Straightforward synthesis of monodisperse Co/Zn-based nanoparticles and their antifungal activities on Botrytis cinerea

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 Co_0.5Zn_0.5Fe₂O₄. 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.

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.

The monodisperse CoFe₂O₄, ZnFe₂O₄, and Co_0.5Zn_0.5Fe₂O₄ 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 Co_0.5Zn_0.5Fe₂O₄ 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.

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, Co_0.5Zn_0.5Fe₂O₄ 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.

The diffraction patterns for the monodisperse CoFe₂O₄, Co_0.5Zn_0.5Fe₂O₄ 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 Co_0.5Zn_0.5Fe₂O₄ 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.

TEM micrographs for CoFe₂O₄, Co_0.5Zn_0.5Fe₂O₄ 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, Co_0.5Zn_0.5Fe₂O₄ 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, Co_0.5Zn_0.5Fe₂O₄ 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, Co_0.5Zn_0.5Fe₂O₄ 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 [11200425] and DIUA 206-2021 from the Dirección de Investigación of Universidad Autónoma de Chile.

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.