The continuous pursuit of a sustainable food system remains a global priority, with increased food security and minimization of postharvest loss as primary objectives. This is a critical issue, as recent estimates from the Food and Agriculture Organization (FAO) Reports on the State of Food Security and Nutrition in the World 2023 indicate that hunger affected a staggering number of individuals ranging from approximately 691 million to 783 million people in 2022, with an approximate midrange of 735 million people. Therefore, there is a need to transform food systems, including storage and food waste management, to provide affordable and healthy diets in order to achieve food security and nutrition goals. This, thus, underscores that addressing postharvest loss is essential to improving food security and sustainability, according to the World Bank.
Developing nations often face severe postharvest losses due to inadequate storage and poor transportation systems. According to the World Bank, Sub-Saharan Africa faces about 45% postharvest loss in horticultural crops, thus highlighting the severe challenges in preserving agricultural produce due to inadequate storage and poor transportation systems 6. Additionally, the FAO estimates that postharvest losses for horticultural crops in the region are about 30–40%. Furthermore, the African Postharvest Losses Information System (APHLIS) estimated that between 20 to 25% of horticultural crops are lost due to handling and storage losses in Sub-Saharan Africa. These losses, encompassing both food wastage and deterioration, impose a significant economic burden on farmers and retailers, while also straining the entire food supply chain, thus exacerbating food insecurity and environmental degradation. Microbial activity, particularly fungal activity, has been identified as a major contributor to postharvest losses. For instance, in 2014, the Kenyan government disposed of approximately 14,000 tonnes of maize contaminated with aflatoxins, a type of mycotoxin produced by fungal infestation in crops. Mycotoxins are highly toxic and pose significant health risks to both humans and animals. The detrimental impact of microbial action has led to the widespread use of synthetic fungicides, which, despite their efficacy, are harmful to human health and the environment. These fungicides are commonly used in underdeveloped and developing countries to manage postharvest diseases affecting produce such as fruits and vegetables.
Fungi are ubiquitously in the environment and have the ability to colonize and degrade different harvested agricultural products. The economic implications of fungal activity are significant, as they compromise the market value of food resources and pose risks to food safety and human health. For example, Botrytis sp. is a well-known pathogen causing gray mold, leading to significant losses in crops like grapes, strawberries, and tomatoes. Penicillium sp. is responsible for blue mold rot, affecting fruits such as apples, pears, and citrus, resulting in substantial economic losses during storage and transportation. Mucor sp. is implicated in the soft rot of various fruits and vegetables, causing considerable postharvest decay. Pilidiella granati is known to cause heart rot in pomegranates, affecting the marketability and economic value of the crop. Consequently, there is an urgent need for safe and environmentally friendly antimicrobial interventions to combat fungal-induced postharvest losses. Although biological agents, chemical fungicides, and natural compounds have been used to mitigate fungal infestations, issues such as antimicrobial resistance, environmental concerns, and the need for novel fungicides necessitate further research.
In recent years, the use of nanoparticles (NPs) has emerged as a promising platform for microbial control in both medicine and agriculture. Metal oxide nanoparticles, particularly zinc oxide nanoparticles (ZnO NPs), have demonstrated significant antimicrobial properties, including antifungal activity. Zinc oxide nanoparticles are favored due to their safety profile, making them suitable for applications in food, agriculture, and medicine. Research has highlighted their potential in various biological applications, including antioxidant, antimicrobial, and anticancer properties. These nanoparticles have semiconducting properties and band gaps between 3.1 and 3.3 eV, making them suitable for biosensor fabrication. Additionally, they are known for their excellent heat resistance and low electrical conductivity.
The green synthesis of ZnO NPs using plant extracts has gained popularity due to its environmental friendliness and biocompatibility. This synthesis route typically involves three essential components: a reducing agent, a stabilizing agent, and a solvent medium. Plant extracts act as both reducing and stabilizing agents due to the presence of various phytochemicals such as phenolic compounds, terpenoids, and amides. These extracts facilitate the reduction of Zn2+ ions by transferring electrons via an extracellular enzyme, NADH reductase, leading to the formation of ZnO NPs.
Recent studies have demonstrated the antifungal properties of ZnO NPs synthesized using various plant extracts. For instance, seed extracts of Trachyspermum ammi have been used to mediate ZnO NP formation, exhibiting remarkable antifungal activity against the pathogen Rhizoctonia solani. Similarly, ZnO NPs synthesized using extracts from Beta vulgaris, Cinnamomum tamala, Cinnamomum verum, and Brassica oleracea have shown effectiveness against microbial strains including Candida albicans and Aspergillus niger.
Therefore, in this report, ZnO NPs were synthesized using aqueous leaf extracts from four medicinal plants, including Syzygium cordatum, Lippia javanica, Bidens pilosa, and Ximenia cafra, whose metabolites have already been profiled, confirming the presence of useful phytochemicals in our previous report. The synthesized nanoparticles were characterized and their antifungal efficacy was evaluated against Botrytis sp., Penicillium sp., Mucor sp. and Pilidiella granati. Additionally, molecular docking studies were conducted to explore the interactions of ZnO NPs with druggable targets, comparing them to the standard fungicide propiconazole. This present work will thus provide insight into the plant-mediated synthesis and application of ZnO NPs against postharvest fruit and vegetable fungi, which can improve their production and consequently result in economic gains in the food industry.
Experimental methods
Materials
All chemicals used, including zinc nitrate, were purchased from Sigma Aldrich and used without further purification. Leaf samples of Syzygium cordatum (1), Lippia javanica (2), Bidens pilosa (3), and Ximenia cafra (4) were collected from the Eswatini Institute for Research in Traditional Medicine, Medical and Indigenous Food Plants (EIRMIP) farm at Mafutseni (026°23´S and 031°31´E) in the Kingdom of Eswatini. A taxonomist identified the plants, and the herbarium specimens were archived as KN1001 (B. pilosa), KN1002 (L. japonica), KN1003 (S. cordatum), and KN1004 (X. cafra). The leaves were oven-dried using a Labotec oven (Labotec (Pty) Ltd, Bavaria, South Africa) at 40 °C for 72 h. After drying, the leaves were ground into a fine powder through a 1 mm sieve using a Kambrook blender (Kambrook, Zhejiang, China) and stored in airtight containers until further use.
Extraction of plant material and green synthesis of zinc oxide nanoparticles
The aqueous extracts of the respective plant materials were obtained using the procedure reported by Bhuyan et al., with slight modifications. Briefly, 5 g of each plant powder was mixed in a 250 mL Erlenmeyer flask with 100 mL distilled water and heated to 60 °C for 10 min with agitation. The resulting mixture was cooled to room temperature and filtered through Whatman No.1 flter paper for further use as in our previous reports.
The synthesis of ZnO-NPs from each respective leaf extract followed the procedure described by Mishra et al.. A 100 mL volume of 2 M zinc nitrate dihydrate solution was prepared with deionised water in a flask and heated at 80 °C until a clear solution was obtained. This was followed by the dropwise addition of 25 mL of each plant extract under continuous stirring. Subsequently, 20 mL of 0.05 M NaOH was slowly added to the solution with continuous stirring. The reaction was maintained at 40 °C for 2 h until an off-white precipitate was formed. The pale white solid pellets were collected through centrifugation at 8000 rpm at 30 °C for 15 min, washed three times with distilled water and once with 100% ethanol, and then oven-dried at 100 °C for 12 h. The dried material was then heated at 450 °C for 4 h. The obtained powder material was cooled to room temperature and stored in air-tight bottles pending further analysis. The zinc oxide nanoparticles (ZnO-NPs) derived from the leaf extracts of Syzygium cordatum, Lippia javanica, Bidens pilosa, and Ximenia cafra were designated as ZnO 1, ZnO 2, ZnO 3, and ZnO 4, respectively.
Characterization of biosynthesized zinc oxide nanoparticles
X‑ray diffraction
X-ray diffraction (XRD) of the synthesized ZnO-NPs was carried out on the Bruker D8 Advance diffractometer (X’Pert PRO-PAN Analytical, Europe) with Cu-K radiation (40 kV, 20 mA) and diffracted beam monochromator. Scanning of compounds was done in the region of 2 θ from 20° to 90°.
UV–visible spectroscopy
UV–vis spectroscopy was used to monitor the reduction of zinc ions to form the ZnO nanoparticles. The synthesized ZnO-NPs were evaluated using Perkin Elmer Lambda 25 UV/Vis spectrophotometer for the absorption measurement in the range of 200–700 nm. The energy gap (E) of the ZnO-NPs was calculated using the following formula: E=hc/λ. where:
λ is the wavelength; h=6.626× 10–34 Js (Plank’s constant); and c=3× 108 m/s.
Zeta potential measurement
The particle size distribution and the average size of the ZnO-NPs were carried out using a Malvern Zeta sizer Nano series analyzer (Malvern Zetasizer, Nano Z500 UK). Briefly, the samples were diluted using Milli-Q water, centrifuged at 10,000×g, and the filtrate was transferred to a cuvette for analysis. For the zeta potential of the bio-inspired zinc oxide nanoparticles, the water dispersant method was used.
Fourier transform infrared spectroscopy
Fourier transform infrared spectroscopy (FTIR) analysis of the green synthesized ZnO nanoparticles was carried out using a Spectrum Two Perkin Elmer. The FTIR spectra transmittance was set at a resolution of 4 cm−1 and recorded in the wave range of 500–4000 cm−1.
Scanning electron microscopy
The surface morphology of the ZnO-NPs was observed using scanning electron microscopy (SEM, SU8010, Hitachi, Japan) at 10 kV. The samples were mounted on double-sided carbon tape and were thoroughly dried by exposing them to a mercury lamp for 5 min. The surface structures of the ZnO-NPs were then viewed and determined using scanning electron microscopy.
Transmission electron microscopy
The size and morphology of the respective ZnO-NPs were further examined by transmission electron microscopy (TEM), operating at 200 kV. The different zinc oxide nanoparticle samples were prepared by suspending them in 2-propanol and sonicating them for 30 min. Ten a drop of the colloidal suspension was mounted onto a carbon-coated copper grid and evaporated under a vacuum. The size and morphology of the ZnO-NPs were then examined under TEM.
Antifungal evaluation of bio‑synthesised ZnO‑NPs
Fungal isolates
Pathogenic plant fungi, namely Botrytis sp. (STEU 7866), Penicillium sp. (STEU 7865), and Pilidiella granati (STEU 7864), were obtained from the Stellenbosch University’s Department of Plant Pathology. The plant pathogen Mucor sp. was isolated from raspberry fruits with symptoms of fruits rotting by pulping the fruit and plating on potato dextrose agar (PDA, Sigma Aldrich, Co). Fast growing fungal species were then consistently isolated and identified as described by Campbell and Johnson and Lopez et al.. All fungal isolates were subsequently grown and maintained on PDA agar.
In vitro antifungal efficacy of nanoparticles
Assessment of fungal susceptibility to the synthesised ZnO-NPs was performed as described by Adjou et al., using the poisoned food technique. Potato dextrose agar was autoclaved (20 min, 121 °C) and cooled to 45 °C. The zinc oxide nanoparticles (ZnO-NPs) designated as ZnO 1, ZnO 2, ZnO 3, and ZnO 4 derived from the leaf extracts of Syzygium cordatum, Lippia javanica, Bidens pilosa, and Ximenia cafra, respectively, were each added to the molten agar to give a final concentration of 200 ppm. The resulting media was aseptically poured into sterile Petri dishes (9 cm) and allowed to cool. Mycelial disks (6 mm) were obtained from 7-day old cultures of each of the four test pathogens using a sterile cork borer and placed on the center of the petri plates. Each of the four ZnO-NPs was tested against each of the four fungal pathogens, and each combination was performed in triplicate. Negative control plates did not contain any antifungal, while the commercial antifungal effecto ® was used as a positive control. Inoculated plates were incubated for 5 days at 25 °C in the dark. Assessment of the fungal growth area on the Petri dishes was done using ImageJ software v 1.48. The susceptibility of each fungal strain was then calculated using the following equation;
where R is the area of growth of fungal mycelia in the negative control, and r is the area of mycelial growth in each of the ZnO-NPs-challenged plates.
Following the susceptibility testing, the fungal species showing the highest resistance was further evaluated against each of the four ZnO-NPs at different concentrations. Specific initial concentrations of each ZnO-NP (50, 100, 200 and 400 ppm) were prepared by adding appropriate amounts of nanoparticle to cooled molten PDA (45 °C) with gentle shaking to disperse the nanoparticle in the medium. The mixture was then poured into sterile petri dishes (9 cm in diameter) under aseptic conditions and allowed to cool. Agar discs (6 mm in diameter) with mycelia from the previously selected fungi (7-day old) were cut from the periphery of actively growing regions using a sterile cork borer and inoculated aseptically at the centre of the petri plates. Negative and positive control plates (without the ZnO-NPs and with effecto®, respectively) were inoculated following the same procedure. Tree replicates were maintained for each treatment, and the readings for the mycelia growth area were taken after five days of incubation at 25 °C. The percentage inhibition of the mycelial growth of the test fungi by the ZnO-NPs was calculated as previously described above.
Broth microdilution assay
The minimum inhibitory concentration of the ZnO-NPs was determined using the broth microdilution assay described by Loo et al.. The preparation of the inoculum for Botrytis sp., Penicillium sp. and Pilidiella granati was done by first sectioning a 5×5 mm agar block from 5-day-old fungal cultures. The section blocks were then aseptically transferred to potato dextrose broth (PDB) containing 0.1% (v/v) Tween 80. After a 72 h incubation, the fungal suspensions were vortexed and adjusted to match the turbidity of a 0.5 McFarland standard corresponding to 1–5×106 CFU/mL. A further 1:10 dilution of the adjusted suspension was made, resulting in a working suspension with a final inoculum size of approximately 1–5×105 CFU/mL. The ZnO 1, ZnO 2, ZnO 3, and ZnO 4 stock solutions were prepared by dissolving in PDB. Two-fold serial dilutions of each ZnO-NP were then made in 96-well plates at double the desired strength for each well with a volume of 100 µL. The PDB was prepared with 0.002% (w/v) resazurin as a fungal growth indicator. Each of the wells with the test substance was then inoculated with 100 µL of the adjusted fungal suspension. The ZnO-NPs’ concentration ranged from 200 to 1.563 µg/mL after adding the inoculum. Fluconazole (200–1.56 µg/mL) was used as the positive control, while sterility and growth control well contained PDB and the inoculum, respectively. after closing the lids, the plates were sealed with parafilm and incubated at 30 °C for 30 h. After incubation, growth in the plates was visually observed, with minimum inhibitory concentrations (MIC) being defined as the lowest concentration of each ZnO-NP, which prevented visible mycelial growth compared to the growth control. Wells with inhibition remained pink, while clear wells indicated fungal activity (growth). Values were expressed as mg/mL.
Molecular docking
Three druggable targets obtained from three fungi Botrytis sp.: ene-reductase OYE4A (PDB ID: 7BLF); Penicillium sp.: 3,4-Dihydroxy-2-butanone 4-phosphate synthase (PDB ID: 1G57), and Mucor sp.: mevalonate 5-diphosphate decarboxylase (PDB ID: 1FI4). They were selected as drug targets in these fungi species based on their critical roles in the survival of these organisms. The 3D structures of these targets, alongside their co-crystallized ligands, were retrieved from the Protein Data Bank (http://www.rscb.org). A standard anti-fungal drug, propiconazole, was used as a reference, and its binding modes to the 7BLF, 1G57 and 1FI4 receptors were compared with the ZnO-NPs. Autodock tools and other software were used to investigate the activity in binding affinity (Kcal/mol). Afterward, the outcomes were compared in binding affinity score for best-docked conformation.
Statistical analysis
Data was subjected to statistical analysis using R software (version 4.1.2; 2021). Antifungal data was presented as mean±SEM. A two-way analysis of variance (ANOVA) was used to check for interaction between the main effects of type of ZnO-NP and type of fungi, and then, between type of ZnO-NP and concentration. Tukey’s post-hoc test was used to separate treatment means. differences were considered significant at p< 0.05.
Results and discussion
Synthesis of ZnO‑NPs using leaf extracts of Syzygium cordatum, Lippia javanica, Bidens pilosa, and Ximenia cafra
The biosynthetic process of converting metal salt to ZnO-NPs using the plant extracts is depicted in Fig. 1. Typically, the synthesis of metal-based nanoparticles using plant extracts involves three reaction stages: a brief incubation period, a growth phase, and a termination period. The nucleation phase, which results in the formation of smaller particles, occurs at a faster rate than the particle growth phase. Bioactive compounds in the extracts, such as polyphenols, facilitate the reduction of Zn2+ metal salt through electron transfer from NADH via the extracellular enzyme known as NADH reductase, which acts as an electron carrier in the formation of ZnO-NPs. Functional groups within the active components of the plant extracts, including amide, amino, and carboxyl groups, possess capping and stabilizing properties that prevent aggregation and agglomeration of the biosynthesized ZnO nanomaterials. Additionally, the presence of π-electrons and carbonyl groups in the molecular structures of these nanoparticles has been linked to the absorption of reducing agents on their surfaces.
Figure 1. Biosynthesis scheme of ZnO-NPs using leaf extracts of Syzygium cordatum, Lippia javanica, Bidens pilosa, and Ximenia cafra.
X‑ray diffraction (XRD) analysis
The XRD diffraction patterns for each synthesized ZnO-NPs are shown in the combined diffractogram in Fig. 2. The indexed peaks in the diffractogram for all the synthesized ZnO particles, using different leaf extracts, have been fully identified as those of bulk ZnO ((JCPDS) Card No. 36–1451). This indicates the presence of the wurtzite crystal structure with distinct diffraction peaks corresponding to the 100, 002, 101, 102, 110, 103, 112, 201 and 202 crystalline planes of hexagonal ZnO. Furthermore, the sharp narrow peaks, along with the absence of unwanted peaks, indicate a high degree of sample crystallization and a lack of impurities. The average size D of the synthesized nanomaterials was determined using Debye Scherrer’s (Eq. (1)):
In this equation, D represents the average size of the crystalline particles in the synthesized ZnO-NPs. The Scherrer constant (k) is 0.93, λ represents the X-ray wavelength utilized (1.5406 Å), β denotes the full width at half-maximum (FWHM) of the peak, and θ represents the Bragg angle. Using this equation, the estimated average sizes were found range between 25 nm (for L. javanica) to 43 nm (for B. pilosa). The average crystalline sizes observed in this study are consistent with those reported in the literature for ZnO synthesized from Mentha pulegium leaf extract (approximately 44.94 nm) and Acacia caesia bark extract (32.32 nm).
Figure 2. XRD diffraction pattern for the ZnO-NPs derived from Syzygium cordatum, Lippia javanica, Bidens pilosa, and Ximenia cafra leaf extracts.
Fourier‑transform infrared spectroscopy (FTIR) analysis
The FTIR spectra collected for all the biosynthesized zinc oxide nanoparticles, designated ZnO 1, ZnO 2, ZnO 3, and ZnO 4, are presented in Fig. 3. Te spectra all showed small bumps around 3500 and 3275 cm-1, which was more pronounced in ZnO 3, attributed to the hydrogen bond stretching vibrations, indicating the presence of OH or H2O, which may be due to humidity upon exposure to the atmosphere, as suggested by Gatou et al.46. It is worth mentioning that this is unexpected after calcination. Furthermore, other vibration bands associated with the peak between 1780 cm-1 and 1658 cm-1 on ZnO 2 could be –C=C– stretching of aromatic rings. Furthermore, the peaks between 1470 cm-1 and 1300cm-1 have been attributed to the presence of the C=O bond and O–H bending. The peaks at around 1020 cm-1 on ZnO 2 and ZnO 3 correspond to the -OH deformation of tertiary alcohols and C–N or C–O bond stretching. The bands identified between 887 cm-1 and 794 cm-1 could relate to the aromatic C-H bending and carboxylic acid. These bonds originate from the plant extract and help stabilize the formation of ZnO nanoparticles, as reported in the literature43. As previously indicated in the literature, peaks with wavenumbers below 500 cm-1, sometimes appearing as a shoulder peak, were observed in the acquired spectra of all the prepared nanoparticles. This observation suggests the presence of a metal–oxygen vibrational band, indicating the formation of zinc and oxygen bonding vibrations. It is worth noting that other studies utilizing different plant extracts such as lemon peel extract and Cymbopogon citratus leaf extract51 have also reported a ZnO peak below 500 cm-1.
UV–visible spectroscopy absorption analysis
Secondary metabolites in plant extracts act as reducing and stabilizing agents, facilitating the reduction of zinc ions to zinc oxide nanoparticles. The absorption spectra (Fig. 4) revealed a strong quantum confinement effect with respect to the bulk ZnO-NPs, showing an absorption shoulder at around 375 nm, which corresponds to an average bandgap of 3.32 eV estimated using the Tauc plot (inset) for all the extracts. A similar observation was reported by previous studies for ZnO-NPs synthesized from an aqueous extract of Deverra tortuosa. The slight shift in the absorption shoulder from one extract to another can be attributed to the different secondary metabolites in the different medicinal plants. A similar observation has been documented in the case of ZnO synthesized using lemon peel extracts, exhibiting a maximum optical absorption band at 358 nm. Likewise, the application of pomegranate peel and seed extracts resulted in the observation of maximum absorption bands at 369 and 372 nm, respectively.
Zeta potential analysis
The zeta potential results of the biosynthesized ZnO-NPs are summarized in Table 1. According to the literature, the zeta potential of biosynthesized nanoparticles depends on the magnitude of the electrostatic charge repulsion of the synthesized nanoparticles in the medium. The zeta potential values for the synthesized ZnO-NPs are 15.4 mv, 24.3 mv, 9.5 mv, and -18.5 mv for ZnO 1, ZnO 2, ZnO 3 and ZnO 4, respectively. As indicated in the literature, zeta potential values of nanoparticles greater than+30 mV or smaller than -30 mV result in stable suspensions with strong repulsive forces between the particles in solution, thereby minimizing aggregation between the nanoparticles. The zeta potential results for ZnO 1, ZnO 2, ZnO 3, and ZnO 4 were all outside the range of+30 mV and -30 mV, indicating weak repulsive forces and limited stability in the solution.
Figure 3. FTIR spectra of ZnO 1, ZnO 2, ZnO 3, and ZnO 4 were synthesized using Syzygium cordatum, Lippia javanica, Bidens pilosa, and Ximenia cafra leaf extracts.
Figure 4. Absorption spectra of ZnO-NPs synthesized using Syzygium cordatum (ZnO 1), Lippia javanica (ZnO 2), Bidens pilosa (ZnO 3), and Ximenia cafra (ZnO 4) extracts. Moreover, the inset shows a representative plot of Tauc’s plots of ZnO 1.
Table 1. Summary zeta potential of ZnO-NPs synthesized using Syzygium cordatum (ZnO 1), Lippia javanica (ZnO 2), Bidens pilosa (ZnO 3), and Ximenia cafra (ZnO 4) extracts.
Morphological studies using TEM and SEM
The morphology of the ZnO nanoparticles, mediated using the various leaf extracts, was investigated using SEM and TEM micrographs, as presented in Figs. 5 and 6. The SEM images showed that the prepared nanoparticles appeared rounded and tended to agglomerate into different clusters, as illustrated in Fig. 5A–D. Furthermore, the Transmission electron microscope (TEM) images shown in Fig. 6A–D revealed that all the nanoparticles possessed a spherical shape with varying sizes and degrees of agglomeration, similar to another report in the literature. From the histogram distribution plots presented in Fig. 7, which were plotted using ImageJ, the average sizes of the biosynthesized ZnO-NPs from the different medicinal plant extracts were estimated to be 47.50, 46.62, 44.02, and 37.47 nm for the ZnO-NPs synthesized using Syzygium cordatum (ZnO 1), Lippia javanica (ZnO 2), Bidens pilosa (ZnO 3), and Ximenia cafra (ZnO 4) extracts, respectively.
Figure 5. Scanning electron microscope micrograph of ZnO-NPs synthesized using (A) Syzygium cordatum (ZnO 1), (B) Lippia javanica (ZnO 2), (C) Bidens pilosa (ZnO 3), and (D) Ximenia cafra (ZnO 4) extracts.
Figure 6. Transmission electron microscope micrographs of ZnO-NPs synthesized using (A) Syzygium cordatum (ZnO 1), (B) Lippia javanica (ZnO 2), (C) Bidens pilosa (ZnO 3), and (D) Ximenia cafra (ZnO 4) extracts.
Susceptibility of fungi to synthesised ZnO‑NPs
In vitro susceptibility testing showed significant interaction between the type of nanoparticle and fungal species (p≤0.05; Table 2). This indicated that the respective ZnO-NPs showed different inhibition intensities depending on the fungal species. The highest susceptibility was shown by Mucor sp. in response to ZnO 2, with the other ZnO-NPs also showing inhibition greater than 50% against the same fungi. Studies by Ouzakar et al. reported that ZnO nanoparticles synthesised by microalgal extracts inhibited the growth of Mucor heimalis responsible for rot in sweet cherries. The inhibition potency of ZnO 2 at 200 ppm against the Mucor sp. was similar to that observed by Ouzakar et al. at 1000 ppm, highlighting the effectiveness of the ZnO-NPs synthesised in this study. The observed differences in potency may be attributed to internal factors such as particle size, with ZnO 2 having a smaller average size (37.47 nm) compared to the nanoparticles applied by Ouzakar et al., which ranged in size from 39 to 51 nm.
Penicillium sp. also showed high susceptibility against all four ZnO-NPs, which was not significantly different from the susceptibility of Mucor sp. vs ZnO 2. Earlier research by He et al. showed that Penicillium expansum was sensitive to ZnO-NP treatment, as the nanoparticles prevented the development of conidiophores and conidia. This inhibition results in the inability of the fungus to produce and disseminate large numbers of asexual spores, leading to the death of fungal hyphae.
Botrytis sp. and Pilidiella granati showed the least susceptibility to the ZnO-NPs under evaluation. He et al. also observed the low susceptibility of Botrytis sp. to ZnO-NPs. Research has shown that Botrytis sp. has developed multiple mutations in target genes, resulting in multiple resistance and overexpression of efflux transporters, rendering multiple drug resistance. However, metallic nanoparticles have shown high efficacy in inhibiting the functioning of efflux pumps, which diminishes the microbial capacity to expel toxins and waste from the cell.
Figure 7. Particle size distribution of ZnO-NPs synthesized using (A) Syzygium cordatum (ZnO 1), (B) Lippia javanica (ZnO 2), (C) Bidens pilosa (ZnO 3), and (D) Ximenia cafra (ZnO 4) extracts.
Table 2. Susceptibility testing of Botrytis sp., Penicilium sp., Mucor sp. and Pilidiella granati to Syzygium cordatum (ZnO 1), Lippia javanica (ZnO 2), Bidens pilosa (ZnO 3) and Ximenia cafra (ZnO 4) zinc oxide nanoparticles at 200 ppm. 1Standard error of mean. 2Nanoparticle. 3Fungi. 4Nanoparticle and Fungi interaction; Least square means with different superscripts are significantly different (P≤0.05). 5Not significant. ***p≤0.001.
Further evaluations were made of ZnO-NPs’ antifungal effects against Botrytis sp., which displayed the least susceptibility. Inhibition of Botrytis sp. mycelial growth was influenced by the type of nanoparticle×concentration interaction (p≤0.05). ZnO 2 at 400 ppm was observed to have the greatest inhibitory potency, with no significant difference from ZnO 2 at 200 ppm (Table 3, Fig. 8). The observed higher potency of ZnO 2 can be attributed to its larger positive zeta potential compared to the other nanoparticles. According to Clogston et al., the zeta potential of nanoparticles influences their attraction to the negative charge present on most cell membranes. This, in turn, influences the ability of the nanoparticles to permeate the membrane and disrupt the functioning of the cell wall. Furthermore, zeta potentials greater than+30 mV and less than − 30 mV are considered strongly cationic and anionic, respectively. As such, the larger cationic zeta potential exhibited by ZnO 2 translates to stronger membrane binding and greater cellular uptake. On the other hand, a zeta potential between − 10 and+10 mV points to a neutral nanoparticle, which could account for the observed lower inhibitory activity for ZnO3.
Table 3. Effect of Syzygium cordatum (ZnO 1), Lippia javanica (ZnO 2), Bidens pilosa (ZnO 3), and Ximenia cafra (ZnO 4) zinc oxide nanoparticles at different concentrations against Botrytis sp. growth inhibition (%). 1Standard error of mean. 2Nanoparticle. 3Concentration. 4Nanoparticle and Concentration interaction; Least square means with different superscripts are significantly different (p≤0.05). ***p≤0.001.
Figure 8. Agar plates showing the effect of Syzygium cordatum (ZnO 1), Lippia javanica (ZnO 2), Bidens pilosa (ZnO 3), and Ximenia cafra (ZnO 4) zinc oxide nanoparticles at 50, 100, 200 and 400 ppm against Botrytis sp.
The MIC values for the ZnO-NPs ranged from 25 to 200 µg/mL, with ZnO 2 being the most effective (Tabe 4). The observed higher potency of ZnO 2 can be attributed to its larger positive zeta potential compared to the other nanoparticles. According to Clogston et al., the zeta potential of nanoparticles influences their attraction to the negative charge present on most cell membranes. This, in turn, influences the ability of the nanoparticles to permeate the membrane and disrupt the functioning of the cell wall. Furthermore, zeta potentials greater than+30 mV and less than − 30 mV are considered strongly cationic and anionic, respectively. As such, the larger cationic zeta potential exhibited by ZnO 2 translates to stronger membrane binding and greater cellular uptake. On the other hand, a zeta potential between − 10 and+10 mV points to a neutral nanoparticle, which could account for the observed lower inhibitory activity for ZnO 3.
Apart from the zeta potential, the smaller size of the ZnO 2 favours greater cellular uptake, where they can exert inhibitory capacity. This is supported by observations from Padmavathy & Vijayaraghavan, who assert that there is a strong inverse relationship between antimicrobial efficacy and particle size of ZnO-NPs. Once inside the cellular matrix, the ZnO-NPs exert antifungal effects by generating reactive oxygen species (ROS) and through the poisoning effect of released Zn2+ ions. The ROS, generated via the photocatalytic and photo-oxidising capacity of ZnO, disrupt fungal cellular structures, inhibit biological macromolecular activity, and prevent DNA replication. This accounts for the observed dose-dependent antifungal effects for all the nanoparticles evaluated. Furthermore, the nanoscale effect translates to a large surface area from which ROS can be generated. This aligns with research by Miri et al., who observed a dose-dependent activity of ZnO-NPs against Candida albicans.
Table 4. MIC values (µg/mL) for Syzygium cordatum (ZnO 1), Lippia javanica (ZnO 2), Bidens pilosa (ZnO 3), and Ximenia cafra (ZnO 4) zinc oxide nanoparticles against Botrytis sp.
Molecular docking discussion
Molecular docking studies based on structure-based drug design (SBDD) were explored to understand the drug target (fungi receptors) with which the ZnO-NPs have the highest affinity and possibly predict the mechanism of action. The binding free energy, ΔG (kcal/mol) of ZnO-NPs and propiconazole against different fungal drug targets are presented in Table 5.
The results revealed a high binding affinity of the standard drug with all the receptors compared to ZnO-NPs. This finding is consistent with the in vitro assay results. However, the ZnO-NPs exhibited some degree of affinity with the receptors. The binding modes of the ZnO-NP and propiconazole with their respective receptors are shown in Fig. 9. It was observed that the ZnO-NPs fit very well into the same active binding sites of the receptors as propiconazole. The nanoparticles had a better binding affinity with Mucor sp. and Penicillium sp. than with Botrytis sp. Additionally, the ZnO-NPs formed a strong H-bond with 1FI4. The O-6 atom of ZnO-NPs interacted with the N-atom of ALA 119 of 1FI4 through a molecular interaction distance of 2.96 Å and an interaction energy of − 1.3 kcal/mol. There was also an ionic interaction between the O-2 atom of the nanoparticle and OD2 of ASP 302 with an interaction energy of 5.2 kcal/mol. Similarly, 1G57 from Mucor sp. formed numerous chemical interactions with the ZnO-NPs. There was a strong H-bond interaction between the O-7 atom of the nanoparticle and the OD2 of ASP 33 with a distance and energy of interaction of 3.44 Å and − 2.3 kcal/mol, respectively. Although the binding modes were similar, the mode of chemical interactions of the nanoparticle was quite different from propiconazole. The O-4 atom of propiconazole formed an H-bond with the N atom of HIS 153 with a distance and energy of interaction of 3.33 Å and − 0.9 kcal/mol. While the standard drug propiconazole exhibited better inhibition, the ZnO-NPs significantly inhibited the biochemical functions of these fungi, indicating their potential application against pathogenic fungi.
Conclusions
In this study, leaf extracts (Syzygium cordatum, Lippia javanica, Bidens pilosa, and Ximenia cafra) were used as mediating agents for the synthesis of ZnO-NPs, designated ZnO 1, ZnO 2, ZnO 3, and ZnO 4, respectively. Using well-documented physicochemical techniques, the characteristics of the synthesized nanoparticles were found to be consistent with the literature, showing the formation of a wurtzite crystal structure of ZnO NPs in all cases and a spherical morphology with varying sizes for all the leaf extracts, ranging between 37 to 47 nm in diameter. Furthermore, the antifungal evaluation demonstrated significant antifungal efficacy against pathogenic fungi responsible for postharvest decay but varies significantly with the different leaf extracts and fungal species, with Mucor sp. showing the highest susceptibility, particularly to ZnO 2, while Penicillium sp. also exhibited high susceptibility across all ZnO-NPs. Conversely, Botrytis sp. and Pilidiella granati showed the least susceptibility, although ZnO 2 at higher concentrations effectively inhibited Botrytis sp. due to its larger positive zeta potential and smaller particle size. In addition, the molecular docking studies confirmed the higher susceptibility of these fungi to the ZnO-NPs. Specifically, ZnO 2 synthesized using Lippia javanica extract with an MIC of 25 µg/ mL, was highly effective in reducing fungal growth. These results highlight the potential of the medicinal plant extracts in green nanotechnology for the control of fungal spoilage. The application of ZnO-NPs synthesized from these extracts offers a promising and environmentally friendly approach to managing postharvest pathology and reducing decay in horticultural commodities. Therefore, based on our findings, it can be concluded that the production of ZnO nanoparticles using plant extracts, particularly Lippia javanica extract, presents a promising solution for combating emerging and resistant plant pathogens. Furthermore, their application for postharvest preservation on fruits and vegetables can effectively manage emerging diseases and reduce waste in an environmentally sustainable manner, thereby addressing the increasing global demand for food. This approach can thus significantly improve food security by minimizing postharvest losses and ensuring the safety and quality of produce during storage and transportation.
Table 5. Binding free energy with drug targets, ΔG (kcal/mol).
Figure 9. Binding modes of ZnO NPs (cyan) and propiconazole (yellow) in the active binding sites of (A) 7BLF (B) 1G57 (C) 1FI4.
