Antifungal activity of TiO2/AgBr nanocomposites on some phytopathogenic fungi

Abstract TiO2/AgBr composites were synthesized by a simple ultrasonic strategy. Various instruments such as SEM, EDX, XRD, and FT‐IR were exploited to investigate their characteristics. Antifungal activities of the as‐obtained samples were assessed through the inactivation of Fusarium graminearum in the spore suspension method and mycelial growth inhibition of F. graminearum, Botrytis cinerea, and Sclerotinia sclerotiorum in the microdilution method. The results represented that the TiO2/AgBr samples possess higher antifungal activities on F. graminearum spores than the pure TiO2. The sample with 20 wt% silver bromide represented the highest inhibitory effect on the growth of F. graminearum so that all fungal spores were degraded in the initial times of the treatment process. The inactivation of fungal spores after 60 min was 35.2%, 97.8%, 98.9%, and 98.7%, in respect, for 5, 10, 20, and 30 weight percent of AgBr in the binary nanocomposites, while the inhibition rate was 13.4% for the pure TiO2. With increasing ultrasound irradiation time for more than 30 min, the inactivation rate constant decreased. It was also found that the antifungal activity of the nanocomposites without calcination was higher than those of the calcined materials. Considering the antifungal potential against phytopathogenic fungi and advantages such as simple synthesis and eco‐friendly nature, it seems that TiO2/AgBr nanocomposites can be used instead of synthetic chemicals after additional field investigations and mass production.


| INTRODUC TI ON
The diminishment of agricultural products caused by different phytopathogenic agents remains a severe challenge in recent years.
Among the pathogens, fungi have the most significant role in crop production losses (Bebber & Gurr, 2015). Fusarium graminearum, as a causal agent of Fusarium head blight, has become a major limiting factor for sustainable wheat production around the world (Davari et al., 2013). Sclerotinia sclerotiorum and Botrytis cinerea are common necrotrophic fungal plant pathogens that can attack a wide range of plant species during crop cultivation and harvested productions (Amselem et al., 2011). Although synthetic fungicides are widely utilized for fungal plant diseases management, some disadvantages such as harmful effects on the environment and human health, high production costs, and appearance of resistant strains have attracted researchers to design more efficient strategies (Bartlett et al., 2002;Joo, 2005;Le & Bach, 2019;Sharma et al., 2009).
Nowadays, the use of nanomaterials as efficient and eco-friendly agents in the control of plant pathogens has been successfully studied (Balaure et al., 2017;Hayles et al., 2017;Khan & Rizvi, 2014;Servin et al., 2015;Singh et al., 2015;Sinha et al., 2017;Worrall et al., 2018). Among the nanomaterials, titanium dioxide (TiO 2 ) has been gained great attention compared to CuO, ZnO, etc. to reduce the fungal diseases, because of its low cost, eco-friendly nature, unique physicochemical properties, and high stability (Darbari et al., 2011;Huang et al., 2013). Despite the appealing features, TiO 2 revealed low antifungal efficiency in practical applications (Beltrán-Partida et al., 2017). To overcome this problem and boost the antifungal activity, the integration of TiO 2 with suitable nanomaterials has been suggested. Silver-based semiconductors are an excellent choice to increase the antifungal activity of TiO 2 due to their high antifungal properties (Liu et al., 2017). Antimicrobial activity of TiO 2 or in binary compositions carried out in medicine or food science. The TiO 2 /ZnO supported in 4A zeolite showed superior activity as antimicrobial agent (Azizi-Lalabadi et al., 2019). Ansari et al. (2020) showed that TiO 2 nanofibers were more active against Gram-negative Pseudomonas aeruginosa cells than Gram-positive Staphylococcus aureus. Also, the antibacterial and antibiofilm results suggested that TiO 2 can be utilized for coating different inanimate objects, in food packaging and in wastewater treatment, and purification for preventing bacterial growth. In other research, the Fedoped TiO 2 /bamboo exhibited a much higher inhibition ability to mold fungi compared with original bamboo and TiO 2 /bamboo, under the natural environment . Based on Kim et al. (2019) results, thorn-like TiO 2 nanoarrays physically punctured the cell membrane of bacteria.
As far as we know, there is no report about the antifungal activity of TiO 2 /AgBr nanocomposites. Given the above discussions, we prepared binary TiO 2 /AgBr nanocomposites by a simple ultrasonicaided method. Then, these nanocomposites were studied by different instruments such as FT-IR, SEM, EDX, and XRD. The antifungal activity of TiO 2 /AgBr nanocomposites was evaluated against F. graminearum, B. cinerea, and S. sclerotiorum. The results exhibited that the TiO 2 /AgBr (20%) sample acts as a highly effective nanocomposite for antifungal property compared to the other samples.

| Instruments
The phase structure of the materials was studied by Philips Xpert XRD, applying CuKα radiation. The morphological features and chemical composition of the samples were characterized by LEO 1430 VP SEM/EDX instrument. The FT-IR spectra were provided by a PerkinElmer Spectrum RX I instrument. The ultrasonic treatment was applied with a Bandelin ultrasound generator HD 3100.

| Preparation of the nanocomposites
All chemicals had high purity, and deionized water was used during this study. The TiO 2 /AgBr nanocomposites, with the AgBr content of 20 wt%, were synthesized as follows: 0.4 g TiO 2 (P25) was first sonicated in water (150 ml) with ultrasonication for 10 min.
Then, 0.09 g AgNO 3 (Loba Chemie) was added into the solution with stirring. Next, afterward, a 20 ml aqueous solution containing 0.054 g NaBr (Loba Chemie) was drop-wisely appended into the solution and followed for 1 hr vigorous stirring and the suspension was sonicated for 60 min. Finally, the produced precipitate was filtered and washed two times with water, and then air-dried at 60°C (Figure 1).

| Effect of nanocomposites on the fungi spores in the bioreactor
The growth of F. graminearum spores was realized on the SNA at 25°C for 7 days. After washing the plates with sterile water and separating spores by mechanical agitation, the spore suspension was set on 1 × 10 4 spores/ml via a hemacytometer. The antifungal effect of the nanocomposite on the F. graminearum spores was carried out according to the related method (Sichel et al., 2007). A wooden enclosure and a two-walled Pyrex reactor were attached to a thermostat and the solution temperature was kept at 25°C. To avoid the adverse light effects, the experiments were performed in the dark condition. The fungal spore suspension and 0.002 g of the nanocomposite were transferred to one reactor, and the fungal spore suspension alone as control was added to the other reactor. At different time intervals, the sampling from each reactor was performed using a micro sampler. Samples were inoculated into pellets containing MEA (Malt Extract Agar) medium and incubated in the dark medium for 15 hr.
Germinated spores were counted and compared with the control using a stereo microscope.

| Effects of nanocomposite on fungi mycelial growth
The nanocomposite was mixed with PDA medium according to the method known as the microdilution (Kaur et al., 2012). A 5-mm mycelial disk from fresh cultures of target fungi was placed in the middle of with PDA medium containing 100, 150, 200, 300, and 400 ppm of the nanocomposite. The inoculated and the control (PDA medium without the nanocomposite) Petri dishes were incubated at 25°C for 16 hr light and 8 hr dark. The growth of the mycelial colony in each fungus was measured daily until filled by the fungi grown in the control plates. It was elongated 4, 5, and 7 days for S. sclerotiorum, B. cinerea, and F. graminearum, respectively. Three replicates were considered for each treatment, and mycelial growth inhibition percentages of different concentrations were calculated using the following formula: where R and r are the diameter of the fungus colony in control and treated plates, respectively. Analysis of variance and probit analysis of inhibition percentage were performed using SPSS software version 24.
Also, the observed rate constant (k obs ) of the inactivation processes over the materials were obtained by the slope of ln (N t /N o ) = −kt, in which N o and N t are the initial and at time of t fungus population (in cfu/ml), respectively.

| RE SULTS
To investigate the phase structure and purity of the materials, the XRD tests were used. The XRD patterns of the as-fabricated samples are shown in Figure 2. For the TiO 2 , the diffraction peaks belong to the tetragonal phase (JCPDS no. 04-0477) (Feizpoor et al., 2019). For the TiO 2 /AgBr nanocomposites, in addition to the peaks belonged to TiO 2 , characteristic peaks of AgBr were also observed (JCPDS no. 79-0149) (Pirhashemi & Habibi-Yangjeh, 2016). Because of the small amount of AgBr in the TiO 2 /AgBr (5%) sample, no characteristic peaks for AgBr were detected in the TiO 2 /AgBr (5%) sample. These patterns show the successful combining of TiO 2 and AgBr to build the TiO 2 /AgBr nanocomposites.
To evaluate the presence of expected elements in the samples, EDX analyses were employed. The corresponding EDX spectra of  (Fang et al., 2017;Zhu et al., 2015). Finally, like to the other articles about silver halides in the FT-IR spectra, the peaks for the Ag-Br bond in 400-4000 cm −1 are not observed (Pirhashemi & Habibi-Yangjeh, 2016).  AgBr nanocomposite exhibited the best antifungal performance.
The preparation time of samples could primarily affect their crystallinity and size of the particles. Thus, the 20% TiO 2 /AgBr sample was fabricated by ultrasonic irradiations for 15, 30, 60, 90, and 120 min. As can be seen in Figure 7b, the antifungal activity reduces with increased preparation time, and the sample fabricated by ultrasonic irradiation for 30 min possesses the highest antifungal activity.
The influence of calcination temperature on the antifungal activity of the 20% TiO 2 /AgBr sample was studied, and the results are shown in Figure 7c be seen that the inactivation rate constant of the noncalcined nanocomposite is much higher than that of the calcined samples.
The TiO 2 /AgBr nanocomposites also has considerable inhibition on the mycelial growth of phytopathogenic fungi (Figure 8)

| D ISCUSS I ON
Briefly, binary TiO 2 /AgBr nanocomposites were synthesized using a facile ultrasonic irradiation route, and they were characterized by with 20 wt% of silver bromide represented the highest inhibitory on the mycelial growth of F. graminearum.
Furthermore, the inactivation rate decreased with increasing ultrasound irradiation time. Reducing the antifungal properties of the nanocomposite by increasing the preparation time can be ascribed to the aggregation of nanoparticles (Hoseinzadeh et al., 2016). It was also found that the antifungal activity of the nanocomposite without calcination was higher than those of the calcined samples.
It is in accordance with the report of Hoseinzadeh et al. (2016) which attributed to the reduction of the nanocomposite surface area at high temperatures due to particle agglomeration and the size growth (Hoseinzadeh et al., 2016;Singh et al., 2014).  Note: nc is a noun calculated. a Because the significant level is greater than 0.05, no heterogeneity factor is used in the calculation of the fiducial limit. b Because the significant level is less than 0.05, a heterogeneity factor is used in the calculation of the fiducial limit.
TA B L E 1 Probit analysis of TiO 2 /AgBr nanocomposite on the mycelial growth inhibition of Fusarium graminearum, Botrytis cinerea, and Sclerotinia sclerotiorum at different exposure times of antifungal mechanism of TiO2/AgBr nanocomposites is suggested for further studies.

ACK N OWLED G M ENT
The authors express special thanks to the University of Mohaghegh Ardabili for their support in this research.

CO N FLI C T O F I NTE R E S T
The authors do not have any conflict of interest to declare. Writing-original draft (equal); Writing-review & editing (equal).

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.