Enhancement of the Antifungal Properties of Zataria Multiora Essential Oil Thorough Combination With Zno Nanomaterial

Fusarium is one of the most important and destructive phytopathogenic fungi on a wide range of host plants. In the present study, to achieve a suitable alternative for high-risk synthetic chemicals, the antifungal effects of ZnO and ZnO-EO (Zataria multiora Boiss essential oil loaded on ZnO) materials were investigated against six isolates of Fusarium. The chemical composition of Z. multiora essential oil (EO) was explored by GC-MS, in which thymol and carvacrol were the main components. The physio-chemical properties of fabricated materials were studied by SEM, BET, FT-IR, TGA, EDX, XRD, and DLS analyses. The mycelial growth inhibitory (MGI) of ZnO and ZnO-EO materials were tested against Fusarium oxysporum f.sp. lycopersici, F. oxysporum f.sp. lentis, F. graminearum, F. graminearum, F. verticillioides, and F. brasilicum in the laboratory conditions. The results showed that ZnO-EO nanocomposite had a fungistatic effect against all tested fungi except F. oxysporum f.sp. lentis and the fungicidal activity against F. graminearum at a concentration of 1000 ppm. The MGI of ZnO-EO nanocomposite was increased by 42.70% compared to the pure ZnO and by 66.33% compared to Z. multiora EO. The MGI of pure ZnO compared to Z. multiora EO was also increased by 23.63%. According ndings, the ZnO-EO nanocomposite can be considered as a bio-rational ecient alternative to conventional chemical fungicides.


Introduction
One of the most destructive agents in agriculture is phytopathogenic fungi. A signi cant portion of plant products is annually lost due to plant diseases, while more than 800 million people suffer from food To the best of our knowledge, there is not any research about the inhibitory of Zataria multi ora essential oil integrated with ZnO nanomaterials on the mycelial growth of pathogenic fungi. Herein, we combined ZnO with Z. multi ora essential oil using one-pot ultrasonic-assisted method, and the antifungal performance of ZnO and ZnO-EO (Zataria multi ora Boiss essential oil loaded on ZnO) materials were investigated against six isolates of Fusarium and the results were discussed.

Preparation and analysis of essential oil
Zataria multi ora essential oils were provided from Barij Essential oil Company (Kashan, Iran). The essential oil was analyzed by a Gas Chromatography (HP-7890B) coupled with a Mass Spectrometer (Agilent-MSD5975C). The length, diameter, and lm thickness of HP-5MS column were 30 m, 025 mm, and 0.25 µm, respectively. Helium (99.99%) was used as carrier gas with a ow rate of 1 mL per minute. Identi cation of each component was made by comparing its retention time and mass spectra fragmentation with those stored in the computer libraries; Wiley 7n 0.1 (Wiley, NY) and NIST (Standard Reference Data, Gaithersburg).

Antifungal effect of multi ora essential oil in laboratory conditions
The antifungal effect of EO on six pathogenic strains of Fusarium was performed by mixing different concentrations of EO with PDA medium. Brie y, the emulsion of the essential oil was prepared with 0.05% (v/v) Tween 80 and blended with sterile PDA at 40-45 ºC to obtain a nal concentration of 600 µl/L of Z. multi ora EO. Then, this medium was divided into 9 cm Petri dishes and allowed to coagulate. A 5 mm mycelial disc from young cultures of target fungi was placed in treated plates. In control plates, only Tween 80 was used. Inoculated plates were sealed with para lm in order to prevent exhaust of the essential oil, and placed in an incubator at 25 ºC. The experiment was performed in three repeats. After 48 hours, the vegetative growth of fungal colonies was measured daily until the surface of the culture medium of control petri dishes was completely occupied by the fungus. The inhibitory percentage of different concentrations of essential oil was determined using Abbott's formula: IP = (C-T / C) × 100, in which IP = inhibitory percentage, C = the mean diameter of the fungus colony in the control treatment, and T = the mean diameter of the fungus colony in the mentioned treatment (7Davari and Ezazi, 2017).

Synthesis of ZnO
For this purpose, 3.65 g Zn (NO 3 ) 2 .6H 2 O (Loba chemie, India) was added to 100 mL of water. The solution was placed on a mixer (Heidolph model, Germany) at 450 rpm for 30 minutes at 25 °C and its pH was adjusted to 10 using NaOH. The prepared solution was then placed in an ultrasonic device (Bandelin model HD 3100) with water circulation within 2 hours. Thereafter, the prepared ZnO was separated by centrifuge and dried in an oven within 24 hours (24Pirhashemi and Habibi-Yangjeh 2017).

Combination of multi ora essential oil with ZnO
To synthesize ZnO-EO (ZnO/essential oil of Z. multi ora), 3 g Zn (NO 3 ).6H 2 O was added to 90 mL water.
Then, 1 mL of Z. multi ora EO was dissolved in 9 mL of ethanol and added to the above solution. The pH of the solution was adjusted to 10 and placed for half an hour on a mixer at 450 rpm. Then, it was ultrasonicated for 2 hours and separated and dried similar to the ZnO sample.
2.6. Characterization of the materials XRD patterns were performed using Cu-K radiation. Surface morphology was assessed using SEM (LEO 1430VP model, Germany) with 15 kV accelerator voltage. A FT-IR was used to examine the vibrational spectra of the samples. For this purpose, a mixture of the material with KBr powder was prepared in 1 to 10 ratio and the position of their peaks was examined in the frequency range from 400 to 4000 cm -1 . Material purity and analysis of elements in the synthesized products were investigated by EDX (Rontec GmbH, Germany). Thermo-gravimetric analysis was used to investigate the differences in thermal stability of samples in the temperature range from 31 to 700 °C with TGA/DTA thermal analysis device under air atmosphere. The BET instrument (BELSORP mini model, Japan) was used to investigate the degree of porosity, and the DLS (Dynamic light scattering; HORIBA model, Japan) was used to estimate the size of the particles.

Antifungal activity of materials
The desired concentrations of ZnO and ZnO-EO materials were prepared and added to PDA in Petri dishes. A 5 mm mycelial disc from young cultures of target fungi was placed in treated plates. At certain time intervals, the mycelial diameters of pathogenic fungi were measured. In this study, the antifungal effects of ZnO and ZnO-EO materials on Fusarium isolates were measured by mixing method with PDA (micro-dilution) at concentrations of 75, 150, 300, 600, and 1000 (along with 2000 ppm for ZnO) in a completely randomized design (CRD) in three replications. Mycelial inhibition percentages for each pathogenic fungi were compared with Tukey's text at p = 0.05.

Results And Discussion
The analysis of Z. multi ora EO represented that thymol (36.18%), carvacrol (32.53%), p-cymene (7.52%), and -Terpinene (5.28%) were the main compounds ( Table 1). The results obtained from comparing the mean inhibitory percentages at 2000 ppm showed that ZnO had the greatest mycelial growth inhibitory on the F. graminearum UM89 (81.43%), while the lowest growth inhibitory was found on F. brasilicum (58.19%) (Fig. 1a). Based on the values of IP 50 , no signi cant difference was observed between the studied fungi, and the pure ZnO had the highest and lowest antifungal activity on the growth of F. graminearum UM89 and F. oxysporum f.sp. lycopercisi, respectively. At a concentration of 1000 ppm, 100% growth inhibitory was observed on all pathogenic fungi except F. oxysporum f.sp. lentis (Fig. 1b  and 3b). In order to determine whether ZnO-EO had fungistatic or fungicidal effects, mycelial discs, which did not grow at 1000 ppm were placed on a PDA without inhibition material and examined after one week. The results showed that it has fungistatic property on the mycelial growth of all fungi except F. graminearum UM89. In fact, the ZnO-EO nanocomposite had a fungicidal effect on F. graminearum UM89. According to probit analysis, there was no signi cant difference between the tested fungi in terms of the effectiveness of ZnO-EO nanocomposite. Also, this composite had the highest and lowest antifungal activity on the mycelial growth of F. graminearum UM89 and F. oxysporum f.sp. lycopercisi, respectively. The comparison of mean inhibitory percentages in mycelial growth of pathogenic fungi is shown in Fig. 2a. As can be observed, the ZnO-EO nanocomposite has the greatest impact on the growth of the studied fungi. Pure Z. multi ora EO at 600 μl/L was compared to the ZnO and ZnO-EO samples on the growth of pathogenic fungi. So that by using a very small amount of essential oil, we are able to signi cantly increase the effect of ZnO NP. In the ZnO-EO, the mycelial growth inhibitory was increased to 42.70% compared to the ZnO. Inhibitory in the ZnO-EO, compared to pure Z. multi ora EO, has also been increased to 66.33%. Mycelial growth inhibitory in ZnO was 23.26% higher than pure Z. multi ora EO ( Fig.  2b and 3a).
The use of essential oils in the control of plant diseases has been proposed as an effective and safe method in recent years (25Raveau et al. 2020). However, the application of EOs can be associated with some limitations such as high cost, evaporation in high temperature, instability in high pressure, and decomposition with oxygen (26Joel et al. 2019). Therefore, in the present study, enhancing the e ciency of Z. multi ora EO was investigated by combination with ZnO (27Patra and Goswami 2012). The main constituents of Z. multi ora EO were thymol and carvacrol. In the previous studies, thymol and carvacrol were also reported as the main constituents of this essential oil  As seen in the obtained XRD patterns, the ZnO-EO sample shows the same XRD pattern of the ZnO sample, con rming that the addition of Zataria multi ora Boiss essential oil on the surface of ZnO did not in uence on the crystal structure of ZnO. Based on the Debye-Scherrer equation, the crystallite sizes for the ZnO and ZnO-EO were estimated to be 22 and 17 nm. The particle size reduction in the binary sample could be due to the capping feature of the metabolites presented in the Zataria multi ora Boiss essential oil, which prevented the crystalline growth of the ZnO. The absence of additional peaks indicates the high purity of the as-obtained samples.
EDS analysis was applied to investigate the elemental composition of the samples and the results are displayed in Fig. 5a. The elements of Zn and O were identi ed on the surface of ZnO sample. Additionally, the elements of Zn, C, and O were represented in the EDS spectrum of the ZnO-EO sample. The results of EDS technique demonstrated that the Zataria multi ora Boiss essential oil have successfully adhered to the surface of ZnO. For detecting the microstructure of the ZnO and ZnO-EO samples, the SEM technique was applied. As observed in the SEM images (Fig. 5b), the ZnO sample illustrates the spindle-shaped structure. After integrating Zataria multi ora Boiss essential oil with ZnO, the morphology of ZnO was markedly changed. So that the binary sample almost presented the spherical-like structure (Fig. 5c).
FT-IR spectroscopy was conducted to explore the chemical bonds of ZnO and ZnO-EO samples. From The porous nature and surface area of the samples were studied by BET analysis. Table 2 and Fig. 7 present the information on porosity and N 2 sorption curves of the ZnO and ZnO-EO samples. The isotherms of the samples are classed as isotherm of type II. The surface areas of ZnO is 11.5 m 2 g -1 with pore volumes and mean pore diameters of 0.161 cm 3 g -1 and 56.1 nm, which are higher than those of ZnO-EO sample (3.50 m 2 g -1 with pore volumes and mean pore diameters of 0.1515 cm 3 g -1 and 7.6 nm). The reduction in S BET for ZnO-EO sample can be related to the occupation of the ZnO surface by the deposited Zataria multi ora Boiss essential oil molecules.
Dynamic light scattering (DLS) is an effective analysis to examine the particle size distribution of the samples. The DLS results of the ZnO and ZnO-EO samples are demonstrated in Fig. 8. The average particle sizes reduced from 118 nm for ZnO to 93 nm for ZnO-EO. The results of DLS analysis of synthesized ZnO sample displayed that the mean size of ZnO is larger than the ZnO-EO. The smallest size is related to ZnO-EO, which has the greatest effect on growth inhibitory on mycelial growth of fungi. Therefore, particles size can affect the inhibitory value of mycelial growth of fungi.
TG analysis could supply information about the thermal stability of the ZnO and ZnO-EO samples (Fig.  9). The ZnO represents weight loss of 2.5% after heating to 600 °C, due to removal of adsorbed water. In the TGA diagram of ZnO-EO, the weight loss is observed in two parts. The rst one is very small and up to about 100 °C. The main reason for this weight loss is the evaporation of water molecules, as seen in the ZnO. The second weight loss is about 17.5%, which occurred at higher temperatures and is assigned to the destruction of organic molecules linked to the surface of ZnO. Therefore, it can be concluded that the active compounds and metabolites of Z. multi ora EO combined with ZnO-EO is nearly 15%.

Conclusion
The present study was aimed to combine the essential oils from Z. multi ora EO to the surface of ZnO and the antifungal activities were investigated against six isolates of Fusarium. For this aim, the ZnO-EO nanocomposite was prepared using a facile ultrasonic-assisted method. The TGA data showed that nearly 15% of the essential oils was combined with the ZnO in the nanocomposite. The XRD, SEM, and DLS analyses displayed that the particle sizes of ZnO-EO nanocomposite is smaller than ZnO, which related to the capping feature of the metabolites presented in the essential oil. The results showed that the antifungal effect of the ZnO after integration with the essential oils from Z. multi ora was severely enhanced. Overall, the results lead to consideration of additional research of the potential antifungal activity of the ZnO-EO nanocomposite and ZnO-NPs on phytopathogenic fungi such as Fusarium species, an e cient, economical and viable antifungal alternative to be used in plant disease management specially under eld conditions.