Nano zinc oxide to treat Colletotrichumsp fungus on orchids

INTRODUCTION

Nano zinc oxide (ZnO NPs) have been shown to effectively control microbial growth [ 1 ]. They are cheaper than silver nanoparticles with very high photocatalytic efficiency [ 2 ] and more biocompatible than other inorganic photocatalyst materials such as titanium dioxide [ 3 ]. Therefore, they are commonly used as an active anti-microbial agent in food packaging [ 4 ], in textiles [ 5 ], paints [ 6 ] and plastics [ 7 ].

In recent decades,   of nanoparticles has gained wide interest because it is environmentally friendly, reliable, cost-effective and does not require high pressure or high energy [8]. Essentially, the process of nanoparticles can be achieved by the addition of reducing agents and/or substances that act as metals such as flavonoids, phenols, enzymes and aldehydes [9, 10] found in plants or microorganisms. animals, including animals [11]. Several studies have reported successful synthesis of ZnO NPs with antibacterial activity from different biological sources, for example, plants [12-14], microorganisms [15], algae [16], and bio-composites such as vegetable peels [17], orange peels [18], banana peels [19], and durian peels [20]. In addition, biosynthesis of nano zinc oxide ZnO NPs was also conducted based on their antifungal activity. For example, spherical ZnO NPNs synthesized by Aspergillus terreus  were reported to have strong inhibitory activity against  Aspergillus niger  (causes black mold on some fruits) , Aspergillus fumigatus  (causes aspergillosis in humans) ) and  Aspergillus aculeatus (plant pathogen) [21]. Furthermore, ZnO NPs using the flower extract of Nyctanthes arbortristis showed effective antifungal activity against the mold species Alternaria (which causes leaf spot disease in plants),  Aspergillus niger , Botrytis cinerea  (which causes mold rot) gray),  Fusarium oxysporum  (a plant rot agent), and  Penicillium expansum (causing green mold) [ 22 ]. Subsequently, ZnO NPs were shown to be highly potent antifungal agents against plant pathogenic fungi.

Colletotrichum spp. is the most common cause of a post-harvest disease called anthracnose. It widely infects several tropical fruits [ 23 , 24 ], many plants and vegetables [ 25 ] including an ornamental plant such as orchids [ 26 , 27 ]. Infection  Colletotrichum sp. on orchids such as C. gloeosporioides [ 28 ] or  C. boninense [ 29 ] causes round, concave leaf spots known as anthracnose or necrotic spots that lead to defoliation and plant death in severe cases. Therefore, anthracnose caused by the fungus Colletotrichum spp. caused serious production losses [ 28 , 30 ]. Although anthracnose is mainly controlled based on fungicide treatment, Colletotrichum spp. has been reported to be able to develop fungicide resistance in some crops [ 31 – 33 ]. Therefore, it is important to find alternative management methods to control the colonization of plant pathogenic fungi such as  Colletotrichum sp.

Therefore, this study aims to chemically synthesize nano zinc oxide ZnO NPs using a crude extract from banana peel (Musa sapientum ) in a large-scale synthesis process. The physical properties of the synthesized ZnO NPs were then characterized for their structure, morphology and absorption spectra using X-ray diffraction (XRD), scanning electron microscopy (SEM) and UV-visible spectrometer, respectively. Furthermore, the surface chemistry of the nanoparticles was also analyzed using a Fourier transform infrared (FTIR) spectrometer. Furthermore, the antifungal activity of the synthesized nano zinc oxide ZnO NPs against  Colletotrichum spp. Dendrobium sp  has been infected. (Sonia Earsakul) [ 34 ] was also determined in both in vitro and in greenhouse tests.

Materials and methods

1. Preparation of raw extract from banana peel

Medium ripe banana peel ( Musa sapientum ) was prepared as previously described in Ruangtong et al. [ 19 ]. In this study, 400% (w/v) crude extract was prepared by extracting 3,200 mg of mashed banana peels in 800 mL of distilled water at room temperature for 1 h under continuous stirring (VELP Scientifica). Next, the crude extract was filtered twice with a filter cloth and then stored at 4 °C until further use.

2. Large-scale synthesis of nano zinc oxide ZnO NPs by chemical method using banana peel extract

First, 800 mL of 2 M zinc acetate solution (Ajax Finechem) was prepared using deionized water. It was then mixed with 800 mL 400% (w/v) banana peel crude extract at 30 °C under continuous stirring with a magnetic stirrer (VELP Scientifica). After 1 h, the mixture was adjusted to pH 12 with 10 M NaOH solution. Next, the precipitates were filtered with Whatman filter paper, grade 4 (GE Healthcare), and then dried in a hot oven (Kelvitron®) at 80 °C overnight. Finally, the white powder was collected, washed several times with distilled water, and further calcined in a hot oven at 80°C until dry. This synthesis was performed independently three times. For each synthesis, the resulting powder mass was measured twice using a four-digit analytical balance (Thomas Scientific).

3. X-ray diffractometer (XRD)

X-ray diffraction patterns were recorded with an X-ray diffractometer (Bruker d8 Advance) using Cu K radiation of wavelength = 0.1541 nm in the scanning range 2 = 20-80oC. Then, phase search was compared using ZnO wurtzite JCPDS number 00036-1451 [ 35 ].

4. UV-VIS . Spectroscopy

The optical absorption spectra of synthetic ZnO dispersed in water (about 500  μ g/mL) were recorded using a UV-VIS spectrometer (SHIMADZU). The measurement spectra range from 300 to 600 nm. Next, the optical band gap of ZnO was determined using a Tauc plot [ 36 ].

5. Scanning electron microscopy (SEM)

Before analysis, nano zinc oxide ZnO particles were mounted on aluminum bases and covered with a gold film. The morphology of ZnO was performed using SEM (FEI). The size of the particles was then analyzed using the ImageJ program [37].

6. Fourier transform infrared spectroscopy (FTIR)

Grounded banana peel and synthesized ZnO NPs were analyzed using Vertex 70, Platinum ATR (Bruker) by collecting spectra at room temperature under atmospheric pressure, at an average of 32 scans with a 4 cm -1  resolution. ATR mode is performed from 200 to 4,000 cm -1 . The IR spectroscopy table (Merck) was then used to identify the functional groups and compound classes.

7. Mushroom isolation

Symptomatic orchid, Dendrobium sp. (Sonia Earsakul), collected in June 2020 to identify the causative agent. The isolation of fungi from orchid leaves was performed according to the tissue culture technique described in Agrios (2005) [ 38 ]. Specifically, infected leaves were cut into 0.5 cm * 0.5 cm, thoroughly cleaned and soaked in a solution containing 10% NaOCl (Clorox® Company, Jiangsu China) for 3 min. Leaves were rinsed with sterile distilled water (SDW) twice and dried on sterile filter paper. Next, the leaves were placed on potato dextrose agar (PDA; Difco Oxford, UK) and incubated at 25 ± 02°C until mycelium was developed on the infected leaves. The mycelium was then cultured to fresh PDA to obtain pure cultures for identification.

8. Identification and Characteristics of  Colletotrichum sp.

Three strains of Colletotrichumsp. including KUFC 021, KUFC 022, and KUFC 023 were obtained, explants were cultured on PDA for 7 days at 25 ± 2°C. Macroscopic features of fungi were observed, such as coloration. Colony color, growth rate, fungal pigment production and fruiting bodies. Microcharacteristics such as acervuli and fellow species traits were examined under stereomicroscopy ( Olympus, Tokyo, Japan) and compound ( Carl Zeiss, Jena, Germany) and compared with those in identification key and species description [ 39 ].

9. Check for pathogenicity

Based on spore formation,  Colletotrichum sp. isolate KUFC 021 was selected for pathogenicity testing on three five-month-old dendrobium plants in a greenhouse. Five needle-injured leaves were inoculated by spraying a 10 6 conidia/mL spore suspension prepared from the isolate. Plants inoculated with SDW served as controls. The inoculated plants were incubated in a humidified chamber at 25 ± 02°C for 48 h and then maintained for 7 days in a greenhouse for symptom assessment [ 26 ].

10. In vitro antifungal activity assay of nano zinc oxide

In the antifungal activity of ZnO NPs for control Colletotrichum sp., Isolated KUFC 021 was tested in vitro  three times on PDA. Mycelial discs (5 mm diameter removed from the edge of 7-day-old explants) were transferred to a modified PDA with ZnO NPs at six different concentrations (5,000, 7,500, 12,500, 15,000, 17,500 and 20,000 mg/L). The experiment was conducted by including a negative control agar plate (no ZnO NPs) and two positive control plates, that is, a PDA plate containing contact fungicide (80% W/W WP mancozeb, Corteva) Ltd., Thailand) and systemic fungicide (carbendazim 50% W/W WP, Erawan Ltd., Thailand). Five repeatable PDA dishes for each treatment were incubated at. The antibacterial activity was assessed by measuring the colony diameter at 7 and 14 days after incubation. The percentage inhibition of mycelia growth was calculated as (A-B/A)*100, where A and B are the diameters of the fungal colonies grown in the negative control plate and the diameters of the fungal colonies grown in plate containing the synthesized ZnO NPs, respectively. Then the EC 50  values ​​of ZnO NP for  Colletotrichum sp. (KUFC 021) was calculated using GraphPad QuickCalcs (GraphPad software).

11. Evaluation of greenhouse antifungal activity from nano zinc oxide

The efficacy of ZnO-synthesized NPNs was evaluated in controlling orchid anthracnose in vivo . One hundred and twenty plants Dendrobium sp. (Sonia Earsakul) were grown in a greenhouse and arranged in a completely randomized design (CRD) for six treatments (positive control, negative control, 1 g/L carbendazim, 1.5 g/L mancozeb, 20 g/L ZnO NPs, and 30 g/L ZnO NPNs) with 20 replicates each treatment. Five treatments were sprayed with spore suspension Colletotrichum sp. (KUFC 021) at   108 conidia/ml while the negative control was sprayed with water. The tree is then covered with a plastic bag for 24 hours. The plastic bags were removed after inoculation, and two different concentrations of ZnO at 20 and 30 g/L were applied to the orchid and compared with 1 g/L carbendazim and 1.5 g/L mancozeb when used Use foliar spray. For positive control, the plants were sprayed with water. The percentages of disease severity index and disease severity [40] were calculated at 7 and 14 days after application. Disease severity is scored on a scale of 1 to 5, where 1 indicates no infection, healthy; Level 2 shows 1-10% infection, leaf area has necrotic lesions; level 3 infected 11-20%, leaf area has dark brown spots with color spots; level 4 shows infection from 21-50%, and the leaf area with dark brown lesions/diseases joins together to form large spots and grade 5 shows more than 50% infection, severely infected leaves turn turns completely brown and forms several concentric rings.

12. Statistical analysis

P data were analyzed using SPSS statistical software (version 22). The effects of different concentrations of synthesized nano zinc oxide ZnO NPs on fungal growth were evaluated by one-way analysis of variance (ANOVA). Duncan’s multiple range test is used to compare differences between treatments. Values ​​less than 0.05 were considered statistically significant.

RESULT

1. Physical characteristics of zinc oxide nanoparticles synthesized from banana peel extract during large-scale synthesis

This study is designed to simplify the biosynthesis of zinc oxide nanoparticles without the need for centrifuges or microwave ovens in the laboratory and aims to obtain high yields of ZnO NPs. . By conducting three independent synthesis times, in each synthesis a white powder was obtained with an average amount of 177.25 ± 8.17g. All three synthesiss provide high crystallinity of ZnO and zincite (JCPDS No. 00-036-1451) without other crystalline impurities as demonstrated in representative XRD spectra (Fig. The illustrative SEM images show that most of the synthesized ZnO is spherical and short oval with an average diameter of 256 ± 40 nm (Fig. The optical properties of the synthesized zinc oxide nanoparticles were then investigated by UV-visible spectroscopy. As a result, ZnO NPs dispersed in deionized water showed an absorption peak centered at ~370 nm (Fig. By using the Tauc plot [36], the calculated optical band gap value was found to be ~2.8 eV. In addition, the FTIR spectrum indicates the presence of several functional groups on the ZnO NPs (Fig. The characteristic peaks obtained between 600 and 450 cm -1  confirm the stretching bonds of Zn-O, while the peak at 3427 cm -1 belongs to the OH segment of the carboxylic acid group present in both ZnO and banana peel NPs. Furthermore, the peaks at 1638 cm -1 , 1536 cm -1 , and 1407 cm -1  of the synthesized ZnO NPs correspond to C=C stretching, NO stretching and OH bending, respectively (Fig. ).

Nano zinc oxide to treat Colletotrichumsp fungus causing anthracnose disease on orchids -

Nano zinc oxide to treat Colletotrichumsp fungus causing anthracnose disease on orchids -

Figure 1. Physical properties of nano zinc oxide from banana peels: (a) XRD analysis, (b) SEM images, (c) UV-vis spectroscopy, and (d) FT-IR spectroscopy.

2. Characteristics of  Colletotrichum sp. Isolation causing anthracnose disease on orchids

Three isolates including KUFC 021, KUFC 022 and KUFC 023 were obtained from infected leaves of  Dendrobium sp. (Sonia Earsakul) with anthracnose symptoms such as numerous black spots on leaves and leaf tips turning brown (Figure 2(a) ). Then, both cultural and morphological traits were examined. After 7 days, the colony color of the isolates was dark brown with an average diameter of 8.5-9 cm (Figure 2(b) ). Cylindrical spores were produced inside black acervuli filled with orange spore masses (Figure 2(c) ). Setae were also produced and found to be seed-shaped with a dark brown color (Figure 2(d)) while appressoria was ovoid to slightly irregular in shape and dark brown in color (Figure 2(e) ). The spores are rod-shaped and colorless, ranging from 12-17×3-6 µm in size (Figure 2(f) ). Taken all together, the morphological characteristics showed that all isolates were  Colletotrichum sp. To confirm the  Colletotrichum  species, it is necessary to determine the molecular characterization for a future experiment.

Nano zinc oxide to treat Colletotrichumsp fungus causing anthracnose disease on orchids -

Nano zinc oxide to treat Colletotrichumsp fungus causing anthracnose disease on orchids -

Figure 2: Characterization of an isolated fungus,  Colletotrichum sp. (KUFC 021) causes anthracnose on orchids, Dendrobium sp. (Sonia Earsakul) (a) infected leaves with symptoms of anthracnose, (b) colonies on PDA plates, (c) acervulus, (d) setae, (e) appresoria, (f) conidia. Scale bars for d and f = 20 M and e = 5µM

In addition, a pathogenicity test was performed. Dark brown lesions develop on leaf margins. On the lesions, concentrically formed salmon-colored spore masses, similar to the symptoms occurring in the field, were observed on the leaves 7 to 10 days after inoculation, whereas these symptoms did not occur. in the control tree (data not shown). The same fungus has been recreated from transplanted plants. Pathogenicity testing showed that isolates of  Colletotrichum sp. (KUFC 021) infected  Dendrobium sp. (Sonia Earsakul), thus satisfying Koch’s postulates.

3. Large-scale synthesis of nano zinc oxide from banana peels with antifungal activity against  Colletotrichum sp. (KUFC 021) In a test tube

To study the antifungal activity of zinc oxide nanoparticles against the fungus Colletotrichum sp. (KUFC 021), the inhibitory effects of different concentrations of nano zinc oxide ZnO NPs dispersed in deionized water on fungal growth were analyzed at different time points. From Figure 3 , the radial growth of  Colletotrichum sp. (KUFC 021) was significantly destroyed by 1,000 mg/mL (manufacturer’s recommended dose) of a commercial systemic fungicide and carbendazim over all time points. In contrast, 1,000 mg/mL of both mancozeb (commercial contact fungicide) and synthetic ZnO NPs failed to inhibit fungal growth at any time point. However, 4,000 mg/mL ZnO NPs moderately inhibited the growth of  Colletotrichumsp. (KUFC 021) on day 7 and the effect was slightly reduced on day 10 and day 14.

Nano zinc oxide to treat Colletotrichumsp fungus causing anthracnose disease on orchids -

Figure 3: Inhibitory effect of synthetic nano zinc oxide against the growth of Colletotrichum sp. (KUFC 021) on the PDA plate.

Next, a further antifungal activity assay was conducted using higher concentrations of nano zinc oxide ZnO NPs to determine the ability to effectively control the 50% growth inhibition (EC 50 ) against the growth of the plant. of  Colletotrichum sp. isolated KUFC 021. As a result, the antifungal activity of the synthesized ZnO NPs was shown in a dose-dependent manner. The best inhibitory effect was observed on day 7 with an EC 50  value of 13,991.6 mg/mL (Figure  4 ). Thereafter, their effects were found to be slightly attenuated by day 9 (EC50=14215.4 mg/ml). On the other hand, the least inhibition was shown on day 3 (EC50 = 17717.5 mg/ml). Therefore, the results show the optimal concentration and time to treat ZnO NPs against  Colletotrichum sp. (KUFC 021).

Nano zinc oxide to treat Colletotrichumsp fungus causing anthracnose disease on orchids -

Figure 4: Antifungal activity of different concentrations of synthesized nano zinc oxide against the growth of Colletotrichum sp. (KUFC 021). (a) Inhibitory effect on the PDA plate at different time points. (b) Quantitative analysis and EC 50  value of the synthesized ZnO NPs.

4. Effect of zinc oxide nanoparticles synthesized from banana peel on the growth of  Colletotrichum sp. (KUFC 021) In Vivo

Next, the antifungal activity of the synthesized ZnO NPs was evaluated in vivo . From Figure 5 , typical symptoms of anthracnose were evident on the leaves of the positive control (+ control) inoculated with  Colletotrichum sp. (KUFC 021) suspended both times. On the other hand, the negative control (-control) plants without fungal infection still had healthy leaves. Under greenhouse conditions, orchid plants treated with ZnO synthetic NPs showed fewer foliar disease symptoms like carbendazim and mancozeb.

Nano zinc oxide to treat Colletotrichumsp fungus causing anthracnose disease on orchids -

Figure 5: Evaluation of antifungal activity of synthesized ZnO NPs on orchid leaves inoculated with Colletotrichumsp. (KUFC 021) under greenhouse conditions.

Disease severity and disease severity index percentile calculations confirm that 30 g/L treatment of synthetic zinc oxide nanoparticles is comparable to the manufacturer’s recommended dose for carbendazim (1 g/L) on day 7 and significantly better on day 14 (Table 1). Furthermore, the effect of the synthesized zinc oxide nanosheets was also equivalent to that of 1.5 g/L mancozeb (Table 1 ).

Nano zinc oxide to treat Colletotrichumsp fungus causing anthracnose disease on orchids -

Table 1: Effect of synthetic zinc oxide nanoparticles against Colletotrichum sp. (KUFC 021) in the greenhouse. Lowercase letters exhibit significant differences as determined by ANOVA, followed by Duncan’s posthoc test.

DISCUSSION

The synthesis conditions of ZnO NPs have been reported to have a significant effect on the size and shape of the particles thereby influencing their physical and biological properties. Important factors include type [41] and concentration of precursors [19], temperature [42], and process [12]. As for the process, biological entities and their concentrations [43] are also important factors affecting the morphology of ZnO NPs. Previously, we synthesized ZnO NPs from banana peel extracts and obtained laboratory-scale average yields (0.5-1.0 g) [19]. In this study, we simplified the synthesis process by using filter paper instead of centrifugation and increased the volume of both percussion and banana peel extract. Here, we obtained an approximately 170-fold increase in the yield of ZnO NPs without the appearance of other crystalline impurities. As this study could generate an average of 177 g per fusion, this fusion condition is feasible to provide effective dosages for greenhouse disease control. With the quality control inspection of the shape and size of the ZnO particles, the production cost is around $0.55 USD. This price is comparable to that of commercial ZnO NPs on the market. More than that,44 , 45 ]. Although the FTIR results also confirm the functional groups of phytochemicals present in the banana peel extract as previously reported [19], a modified procedure for this synthesis leads to new morphology of the NPs. ZnO with round shape 256 ± 40 nm..

Many studies have shown that ZnO particles have strong antibacterial activity while some studies have mentioned their antifungal effects. This study evaluated for the first time the antifungal activity of ZnO NPs from banana peel extract against Colletotrichum sp. Our synthetic ZnO NPs significantly inhibited the growth of  Colletotrichum sp. (KUFC 021) and drastically reduced anthracnose symptoms. Although their inhibitory effect was weaker than that of carbendazim and mancozeb, the inhibitory effect of the synthesized ZnO NPs was persistent against  Colletotrichumsp. (KUFC 021) was observed in the greenhouse, and it tended to outperform both mancozeb and carbendazim. In addition, the toxicity of both carbendazim and mancozeb has raised serious safety concerns. For example, carbendazim has been reported to induce toxicity on testes and immature sperm in male rats [ 46 ] and even its low dose (10 mM or 1.91 g/L) can affect significantly to the liver and hematology of the mouse model [ 47 ]. Similarly, mancozeb has been shown to induce oxidative stress in mammalian cell lines, thyroid disease [ 48 ], acute neurotoxic effects, and mitochondrial dysfunction in laboratory animals. mature [ 49]. Notably, carbendazim and mancozeb are widely used fungicides to control fungal diseases of orchids [ 50 ]. Therefore, it is very important to develop safe alternative fungicides. De la Rosa-García et al. (2018) showed that ZnO NPs chemically synthesized (size ~26-37 nm) by co-precipitation and hydrothermal significantly inhibited the growth of  C. gloeosporioides  isolated from avocado with The minimum inhibitory concentration was 0.312 mg/mL on the PDA plate [ 51 ]. Furthermore, Pariona et al. (2020) report that 1 mg/mL of chemically synthesized ZnO NPN with platelet shape (size 256 ± 40 nm) using hydrothermal method prevented the growth of C. gloeosporioides  on the plates PDA by 60% inhibition, stronger than the rod-shaped (size 780×142 nm) and spherical (size 18 ± 2 nm) ZnO NPs [ 52 ].

In addition, foliar application of ZnO NPs has been reported to enhance plant growth, fruit yield and biomass accumulation in different crops such as habanero pepper [53], maize [54] ], wheat [ 55 ], and foxtail millet [ 56 ] . However, depending on plant size, plant type, NP concentration, exposure time, and plant species, NPs can induce phytotoxicity, cytotoxicity, genotoxicity, or oxygen stress chemistry in plants [57, 58]. Although the zinc oxide nano-treated orchid plants appeared healthy throughout our experiment, a long-term investigation is needed to determine the impact of ZnO  NPNs on growth. of the orchid.

Taken together, the appropriate morphology of ZnO NPs could serve as a novel antifungal agent. We know that our ZnO-synthetic NPN requires high doses to suppress the growth of Colletotrichum sp. We are currently performing experiments using different biomaterials for synthesis with combinations of metal doping to improve the physical and biological properties of the synthesized ZnO NPs.

CONCLUSION

This study highlights the large-scale synthesis of zinc oxide nanoparticles from banana peel extracts by green synthesis. The synthesized zinc oxide nanosheets have a circular shape, size 256 ± 40 nm, obtained without other crystalline impurities and have an average energy band gap of ~2.8 eV. High doses of the synthesized ZnO NPs significantly suppressed the growth of isolated  Colletotrichum sp. (KUFC 021) from orchids in culture plates. Furthermore, they significantly reduced anthracnose symptoms on leaves inoculated with the fungus Colletotrichum sp. (KUFC 021) under greenhouse conditions.

Reference Source: Large Scale Synthesis of Green Synthesized Zinc Oxide Nanoparticles from Banana Peel Extracts and Their Inhibitory Effects against Colletotrichum sp., Isolate KUFC 021, Causal Agent of Anthracnose on Dendrobium Orchid Nattanan Panjaworayan T-Thienprasert,1 Jiraroj T-Thienprasert,2,3 Jittiporn Ruangtong,1 Thitiradsadakorn Jaithon,1 Pattana Srifah Huehne,4 and Onuma Piasai