Green Pesticide High Activity Based on TiO₂ Nanosuspension Incorporated Silver Microspheres Against Phytophthora palmivora

Abstract

Cocoa pod production has experienced a significant decline due to attacks by the Phytophthora palmivora (P. palmivora) fungus, which is the main cause of cocoa pod rot. To overcome this problem, Titanium dioxide (TiO₂) was chosen because of its potential as an antifungal, and its activity can be increased by adding silver nanoparticles (AgNPs). This research aims to determine the antifungal properties of TiO₂–Ag nanosuspension on the growth of P. palmivora under exposure to UV, Visible and without irradiation. The sol–gel process was used to synthesize TiO₂, and ultrasonics was used to integrate silver nanoparticles into TiO₂. Characterization of UV–Vis diffuse reflectance spectroscopy (UV-DRS) shows a change in the energy gap from 3.24 to 2.82 eV. The Fourier confirmed the crystal structure of the TiO₂-Ag anatase transform infrared spectroscopy (FTIR) spectrum, which showed the stretching vibration peak of the Ti–O and Ag–O bonds (463.88 cm⁻¹). Particle size analysis (PSA) characterization revealed that the nanoscale of TiO₂–Ag was 92.4 nm. The disc diffusion method was used to test the antifungal inhibitory of 0.1%, 0.3%, and 0.5% TiO₂–Ag against P. palmivora. The antifungal activity of the TiO₂–Ag showed strong resistance under exposure to visible light, and the optimum concentration of TiO₂–Ag was 0.5%.

Introduction

Cocoa (Theobroma cacao) is a significant tropical plant in the plantation industry. However, cocoa plant productivity is often disrupted by pathogen attacks, one of which is P. palmivora, which causes cocoa fruit rot. The disease caused by P. palmivora, often known as cocoa fruit rot, can result in significant losses in cocoa production, reduce fruit quality, and hinder plant growth. Therefore, controlling P. palmivora disease becomes crucial [1, 2]. One emerging approach in plant disease control is the utilization of TiO₂ nanoparticles due to their advantages.

Previous research has demonstrated that TiO₂ has potential as an antimicrobial material [3–7]. TiO₂ has antimicrobial effects through photocatalysis, accelerating redox reactions and generating reactive oxygen species (ROS) when exposed to UV and sunlight [3, 8]. The formation of ROS leads to the inactivation of enzymes and proteins on the bacterial cell membrane, as well as an increase in the permeability of reactive species, allowing nanoparticles to easily enter the cell and disrupt the balance of transportation and energy respiration, ultimately resulting in the death of microorganisms [9, 10]. One of the most extensively developed forms of TiO₂ nanoparticles is the TiO₂ nanoparticle suspension.

Suspension is a system in which solid particles are evenly distributed in a dispersing liquid medium. Controlling the dispersion and aggregation of nanoparticles is crucial for optimizing the advantages of nanoscale TiO₂ particles [11]. The TiO₂ suspension can be applied by spraying it on the surface of cocoa plants to prevent or treat P. Palmivora infection [10, 12, 13].

TiO₂ can be modified by doping metal and non-metal compounds for broader use as an antifungal. This modification has significantly improved antifungal activity [14, 15]. The choice of Ag Nanoparticles (AgNPs) as a modification for TiO₂ in this study is due to their large surface area, facilitating more intense interactions with microbes making them more effective as antifungal agents [13, 16]. Based on several relevant studies, AgNPs have demonstrated excellent performance against some fungi, such as Fusarium oxysporum, P. parasitica, P. infestans, P. palmivora, P. cinnamomi, P. tropicalis, P. capsici, and P. katsurae [17–20].

Experimental Method

Synthesis of TiO₂ Nanoparticles

This synthesis is based on previous research [21–25]. It involves mixing 15 mL of ethanol, 2 mL of distilled water, and 1 mL of 0.5 M acetic acid and then adding this mixture to a reflux flask containing 6 mL of titanium tetraisopropoxide (TTIP) as the TiO₂ precursor, 0.5 mL of acetylacetone, and 15 mL of ethanol. The solution is refluxed for 3 h at 100 °C to form a TiO₂ sol, and then 4 mL of the obtained TiO₂ sol is dissolved in 96 mL of distilled water to obtain a 4% TiO₂ Sol suspension.

Synthesis of TiO₂–AgNPs

This synthesis refers to the research [21–24, 26] by mixing 15 mL of ethanol, 2 mL of distilled water, and 1 mL of 0.5 M acetic acid, then introducing it into a reflux flask containing 6 mL of titanium tetra-isopropoxide (TTIP) as a TiO₂ precursor, 0.5 mL of acetylacetone, and 15 mL of ethanol. Next, reflux the solution for 3 h at a temperature of 100 °C to form TiO₂ sol and dissolve 4 mL of the obtained TiO₂ sol in 96 mL of distilled water to get a 4% TiO₂ suspension.

The preparation of Ag dopants follows previous research [21–24]. Before doping, silver nitrate (AgNO₃) is dissolved in 100 mL of distilled water with a 1% concentration, then heated at 60 °C for 2 h and sonicated at 40 kHz for 2 h. The silver nitrate solution is then re-dissolved with variations of 0.1, 0.3, and 0.5% concentrations. The resulting AgNO₃ solution is a silver (Ag) or doping source. The 4% TiO₂ sol–gel is prepared, silver is added, followed by a simple hydrothermal treatment with a magnetic stirrer for one hour.

Characterization

The energy gap of the TiO₂–Ag nanosuspension is characterized using Ultraviolet–Visible Diffuse Reflectance Spectroscopy (UV–Vis DRS) within a wavelength range of 200–800 nm. The particle size of the TiO₂–Ag nanosuspension is described using Particle Size Analysis (PSA). Fourier-transform infrared (FT–IR) spectra, measured using a Shimadzu Varian 4300 spectrophotometer, are used to identify chemical bonds and functional groups.

Antifungal Activity Test of TiO₂–Ag

The initial evaluation of the antifungal activity of TiO₂–Ag suspension against P. palmivora was conducted using the disc diffusion method. The experiment was conducted by marking a Petri dish and dividing it into three sections labeled 0.1, 0.3, and 0.5%. Next, 15 mL of Potato Dextrose Agar (PDA) is poured into each petri dish and left to solidify. The disc paper is placed at the marked point, and the P. palmivora inoculum is evenly spread on the surface of the media. TiO₂–Ag suspensions with 0.1, 0.3, and 0.5% were applied to marked paper discs. The incubation period lasts five days under UV, visible, and radiation. The positive control (+) uses PDA and Dithane M–45, whereas the negative control (−) is performed using distilled water and TiO₂ solution.

Result and Discussion

Synthesis of TiO₂–AgNPs

In this study, a TiO₂–Ag suspension was prepared using TTIP as the TiO₂ precursor and alkoxide source and AgNO₃ as the silver (Ag) source. The process begins by combining TTIP with a solvent such as ethanol. This process is known as sol–gel, in which the sol–gel reaction produces a sol or a suspension of fine particles. Adding ethanol serves as an inhibitor of precursor hydrolysis, promoting the formation of a more stable solution and maintaining the reactivity of alkoxide metals while preventing excessive hydrolysis. Asetil acetate is added to form the mesostructure of TiO₂ anatase and imparts a yellow color to the solution [27–29] (Fig. 1a).

Fig. 1.

a Sol TiO₂, b TiO₂-Ag suspension

TiO₂–Ag suspension is produced using two main processes, namely sonication and hydrothermal treatment. AgNPs were obtained from AgNO₃ in H₂O, which was irradiated using ultrasonic waves at 40 kHz. Sonication using ultrasonic waves increases the amount of AgNPs, changes the average molecular mass viscosity, and reduces the particle size [30]. After that, the prepared AgNPs solution and TiO₂ sol were mixed and subjected to hydrothermal treatment until a homogeneous white TiO₂–Ag suspension was formed (Fig. 1b).

UV–Vis DRS Characterization

Characterization using UV–Vis Diffuse Reflectance Spectroscopy (UV–Vis DRS) was employed to determine the band gap energy, which was calculated using the Kubelka–Munk equation. Figure 2 shows that the band gap energy of TiO₂ is 3.24 eV, with a wavelength of 439 nm, corresponding to the UV region. This indicates that the synthesis of TiO2 from the TTIP precursor using the sol–gel method has been successfully performed. Meanwhile, in TiO₂–Ag, the band gap energy obtained is 2.82 eV, with a 439 nm wavelength corresponding to the visible region. The spectral data obtained is consistent with previous research [23]. A table of band gap energy results for TiO₂–Ag from various literature sources is also provided in Table 1.

Fig. 2.

Plot the visible transmittance and band gap of TiO₂ and TiO₂-Ag

Table 1.

Band gap energies of TiO₂-Ag compounds

Compounds	Method	Band gap (eV)	Ref.
TiO2–Ag	Pechini	2.77	[31]
TiO2–Ag	Sol–gel	2.97	[32]
TiO2–Ag	Sol–gel	2.80	[33]
TiO2–Ag	Sonication	2.85	[23]
TiO2–Ag	Combination of (Sol–gel, Hydrothermal, and Sonication)	2.82	This work

Table 1 compares the band gap energies of Ag–TiO₂ nanosuspensions from various literature sources, falling within the 2.7–2.9 eV range. This indicates a reduction in the band gap of TiO₂–Ag due to the incorporation of silver dopants into the TiO₂ structure. The data obtained is consistent with previous research [23], indicating the successful synthesis of TiO₂–Ag. The determined band gap energy suggests that the TiO₂ material possesses photocatalytic properties, making it suitable for various photocatalytic applications in the visible light range.

PSA Characterization

The Particle Size Analyzer (PSA) is a device used to determine the particle size distribution of TiO₂–Ag nanoparticles. PSA observations (Fig. 3) show that the average particle size of undoped TiO₂ and Ag-doped TiO₂ is approximately 617.5 and 92.4 nm, respectively.

Fig. 3.

Characterization by PSA: (A) TiO₂, (B) TiO₂–Ag

Table 2 presents particle sizes according to [34], indicating that the particle size of TiO₂–Ag compounds is smaller than TiO₂–Sr. This table also reveals that the particle sizes of TiO₂–rGOCu, TiO₂–SiO₂, TiO₂–CaCO₃, and TiO₂–ZnO [35] are larger, suggesting Ag dopants effectively doping titanium dioxide (Table 3).

Table 2.

The particle size range of TiO₂ doped with other elements

Compounds	Method	Particle Size (nm)	Ref.
TiO2–ZnO	Sol–gel	983	[35]
N–TiO2/SiO2	Solvothermal	200	[36]
TiO2–rGOCu	Hydrothermal	226	[37]
TiO2–Sr	Hydrothermal Sol–Gel	200	[34]
TiO2–Ag	Combination of (Sol–Gel, Hydrothermal, and Sonication)	92.4	This work

Table 3.

The reference FTIR of TiO₂-Ag

Functional Groups	Reference Wavenumber (cm−1)	Wavenumber (cm−1)	Reference
Ti–O	800–400	711.73	[38, 39]
O–H	3600–3200	3485.37	[40–42]
C–H	3000–2800	2794.85	[36]
C=O	1900–1650	1797.66	[43]
Ag–O	670–400	453.27	[44]
Ag–TiO2	1630	1633.71	[45]
C–O	1200–1000	1045	[40]
C–H	1395–1120	1382.96	[41]

FTIR Characterization

The spectrum of FTIR TiO₂–Ag nanosuspension is shown in Fig. 4. The peak width at 3485.37 cm⁻¹ in the spectrum can be attributed to the strong intensity of the O–H bond stretching vibration, originating from water molecules, alcohols, and the Ti–OH hydroxyl group [46]. The peak at 1797.71 cm⁻¹ corresponds to the asymmetric stretching vibration of the C=O bond in titanium carboxylate derived from the alkane and carboxylate groups [43].

Fig. 4.

Characterization of FTIR for TiO₂ and TiO₂-Ag

Pure TiO₂ exhibits stretching vibrations of titania Ti in the range of 800–400 cm⁻¹ [38, 39]. The peak at 711.73 cm⁻¹ is associated with the stretching vibration of the Ti–O bond, a characteristic feature of TiO₂ [46]. The standard spectrum of TiO₂, with a peak at 463.88 cm⁻¹, is associated with the vibrational bonding of Ti–O in the structure of TiO₂ (anatase titania), indicating that the organic ligand has been completely removed after heating at 450 °C [47]. In addition, the observed TiO₂–Ag nanosuspension in the 670–400 cm⁻¹ can be attributed to the overlap between Ti–O–Ti and Ag–O bonds [44]. After adding Ag at a concentration of 0.5%, it was seen that the lattice vibration of TiO₂ shifted from 445  to 453.27 cm⁻¹, indicating the formation of TiO₂–Ag bonding [44]. A new peak at 1633.71 cm⁻¹ is also visible, believed to be the peak of Ag metal absorption as per literature [45]. The peak observed at 1045 cm⁻¹ is characteristic of C–O extended vibrations [40].

Antifungal Activity Test Against P. palmivora

The antifungal assay aims to determine the effects of TiO₂–Ag nanosuspension on the growth of P. palmivora. Our study conducted an antifungal test under exposure to UV, visible light, and un-irradiation. The concentration of TiO₂–Ag nanosuspension, namely 0.1, 0.3, and 0.5%, was selected based on literature [23, 48]. The incubation period of the fungus lasts for 5 days, and observations are made when the fungus has spread throughout the entire surface of the medium (Fig. 5). The observation results indicate the presence of inhibitory zones at each concentration [48, 49].

Fig. 5.

Observation of Antifungal Activity against P. palmivora

For quantitative analysis, the diameter of the growth inhibition zone of P. palmivora was recorded for each observation in Table 4. After observing for five days, there was an increase corresponding to the concentration escalation of TiO₂–Ag nanosuspension at 0.1, 0.3, and 0.5%, towards the growth of P. palmivora under exposure to UV, visible, and un-irradiation. The Ag in TiO₂ has antifungal properties [23, 50, 51]. The higher the concentration of TiO₂–Ag, the greater the number of Ag particles within TiO₂. As a result, the resulting inhibitory zone becomes increasingly extensive. Furthermore, even without irradiation, TiO₂–Ag has antifungal effects. This is based on the nano-scale size of TiO₂–Ag (92.4 nm, Fig. 3), which is less than 100 nm. It can easily penetrate fungal membranes and inhibit fungal DNA [50, 51].

Table 4.

Inhibitory strength of TiO₂–Ag

Test Samples	Inhibition zone diameter (mm)			Inhibition Strength
	Un-Irradiation	UV	Vis	Un-Irradiation	UV	Vis
TiO2–Ag 0.1%	1	1.5	6.5	Weak	Weak	Medium
TiO2–Ag 0.3%	3	4	16.5	Weak	Weak	Strong
TiO2–Ag 0.5%	5.5	5	23	Medium	Weak	Very Strong
TiO2 1% (K+)	4.5	4.5	8	Weak	Weak	Medium
Dithane M45 4,88% (K+)	22.5	28	16.0	Very Strong	Very Strong	Strong
Aquades	0	0	0	None	None	None

The increase in the inhibition zone diameter has also been observed in the growth medium of P. palmivora due to radiation under visible light. The inhibitory zone of TiO₂–Ag suspension under visible light radiation is, on average, ~ four times larger than under UV exposure and un-irradiation. The consecutive concentrations of TiO₂–Ag nanosuspension, namely 0.1, 0.3, and 0.5%, without quantitative irradiation of UV and visible light, exhibited inhibitory zones of growth for P. palmivora measuring 1, 1.5, and 6.5 mm; 3, 4, and 16.5 mm; and 5.5, 5, and 23 mm, respectively. The photocatalytic activity of TiO₂–Ag under visible light exposure occurs due to Ag doping [52, 53]. In addition to serving as an antifungal agent, Ag can also influence the photocatalytic activity of TiO₂ materials. The energy gap of TiO₂ often falls inside the UV range, allowing it only to absorb UV radiation. However, doping Ag can influence the energy gap, particularly in the conduction energy band. Ag doping induces a downward shift in the energy gap (Fig. 1.), enabling visible light absorption by TiO₂–Ag. This can enhance the efficiency of electron–hole pair (e⁻/h⁺) formation in TiO₂–Ag [54]. This pair h⁺, can participate in redox reactions with organic substrates on the fungal cell membrane, resulting in a stronger antifungal effect [55].

A comparison graph of measurements of the TiO₂–Ag inhibition zone on the growth of P. palmivora under exposure to UV light, visible light, and no radiation is depicted in Fig. 6. This graph shows that radiation under visible light is more effective than ultraviolet and without radiation. This proves that the green TiO₂–Ag nanosuspension pesticide has been successful. Further research is needed to confirm whether the antifungal effect of TiO₂–Ag nanosuspension is fungistatic or fungicidal.

Fig. 6.

Comparison of TiO₂-Ag Inhibition Measurements under exposure to UV, visible and un-irradiation

Conclusion

This research concludes that the synthesis of TiO₂–Ag nanosuspension has been successfully carried out. This is proven by the change in gap energy shift towards lower energy due to Ag doping on the TiO₂ lattice from 3.24 to 2.82 eV and having a size of 92.4 nm. In addition, FTIR characterization of TiO₂–Ag at the peak of 670 to 400 cm⁻¹ has been confirmed as an overlap between the Ti–O–Ti and Ag–O bonds at the peak of 453.27 cm⁻¹, indicating the formation of a TiO₂–Ag bond. Performance tests of TiO₂–Ag nanosuspension in inhibiting the growth of P. palmivora show that the antifungal activity of TiO₂–Ag can effectively inhibit the growth of P. palmivora under visible light.

Author’s Contribution

ZA: Writing-original draft, Investigation, Formal analysis, Data curation, Conceptualization. AZAI: Investigation. ME: Formal analysis, Investigation. AHW: Formal analysis, validation, methodology. II: Formal analysis, Investigation. MN: Conceptualization, Writing-original draft, Data curation. MM: Methodology, Conceptualization, Data curation.

Data Availability

The datasets generated during and analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Conflict of interest

Zul Arham declares that he has no conflict of interest. Annisa Zalfa Ikhwan declares that she has no conflict of interest. Muhammad Edihar declares that he has no conflict of interest. Abdul Haris Watoni declares that he has no conflict of interest. Irwan Irwan declares that he has no conflict of interest. Muhammad Nurdin declares that he has no conflict of interest. Maulidiyah Maulidiyah declares that she has no conflict of interest.

Consent for Publication

The manuscript in full has not been published anywhere.

Human and Animal Participants

This study does not contain experiments that involve humans or animals other than the authors who performed the work.