. The integrated solution is then exposed to UV and visible light. To assess effectiveness of TiO₂ NPs degradation, the irradiation samples were removed (5 mL) at regular intervals (20 min). The following equation was applied to measure the effectiveness of photo-catalytic dye degradation:

MB dye degradation percentage  = ${\displaystyle \frac{\mathrm{C}0-\mathrm{C}}{\mathrm{C}}}$

Where C₀ = Initial dye absorbance at time 0 min; C = Dye absorbance with light at regular intervals of time (20 min).

2.5. Maintenance of J2s inoculum of M. incognita

Meloidogyne spp.-infected brinjal roots were taken from surrounding fields in the Aligarh district. The perineal pattern helps to identify M. incognita (29). At the Botany Department, J2s are preserved on brinjal and kept in the greenhouse. In order to prevent egg masses from separating from the roots, brinjal plants were gently pulled before being thoroughly washed in DDW to eliminate all soil debris. Employing clean forceps, we gently detached egg masses from the roots. After being thoroughly washed with DDW, collected egg masses were emptied into 25 m pore-size mesh sieves that crossed tissue layers and then set in petri dishes with water. The petri dishes were then placed in a BOD incubator to hatch. The newly hatched J2s dropped to the bottom of petri dishes after passing through a sieve, while the mesh contained egg masses. Daily collections of suspension containing J2s were made, and DDW was added. Freshly hatched J2s had their concentration standardized as needed and preserved for further study.

2.6. Mortality test

Different TiO₂ NPs concentrations (125, 250, 375 and 500 ppm) were tested for nematicidal activity against J2s of M. incognita, and LC50 values were derived for each treatment. To find out TiO₂ NPs affected the mortality of J2s, 8 mL of TiO₂ NPs of various concentrations were added to petri dishes holding 2 mL of DDW containing 65 newly hatched J2s. Petri dishes containing solely DDW are employed as control. A dissecting microscope assessed J2 mortality after 12, 24, 36, and 48 h of incubation. Petri dishes are covered with parafilm to avoid evaporation. For each treatment, six replications were taken. If the J2s moved or took on a zigzag shape, they were deemed alive (30). However, when inspected with a very sharp needle after being transferred to tap water, dead J2s exhibited no motility (31). The LC50 values of every treatment were established using probit analysis (32, 33). The following formula was used to calculate the per cent mortality for each treatment. (1) $\mathrm{Percent}\mathrm{mortality}\mathrm{of}\mathrm{J}2\mathrm{s}=\left({\displaystyle \frac{\mathrm{Total}\mathrm{J}2\mathrm{s}-\mathrm{Dead}\mathrm{J}2\mathrm{s}}{\mathrm{Total}\mathrm{J}2\mathrm{s}}}\right)\times 100$(1)

2.7. Hatching bioassay

Following the egg mass dipping method, the J2s hatching inhibitory capacity of various TiO₂ NP concentrations (125, 250, 375 and 500 ppm) was assessed. Five egg masses of a median size were manually removed from M. incognita-infected brinjal roots and positioned into petri dishes having 10 mL TiO₂ NPs in five varying concentrations. To avoid evaporation, parafilm was used to wrap the petri dishes securely and then set to 28°C securely. Five egg masses are put into petri dishes. Egg masses in DDW were used as control. There were six replicates of each treatment. Hatched J2s were counted by applying a binocular microscope to determine the hatching rates. The following formula determines per cent inhibition in J2s hatching (34). (2) $\mathrm{Percent}\mathrm{hatching}\mathrm{inhibition}=\left({\displaystyle \frac{{\mathrm{C}}_0-{\mathrm{T}}_{\propto }}{{\mathrm{C}}_0}}\right)\times 100$(2) where, C₀ = Number of J2s hatched in DDW; T_α = Number of J2s hatched in every concentration of TiO₂ NPs.

2.8. Pot assay and experimental plan

The glasshouse experiment using pots was carried out to ascertain nematode-toxic effectiveness of TiO₂ NPs against M incognita on carrots. From the market, carrot seeds ‘red beauty’ variety were bought. Mercuric chloride (0.02%) was used to sterilize carrot seeds, which were immediately washed thrice in running water. Pots made of clay (15 cm in diameter) were supplied with 1 kg of autoclaved loam and farmyard manure in a 3:1 ratio. In pots, five sterilized carrot seeds were planted. We carefully removed undesirable seedlings after germination, leaving the healthiest one in each pot. Around the seedling's root, we drilled 2–3 holes and injected 2500 J2s. A pipette was used to apply 10 ml of various TiO₂ NP concentrations (125, 250, 375, and 500) near the seedling's root after two days of J2s inoculation. Plants were given enough water and irrigation during the trial. The treatments consist of (a) Control (None TiO₂ NPs and J2s); (b) Untreated inoculated control (J2s only); (c) 10 mL f 125 ppm of TiO₂ NPs + 2500 J2s; (d) 10 mL of 250 ppm of TiO₂ NPs + 2500 J2s; (e) 10 mL of 375 ppm of TiO₂ NPs + 2500 J2s; (f) 10 mL of 500 ppm of TiO₂ NPs + 2500 J2s.

2.9. Determination of growth and physiological parameters

Plants were harvested after being fully grown, washed with running water to get out clinging dirt, and assessed for growth and physiological traits. In the case of growth characteristics (root and shoot length, root and shoot fresh weight), photosynthetic traits viz., chlorophyll and carotenoid content (mg/g) considered following techniques outlined by Mackinney (35) and MacLachlan and Zalik (36), respectively.

2.10. Estimation of pathological parameter (RKI and J2s population)

The RKI was computed using a 0–5 scale developed by Taylor and Sasser (37). Cobb's sieving and decanting approach (38) employed to compute final number of J2s in 250 g of soil at harvest time using an updated Baermann's funnel methodology (39).

2.11. Statistical analysis

The acquired data, which included a wide range of features under investigation, underwent statistical analysis by R software (version 2.14.1). It was determined which of the examined attributes predict significant differences (p = 0.05) via Duncan's Multiple Range Test (DMRT). An ANOVA was performed using OPSTAT (40). The LC50 values for each treatment were calculated using OPSTAT (40).

3. Results

3.1. X ray diffraction

The X-ray diffractometer (XRD) system was utilized to analyze crystal structure, strain, purity, and average crystallite size of bio-synthesized rutile TiO₂ NPs; Figure 2 exhibits the XRD outline of the sample. The bio-synthesized rutile phase of TiO₂ NPs had an average crystallite size of 24 nm, corresponding to three maximum (27.43°, 36.08° and 54.32°) intense peaks as seen in the XRD outline of TiO₂ NPs. The occurrence of diffraction angle (2Θ) at 27.43°, 36.08°, 39.18°, 41.24°, 44.04°, 54.32°, 56.62°, 62.77°, 64.04°, 69.04°, 69.81° and 76.55° represents the Braggs refection plane of (110), (101), (200), (111), (210), (211), (220), (002), (221), (301), (112) and (202) respectively corresponds to rutile phase of TiO₂. The green synthesized TiO₂ NPs using jamun leaves exhibited higher intensity peaks of rutile TiO₂ NPs due to poly-phenolic compounds in the plant extract. XRD average crystallite size and TEM particle size are tabulated in Table 1.

Figure 2. XRD pattern of rutile TiO₂ NPs.

Table 1. Illustrating all parameters of TiO₂ NPs.

Sample Lattice Effective Avg. Crystallite Avg. Particle constant (Å) strain (ɳ) size (nm) XRD size (nm) TEM a = b c ×10^−3
TiO2 NPs	4.59402.9580	1.6	24	21.50

3.2. Fourier-transform infrared spectroscopy

According to Figure 3, FT-IR spectroscopy has been utilized to examine different functional groups present in the chemical composition of TiO₂ NPs. When FT-IR analysis was used to establish the function of phyto-constituents in plant extract that were in charge of capping and stabilizing the TiO₂ NPs, it revealed a variety of bands, particularly between 400 and 4000 cm-1, which indicated the existence of several functional groups on TiO₂ NPs (Table 2).

Figure 3. TiO₂ NP formation is confirmed by the creation of Ti–O–Ti and Ti–O vibration bonds.

Table 2. FT-IR tentative frequency of TiO₂ NPs.

S.N.	Wave number (cm^−1)	Band assignment
1.	3471	O-H stretching vibration
2.	1641	C-H bending vibration
3.	1034	C-O aromatic stretching vibration
4.	908	Ti-O-Ti bending vibration
5.	559	Ti-O bending vibration

3.3. UV-Visible diffuse reflectance spectrophotometer and photoluminescence spectroscopy

TiO₂ NPs were characterized using UV-visible spectroscopy. The final evidence for the fabrication of TiO₂ NPs was the initial transformation from brown to white. At first, the reaction mixture's UV-vis absorbance was barely detectable. As soon as color shifted, the absorption band at 402 nm was observed, as seen in Figure 4. Photoluminescence (PL) of TiO₂ NPs spans in the spectral range 325–550 nm (Figure 5). In the high-energy region, PL exhibits indirect band-to-band recombination between band gaps (red line).

Figure 4. UV–Vis DRS spectroscopy, TiO₂ NPs optically active region at (A) 402 nm exhibits visible region spectra and (B) band gap of 2.99 eV using Kubelka Munk function.

Figure 5. Photoluminescence spectrum (PL) of Jamun leaves extract prepared TiO₂ NPs annealed at 650°C. Photoluminescence spectra of TiO₂ (black-original, yellow-fitted curve) and its Gauss fit of TiO₂ emission band corresponding to contributing (red) indirect band to band recombination, (green) excitons, (blue and cyan) trapped electrons from defects and O vacancies and (magenta) trapped holes.

3.4. SEM and TEM-SAED analysis

The exterior appearance of synthesized TiO₂ NPs was evaluated using scanning electron microscopy (SEM). It is evident from Figure 6(a)–(d) TiO₂ NPs are diverse in shape and size, representing non-uniform size distribution. TEM was performed to give more information on the size, shape, and morphology of synthesized TiO₂ NPs. The TEM images illustrate that the shape of synthesized TiO₂ NPs was round and polydisperse with widths in the 12−30 nm range (Figure 7).

Figure 6. TiO₂ NPs SEM structures with different magnifications (a) 2500x (b) 5000x (c) 10000x (d) 30000x and scale at 15 kV.

Figure 7. (A) Spherical shaped nanoparticles of rutile TiO₂ annealed at 650°C (B) Interplanar spacing of 0.247 nm was observed (C) SAED pattern confirming polycrystalline nature of rutile TiO₂ (D) Particle size distribution curve.

3.5. Study of photo-catalytic activity

Figure 8(a) displays the green-produced TiO₂ NPs in contrast to methylene blue dye under visible light. Due to the catalyst's reactive sites, exposure to visible light gradually accelerated the degradation rate. Because their OH radicals cause the dye molecules to oxidize after exposure to visible light for longer than 60 min, they exhibit more significant degradation. The electron–hole pair recombination that plant bio-molecules supported led to potent oxidation and reduction of dye molecules over the catalyst. With the migration of the concerned band's electrons and holes, this procedure lasts 60–120 min. The potential for UV radiation, additional effectual than visible light radiation, is supplied by obtained pseudo-first-order kinetics. The electron mobilization and evoked electron–hole pair caused by visible light help improve degradation efficiency. After being exposed to visible light for 20 min, TiO₂ NPs showed a disintegration rate of more than 80%. TiO₂ NPs degraded more quickly when exposed to visible light (86.60%). Figure 8(c) shows how the spectrum determined photocatalyst efficiency (C/C0). The degradation efficiency is influenced by catalyst dosage, pH, light intensity, and dye content. Due to their wide band gap and e-h pair limitation characteristic, TiO₂ NPs are widely used in photo-catalytic activity. According to UV-visible NIR investigation, rutile TiO₂ NPs showed a broad band gap (2.99 eV), smaller crystallite size, and large surface area. Reusability and stability are critical for photocatalysts to be used in practical applications. Here, we evaluated the reusability and stability performance of TiO₂ NPs. The decomposition per cent of MB was observed for three successive cycles, where each process was 120 min with 86.60% after three cycles Figure 8(b). The results show that the decomposition efficiency was 88.78% in first cycle and remained at 86.60% after three consecutive cycles. The minor decrease in performance (2.18%) was due to a loss of catalyst during recycling. The photodegradation ratio (C/C0) of MB dye vs. time is plotted in Figure 8(c). With increasing exposure time, the dye percentage decreased almost linearly. The degradation kinetics were calculated according to the equation ln (C0/Ct) = kt (Figure 8(d)).

Figure 8. (a) The photocatalytic performance of MB dye decomposition in presence of TiO₂ NPs under visible light (b) Degradation percentage of MB dye by photocatalyst TiO₂ after 3 cycles (c) Photocatalytic performance, as represented by variation of C/C₀ with time (d) The corresponding kinetic plots for dye degradation.

where C₀ is starting concentration; C is concentration at irradiation time interval t; k is rate constant (min⁻¹). Further, we calculated rate constant for three reusability cycles. The k’s were 0.0484, 0.0483, and 0.0482 min⁻¹ for three consecutive recycles. These results show that rate constant for three cycles was not substantially reduced.

When exposed to visible light on the surface of the photocatalyst, excitation of electrons from the valence band to the conduction band takes place (Figure 9). The holes may be produced in a valence band. The photogenerated holes may combine with water molecules to create hydroxyl radicals. Now, electrons present in the conduction band combine with atmospheric oxygen (or supplied oxygen) to generate superoxide radicals. Thus, photogenerated holes, hydroxyl radicals, and superoxide radicals play a significant role in the degradation of methylene blue. The mechanism of TiO₂ degradation in solar light is shown in Figure 9.

Figure 9. Photo-degradation mechanism of methylene blue (MB) using TiO₂ NPs synthesized by S. cumini leaves extract.

3.6. Effect of tiO₂ NPs on J2s mortality and egg hatching

The present study used a direct-contact approach to analyze J2 mortality in TiO₂ NPs. Substantial variations were found between the concentrations (125, 250, 375 and 500 ppm) in case of J2s mortality. Each concentration showed some degree of toxicity against J2s. Generally speaking, mortality of J2s rose through longer exposure times and higher TiO₂ NP concentrations. During 48 h of exposure, the 500 ppm was found to be very toxic to J2s, which was found to be substantial compared to other concentrations. Similarly, the incubation period had an enormous impact on the % mortality of J2s and peaked following 48 h. But 125 ppm also predicts a considerable increase in J2s mortality compared to control. In DDW (control), there were no known J2s fatalities. An increase in J2s mortality was seen as concentration strength rose from 100 to 500 ppm (Table 3). After 48 h of exposure, LC50-232.40 showed higher J2s mortality than it did after 36 h (LC50-385.95), 24 h (LC50-589.58), and 12 h (LC50-985.63) of exposure (Table 4). Our study found that the treatment's toxicity to M. incognita J2s was higher with lower LC50 values and that treatment with higher LC50 value was less toxic to J2s. According to the results, all concentrations were extremely hazardous to J2s and showed mortality rates ranging from 9% to 69% (Table 3). Table 3 shows the individual harmful effects of various TiO₂ NPs concentrations (125, 250, 375 and 500 ppm) on J2 mortality.

Table 3. Impact of varying TiO₂ NPs concentrations on J2s mortality after 12, 24, 36, and 48 h of exposure.

Treatment	Concentrations (ppm)	Number of dead J2s (Mean ± Standard error) at different time intervals (Hours)
		12	24	36	48
	125	10 ± 1.15 (15.38%)	16 ± 1.54 (24.61%)	20 ± 1.73 (30.76%)	26 ± 1.52 (40.00%)
	250	15 ± 1.54 (23.07%)	21 ± 1.52 (32.30%)	24 ± 1.54 (36.92%)	31 ± 2.08 (47.69%)
	375	19 ± 1.73 (29.23%)	26 ± 1.52 (40.00%)	30 ± 1.73 (46.15%)	37 ± 2.08 (56.92%)
	500	25 ± 1.73 (38.46%)	32 ± 1.52 (49.23%)	39 ± 2.08 (60.00%)	45 ± 2.30 (69.23%)
	DDW	0 ± 0 (0%)	0 ± 0 (%)	0 ± 0 (%)	0± (%)
Degrees of freedom		3	3	3	3
Sum of Squares		362.25	422.25	614.25	602.25
Mean Squares		120.75	140.75	204.75	200.75
F-Calculated		25.42	23.45	22.75	22.30
Significance		0.00019	0.00026	0.00029	0.00031

The percentage of J2s mortality over control is represented by values in parenthesis.

The numbers that are provided without parentheses indicates killed J2s at various concentrations of TiO₂ NPs.

Table 4. LC 50 values of TiO₂ NPs at different exposure against J2s.

Treatment	Exposure Period (hours)	LC50 value in ppm (95% CL)
TiO2 NPs	12	985.63
	24	589.58
	36	385.95
	48	232.40

LC50 – Lethal concentration showed 50% mortality after 12, 24, 36 and 48 h at 95% confidence limits.

CL – Confidence limit.

In this bioassay, the straight contact method was followed to analyze the suppression of egg hatching caused by TiO₂ NPs. A noteworthy differentiation was established among concentrations (125, 250, 375 and 500 ppm). Compared to the control, all doses dramatically decreased egg hatching (Figure 10). Along with an increase in concentration strength, inhibition of egg hatching increased. But compared to the control, 100 ppm also showed considerable variations in hatching inhibition. From 125 to 500 ppm of TiO₂ NPs, a proportional rise in the inhibition of egg hatching in a step-by-step fashion. After a 5-day incubation period, 500 ppm induced the most excellent egg-hatching inhibition, which was then followed by 375, 250, and 125 ppm. Figure 10 shows the individual inhibitory effect of various TiO₂ NP concentrations on egg hatching.

Figure 10. Effect of different concentrations of TiO₂ NPs on J2s hatching after 5 days of exposure. The treatment with same letter is not significant and treatment with different letter (like a, b, c, d, e, f …  …  …  …) are significant (P > 0.05) according to DMRT.

3.7. Nematicidal effect of TiO₂ NPs against M. incognita in-planta trial

Carrot development characteristics, including plant length and fresh weight, considerably improved at all TiO₂ NP concentrations (125, 250, 375, and 500 ppm). The 500 ppm treatment demonstrated the most significant improvement in developing the above parameters among all applied treatments. Compared to the pot inoculated with only J2s, other treatments (125, 250, and 375 ppm) also improved all growth parameters. When varied quantities of TiO₂ NPs were applied, the carrot plants responded in various ways (Figure 11). Similarly, a considerable increase in carotenoid and chlorophyll concentration was seen at 500 ppm and in contrast to pot infected with only J2s, other treatments, including 125, 250, and 375 ppm also showed an increase in chlorophyll and carotenoid content (Figure 12). Only J2s-inoculated plants showed the most significant decrease in carotenoid and chlorophyll levels.

Figure 11. Nematicidal effect of varying TiO₂ NPs concentrations on growth parameters of J2s inoculated carrot plants. The treatment with same letter is not significant and treatment with different letter (like a, b, c, d, e, f …  …  …  …) are significant (P > 0.05) according to DMRT.

Figure 12. Nematicidal effect of varying TiO₂ NPs concentrations on physiological and pathological parameters of J2s inoculated carrot plants. The treatment with same letter is not significant and treatment with different letter (like a, b, c, d, e, f …  …  …  …) are significant (P > 0.05) according to DMRT.

When different TiO₂ NP concentrations (125, 250, 375, and 500 ppm) were administered, the pathogenic parameters (RKI and J2s population) dramatically decreased in comparison to the pot inoculated with only J2s (Figure 12). RKI was reduced to the greatest extent at 500, 375, 250 ppm, and to the least at 125 ppm. Similarly, the J2 population decreased most dramatically at 500, 375, 300, and 125 ppm. Only those pots that received an inoculation of 2500 J2s displayed the highest RKI and J2s populations.

4. Discussion

Most nanoparticles are synthesized via time-consuming, dangerous, physical and chemical processes, but safer, more scalable green synthesis techniques have recently been discovered. The bio-reduction and capping processes involve secondary metabolites in organisms like microorganisms and plants (41). Limon-citrus extract acts as a capping/reducing agent for synthesizing titanium dioxide nanoparticles and showed antibacterial activity against Escherichia coli (42). The diffraction angle at 27.43° of (101) plane shows the highest crystalline nature of rutile phase TiO₂ NPs. The XRD structure of rutile phase TiO₂ NPs shows consistency with JCPDS file: 96-153-4782, showing tetragonal structure of the crystal (43). The average crystallite size of rutile TiO₂ NPs was calculated using the Debye Scherer rule (44). Interestingly, compared to the chemical method, the green synthesis process appears to yield a more thermodynamically stable anatase phase (42). The intense broad peak at 3471 cm−1 is due to O–H stretching vibration (45). The small band around 1641 cm−1 shows C–H functional group bending vibration (46). The narrow band at 1034 cm−1exhibits C–O stretching vibration of aromatic group (47). The intense band at 559 and 908 cm−1 showed the presence of Ti–O and Ti–O-Ti bending vibrations (48). The Ti–O–Ti bonds and Ti–O metal oxide bonds further confirm the existence of TiO₂ in fabricated TiO₂ NPs. However, plant phytochemicals are mainly responsible for reducing titanium salt to stable TiO₂ NPs (49). The hydroxyl groups (O-H) found between 3450–3712 cm−1 in TiO₂ NPs increase the photocatalytic performance. It has been observed that the IR frequency of TiO₂ NPs slightly changes compared to green and chemically synthesized TiO₂ NPs.

The UV–Vis reflectance spectrum of TiO₂ NPs at 402 nm indicates the electronic transition of charge between O 2p state and Ti 3d state (49). In the plant-mediated synthesis of NPs, the colloidal solution color changes from white to yellowish-grey, indicating the synthesis of TiO₂ NPs. The white color dispersion confirms TiO₂ NP formation during the biochemical process. The distinct absorption peak exhibits a change in crystallite phase and average crystallite size (50). The different absorbance peak between 385–405 nm regions indicates the formation of TiO₂ NPs. Furthermore, TiO₂ NP absorbance spectra matched well with previous reports (51). The band gap was calculated at 2.99 eV using the Kubelka Munk function, which is close to the reported value (52). In the high-energy region, PL exhibits indirect band-to-band recombination between band gaps (red line). However, excitons that originate from surface oxygen vacancies and defects (cyan and blue lines) are what caused the majority of PL intensity in the low energy range (green line) (53).

SEM shows vivid surface morphology with negligible agglomeration. The NPs in the TiO₂ rutile TEM micrograph are spherical. The TEM image indicates that the particle size of TiO₂ does not vary significantly as reaction time, temperature, and other environmental parameters are maintained. The polycrystalline nature of samples indicated by selected area electron diffraction (SAED) which validates crystallinity nature of the produced TiO₂ NPs as previously validated by XRD structure (54, 55). Due to their OH radicals, TiO₂ degrades more quickly in visible light radiation (56). Although degradation caused by visible light is less, adding metal compounds boosted catalytic efficiency. The ability of dye molecules to oxidize was boosted by creating free radicals. In visible light irradiation, radical forms are elicited. The superoxides and radicals produced by the current visible light irradiation catalytic activity showed stronger photo-catalytic dye degradation activity than those from earlier reported works. The present study, which concentrated on visible light irradiation, shows that biogenic TiO₂ NPs had a more remarkable ability to degrade the MB dye (57). Various authors have reported the photocatalytic activity of green-mediated TiO₂ NPs to reduce different dyes and compounds (58, 59). Compared with chemically synthesized TiO₂, green-mediated NPs showed excellent photocatalytic potential (41).

Our mortality and hatching studies proved that all concentrations (125, 250, 375 and 500 ppm) caused hazardous effects on J2s and egg masses of M. incognita. TiO₂ nanoparticles’ harmful impact on nematodes such as M. incognita may result from an assortment of mechanisms, viz., disruption of several cellular processes, including oxidative stress response, production of ATP & membrane permeability (60, 61). Our study's findings imply that RKNs, which reduce agricultural yields, might be combated directly or indirectly using green synthetic TiO₂ NPs. According to Tauseef et al. (62), copper oxide NPs enhance cowpea growth and physiological indices while lowering galls and other M. incognita-induced pathological attributes. As a naturally occurring lipid-soluble pigment, lutein is a carotenoid that has been shown to have antibacterial and anticancer properties, nanoencapsulated by nanomaterials with various morphologies to enhance its therapeutic potential by boosting bioavailability (63). Primary and secondary metabolites can use intracellular or extracellular pathways to reduce metal ions and stabilize resultant NPs (64). By supporting green technologies, some scientific disciplines, such as chemistry and nanotechnology, contribute significantly to environmental sustainability (65). Key sustainability issues, including climate change, water purification, sustainable agriculture, food production, natural resources, and renewable energy, can all be helped by green nanotechnology (65). According to the findings above, TiO₂ NPs have outstanding nematostatic capability and nematicidal effectiveness, which may be helpful in the long-term management of RKNs. Regarding the mode of action of NPs, it was shown that NPs work by causing cellular processes to break down, which allows them to penetrate nematode egg cell walls (66). Nguyen et al. (67) reported that metal-based nanoparticles exhibit antimicrobial mechanisms such as the release of harmful ions, alterations in protons that cause damage, and cell walls. In their in vitro investigation, Pragathiswaran et al. (68, 69) showed that TiO₂@ZnO-Au nanocomposites showed noteworthy antibacterial activity against Candida albicans, Staphylococcus aureus, and Escherichia coli. According to the findings, bio-synthesized TiO₂ NPs at proper concentration effectively killed the J2s and decreased root-knot infestation in carrot plants. Therefore, using TiO₂ NPs may be a useful long-term strategy for managing M. incognita for sustainable agricultural production.

5. Conclusions

In the current work, stable TiO₂ NPs were synthesized using S. cumini aqueous leaf extract, evaluated, and their effectiveness against M. incognita. TiO₂ NPs have nematicidal activity, which could make them a safer substitute for chemical nematicides that carry a high risk. Based on observed data, the application of TiO₂ NPs for combating RKN, M. incognita, could be considered a promising additive, a very safe material regarding genotoxicity, compared with the chemical nematicides. FT-IR results confirmed stabilized TiO₂ NPs, and the appearance of some functional groups. As demonstrated by TEM images, TiO₂ NPs were round and poly-disperse with 12−30 nm widths. Our findings indicate that biosynthesis of TiO₂ NPs via simple, effective, and easy pathways would be environmentally friendly and helpful to manage nematode infection in agriculture crops sustainably due to their nematicidal activity. The findings of our study suggested that the conversion of plant extracts to nanoparticles greatly enhanced its nematicidal activity against the significant plant pathogenic nematodes. Using such a technique in RKNS control could gain a new trend, a safe and effective nematode management program. So, more research is needed to develop bio-fabricated green nanoparticles that kill nematodes while having biodegradation modes of action before they can be recommended for field application and IPM programs.

Author contributions

Amir Khan, Azam Raza and Absar Ahmad: Conceptualization conceived and designed experiments and drafted manuscript. Amir Khan and Azam Raza: Responsible for software analysis. Amir Khan, Azam Raza, Faheem Ahmad and Absar Ahmad: Responsible for visualization. Amir Khan, Azam Raza, Abeer Hashem, Graciela Dolores Avila-Quezada, Elsayed Fathi Abd_Allah, Faheem Ahmad and Absar Ahmad: Critically revised the manuscript. Elsayed Fathi Abd_Allah: Funding. All authors read and agreed to publish version of manuscript.

Acknowledgements

The authors would like to extend their sincere appreciation to the Researchers Supporting Project Number (RSP2024R356), King Saud University, Riyadh, Saudi Arabia and Department of Biotechnology, Government of India, (COE, BT/PR1-3584/COE/34/29/2015), Interdisciplinary Nanotechnology Centre, Aligarh Muslim University.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The authors would like to extend their sincere appreciation to the Researchers Supporting Project Number (RSP2024R356), King Saud University, Riyadh, Saudi Arabia.