1 Introduction

Plant diseases are considered one of the most agricultural problems around the worldwide, affecting agricultural production as well as the quality of the product [1]. There are some diseases affecting the plant, especially economic crop plants which negatively affect the plant at different stages of plant growth, as well as the plant production of crop significantly [2, 3]. Root rot and wilting diseases are caused by a group of fungi latent in the soil [4, 5]. One of these diseases is Fusarium wilt disease which causes a clear impact on plant’s autoimmunity and thus affects the efficiency and quantity of the crop during the planting season [6,7,8]. The pathogen spreads within the infected plant starting with the roots during the planting process and blocking the vessels which transport water and nutrients from the root to the rest of the plant parts causing the plant and leaves to dissolve [8]. It is difficult and harmful to completely control chemically, so the most appropriate solution was the trend to use a high efficiency biological method to control Fusarium wilt disease as well as be less expensive and ecofriendly [9,10,11,12,13]. Recently, the phenomenon of climatic changes has exacerbated, which has led to the rapid spread of plant pathogens [14]. The attention of researchers has turned to finding safe and environmentally friendly solutions to reduce the spread of plant diseases as safe alternatives to chemical pesticides [15,16,17,18,19,20].

The use of nano-biotechnology in resisting pathogens and stimulating plant physiological immunity showed important and strong results in combating fungal diseases because it enables the plant to improve systemic immunity and disease resistance and increase productivity [21,22,23]. Biological approaches to NPs and nanocrystal synthesis have been extended to intact biological particles [24, 25]. In the last period, biological synthesis of metal NPs using plant, bacteria, and fungi has received great much attention due to it being safe as well as ecofriendly [26,27,28,29,30,31]. The utilization of fungal platforms as promising candidates for the synthesis of nanomaterials had been reported in the recent applications [32,33,34,35], due to the enrichment of different secondary metabolites in the fungal extract which give the promising light for the green synthesis of different nanocomposites.

Previous studies reported that fungal extracts contain macromolecules such as phenolics, flavonoids, alkaloid carbohydrates, and tannins which have roles in biosynthesis of metal NPs as safe reducing and stabilizing agents [36]. Zinc is an essential element in plant growth and oxidation of sugars in plants and has a major role in the formation of chlorophyll and in photosynthesis [37,38,39,40]. It is worth noting that several studies have demonstrated the role of nano-zinc in inducing plant immunity against fungal disease [41, 42]. Metal oxide NPs such as ZnO and CuO NPs possess a promising application in biological fields due to the reduced toxicity and bioactivity. For example, the fabricated carboxymethyl cellulose-ZnO NP films could be effectively utilized as protective edible coating films of food products [43]. Additionally, the supplementation of ZnO NPs to broiler diet at 40 or 60 mg/kg improved productive performance and birds’ physiological status, and the lower level Zn (40 mg/kg diet) revealed promising results and can be used as an effective feed additive in broilers [44]. Finally, the promising wound healing [45], anti-inflammatory [46], food packaging [47], antimicrobial activity [48, 49], and acetylcholinesterase inhibition potentials of the synthesized ZnO nanocomposites were noted in the recent publication [50].

The main objective of this research was to myco-synthesize bimetallic ZnO-CuO NPs using Aspergillus fumigatus and compare it with monometallic ZnO NPs and CuO NPs as biofungicide against Fusarium oxysporum that causes Fusarium wilt disease and also to study the effect of ZnO-CuO NPs, ZnO NPs, and CuO NPs on F. oxysporum under transmission electron microscope.

2 Materials and methods

2.1 Materials

Potato dextrose agar (PDA) and potato dextrose broth (PDB) were purchased from Oxoid, England. Zinc nitrate and Copper sulphate, isopropyl alcohol, lead citrate and uranyl acetate, ethyl alcohol, and glutaraldehyde were purchased from Sigma-Aldrich, Germany.

2.2 Isolation and screening of fungi

Fungi were isolated from soil samples collected from Menoufia and Qalyubiyya Governorates. All soil samples were cultured on potato dextrose agar (PDA) plates and incubated at 28 ± 2 °C for 6 days. Purification of fungi was carried out using PDA plates where each fungus re-cultured on agar surface of PDA [51,52,53]. All fungal isolates were cultured on potato dextrose broth (PDB) and incubated at 28 ± 2 °C, 150 rpm for 7 days. Then, the biomass of fungi was separated using filtration method at the end of incubation period. Whatman filter paper no. 1 was used for the filtering process in order to produce filtrates free of cells. For the production of ZnO, CuO, and ZnO-CuO NPs, all fungal filtrates were utilized. All prepared solutions were tested for biosynthesis of ZnO-CuO NPs, ZnO NPs, and CuO NPs using UV-Vis to select the most potent fungus which has a great ability to biosynthesize NPs.

2.3 Identification of the most potent fungus

Both morphological and genetic identification were carried out to identify the most potent fungal isolate. The morphological properties (color, texture, and look) of the fungi were observed using a light microscope, along with microscopic characteristics. Maxima Hot Start PCR Master Mix (Thermo; K1051) was used to conduct the PCR. According to the methodology employed by Visagie et al. [54], the primers utilized were forward ITS1-F (5′-TCCGTAGGTGAACCTGCGG-3′) and reverse ITS4-R (5′-TCCTCCGCTTATTGATATGC-3′). Phylogenetic tree was constructed using the MEGA11 software.

2.4 Preparation of ZnO NPs, CuO NPs, and bimetallic ZnO-CuO NPs

The biogenesis of ZnO-CuO NPs used the precise quantity of salts. More specifically, 10 ml of (2.0 mM) \(\mathrm{Zn}\;{\left({\mathrm{NO}}_3\right)}_2\) and 10 ml of (2.0 mM) CuSO4 were mixed for about 30 min at room temperature, and 80 ml of the fungal filtrate was then added to them. After creating the combined solution, we measured the pH of the mixture and discovered that it was 7.2. The reaction conditions were set as the incubation temperature set at 30 °C and reaction period approximately 24 h under agitation (500 rpm) in a shaking incubator in order to produce the most successful synthesis of ZnO-CuO NPs [55]. After the complete incubation, color shift was noticed and recorded it as a subtle brown, verifying the bio-formation of ZnO-CuO NPs.

2.5 Characterization of ZnO NPs, CuO NPs, and bimetallic ZnO-CuO NPs

The optical properties of the tested bimetallic ZnO-CuO NPs were examined using a UV-Vis spectrophotometer (JASCO V-560) set to a specified wavelength range between 190 and 900 nm. Using dynamic light scattering (DLS-PSS-NICOMP 380, USA), the generated bimetallic ZnO-CuO NPs’ moderate particle size distribution was evaluated. To determine the precise produced shape and the mean and accurate particle size to establish their formation at the nanomaterial scale, HR-TEM, JEM2100, JEOL, Japan, was used. While X-ray diffraction analysis (XRD-6000, Shimadzu Scientific Instruments, Japan) was used to determine the crystalline nature and the crystal size of the synthesized NPs, it was adjusted with the XRD-6000 lists, including outstanding austenite quantitation, crystallinity estimation, stress examination, and crystallite size/lattice strain matters. The investigation of the extended X-ray diffraction models employed Cu-Kα target and nickel filter. Working by a Cu anode at 50.0 mA and 40.0 kV in the state of 2θ value inside 20° and 100° with a flow of 2°/min, the intensity of the diffracted X-rays was estimated as a function of the diffracted angle 2θ. The final step was to evaluate the surface appearance and precise out-surface shape of the synthesized bimetallic ZnO-CuO NPs using SEM, ZEISS, EVO-MA10, Germany.

2.6 Control of F. oxysporum ZnO-CuO NPs, ZnO NPs, and CuO NPs

2.6.1 Pathogen, inoculum, and culture conditions

Fusarium oxysporum RCMB (008002) was purchased from The Regional Centre for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt. The Koch’s postulate was proven for the pathogen that was identified as the causative agent of Fusarium wilt of eggplant after F. oxysporum was grown on PDA plates and incubated for 3–5 days at 25 °C [56], and the pathogen was subsequently stored at 4 °C for further use.

2.6.2 In vitro assessment of antifungal activity and growth inhibition

Well diffusion method

The antifungal activity of biosynthesized ZnO NPs, CuO NPs, and ZnO-CuO NPs was evaluated using the well diffusion method according to the method used by Shubharani, Mahesh, and VN [57] with minor modifications. Fusarium oxysporum was inoculated onto PDB medium and cultured for 3–5 days at a temperature of 28 ± 2 °C. Fungal inoculum of F. oxysporum was distributed on the surface of PDA plates. Then, wells with 8 mm diameter were made using sterile cork-borer on each agar plate (90 mm). A total of 100 µl of each ZnO-CuO NPs, ZnO NPs, CuO NPs, copper acetate, and zinc acetate at concentration 1000 µg/ml was put in wells individually. Likewise, 100 µl of cell free filtrate was tested. To detect minimum inhibitory concentration for each compound, different concentrations of ZnO-CuO NPs, ZnO NPs, and CuO NPs (62.5–1000 µg/ml) were assessed. The culture plates were incubated at 25 °C for 5 days and the zones of inhibition were observed and measured.

Radial growth method

Different concentrations of ZnO-CuO NPs, ZnO NPs, and CuO NPs (62.5, 125, 250, 500, and 1000 µg/ml) were carried out for evaluation radial growth of F. oxysporum according to the method used by Joshi, De Britto, Jogaiah, and Ito [58] with minor modifications. Inhibition percentage of F. oxysporum growth was calculated using the following equation:

$$\mathrm{Inhibition}\;\mathrm{of}\;\mathrm{pathogen}\;\mathrm{growth}\;(\%)=\frac{\mathrm{growth}\;\mathrm{of}\;\mathrm{control}-\mathrm{growth}\;\mathrm{of}\;\mathrm{treated}}{\mathrm{growth}\;\mathrm{of}\;\mathrm{control}}\times100$$

Ultrastructure study of control and treated F. oxysporum

After washing control and treated F. oxysporum with distilled water and centrifuging 7-day-old cultures, samples were then fixed in 3% glutaraldehyde, rinsed in phosphate buffer, and post-fixed in potassium permanganate solution for 5 min at room temperature. Samples were dehydrated for 15 min in each ethanol dilution, ranging from 10 to 90%, and then for 30 min in absolute ethanol. Through a graded series of injections of epoxy resin and acetone, samples were finally immersed in pure resin. On copper grids, extremely thin pieces were gathered. After that, sections were doubly stained with lead citrate and uranyl acetate [59]. A JEOL-JEM 1010 transmission electron microscope operating at 70 kV was used at the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University [60].

2.7 Statistical analysis

All results were calculated as means of three replicates and standard deviations, and the data were then subjected to analysis of variance means using the Minitab 18 software.

3 Results and discussion

3.1 Isolation and identification of fungi

In the current study, thirty-one fungal isolates were isolated from soil samples. All fungal isolates were tested for ZnO-CuO NPs, ZnO NPs, and CuO NP biosynthesis using UV-Vis. Results revealed that, fungal isolate S20 was the most potent for biosynthesis of ZnO-CuO NPs, ZnO NPs, and CuO NPs (data not shown). Therefore, fungal isolate S20 was selected for further experiment; this fungal isolate was identified morphologically then genetically. Morphological identification illustrated that fungal isolate S20 appeared blue-green in color with growth diameter of 30–40 mm as shown in Fig. 1A. Moreover, the head appeared small as well as the vesicle, the phialides are uniserrate, conidiophore is hyaline and unbranched (Fig. 1B), and conidia are circular to oval in shape (Fig. 1C).

Fig. 1
figure 1

Identification of A. fumigatus S20 (A-D). A Surface culture on PDA. B Conidiophore and head under bright field microscope (400 ×). C Under bright field microscope (800 ×). D Phylogenetic tree with accession number OQ519856

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The ITS region of the fungal isolate S20 was used for molecular identification to support the morphological identification. Phylogenetic tree was built using the MEGA 11 software as shown in Fig. 4C. Based on alignment search tool (https://blast.ncbi.nlm.nih.gov), the ITS rRNA homology value of strain MTMA 5 showed similarity (98%) with A. fumigatus. The obtained sequence was recorded in GenBank under accession OQ519856.

3.2 Myco-synthesis of ZnO NPs, CuO NPs, and ZnO-CuO NPs

The fungal production of bimetallic ZnO-CuO NPs was observed in this study by a change in solution color to faint brown color, which operated as a reducing agent or capping agent to reduce zinc nitrate and copper sulfate into ZnO-CuO NPs and stabilize them in colloidal form. The bioactive metabolites from fungal extract serve as a capping agent, preventing nanoparticle aggregation and altering their biological activity [61, 62].

Kumaravel J. [63], indicated that the fungal metabolites from Metarhizium anisopliae have a higher content which help in the reduction of zinc and copper ions to bio-nano formulation of bimetallic ZnO-CuO NPs. The main active metabolites present in fungal extract are responsible for bio-reduction of NPs [64].

Šebesta, Vojtková, Cyprichová, Ingle, Urík, and Kolenčík [65] reported that, the fungal filtrate has enzymes which have ability to reduce metal ions to elemental metal (Mo) on a nanometric scale. During biosynthesis of metals, biomolecules can interact with metal ions to form complex electron transport pathways, where NADPH/NADH convert to NADP+/NAD+ [66, 67]. Due to the ease of downstream processing and biomass treatments, as well as higher productivity than bacteria, fungal production of NPs is a simple and easy technique [68].

3.3 Characterization of ZnO NPs, CuO NPs, and ZnO-CuO NPs

The capacity of the fungal filtrate to synthesize ZnO NPs, CuO NPs, and ZnO-CuO NPs was evaluated. The fungal filtrate first seemed to be brown, but as ZnO NPs, CuO NPs, and ZnO-CuO NPs were synthesized, the color changed to a faint brown, deep brown, and off-white, respectively. The generated light brown hue was attributed to the stimulation of the surface Plasmon resonance of biogenic ZnO-CuO NPs and provided a reliable spectroscopic signal of their manifestation [69].

The experimental peak was present in the spectra (Fig. 2) due to the O. D. (1.03; diluted five times), and the UV-Vis investigations revealed that the myco-produced ZnO-CuO NPs were small and visible at 375.0 nm. Finally, for the bio-produced CuO NPs, the (O. D.) was identified at 0.75 (diluted 5 times) at a specific wavelength at 320 nm as shown in Fig. 2. This is similar to the case for ZnO NPs, where the (O. D.) was found at 0.76 (diluted 5 times) at a specific wavelength at 400 nm.

Fig. 2
figure 2

UV-Vis spectrum of the myco-synthesized ZnO NPs, CuO NPs, and bimetallic ZnO-CuO NPs (diluted 5 times)

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The brown color’s intensity matched the produced fungal filtrate’s capacity to biosynthesize ZnO-CuO NPs [70, 71]. The intensity, dimension, morphological surfaces, structure, and dielectric properties of any manufactured NPs typically affect surface Plasmon resonance (SPR) [72, 73].

DLS examination was carried out to determine the hydrodynamic radius, particle size distribution, and polydispersity index (PDI) of biosynthesized ZnO NPs, CuO NPs, and ZnO-CuO NPs. The obtained results were compared with the HR-TEM investigation to determine the average size of these NPs [74]. As shown in Fig. 3A, the HR-TEM picture showed the spheroidal morphologies of considerably poly-dispersed ZnO NPs with common sizes ranging from 14.73 to 72.07 nm, and a mean diameter estimated to be 42.611.5 nm. The similar circumstance was seen in the biosynthesized CuO NPs, which were circular in form and ranged in size from 12.89 to 91.98 nm, with an average diameter of 50.922.1 nm (Fig. 3B).

Fig. 3
figure 3

HR-TEM images of the myco-synthesized ZnO NPs (A), CuO NPs (B), and bimetallic ZnO-CuO NPs (C)

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Finally, the HR-TEM image in Fig. 3C for the synthetic bimetallic ZnO-CuO NPs revealed that the particles were semi-spherical and that their sizes ranged from 11.85 to 93.58 nm, with an average diameter of 54.18 ± 1.9 nm. The provided poly-dispersed NPs were intended to decrease, stabilize, and act as capping agents for the generated fungal filtrate that was rich in protein and amino acids, among other things [75].

The line spacing was exactly the same, resulting in one grade system, as shown by the HR-TEM image result (Fig. 3C). It demonstrated that copper was uniformly distributed throughout the zinc matrix, producing a unique alloy. As shown and similar in the published article, the created radical-multi-position of fungal filtrate may generate concurrent decrease of Zn and Cu [76].

After comparison with publications in the literature on intermediate particle size and form, it was found that our combination ZnO-CuO NPs were poly-dispersed, varied in size, and mainly had spheroidal particles as their predominant shape. Wide-ranging forms may have been developed in that work [77], while the created NPs were all about orbicular or sphere-shaped, and other morphologies may be seen due to the synthetic process from extract, which is why the anisotropic form had been identified. Due to the fact that only the most practicable reducing and capping agents (fungal filtrate) were used in our work, a stable form is polydisplayed.

According to the DLS method, the typical particle size distribution for ZnO NPs, CuO NPs, and ZnO-CuO NPs, which were biosynthesized by the produced fungal filtrate, was calculated to be 85.52 nm, 90.85 nm, and 92.85 nm, respectively (Fig. 4).

Fig. 4
figure 4

DLS analysis of the myco-synthesized ZnO NPs (A), CuO NPs (B), and bimetallic ZnO-CuO NPs (C)

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According to International Standards Organizations (ISOs), samples are deemed to be monodisperse when the polydispersity index (PDI) findings are less than 0.05. In contrast, PDI outcomes of greater than 0.7 are intended to produce particles with a polydispersity distribution [78]. According to our findings, the PDI values for ZnO, CuO, and ZnO-CuO NPs, respectively, were 0.72, 0.79, and 0.95. According to the current values, the biosynthesized NPs were a reasonable range of polymers.

According to the data, the mean and predominant size determined by DLS analysis was larger than the particles’ estimated sizes determined by HR-TEM imaging. According to the following reference, the causes include the hydrodynamic radius within the biosynthesized ZnO NPs, CuO NPs, and bimetallic ZnO-CuO NPs and the water layers surrounding them with regard to the significant sizes of the biosynthesized NPs [79].

Utilizing the SEM technique, the surface characteristics and surface form of the generated biosynthesized NPs were examined. The corresponding brilliant spherical particles were found within the prepared fungal filtrate and the SEM image of ZnO NPs biosynthesized by the prepared fungal filtrate had varying boundary size (Fig. 5A). The same pattern was found for the biosynthesized CuO NPs, which are depicted in Fig. 5B as isolated, rounded, brilliant particles on the fungal filtrate.

Fig. 5
figure 5

SEM images of the myco-synthesized ZnO NPs (A), CuO NPs (B), and bimetallic ZnO-CuO NPs (C and D)

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Additionally, the combined ZnO-CuO NPs and filtrate SEM results (Fig. 5C) show consistent ZnO-CuO NP surfaces with a transparent surface appearance. It was discovered that ZnO-CuO NPs were essentially separated as spheroidal particles fused with one another across the generated fungal filtrate, which appears as illuminated NPs merged and capped with the filtrate (Fig. 5D).

The synthesized ZnO-CuO NPs (in our study) were uniformly disseminated with limited size and the exact spherical formation, according to a comparison with previous studies on the topic of morphological form. Muhammad Mohsin et al. [80] produced bimetallic silver and gold core-shell NPs using the citrate reduction method at various pH and temperature levels. The pH and temperature play a significant function in the synthetic technique since the approved morphological shape and border size suggested that they have maintained size varying from 50 to 65 nm and appear as spheroidal particles.

Figure 6 shows XRD analyses for the biosynthesized NPs. The precursor (fungal filtrate) and the biosynthesized ZnO, CuO, and ZnO-CuO NPs are represented by amorphous and crystal arrangements in the generated NPs, respectively. It should be emphasized that 2Ɵ refers to the amorphous fungal filtrate (amino acids, protein, etc.) in the temperature range of 5 to 25° [81]. Figure 6 shows the ZnO NPs’ XRD diffraction peaks, which included peaks at 2Ɵ = 27.25°, 32.25°, 46.86°, 57.80°, 67.09°, and 76.25°. These peaks were complemented by the JCPDS card number 361451, and they corresponded to (002), (101), (102), (110), (103), and (201) Bragg’s reflections [82]. However, the XRD of the biosynthesized CuO NPs showed distinct peaks at 2Ɵ = 31.25°, 36.09°, 39.85°, 51.80°, 59.49°, 67.58°, and 70.99° that were complemented by a characteristic card JCPDS number 892531 and corresponded to Bragg’s reflections (110), (002), (200), (202), (020), (022), and (220) [83].

Fig. 6
figure 6

XRD analysis of the myco-synthesized ZnO NPs, CuO NPs, and bimetallic ZnO-CuO NPs

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In addition, Fig. 6 shows the XRD results of the biosynthesized bimetallic ZnO-CuO NPs and highlights the XRD diffraction peaks of ZnO NPs, including peaks at 2Ɵ = 27.50°, 31.15°, 45.15°, 56.89°, 67.98°, and 75.25°, which are supplemented with a typical card JCPDS number 361451, and correspond to (002), (101), (102), (110), (103), and (201) Bragg’s reflections [82].

They also contain the CuO NP diffraction peaks at 2Ɵ = 30.19°, 36.19°, 40.12°, 52.09°, 58.40°, 67.40°, and 71.19°, which are complemented by the usual card JCPDS number 892531 and correspond to Bragg’s reflections at angles (110), (002), (200), (202), (020), (022), and (220) [83].

The available XRD data (Fig. 6) shows that the synthesized ZnO-CuO NPs were crystallized and had a face-centered cubic (fcc) crystalline structure. The generated bimetallic NPs were highly crystalline and coupled with amorphous fungal filtrate, improving their diffusion in the solution for improved application, according to the XRD data [84].

Finally, the equation of Williamson-Hall (W H) was used to define the intermediate crystallite size of ZnO NPs, CuO NPs, and bimetallic ZnO-CuO NPs [85, 86], and provided to Eq. 1 were found to be 30.59 nm, 44.98 nm, and 49.58 nm, respectively.

$$\mathrm\beta\;\cos\;\mathrm\theta=\frac{\mathrm k\lambda}{{\mathrm D}_{\mathrm W-\mathrm H}}+4\mathrm\varepsilon\;\sin\;\mathrm\theta$$
(1)

3.4 Antifungal activity of ZnO-CuO NPs, ZnO NPs, and CuO NPs against F. oxysporum

3.4.1 Inhibition zones and minimum inhibitory concentrations

Monometallic NPs have been used frequently for controlling fungal plant diseases [87,88,89], but fungi have acquired resistance against most of the NPs. Therefore, bimetallic NPs have received much attention in the last period.

In the current study, antifungal activities of ZnO-CuO NPs, ZnO NPs and CuO NPs, zinc acetate, copper acetate, and CFF were evaluated toward F. oxysporum using agar well diffusion method (Fig. 7). Results revealed that, ZnO-CuO NPs exhibited the highest antifungal activity among other prepared NPs but start materials (zinc acetate, copper acetate, and CFF) did not give any activity. Moreover, ZnO-CuO NPs had promising antifungal activity against F. oxysporum where inhibition zone was 22.8 ± 0.76 mm at concentration 1000 µg/ml (Fig. 7A), and MIC was 125 µg/ml (Fig. 7B). Furthermore, both ZnO NPs and CuO NPs exhibited antifungal activity but lower than ZnO-CuO NPs where inhibition zones at concentration 1000 µg/ml were 11.3 ± 0.57 and 15.4 ± 0.69 mm respectively, and MICs were 1000 and 500 µg/ml respectively. This confirms bimetallic NPs have antifungal activity better than single metal NPs. Previous studies reported that, bimetallic NPs exhibit antimicrobial activity more than metal NPs individually [90,91,92].

Fig. 7
figure 7

Antifungal activity of bimetallic ZnO-CuO NPs, ZnO NPs, and CuO NPs using agar well diffusion method (A) and MIC results (B)

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Bimetallic NPs synthesized with different biological methods (plant extracts, bacteria, viruses, yeasts, fungi) have more potential than monometallic NPs synthesized with biological methods, due to an additional degree of freedom [93].

Al-Dhabaan, Shoala, Ali, Alaa, Abd-Elsalam, and Abd-Elsalam [90] prepared nanocomposite based on bimetallic zinc-copper NPs and chitosan, and found this nanocomposite has antifungal activity higher than monometallic zinc, copper NPs. Mechanism of action of ZnO-CuO NPs may be attributed to electrostatic interaction which cause cell membrane damage, disruption of proteins and enzymes, ROS generation and oxidative stress, protein binding which leads to homeostasis disturbance (electron transport chain disruption), signal transduction inhibition, and genotoxicity [94, 95]. Also, the efficacy may be attributed to roughness of external surface of NPs which cause damaging the cell wall, and these lead to penetrate NPs to plasma membrane [96].

3.4.2 Radial growth and growth inhibition percentages

Radial growths of F. oxysporum at different concentrations of ZnO-CuO NPs, ZnO NPs, and CuO NPs were assessed as illustrated in Fig. 8. Linear growth of F. oxysporum was carried out to determine the growth inhibition percentage for each concentration of ZnO-CuO NPs, ZnO NPs, and CuO NPs. Generally, the growth diameter is increased with decreasing the concentration of NPs, but inhibition percentage is decreased.

Fig. 8
figure 8

Radial growth of F. oxysporum at different concentrations of ZnO-CuO NPs (A); growth diameters at different concentrations of bimetallic ZnO-CuO NPs, ZnO NPs, and CuO NPs (B); growth inhibition percentages (C)

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Results showed that radial growth of F. oxysporum at different concentrations of ZnO-CuO NPs was lower than ZnO NPs and CuO NPs (Fig. 8A and B), and this means that ZnO-CuO NPs are the most powerful among others. Results illustrated that, growth diameters of F. oxysporum at different concentrations of ZnO-CuO NPs 1000, 500, 250, 125, and 62.5 µg/ml were 10, 31, 53, 82, and 90 mm respectively (Fig. 8A). Also, growth inhibition percentages of F. oxysporum at different concentrations of ZnO-CuO NPs 1000, 500, 250, and 125 µg/ml were 88.9, 65.5, 41.1, and 8.9% respectively, where the highest inhibition was 88.9% at concentration of 1000 µg/ml, while the lowest inhibition was 8.9% at concentration of 125 µg/ml (Fig. 8C).

On the other hand, both ZnO NPs and CuO NPs affect slightly on the growth of F. oxysporum, where growth diameter in the case of ZnO NPs was 90 mm at concentrations of 62.5–500 µg/ml, and also the case of CuO NPs was 90 mm at concentrations of 62.5–250 µg/ml. Furthermore, higher concentrations of both ZnO NPs and CuO NPs affect slightly on growth inhibition where inhibition percentages at concentrations of 1000 and 500 µg/ml were 12.2 and 22.2% and 0 and 10% respectively (Fig. 8C). On other hand, concentrations lower than 500 µg/ml in both ZnO NPs and CuO NPs did not inhibit the growth of F. oxysporum.

Yehia and Ahmed [97] reported that growth inhibition of ZnO NPs at concentration of 12 mg/l against F. oxysporum was 77%. As reported by Yehia and Ahmed [97], ZnO NPs were shown to provide a 77% growth inhibition against F. oxysporum at a dose of 12 mg/l. Viet, Nguyen, Cao, and Hieu [98] revealed that copper NP solution at concentration of 450 ppm after 3 days could inhibit Fusarium sp. with 67.38%, while it was 93.98% after 9 days. From these data, bimetallic ZnO-CuO NPs exhibited antifungal activity higher than their monometallic form.

3.4.3 Ultrastructure of control and treated F. oxysporum

The ultrathin section technique was carried out on treated F. oxysporum with ZnO NPs, CuO NPs, and ZnO-CuO NPs as well as control. Results illustrated that, ultrathin sections of control F. oxysporum showed relatively large intercellular spaces (IS) (Fig. 9A). Also, results showed uniformly thin cell wall (CW) and contain nucleus (N), mitochondria (M), and vacuole (Vc), where these results agree with Al-Surhanee [99]. Treating of F. oxysporum with ZnO NPs led to partial destruction in cell wall and plasma membrane, the nucleus appeared as small size and damaged shape, and the chromatin materials distributed with several dark stained bodies in cytoplasm (Fig. 9B). Zinc-containing compounds were used in the past as fungicides and proved highly effective, such as ethylene thiocarbamate zinc [100]. Moreover, treated F. oxysporum with CuO NPs appeared more destructive, as all cell contents appeared abnormal, and the nucleus and succulent vacuoles disappeared (Fig. 9C). Furthermore, treated F. oxysporum with ZnO-CuO NPs, a clear destruction was found in all cell contents and disintegration of the cell wall as well as destruction of the plasma membrane. Also, the nucleus appeared as small size and damaged shape and the chromatin materials distributed with several dark stained bodies in cytoplasm (Fig. 9D). These ultrastructural changes in fusarium cells could have negative effects on cell wall, nucleus, and mitochondria and could be partially responsible for the decrease in respiration and vital processes. The toxic and destructive effect of copper is attributed to the copper ions on the mushroom protoplasm, which destroys the nucleus and prevents the germination of spores [101, 102].

Fig. 9
figure 9

Ultrastructure of F. oxysporum: control (A); treated with ZnO NPs (B); treated with CuO NPs (C); treated with bimetallic ZnO-CuO NPs (D)

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4 Conclusion

In this study, bimetallic ZnO-CuO NPs were successfully myco-synthesized using A. fumigatus through ecofriendly and cost-effective method. This study used a reducing or stabilizing agent (to reduce metal ions into bimetallic ZnO-CuO NPs and fix them in the colloidal state) to observe the fungal synthesis of bimetallic ZnO-CuO NPs. The solution color changed to a dim brown tone. The line spacing was the same, and a one-phase network was seen, according to HR-TEM imaging. It was emphasized that copper was evenly distributed throughout the zinc matrix to produce an alloy. The radical-multi-position of fungal filtrate may cause simultaneous reduction of both Zn and Cu as mentioned in the published article. The present XRD data indicates that the produced ZnO-CuO NPs were crystal in character and delivered the face-centered cubic (fcc) crystalline design. The XRD results indicate that the produced bimetallic NPs were highly crystalline and conjugated with amorphous fungal filtrate increasing its distribution in the solution for better application. The prepared bimetallic ZnO-CuO NPs had promising antifungal activity against F. oxysporum causing fusarial wilt diseases. To confirm the effect of bimetallic zinc-copper NPs on F. oxysporum, ultrastructure study was carried out using TEM, where results illustrated that the treating of F. oxysporum with ZnO NPs led to partial destruction in cell wall and plasma membrane, the nucleus appeared as small size and damaged shape, and the chromatin materials distributed with several dark stained bodies in cytoplasm. Furthermore, treated F. oxysporum with bimetallic ZnO-CuO NPs, a clear destruction was found in all cell contents and disintegration in the cell wall as well as destruction of the plasma membrane. Also, the nucleus appeared as small size and damaged shape and the chromatin materials distributed with several dark stained bodies in cytoplasm. Finally, the myco-synthesized bimetallic ZnO-CuO NPs which have antifungal activity against F. oxysporum can be used in controlling fusarial wilt disease after in vivo studies.