Herein, we have displayed an easy way to produce monodisperse spinel nanoparticles (NPs) and the antifungal activity of CoFe2O4, Co0.5Zn0.5Fe2O4 and ZnFe2O4 nanostructures. Firstly, the structural, morphological and magnetic properties of each NP were investigated through x-ray diffraction (XRD), Transmission Electron Microscopy (TEM) and Vibrating Sample Magnetometer (VSM). The XRD data showed diffraction peaks related to the crystalline spinel phase. The TEM micrographs displayed monodisperse NPs with spherical morphology. The average sizes of CoFe2O4, Co0.5Zn0.5Fe2O4 and ZnFe2O4 NPs were 6.87 ± 0.05 nm, 5.18 ± 0.01 nm and 11.52 ± 0.09 nm, respectively. The VSM data indicated that the nanostructures are superparamagnetic at room temperature. Afterward, the antifungal properties of the Co/Zn-based ferrite NPs against Botrytis cinerea were tested. So, the inhibition of mycelial growth by different concentrations (45 – 360 ppm) of NPs was measured. The most effective nanostructure was CoFe2O4, with an EC50 value of 265 ppm. Further, to elucidate how the NPs are affecting B. cinerea, reactive oxygen species (ROS) production was measured. The results indicated that the CoFe2O4 monodisperse NPs could induce a burst of ROS in B. cinerea, promoting cellular damage.
In the last decades, fungicides have extensively been used to combat fungal diseases on various plant species. However, many pathogens have developed resistance to multiple chemical substances with antifungal activity. For instance, the causal agent of grey mould disease, Botrytis cinerea is a well-known strain that caused losses of fruit, vegetables and ornamental plants of commercial importance worldwide. It was found that this pathogenic fungus becomes resistant to benzimidazole and dicarboximide fungicides.1 Therefore, their efficiency in controlling fungal infection was negatively affected.
Consequently, it is fair to assume that there is a great demand for novel strategies and antifungal agents to control resistant fungal infections effectively. In this sense, nanoparticles (NPs) with different compositions have drawn attention due to their unique antimicrobial properties. Many reports have demonstrated the significant effect of inorganic NPs as an antifungal agent.2–4 Some examples include metallic nanostructures, like Ag,5,6 Pd7 and Cu NPs.8,9 However, metal oxide can also be mentioned.10–12 In addition, this kind of inorganic substance improves safety compared to the organic compounds used to control plant diseases.13 In this sense, the literature shows ZnO as one of the best candidates since it is a non-toxic material and allows many technological applications, such as sunscreens, textiles and beauty products.14 Moreover, several publications regarding the action of ZnO as an antifungal agent may be easily found in the literature.15,16
The antifungal activities of other metal oxide nanomaterials have also been investigated. For instance, Hathout et al.17 synthesized CoFe2O4 NPs and its fungicide properties were tested against Aspergillus flavus and Aspergillus ochraceus. According to the results, this kind of NPs is a potential nanomaterial to control plant diseases. Also, substituted CoFe2O428 and ZnFe2O4 NPs18 and were investigated against Candida albicans, a fungal pathogen associated to superficial and systemic infections. According to the results observed in these publications, these nanomaterials may be used as antifungal agents. Furthermore, it is interesting to note that the iron oxide-based structure called spinel is a common feature among the NPs used. This type of nanostructure is also known as ferrites and considers a chemical formula of AB2O4 or AO.B2O3, where A and B denote divalent and trivalent cations, respectively. The distribution of these cations in tetrahedral (T) and octahedral (O) sites of the spinel strongly affect the magnetic properties of the spinel. In this structure, the ferrite can be called normal or inverse depending on the occupation of Fe3+ ions. Given these characteristics, the spinel structure is versatile, allowing the production of several metal oxide compositions, which increases the potential of these kinds of NPs as antifungal agents.
Here, we investigated the fungicidal properties of monodisperse spinel-type NPs against Botrytis cinerea strain. Thus, a straightforward solvothermal methodology synthesized nanostructures with a general composition of CoFe2O4, ZnFe2O4 and Co0.5Zn0.5Fe2O4. XRD data evidenced the crystalline nature, while TEM displayed the sphere-type NPs with an average size of 5.1 – 11.5 nm. Furthermore, the VSM performed at 5, and 300 K indicated that the replacement of Co2+ by Zn2+ decreases the magnetic interaction between the nanostructures. Regarding the inhibition experiments, the results indicate that CoFe2O4 NPs were found to reduce the mycelium growth of B. cinerea by nearly 60% after 48h of incubation at 360 ppm. This demonstrates the potential of these NPs as antifungal agents.
II. MATERIALS AND METHODS
Iron (III) acetylacetonate (Fe(C5H7O2)3, 97%, Aldrich, Fe(acac)3), cobalt (II) acetylacetonate (Co(C5H7O2)2, 97%, Aldrich, Co(acac)2), zinc (II) acetylacetonate hydrate (Zn(C5H7O2)2, Aldrich, Zn(acac)2), toluene (C6H5CH3, 99.9%, J. T. Baker), oleic acid (C18H34O2, 90%, Aldrich, OAc), oleylamine (C18H37N, 70%, Aldrich, OAm), ethanol (C2H6O, 99.9%, J. T. Baker) and hexane (C6H14, 98.7%, BioslabChile). All reagents were used as received.
Potato dextrose agar (PDA), Becton Dickinson, ROS-gloTM H2O2 assay, Promega, Menadione, Sigma-Aldrich. Hexane (C6H14) Merck.
B. Synthesis of monodisperse Co/Zn-based ferrite NPs
The monodisperse CoFe2O4, ZnFe2O4, and Co0.5Zn0.5Fe2O4 nanoparticles were prepared using a stainless-steel reaction kettle with a Teflon-liner (20 ml) according to the procedure described by Jiang and Peng19 First, the total concentration of the metallic precursors was set at 0.75 mmol. Therefore, 0.5 mmol of Fe(acac)3 and 0.25 mmol of the corresponding metal precursor were initially dissolved in 9 mL of toluene. Then, 0.5 mL of OAc and 3.0 mL of OAm, were added under vigorous magnetic stirring. The resultant mixture was further stirred for 20 minutes at room temperature after the addition, and the red solution was conducted into the Teflon-liner. The reactor was then placed into a muffle and warmed up to 200 °C. The solvothermal reaction was carried out for 20 h. Subsequently, the mixture was allowed to cool naturally, and the obtained NPs were washed using ethanol and hexane. Finally, the clean NPs were dispersed in hexane and stored at 4 °C for further characterization and use. To synthesize Co0.5Zn0.5Fe2O4 NPs, the solution containing the metallic precursors was prepared with 0.5 mmol of Fe(acac)3, 0.125 mmol of Co(acac)2 and 0.125 mmol of Zn(acac)3. After this, the same methodology was strictly followed.
X-ray patterns of the synthesized nanoparticles were collected using a Bruker D2 Phaser diffractometer with CoKα radiation (λ = 1.7880 Å), using a 2Ɵ range of 10 - 90° and a scanning rate of 2° min−1. Transmission electron microscopy (TEM) images of monodisperse nanoparticles were obtained using a Hitachi® HT7700 TEM system operating at an accelerating voltage of 120 kV. The magnetic curves were obtained using a vibrating sample magnetometer (Cryogenic VSM 5 T system) at 5 and 300 K.
D. Antifungal activities
To evaluate the antifungal activity, the Botrytis cinerea isolate B05.10 used in this study was maintained and grown under the conditions previously described.20 The Fungitoxicity of the monodisperse nanoparticles and commercial fungicide BC-1000 was evaluated using the radial growth test on potato dextrose agar. CoFe2O4, Co0.5Zn0.5Fe2O4 and ZnFe2O4 were dissolved in hexane to obtain different final concentrations (45, 90, 180, 360 ppm). The NPs were completely soluble in all concentrations, and tested with a similar procedure described by Carrasco et al.20 Significant differences were evaluated with a two-way analysis of variance (Tukey’s test; p < 0.05). The half maximal effective concentration, EC50 values of both fungicides for mycelial growth of B. cinerea isolates were analyzed through the PROBIT Test using the MINITAB V.16 program (Minitab, Inc., State College, PA, USA). Finally, the production of nanoparticles-induced reactive oxygen species (ROS) was evaluated using ROS-GloTM H2O2 assay kit (Promega, Madison, WI, USA).21 Spores (77.5 µL) were inoculated in a 96-well plate at 1 × 105 spores/mL/well. Each well was then incubated in the presence of 2.5 µL CoFe2O4 nanoparticles (250 ppm), 20 µL of buffer substrate H2O2, and cultured for 3 h at 21 °C. After this incubation period, 100 µL of ROS-GloTM reagent was added to each well and incubated for 20 min at room temperature. ROS production was measured using a luminometer (Tecan Infinite M200 PRO). Menadione (Sigma-Aldrich, Germany) (10 ppm) was used as a positive control, following the manufacturer’s instructions. The results were subjected to Student’s t-test to verify the existence of differences at the 0.05 significance level.
III. RESULTS AND DISCUSSION
The diffraction patterns for the monodisperse CoFe2O4, Co0.5Zn0.5Fe2O4 and ZnFe2O4 NPs are displayed in Fig. 1. The black dots indicate the experimental data (Exp), while the red and blue lines represent the calculated data (Cal), as well as the difference (Diff) between the Exp and Cal data, respectively. It was possible to observe peaks at 35, 41, 52, 57, 64, 67 and 74°, which are characteristics of the spinel phase (Inorganic Crystal Structure Database code – ICSD-028511). CoFe2O4 and Co0.5Zn0.5Fe2O4 NPs presented broader diffraction peaks compared to ZnFe2O4. This fact may indicate the presence of an amorphous character and the small average size of the nanoparticle.22 To clarify this and further investigate the structure, the refinement of Rietveld was carried out.23,24 This computational methodology estimates an approximation of the structural model for a real structure.
The structural data from XRD experiments were treated using DBWTools, version 2.3 software25 and listed in Table I. The network parameter values increased as long as Zn2+ ions replaced the Co2+ ions in the cubic cell of the spinel structure. Therefore, the behavior of the lattice constant can be explained based on the difference in the ionic radio of both mentioned cations. The literature26 reports Zn2+ and Co2+ ionic sizes as 0.82 and 0.78 A, respectively. So, the replacement of a larger ion in the ferrite network can cause an increase in the lattice parameter.
|Samples .||Network parameter (A) .||Rwp (%) .||Rexp (%) .||χ2 .|
|Samples .||Network parameter (A) .||Rwp (%) .||Rexp (%) .||χ2 .|
TEM micrographs for CoFe2O4, Co0.5Zn0.5Fe2O4 and ZnFe2O4 NPs are shown in Figs. 1(d)–1(i). It is possible to observe that there are nearly spherical and monodisperse, also using a log-normal function to fit the data, the particle size value was found to be 6.87 ± 0.05 nm, 5.18 ± 0.01 nm and 11.52 ± 0.09 nm, respectively.
Magnetization curves of the NPs at room temperature and 5 K are shown in Figs. 1(j)–1(l). It is possible to observe the superparamagnetic behavior at 300 K. At this temperature, the saturation magnetization Ms for the three samples measured at 4 T was 1.58, 33.52 and 11.48 emu/g, while at 5 K, these values increase for each sample to 1.70, 39.06 and 30.98 emu/g for CoFe2O4, Co0.5Zn0.5Fe2O4 and ZnFe2O4, respectively. Also, it is possible to observe that the NPs lose the superparamagnetic behavior at low temperatures under the blocking temperature. The previous result indicates that at low temperatures, the magnetic interaction is stronger in the samples without the Zn component, which may be explained by the presence of the diamagnetic behavior characteristic of Zn2+. The remaining magnetization in the NPs is also affected by the effect of the temperature that gives a ferromagnetic behavior in the magnetization curves due to the presence of cobalt in the spinel structure.
To evaluate the fungicidal effect of the nanoparticles on B. cinerea isolate, the inhibition of mycelial growth by different concentrations of nanoparticles was measured. In addition, antifungal activity was assessed in radial growth measurements in PDA media (Fig. 2(a)).
It can be observed that only CoFe2O4 NPs were able to affect the growth of Botrytis in a dose-dependent manner. On the other hand, ZnFe2O4 and CoZnFe2O4 nanostructures did not show an evident effect on hyphal development. The percentage of hyphal growth inhibition at different nanoparticle concentrations is in Figs. 2(b)–2(d). The results indicate that CoFe2O4 particles reduce the B. cinerea mycelium growth by almost 60% after two days of incubation at 360 ppm. Meanwhile, ZnFe2O4 and CoZnFe2O4 reported an average of 2 and 3% of inhibition, respectively. Considering this, it is only possible to calculate the EC50 values for the CoFe2O4 corresponding to 265 ppm.
To clarify the action mechanism of the NPs, reactive oxygen species (ROS) production was measured. The effect of nanoparticles on the production of reactive oxygen species was examined by incubating B. cinerea conidia in the presence of CoFe2O4 for 3 h at 21 °C. The production of ROS was evaluated using ROS-Glo™ H2O2 Assay as described by Robles-Kelly et al.21 Figure 2(e) shows the luminescence yields measured in the presence of the nanoparticles (250 ppm) and menadione used as a positive control. The results indicate that the CoFe2O4 NPs can induce a burst of ROS in B. cinerea, promoting cellular damage. Also, most of the mass Botrytis cinerea cell walls are constituted by several sugars and proteins. Although, chitin and uronic acids have been detected. It has been reported that the cell wall of B. cinerea undergoes significant modifications during growth, possibly becoming more extensively covalently cross-linked as a result of the aging of mycelia or in response to decreasing nutrient supply or as a consequence of increasing culture density.27 Considering that the electronegativity and size of Zn are lower than Co should explain the lowest values of EC50 obtained by Zn since the ability to cross-link with the cell wall covalently prevents damage to the fungi.
To conclude, biosynthesized nanoparticles could be used effectively against Botrytis to protect the various crop plants and their products instead of using commercially available synthetic pesticides, which show higher toxicity to humans.
In summary, the solvothermal route produced Co/Zn-based ferrite NPs with high purity since the XRD patterns only displayed diffraction peaks related to the spinel phase. Further, the synthesis resulted in monodisperse NPs with a spherical shape, as can be observed through TEM micrographs. The average size was calculated to be 6.87 ± 0.05 nm, 5.18 ± 0.01 nm and 11.52 ± 0.09 nm for CoFe2O4, Co0.5Zn0.5Fe2O4 and ZnFe2O4 NPs, respectively. The VSM data evidence the absence of hysteresis at room temperature, which indicates the nanostructures are superparamagnetic. Additionally, the antifungal properties were tested against Botrytis cinerea. Interestingly, Co0.5Zn0.5Fe2O4 and ZnFe2O4 did not show significant (no more than 5% inhibition) effect on the growth of B. cinerea in a dose-dependent manner. On the other hand, the CoFe2O4 NPs could inhibit 60% after two days of incubation at 360 ppm. Moreover, the results indicate that the Co-based ferrite produced a burst of ROS in B. cinerea, which evidences cellular damage. Thus, even with the same crystallographic structure, a high amount of Co2+ in the spinel structure seems to enhance the fungicide effect.
This work was supported by the Fondo de Innovación para la Competitividad-Minecon [ICM P10-061-F]; the Basal Funding for Scientific and Technological Centers [project AFB180001]; FONDECYT  and DIUA 206-2021 from the Dirección de Investigación of Universidad Autónoma de Chile.
Conflict of Interest
The authors have no conflicts to disclose.
Rafael M. Freire: Conceptualization (equal); Formal analysis (equal); Investigation (equal); Methodology (equal); Writing – original draft (lead). Evelyn Silva-Moreno: Conceptualization (equal); Formal analysis (equal); Investigation (equal); Methodology (equal); Writing – original draft (equal). Christian Robles-Kelly: Data curation (equal); Investigation (equal). Claudia D. Infante: Formal analysis (equal); Investigation (equal); Methodology (equal); Writing – original draft (supporting). Juliano C. Denardin: Conceptualization (supporting); Investigation (supporting). Sebastian Michea: Conceptualization (equal); Investigation (equal); Methodology (equal); Supervision (equal); Writing – review & editing (lead).
The data that support the findings of this study are available from the corresponding author upon reasonable request.