In this work, a synthetic method was designed to obtain nano zinc oxide (ZnO NPs) in a controlled and reproducible manner. The obtained nanoparticles were characterized using infrared spectroscopy, X-ray diffraction and transmission electron microscopy (TEM). In addition, we determined the antifungal activity in vitro of the synthesized zinc oxide nanoparticles, examining their effects on Erythricium salmonicolor pink disease causality. To determine the effect of the amount of zinc precursor used in the synthesis of zinc oxide nanocomposites on the antifungal activity, 0.1 and 0.15 M zinc acetate concentrations were examined. To study the inactivation of mycelium growth, different concentrations of ZnO NPs of two types of synthetic samples were used. The inhibitory effect on fungal growth was determined by measuring the growth area as a function of time. The morphological change was observed by high resolution optical microscope (HROM), while TEM was used to observe the changes in its superstructure. The results showed that a concentration of 9 mmol L −1 for the sample obtained from the 0.15 M system and at 12 mmol L −1 for the 0.1 M system significantly inhibited the growth of E salmonicolor . In the HROM images, a distortion was observed in the growth pattern: a significant thinning of the hyphae filaments and a tendency to agglomerate. TEM images show liquefaction of the cytoplasmic component, making it less electron-dense, in the presence of several vacuoles and significant detachment of the cell wall.
Zinc oxide (ZnO) is one of the inorganic compounds of greatest scientific and technological importance (Klingshirn 2007a , b ; Özgür et al. 2005 ; Pearton et al. 2005 ), a condition that is continuously reinforced by opening up new technologies where the function of ZnO can take on ever more interesting roles (Moezzi et al. 2012 ) due to its optical properties (Djurišić and Leung 2006 ; Jagadish et al. Pearton 2011 ; Klingshirn et al. 2010 ; Morkoç and Özgür 2008 ), its semiconductor nature (Janotti and Van de Walle 2009 ; Klingshirn et al. 2010; Morkoç and Özgür 2008 ), and physicochemical surface properties ( Wöll 2007 ). ZnO is a direct band gap semiconductor (Vogel et al. 1995 ) with an experimental energy value of 3.37 eV. Due to its wide band gap and large exciton binding energy of 60 meV at room temperature, this oxide is attractive for applications such as optoelectronic devices and use as optically degradable materials. Based on the mentioned features and others fully indicated and described in the literature (Jagadish and Pearton 2011 ; Klingshirn et al. 2010 ; Moezzi et al. 2012 ; Morkoç and Özgür 2008), the applications The technological uses of ZnO are extremely wide and varied, notably with mass use as a photoconductive component (Blakeslee et al. 1962 ), as an activator in the vulcanization of industrial rubber. industry (Nieuwenhuizen 2001 ), and in pigments and coatings (Auer et al. 1998 ), among others. Since ZnO is generally classified as a non-toxic material (Patnaik 2003 ), it is used in a wide variety of cosmetic products, including moisturizers, lip products, mineral makeup bases, face powders, ointment, lotion, and hand cream ( Nohynek et al. 2007). The production of nanoparticles and nanostructures in general has led to a growing interest in ZnO, taking into account potential uses in areas such as environmental remediation (Kisch 2015 ; Lead and Smith 2009). ; Lu and Pichat 2013 ).
Regarding the production of ZnO, oxides have been synthesized by several methods (Kołodziejczak-Radzimska and Jesionowski 2014 ), including the following: precipitation (Rodríguez-Páez et al. 2001 ); Polymer complex pechini ( Avila et al. 2004 ), combustion (Guo and Peng 2015 ), sol–gel (Alwan et al. 2015 ); hydrothermal (Hardy et al. 2009 ), mechanization (Anand et al. 2014 ), hydrogen/solvo-thermal (Yan and Chuan-Shang 2009 ), and polyol processes (Dakhlaoui et al. 2009 ), among these other process.
Due to their nano-size, the different morphologies they can have and the high specific surface area, the nanoparticles show high chemical reactivity, high surface adsorption capacity and surface charge. high. These are factors that allow them to interact very effectively with biological systems, causing significant toxicity (Cassaignon and Colbeau 2013 ; Kahru and Dubourguier 2010 ; Ray et al. 2009). Although much progress has been made in understanding the toxic and environmental effects — both direct and indirect — produced by interactions with nanoparticles, this topic has not been fully explored. . General principles are required to be drawn from case studies (from relevant, environmental examples) to determine the behavior of nanoparticles as well as their biological effects (Cassaignon and Colbeau 2013) ; Houdy et al. 2011 ; Rahman et al. 2013 ).
There is currently great scientific and technological interest in nanoparticles and metal oxides, including ZnO (Bréchignac et al. 2007 ; Klabunde and Richards 2009 ). Knowledge of their biological effects has generated a great deal of interest, leading to the special name “nanotoxicology”, a term coined in 2005 by Oberdörster et al. ( 2005 ). Many articles have been published and very comprehensive compilations on the subject (Bréchignac et al. 2007 ; Zucolotto et al. 2013 ). Considering their toxicity, the potential use of nanoparticles to control phytopathogenic fungi has generated great interest (Ram et al. 2011 ; Zucolotto et al. 2013), spurred on by studies previously showed effective antifungal activity of various nanoparticle materials including silver (Kumar et al. 2008 ), copper (Cioffi et al. 2005 ; León et al. 2011 ), titanium dioxide (Maneerat and Hayata) 2006 ), and zinc oxide (Lipovsky et al. 2011 ). In particular, when working in aqueous systems or those in which it is possible to dissolve ZnO NPs and thereby generate Zn 2+ in the environment, two effects are considered when analyzing the toxicity of ZnO. : the first effect is particle dependent and the second is driven by Zn 2+ dissolution , the mechanisms will have different modes of action, as demonstrated by Poynton et al. (2010 ) worked with D. magna . Other factors that may affect the solubility of nano zinc oxide ZnO NPs and thus the contribution of Zn 2+ ions to oxide toxicity are the presence of some Cl – and SO 4 2− anions such as term (Ma et al. 2013 ), temperature (Reed et al. 2012 ), the presence of organic material (Miao et al. 2010 ) – very important for the present study – and the presence of phosphates in the environment, as pointed out by Mingua et al. ( 2010 ) and Reed et al. ( 2012 ) in their studies.
Although there is no consensus on the exact mechanism underlying the toxicity induced by nano zinc oxide ZnO NPs, the most accepted view is that this toxicity is usually mediated by ROS which will be produced on the surface of the particles. . These species may arise due to the electronic properties of semiconductor materials and/or the ability of ZnO NPs to disrupt electron transfer processes in biological systems, which can occur at the inner membrane of mitochondria (Xia et al. 2008 ), such as the reaction of the surface of ZnO NPs with water, generating hydroxyl radicals (OH.) (Sapkota et al. 2011 ) or H 2 O 2 (Sawai et al. associates 1998 ).
Furthermore, different mechanisms should be proposed for different fungi, as illustrated in the study of Botrytis cinerea and Penicillium expansum by Raman spectroscopy. In this study (He et al. 2011 ), the researchers showed that in the Raman spectrum corresponding to the fungus B. cinerea , the intensity of the bands associated with nucleic acids and carbohydrates increased significantly when treated. treated with ZnO NPs, whereas this was not the case with the protein and lipid bands. Sharma et al. ( 2010 ) investigated the antifungal properties of ZnO nanoparticles, synthesized with and without surfactant, under different reaction conditions, on strains of Fusariumsp. The antifungal activity of these NPs was compared with that of fungi treated with traditional antifungal using copper sulphate. The results indicate that the antifungal activity of ZnO NPs depends on the concentration and size of the NPs. The second property is determined by the different reaction conditions used in their synthesis. Mr and his associates. ( 2011 ) then tested the antifungal effects of ZnO NPs and their mode of action on two postharvest pathogenic fungi: Botrytis cinerea and Penicillium expansum . The results obtained showed that at concentrations above 3 mmol L −1, the growth of these pathogens was significantly inhibited. Furthermore, the findings indicated that the antifungal activity of ZnO NPs was different for B. cinerea and P. expansum , since in the first case growth was inhibited because the nanoparticles directly affected direct to cellular functions, causing distortion in the mycelium, whereas in P. expansum there is no growth of fellow and spore cells, which eventually leads to the death of the mycelium.
Based on all the foregoing and the antifungal activity of ZnO particles, this work aimed to investigate the effect of these nanoparticles on a fungus affecting coffee plants: E. salmonicolor . Commercially, coffee is a product of great economic importance to Brazil, Vietnam, Colombia, Indonesia, Ethiopia, Peru, India, Honduras, Mexico and Costa Rica, among others. These countries are the main producers, with an annual global production of about 6.3 million tons per year (Parras et al. 2007 ). In Colombia, the third largest producer in the world, coffee ( Coffea arabica L.) is the second most important resource after oil, with production reaching 10.9 million bags (FNC 2014), making it become an important resource for economic growth and industrial development (Naranjo et al. 2011 ). Meanwhile, fungi, including E. salmonicolor , which are the main cause of several plant diseases (Galvis-García 2002 ) can persist in coffee plantations, severely reducing yields crops and can sometimes destroy entire crops (Rodríguez 2001 ). E. salmonicolor is the cause of a disease known as “pink disease” that yellows and wilts leaves, stems and fruit, leading to plant death (Galvis-García 2002). To address this issue, research on novel antifungal alternatives is needed, for example taking into account the use of nanotechnology, namely nanoparticles, allowing physicochemical control of fungi without alter the final harvest.
In this paper, zinc oxide nanoparticles are synthesized by a chemical pathway and display certain characteristics as determined by the synthesis conditions. Zinc oxide nanoparticles were used to perform a systematic in vitro study that looked at several different concentrations to determine the antifungal effect against E. salmonicolor. The study also evaluated the influence of various synthesis parameters, including the initial concentration of precursors, on the fungicidal ability of ZnO nanoparticles.
1. Synthesis of nano zinc oxide
To synthesize ZnO nanoparticles, the sol-gel method was used. For this, 13.1694 g zinc acetate di-hydrate ((CH 3 COO) 2 · Zn · 2H 2 O — Merck) was used as the precursor, and 0.07289 g of the surfactant, cetyltrimethyl ammonium bromide (((C 16 H 33 ) N (CH3) 3) Br — Merck), was used to control particle growth. To facilitate solid phase nucleation, by hydrolysis and condensation reaction, an analytical ethanol solution (400 mL) was prepared at a precursor concentration [0.15 M] to Disperse the previously indicated amount of zinc acetate, together with the surfactant, and establish the working pH of the system (pH 8.5) by adding dropwise distilled water and ammonium hydroxide (NH 4 OH-) Baker Analyzed) into solution. The system was heated to ~70°C and maintained under constant stirring for 6 h. The suspension was then allowed to last for 3 days. At the end of this period, the suspension was centrifuged at 5000 rpm for 30 min, and after drying the sample was calcined at 450 °C in a furnace for 2 h. Figure 1 shows the synthesis scheme used in this work to obtain nano zinc oxide ZnO-NPs.
Figure 1. Synthetic procedure used to obtain ZnO-NP
The same procedure was followed to obtain a [0.1 M] solution, where the appropriate amount of zinc was dissolved with a surfactant in 600 mL of analyte ethanol. In summary, the following factors were taken as variables of the synthesis: concentration of precursors and solvent volume. This produces two systems known as ZnO NP 0.15/400 (13.1694 g zinc acetate di-hydrate in 400 mL to give a concentration of [0.15 M]) and ZnO NP 0.1/600 (13,1694 g zinc acetate di-hydrate in 600 mL, giving a concentration of [0.1 M]).
2. Characterization of synthesized nano zinc oxide
When samples were collected through the synthesis pathway described above (Figure 1 ), they were characterized using, for example, IR spectroscopy, transmission electron microscopy (TEM), and diffraction. X-ray (XRD).
IR . Spectrum
To determine the different functional groups, the sample to be analyzed is obtained by mixing dry KBr with the synthetic solid, at a concentration of about 10%. Scans were performed between 4000 and 400 cm -1 using a Thermo Nicolet IR 200 spectrometer.
To determine the size and morphology of the synthesized nano zinc oxide, they were suspended in 1 mL of ethanol and placed in an ultrasonic bath for 1 h. Then, with a pasteur pipette, a small amount was taken and deposited on a nickel grid previously coated with formvar film for observation in a Jeol model JEM 1200 EX transmission electron microscope.
To determine the crystalline phases present in solid samples, X-ray diffraction patterns of the samples of interest are obtained in powder form. They were recorded with a Bruker-style D8 ADVANCE diffractometer using Kα radiation from Cu (λ = 1.542 Å) in the range of 10–70 in 2 θ .
3. Multiplication of fungal strains E. salmonicolor in the laboratory
Bacterial strains E. salmonicolor were donated by the National Coffee Research Center (Cenicafe) in Chichina, Caldas, Colombia. These strains were replicated and grown in culture, potato dextrose agar (PDA) + oxytetracycline base. Media were autoclaved at 121 °C and then, in a laminar flow fume hood, they were poured into sterile petri dishes, using 20 mL per dish. Finally, they were incubated for 3 days at 25°C, to ensure sterility, as recommended by the protocol for this type of test (Lane et al. 2012 ). At the end of the 3-day incubation, seed was seeded, a dish of mycelium 1.5 cm in diameter was inoculated in the center of each petri dish and the strains were then incubated to ensure growth (about 16 days). under laboratory conditions (25°C).
The fungi are maintained using a periodic replanting technique that allows the cultures to be short-lived. This technique is based on transferring growth from dry or old media to fresh media, creating optimal conditions for fungal growth. In this way, a high risk of contamination and variation in the characteristics of strains, which exhibit major drawbacks, is avoided (Aleman et al. 2005 ).
Evaluation of the antifungal effect of zinc oxide nanoparticles on the in vitro growth of E. salmonicolor
4. Preparation of fungal preparations and biological testing in culture with nano zinc oxide
To determine the inhibition of the growth of the mycelium under study, taking into account the influence of nano zinc oxide ZnO NPs, solid cultures were prepared for the strain using the method mentioned in “ Replication fungal strains E salmonicolor in the laboratory”. The treatments evaluated were (1) untreated culture medium (control); (2) culture medium + copper oxychloride (23.41 mmol L −1 ) (fungicide); (3) culture medium + ZnO NP (12 mmol L −1 ); (4) culture medium + ZnO NP (9 mmol L −1 ); (5) culture medium + ZnO NP (6 mmol L −1 ), and (6) culture medium + ZnO NP (3 mmol L −1). Meanwhile, it is important to clarify that the fungicide, copper oxychloride (Cu 2 (OH) 3 Cl), was used as a “standard” in the study, as it is often used as a method prevention of rosacea (Galvis-García 2002 ).
Different concentrations of nano zinc oxide ZnO NPs were added to the culture medium and subjected to ultrasonic treatment to ensure their dispersion in the medium; They were then poured into a petri dish, the medium was allowed to solidify, and finally, the systems were incubated for 3 days (see “ Results and discussion ”).
To ensure uniformity and reproducibility during seeding, a 16-day-old fungus was used, from which samples were obtained using a 1.5 cm diameter hole, to ensure the survival of growth structures. The mycelium was then inoculated into the center of each treated petri dish. To obtain reliable results, the experiment was performed three times.
Seven days were allowed to elapse after sowing, to ensure adequate fungal growth, and then photograph the explants every 3 days. These records were then fed to an image analysis system, “Image Analyzer pro” to measure the area of fungal growth in the Petri dish, which continued until day 25, to observe the activity of the methods. treatment over time.
5. Percentage (%) inhibition
Inhibition of mycelium growth was determined based on the fungal growth area, measured in cm 2 and expressed as percentage inhibition, a parameter calculated using the following formula proposed by Pandey et al. export. ( 1982 ):
% Inhibition=(Growth of control−Growth of treatment)×100/ Growth of control
Determination of morphological and ultrastructural lesions of fungi using high resolution optical microscopy (HROM) and electron microscopy (TEM).
6. Sample handling and preparation
The E. salmonicolor, samples, used for ultrastructural analysis of mycelium, were processed according to standard TEM process techniques (Bozzola and Russell 1999 ). Small fungal samples were placed in 1 mL vials, fixed overnight in a 2.5% glutaraldehyde mixture at 4 °C. The next day, the fixator was removed and the samples were washed three times with phosphate buffer (PBS). ) for 5 minutes each time. They were then fixed with 1% osmium tetroxide (OsO 4 ) for 1 h at room temperature and washed again with buffer, three times for the same 5 min each.
The post-fixed samples were dehydrated with ethanol in increasing concentrations of 30, 50, 70, 80, 90, 95 and 100%, and left in each alcohol concentration for 10 min. Pre-soaking was performed with a mixture of alcohol and white resin LR in a ratio of 3:1, 1:1 and 1:3, the first two ratios for 45 minutes each, and the last one for 1 hour. .
Finally, the samples were placed in gelatin capsules, labeled, embedded in LR white resin, and polymerized in a UV chamber at room temperature for 48 h. Once the samples were polymerized, the capsules were taken and etched with a knife to remove excess resin and thus obtained semi-fine fractions of size 200–300 nm and ultrafine fractions of 40–60 nm. Semi-fine and ultra-fine sections were obtained with a glass knife with the help of a Leica micromachine, model Ultracut R.
7. High resolution optical microscope
Using clear duct tape, samples were taken directly from the culture, keeping in mind all treatments and their respective controls. The imprint is then placed on a slide, along with a drop of Lactophenol blue. They were then observed with HROM (Nikon Microphot). The images of interest were recorded with a Nikon Digital Sight DS-2Mv connected to a microscope, using the “Nis Elements” program for imaging.
8. Analysis of thin faces..
Semi-thin sections with a thickness of 200–300 nm were fixed, using heat, on slides by staining with toluidine blue, burning the plate and washing with distilled water. They were then observed in a Nikon Microphot light microscope using 40 × and 100 × objectives to select the region of interest where the greatest number of mycelial structures were found arranged horizontally and vertically. This area has been marked and re-engraved to obtain ultra-thin sections.
9. Electron Microscopy
Ultrastructural analysis and characterization of the effect of zinc oxide nanoparticles on the pathogen E. salmonicolor was performed by observing microscopy images taken at different magnifications, using transmission electron microscopy. via Jeol model JEM 1200 EX, operating at 80 kV (Bozzola and Russell 1999 ).
10. Contrast with uranyl acetate – citrate lead.
Ultrathin sections, 40-60 nm thick and gray to silver in color, are placed together on formvar film-coated copper grids. They were counterstained with 4% uranyl acetate for 20 min, using flotation in a dark and humid chamber. Sections were washed with drops of distilled water and then placed in contact with a drop of lead citrate for 10 min in a humidified chamber containing sodium hydroxide (NaOH) pellets. Finally, sections were washed with distilled water, dried with filter paper, and placed in the TEM sample holder for observation (Bozzola and Russell 1999 ).
11. Statistical analysis
A statistical study was performed on the data regarding the fungal growth area (measured in cm 2), a parameter recorded periodically. To determine whether the observed difference in this parameter was statistically significant, a hypothesis test was conducted using a completely randomized block design to compare the four concentrations of ZnO NP, a concentration of copper oxychloride (control fungicide) and control, constituting the treatments considered in the study (6 in total), and in the seven blocks (day), the activity of Treatments over time were examined at 7, 10, 13, 16, 19, 22 and 25 days. All data were analyzed for distributional fit and homogeneity of variance, and since these two criteria were met, two-way ANOVA test was used, which was performed in BioEstat 5.3 (Zar 2014) program; Graphics were built using the program Graph Pad prism 5 (Lieber et al. 2006 ).
Results and Discussion
1. Characterization of synthesized nano zinc oxide
For the synthesis of nano zinc oxide, the amount in grams of the zinc precursor (Zn(CH 3 COOH) 2 ) and the volume of the solvent (ethanol) were taken as variables, i.e. the initial concentration of the precursor. substance is considered as a variable. . According to the method indicated, the nanoparticles shown in Figure 2 a were obtained, using a 0.15-M concentration of the precursor in a 400 mL volume of ethanol; This ceramic powder is called ZnO NP 0.15/400.
Figure 2 Microscopic images of a ZnO NP 0.15/400 and b ZnO NP 0.1/600 observed by TEM
The images in Figure 2 a1 show that the synthesized nanoparticles have two morphologies: (a) spherical and (b) lenticular, although a closer look, in Figure 2 a2, the needles are formed by orderly aggregation. of nano zinc oxide, a referenced and studied growth mechanism, by Bogush and Zukoski ( 1991 ). The size of these nanoparticles ranges from 20 to 35 nm.
Meanwhile, in search of a better dispersion of the nanoparticles, the concentration of the precursor was adjusted, to 0.1 M, increasing the solvent volume during synthesis to 600 mL and maintaining zero change the amount of surfactant used; this sample is called ZnO NP 0.1/600 and its morphology and particle size are observed in Fig. Here, the size of the nanoparticles ranges from 30 to 45 nm.
It should be noted that during the observation of 0.1/600 zinc oxide nanopowder, using TEM, the electrons in the beam spurred reactions that caused the nanoparticles to self-group in a particular way, as demonstrated shown in Figure 2 b2, creating “clusters” containing a finite number of nanoparticles.
By varying the synthesis conditions, the obtained ceramic powders show some distinct differences, including their agglomeration states, as illustrated in Figure 2 : while the ZnO NP sample 0.15/ 400 produces soft agglomerates, hard clusters or agglomerations are seen. with ZnO NP 0.1/600. Moreover, the color of the synthesized samples also changes depending on the synthesis conditions (Figure 3 ), 0.15/400 ZnO NP is white with a color darker than one bit (Figure 3 a), gray for ZnO NP/0.1 600 (Figure 3b), a feature indicating that the defect structure in the solid is different. Since ZnO is a non-polar solid, it can be found in a variety of colors including white, light green, light yellow, brown, gray and even pink, depending on the concentration of defects in the solid. , a condition largely determined by the amount of oxygen present in its crystal structure (Greenwood and Earnshaw 1997 ).
Figure 3 Color of ZnO NP powder obtained after heat treatment; a ZnO NP nano zinc oxide 0.15/400 and b ZnO NP 0.1 / 600
Figure 4 shows the IR spectrum obtained for the samples under consideration. In the spectrum, it is evident the presence of hydroxyl groups in the band at ~3450 cm −1 and of water molecules in the bending band at ~1630 cm −1, as well as the absence of bindable bands with the organic phase of the solid, although organic compounds were used in the synthesis of zinc oxide nanoparticles. This indicates that heat treatment at 450 °C is effective in removing the organic phase.
Figure 4 Infrared spectra for 0.15/400 zinc oxide nanosample and 0.1/600 . nano zinc oxide ZnO-NPs
Since the special interest of this study lies in the presence of Zn–O and Zn–OH bonds associated with the Zn 2+ cation, and their bands are mainly found between 1000 and 400 cm −1 , this specific region of the IR spectrum was used to analyze the differences caused by changes in the synthesis parameters (Figure 5 ).
Figure 5 IR spectrum from 1200 to 400 cm −1 for sample ZnO NP 0.15/400 and b ZnO NP 0.1/600
The spectrum from 1200 to 400 cm −1 in Figure 5, corresponding to the two zinc oxide nanosolids of interest, shows a clear difference in the number of bands and their positions: while for the ZnO sample NP 0 ,15/400 has two bands (Figure 5 a), one at 450 cm −1 and one at 500 cm −1 , as well as the shoulder at 560 cm −1 , the 0.1/600 ZnO NP shows a single band at 450 cm −1 and one shoulder at 500 cm −1 (Figure 5b). This difference in the spectrum suggests that there have been changes in the environment of the Zn-O and Zn-OH functional groups, mainly in their number and/or arrangement, at the internal and surface levels. On the face of solids, the changes are driven by modifications in the parameters of the synthesis (Socrates 2004 ). This seems to suggest, given the structure-property relationship exhibited by the material, that samples can undergo changes in their physical and chemical properties.
Figure 6 , shows the diffraction plots corresponding to the two samples under consideration. The peaks appearing there correspond to ZnO (PDF 79-206), which is the only crystalline phase present, and the thinness of the peak indicates good crystallization of the samples. In contrast to the IR spectrum in Fig. 4 and 5 , in the diffraction plots in Figure 6 there is no obvious difference between the spectra, indicating that there is no long-range change in the structure of the samples. This leads to the conclusion that the changes observed in the samples, brought about by variations in the synthesis parameters, are of a more localized type, as indicated by IR spectroscopy (Fig. .
Figure 6 X-ray diffraction plot corresponding to sample ZnO NP 0.15/400 and b ZnO NP 0.1/600
2. Multiplication of plant pathogenic fungi E. salmonicolor in the laboratory
When the phytopathogenic fungus was inoculated and allowed to grow in its respective culture, it was found that at 16 days its growth was optimal. This result demonstrates that the culture medium, previously indicated, is the most suitable for its growth (Lane et al. 2012 ). Furthermore, it should be noted that for E. salmonicolor , the use of the antibiotic oxytetracycline, used in an amount of 0.03 g/L, reduced the risk of contamination and the permissible culture purity (Kuang) -Ren and Tzeng 2009 ).
The fungal maintenance method used in this study is periodic replanting, a technique that ensures good survival of the explants in the short term. However, for more reliable results, other methods for maintaining microbial strains should be sought, such as that recommended by Aleman et al. ( 2005 ) and Huertas et al. ( 2006 ), which ensures the viability, purity, and genetic stability of cultures.
3. Evaluation of the antifungal effect of ZnO NPs on the culture of E. salmonicolor bacteria in vitro
Growth of E. salmonicolor in the 0.15/400 ZnO NP system
Figure 7 comparison of control, fungicide treatment and 9 mmol L −1 treatment with ZnO NP 0.15/400. Picture shows the macroscopic growth characteristics of E. salmonicolor at 16 days of age. on potato dextrose agar-based cultures with oxytetracycline. Looking at the photographs, the effect of this ZnO NPs treatment on inhibiting fungal growth is clear. According to these results, at 16 days, fungicide treatment did not inhibit or retard fungal growth, while treatment with nano zinc oxide ZnO NPs did. As a result of this initial observation, morphological changes were evident in the form, margins, texture and areas of fungal growth.
Figure 7 Macro-growth of E. salmonicolor fungus at 16 days of age: control , b fungicide treatment and c treatment with 9 mmol L -1 concentration of nanoparticles of ZnO NP 0 system, 15 / 400
To determine the percentage inhibition of the growth of E. salmonicolor caused by treatment with nano zinc oxide ZnO NP 0.15/400, taking control drugs and fungicides as reference, people used The methodology and equations are shown in ” Percentage (%) inhibition ” ; The results obtained are shown in Table 1 . Fungal area measurements were performed on the indicated follow-up days and they were used to calculate the percentage inhibition, showing that ZnO NP 0.15/400 exerted a significant inhibitory effect on fungal growth when compared with the control (Table 1 ).
Table 1 Percent (%) inhibition of fungal growth of bacteria E. salmonicolor when using 0.15/400 ZnO NP nano zinc oxide
Observing the results in Table 1 , it is clear that the highest percentage of inhibition was found at day 10, with an inhibition of 84.9%, decreasing over time until the end of the experiment. On day 25, 64.3% inhibition was achieved. This suggests that the NPs initially showed high antifungal activity, which decreased over time without ever completely losing their inhibitory effect: the lowest percentage was 23.5% for the concentration. 3 mmol L -1 on day 22, compared with 21.9% obtained with fungicide treatment on the same day. Considering the percentage inhibition values recorded in Table 1, it can be concluded that the presence of 0.15/400 zinc oxide nanoparticle in the explants significantly affected the growth of E. salmonicolor .
Based on the above and according to the two-way analysis of ANOVA used in this study for E. salmonicolor bacteria, when it was treated, a significant difference was found in fungal growth: ANOVA was F = 25.6447 and a value of p <0.0001. The greatest difference in fungal growth was found in the treatments with NPs at 12.9 and 6 mmol L −1 , with the 9 mmol L −1 treatment striking because it better controlled the growth of the fungus. growth of mycelium, while at the concentration of 3 mmol L -1 there was no significant difference between the effects of ZnO NPs and fungicides.
Significant differences were also observed between blocks (days), with an ANOVA of F = 30.7202 and a value of p <0.0001. In Figure 8 , all the results of the tests conducted can be seen, showing that on days 10, 19 and 25, a concentration of 9 mmol L -1 of the 0.15/ZnO NP system 400 went on to show significant inhibition of mycelium growth. .
Figure 8 Antifungal activity of different concentrations of nano zinc oxide ZnO NP 0.15/400 against E. salmonicolor
E. salmonicolor growth on systems with 0.1/600 nano zinc oxide
To determine the effect of the synthesis parameters on the antifungal activity of ZnO NPs, a similar test was performed with the 0.15/400 ZnO NP samples described above, but using the nanoparticles from the ZnO NP 0.1/600 system. For this, the growth of E. salmonicolor was recorded and the percentage inhibition was calculated using these data. . Figure 9 shows the results obtained by treatment with 12 mmol L −1 of 0.1/600 ZnO NPs, in which the inhibitory effect using these new nanoparticles is the most obvious. The results in Figure 9 showed inhibition of the growth of E. salmonicolor up to day 10, at which point it was clear that the NP used here was less effective in controlling mycelial growth than the NPs used here. with nanoparticles of the 0.15/400 ZnO NP system (Fig. It is important to note that in this trial it was not possible to record data for all of the suggested dates, as by day 16 the fungus covered the entire petri dish. Therefore, a statistical study was not performed because there were insufficient data to obtain a reliable result.
Figure 9 Microgrowth of E. salmonicolor bacteria at 10 days of age: control b fungicide treatment and treatment with 12 mmol L −1 of ZnO NP 0.1/600
Table 2 shows the values obtained for the percent inhibition of fungal growth at E. salmonicolor in the cultures treated with nanoparticles from the 0.1/600 ZnO NP system, as a reference. for fungicide control and treatment. The results indicated that the highest percentage inhibition was 71.3% on day 7, compared with 51.1% on day 10, compared with fungicide treatment, which was 66.8% on day 7 and 46.7% at day 10. These data suggest a favorable effect of NP on controlling the growth of mycelium E salmonicolor . However, if the data in Table 2 (ZnO NP 0.1 / 600) are compared with the data in Table 1(ZnO NP 0.15/400), a significant difference is observed, the inhibition of more efficient mechanism for the second system.
Table 2 Percent (%) growth inhibition of mycelium E. salmonicolor when nanoparticles from the 0.1/600 ZnO NP nano zinc oxide system were used in the treatment
The less favorable results obtained with the 0.1/600 ZnO NP sample (Figure 9 ; Table 2 ) could be explained compared with the 0.15/400 ZnO NP sample (Figure 7 ; Table 1 ) when considering the formation. clumps or hard agglomeration , as shown in Figure 2 . These would reduce the surface area of ZnO, leading to a decrease in its antifungal activity, as can be seen in Figure 9 , where the fungus almost completely filled the petri dish at 10 days of age. Furthermore, there is evidence that the nature of each of the two ZnO-synthesized NPN systems is different, as determined by changes in the synthesis conditions. First, the samples have different colors (Figure 3). This can be caused by variations in the concentration and nature of the defects, a condition that can affect the function of the ceramic powder (Greenwood and Earnshaw 1997 ). Second, the IR spectra corresponding to these two samples (Figures 4 , 5 ) show differences in the positions of the bands, which implies changes in the structure of the solid, although chemically similar. learning and structure (Figure 6 ).
Meanwhile, the fungicide treatment was not significant and did not produce significant inhibition of the growth of the studied fungi. This may be explained when considering that conditions of laboratory use are different from those used in the field, which may lead to a change in the mode of inhibitory action for the fungus studied. . Based on the obtained results, it can be concluded that in the in vitro conditions in which the test was conducted, ZnO NPs inhibited the mycelium growth of E. salmonicolor more than the insecticides. The control mushroom used, copper oxychloride. However, for more conclusive data on this antifungal effect, further trials with other fungicides used to control pink disease are needed.
4. Determination of morphological and ultrastructural lesions to fungi identified using HROM and TEM
Given that 0.15/400 zinc oxide nanoparticles were the ones that showed the highest antifungal activity against E. salmonicolor , the morphological and ultrastructural changes of the fungi interacting with them were analyzed. accumulate.
5. Morphological changes of E. salmonicolor observed with HROM
Figure 10 shows the images of mycelium obtained through the imprinting process described in “ High-Resolution Optical Microscopy ”. The hyphae are considered to have smooth walls, a “retinal” structure, and a clear septum (Figure 10 a) that stain better than the interior of the mycelium.
Figure 10 Image of mycelial structure of E. salmonicolor . a Control, b treated with fungicide, c treated with 12 mmol/L , d 9 mmol/ L and e 6 mmol /L ZnO NP nano zinc oxide 0.15/400
In figure 10 b, it can be seen that the filaments of the mycelium tend to clump and the dye used has a stronger affinity for the interior, that is, no septum is visible. In treatments 1, 9 and 6 mmol L −1 with ZnO NP 0.15/400, there was a distortion in growth patterns as a result, as the filaments of the mycelium were significantly thinner and tended to clump together. block (Figure 10 c – e). From the observations, it was clear that 0.15/400 nanozinc oxide inhibited the growth of E. salmonicolor .
6. Ultrastructural changes of E. salmonicolor observed by TEM
In E. salmonicolor sections at 16 days of growth, the presence of organelles such as vacuoles (V) and mitochondria (Mit) in the conserved cytoplasm (Cyt) was observed. identified cell (Cw), making it more electron dense (Romero de Pérez 2003 ) when observing it in TEM, Figure 11 a, during fungicide treatment, fractionation was observed of the cell wall, an alteration that cannot be considered relevant because its structure is similar to that of the control, Figure 11 b. Finally, in fungal sections treated with ZnO NP 0.15/400 (9 mmol L −1), a uniform thickening of the cell wall was observed, with noticeable liquefaction of the intracellular substances. cytoplasm, making it less electron-dense, Fig. A similar effect was also produced with 12 mmol L −1 ZnO NP 0.15/400, where some vacuole count (V) and cell wall detachment (Cw) were also seen, Fig. d.
Figure 11 TEM sections of E. salmonicolor bacteria: a Control, and treated with b copper oxychloride fungicide, c ZnO NP 0.15/400 at 9 mmol L −1 , and d ZnO NP 0.15 /400 at 12 mmol L −1 [vacuum (V), mitochondria (Mit), cell wall (Cw) and cytoplasm (Cyt)]
In summary, the observed effects on the interaction of 0.15/400 nano zinc oxide nanosystems on E. salmonicolor correspond to major to significant morphological changes in the mycelium. (Figure 10 , 11 ). Collectively, the results obtained in this study provide interesting elements to suggest an explanatory and explanatory mechanism for the toxicity of ZnO NPs (Buerki-Thurnherr et al. 2012 ; Sharma 2011), especially their antifungal activity. Taking into account the usual criteria used to classify antifungal compounds, an important aspect to consider is the “active site” of the nanoparticles (on the cell membrane, cell wall, DNA or RNA), as well as its surface features. Therefore, when providing a possible mechanism to account for the antifungal activity of ZnO NPs, the wall of the fungus can be considered as a target, also taking into account the surface physicochemical properties of ZnO (Altunbek et al. et al. 2014 ; Wöll 2007 ) , as well as its other physicochemical properties – eg size and shape – may also influence the toxicity of this oxide (Mu et al. 2014). The fungal wall, in controlling cell permeability, is the part of the cell that interacts with the external environment, and thus with the ZnO NPs present in the fungal culture media of interest in this work. This part of the fungal cell is composed mainly of polysaccharides and proteins. Specifically, there are β-1,3- D -glucan and β-1,6- D -glucan macroproteins, chitin, proteins and lipids, and among the polysaccharides, chitin, glucan and mannan or galactomannan (Pontón 2008 ) predominate. position .
A possible mechanism of the antifungal activity of ZnO-NPs may account for the effect of ROS and/or Zn 2+ on N -acetylglucosamine ( N -acetyl- D -glucose-2-amine) or on β -1 3- D -glucan syntax (FKs1p). ROS is produced by nanoparticles (Lipovsky et al. 2009 , 2011 ), while Zn 2+ is a product of the dissolution of nanoparticles in the culture medium (Lv et al. 2012 ; Mudunkotuwa et al. 2012) ; Reed et al 2012 ; Xia et al. 2008 ) . N-acetylglucosamine is involved in the synthesis of chitin (a polysaccharide of great importance in the structure of the cell wall), while the synthesis of β-1 3- D -glucan participates in the synthesis of β-1 ,3- D -glucan (another important component of the cell wall in fungi). This would replicate the action of penicillin on the bacterial cell wall (Park and Stromistger 1957 ).
Although the impact of nanoparticles on biological systems has been the subject of research (Colvin and Kulinowski 2007 ; Klein 2007 ; Rahman et al. 2013 ), more research is needed to examine the effects of oxides. nanozinc for chitin and glucan, due to the effects observed during this work of ZnO-NPs on bacteria E. salmonicolor , a significant thickening of the cell wall and liquefaction of the substances inside the cytoplasm (Figures 10 , 11). Such studies must consider the chemical composition of the wall and determine how ZnO NPs can control or inhibit the synthesis of chitin and glucan enzymes, referencing the knowledge of inhibitors classical antifungal glucan (Douglas 2001 ; Romero et al. 2005 ) and chitin synthesis (Gongora 2002 ; Merzendorfer 2006 ), with particular attention to the toxicity peculiarities of nanoparticles (Zucolotto et al. 2013 ).
Recent work indicates that nanoparticles induce oxidative stress (Xia et al. 2008 ). Adverse effects appear to be caused either directly on the target tissue, by toxicity of the ROS derivatives, or indirectly by the effect of these derivatives on the production of immune system mediators and inflammation, mainly pro-inflammatory. -toxin. This oxidative stress can disable antiproteases while also activating metalloproteases, thereby encouraging uncontrolled proteolysis and cell destruction. Some authors (L’Azou and Marano 2011) suggest that inflammation is a major response and that oxidative stress is a consequence of this. As a result, the nanoparticles will be recognized by the body as foreign matter that needs to be eliminated by an inflammatory response, such as an increase in the size of the vacuoles and/or of the cell wall, as shown in Fig. 11 . The interaction between the nanoparticles and the proteins of the biological system plays a decisive role in their ability to be recognized by the cells of the immune system responsible for their elimination, as well as the tissue of the cell wall. , the target of nanoparticles, can emit anti-inflammatory signals. Inflammation can accelerate ROS production and reduce antioxidant defenses, promoting oxidative stress and related tissue damage.
A method has been developed that allows the synthesis of zinc oxide nanoparticles in a controlled and reproducible manner. These nanoparticles demonstrated antifungal effects against Erythricium salmonicolor bacteria, a pathogen that causes a coffee plant disease known as pink disease. Based on the results, to ensure the full antifungal function of zinc oxide nanoparticles, the synthesis parameters need to be tightly controlled: any of these changes can affect the particle behavior. Nano zinc oxide with sizes between 20 and 45 nm showed significant antifungal activity on the mycelium growth of E. salmonicolor in vitro and using these sizes at a concentration of 6 mmol L -1 caused a significant inhibition. The 0.15/400 system ZnO NP nanoparticles induce liquefaction of substances within the cytoplasm, making the cytoplasm less densely electron-dense and inducing significant detachment of the fungal cell wall. These effects need to be further investigated as they can be shown to be representative when considering the mechanism of action of ZnO NPs on pathogenic, destructive fungi.
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