Introduction

Nanotechnology is the multidisciplinary science concerned with the creation and modification of materials in a nano-size range between 1 and 100 nm. Nanotechnology is greatly affected by various disciplines especially physics, chemistry, and biology [1,2,3,4,5]. This technology provides great opportunities by changing the properties of materials significantly when they are in nano-size. When the dimension of the materials is getting closer to nano-size, quantum physics plays an important role instead of conventional laws of physics which adds different and unique properties to the material [6, 7]. Nowadays, biological synthesis (plant–algae–fungi–actinomycetes, bacteria, and viruses) method of NPs is preferred because it is safe, clean, cheap, and easily scaled up procedure for well-built scale NPs [8,9,10].

The fungal-intermediated green formation of NPs has many advantages including simple and easy scaling up, easy processing, economic viability, biomass processing, and recovery of considerable surface distances with the optimal outgrowth of mycelia [11,12,13,14]. Furthermore, from an economic overview, the utilization of fungal biomass filtrate containing different metabolites as a green approach for the fabrication of metallic NPs is preferred over other biological methods [6, 8, 15, 16]. Up to now, numerous metal oxides as CuO and ZnO nanoparticles have been bio-synthesized utilizing fungal cell extract [6, 17]. Such metal oxides are generally recognized as safe materials by the US food and drug administration as they are non-toxic and biocompatible for human beings and animals [18, 19]. ZnO-NPs have unique characteristics including electrical conductivity, UV-protection, photochemical beside antiviral, anticancer, antifungal, and antibacterial activities [12, 20, 21]. CuO-NPs have received great attention by numerous researchers for its chemical and biological demeanor which can be returned to their morphology [22,21,22,25]. CuO-NPs have been utilized in diverse biological applications like drug delivery and anticancer [26], antifungal, antioxidant, and antibacterial activities [27].

One of the major problems facing the medical field is the infectious diseases that lead to the death of many people worldwide [28]. The wrong use of antibiotics and the lack of capabilities and scientific tools to develop such drugs had led to the emergence of mutations and generations of microbes that are resistant to such drugs [29]. Moreover, the bacterial cells that emerged inside the biofilm matrix exhibit more resistance to the available antimicrobial therapy in addition to hosting the immune responses. Such a complex network enhances the ability of bacteria to gain resistance against a wide range of antibiotics, and thus makes traditional antibiotic therapy inactive [30]. Therefore, it was necessary to search for new treatment strategies such as the use of nanotechnology to provide a new vision in treatment and limiting the spread of pathogens. To fulfill this target, this work was conducted to synthesize and characterize ZnO-NPs and CuO-NPs through employing the metabolites (proteins) secreted by Penicillium chrysogenum. Studying the antibacterial, antifungal, and antibiofilm activities of the biosynthesized ZnO-NPs and CuO-NPs to be applied as smart nano-materials to be used in the medical field and limit the spread of some pathogenic microbes such as Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli (bacterial strains), Fusarium solani, Fusarium oxysporum, Sclerotium sclerotia, and Aspergillus terreus (fungal strains) will be investigated.

Materials and Methods

Materials

Zinc acetate dihydrate (Zn(CH3COO)2·2H2O) and Copper acetate monohydrate (Cu(CH3COO)2·2H2O) were purchased from Sigma-Aldrich for chemicals and used as precursors for preparation ZnO and CuO nanoparticles.

Fungal Strain

Penicillium chrysogenum MF318506 strain was used to synthesis ZnO-NPs and CuO-NPs. This strain was characterized and identified as described previously [11].

Synthesis of ZnO-NPs and CuO-NPs by Penicillium chrysogenum Filtrate

Penicillium chrysogenum MF318506 was grown up in 250 mL Erlenmeyer flask containing 100 mL Czapek Dox (CD) fermentative broth medium containing (g/L): 2.0 NaNO3, 0.5 MgSO4.7H2O, 0.5 KCl, 0.5 KH2PO4, 0.001 FeSO4, and 20.0 sucrose for 7 days at pH 6.0, 30 °C, and shaking at 150 rpm. The fungal biomass was separated by filtration method at the end of the incubation period using Whatman filter paper No. 1. The biomass filtrate of P. chrysogenum was used for ZnO-NPs and CuO-NPs formation as the following: Two (2 millimole) of zinc acetate and copper acetate were incubated separately and mixed with biomass filtrate of P. chrysogenum at 30 °C for 48 h on the shaker at 150 rpm [6, 31]. White and green dark colors were appeared for ZnO-NP and CuO-NP synthesis at the end incubation period, respectively. The ZnO-NPs and CuO-NPs were separated and dried at 80 °C for 48 h. The ZnO-NP and CuO-NP products were eventually collected and subjected for further investigation.

Characterization of ZnO-NPs and CuO-NPs

Biosynthesis of ZnO-NP and CuO-NP colloids were routinely monitored using UV–vis spectra on JASCO V-630 UV-VIS Spectrophotometer (JASCO INTERNATIONAL CO., LTD) at wavelengths of 200–800 nm to detect intense absorption peak which related to surface plasmon excitation. The particle shape and size of the biosynthesized ZnO-NPs and CuO-NPs were preliminarily characterized using transmission electronic microscope (TEM) (JEOL-2100) measurements through drop coating process in which a drop of solution containing NPs was placed on the coated-carbon copper grids and kept under vacuum desiccation for overnight before loading them onto a specimen holder. Moreover, the functional groups present in myco-synthesized NPs molecules were analyzed using Fourier transform infrared (FTIR) spectroscopy (JASCO, FT/IR- 6100). The samples NPs were mixed with KBr and filled into discs at high pressure. These discs were scanned in the range of 400 to 4000 cm−1 to obtain FTIR spectra. The elemental composition of biosynthesized ZnO-NPs and CuO-NPs was investigated by EDX analysis (JEOL JSM- 6510 LV). The crystalline structure of ZnO-NPs and CuO-NPs was characterized by XRD analysis (XRD, X PERT PRO-PAN Analytical) [1, 6, 24, 32].

Biological Activities of the Biosynthesized ZnO-NPs and CuO-NPs

In Vitro Antibacterial Activity and MIC Determination

Antimicrobial activities of the biosynthesized ZnO-NPs and CuO-NPs were evaluated according to CLSI guideline using agar well diffusion assay by Muller Hinton agar plates versus human pathogenic bacterial strains: Staphylococcus aureus ATCC23235 and Bacillus subtilis ATCC6051 which represent Gram-positive bacteria and Pseudomonas aeruginosa ATCC9027, Salmonella typhimurium ATCC14028, and Escherichia coli ATCC8739 which represent Gram-negative bacteria. The diameter of inhibition zone was determined by millimeter (mm) [33, 34]. Micro-dilution method in micro-titer plates (MTP) performed to determine the minimum inhibitory concentration (MIC) of active ZnO-NPs and CuO-NPs against tested pathogenic strains. Accordingly, ZnO-NPs and CuO-NPs were diluted in 1% DMSO at various concentrations (0.009–5.0 mg/ml, w/v) and tested against the pathogenic strains. Briefly, 1: 100 (v/v) of overnight cultures of the test strains were added to 200 μl of Muller Hinton broth media distributed in the wells of MTP with/without ZnO-NPs and CuO-NPs. The plates were then incubated with shaking (120 rpm) for 24 h at 37 °C. MIC was determined as the lowest concentration of ZnO-NPs and CuO-NPs which inhibit 100% of pathogenic strains [35]. All experiments were performed in triplicates.

In Vitro Biofilm Inhibition Assay

To assess the ability of ZnO-NPs and CuO-NPs to inhibit or prevent bacterial biofilm formation, micro-titer plate assay was used against Staphylococcus aureus ATCC29213 and Pseudomonas aeruginosa ATCC9027, a known biofilm-producing strain. Different concentrations (0.01–3.0 mg/mL) of ZnO-NPs and CuO-NPs were loaded in flat bottom MTP containing Tryptic-Soy broth media (TSB) supplemented with 1% glucose and mixed well. Overnight cultures of the tested strains were separately diluted at 1:100 (v/v) in TSB (5 × 105 CFU/ml), loaded into the wells, and then the plates were incubated at 37 °C for 48 h [36]. After incubation period, the planktonic cells were transferred without disturbing the biofilm and cell growth was read by ELIZA reader (Tecan Elx800, USA) at 620 nm. The plates were washed three times with sterilized phosphate-buffered saline (PBS) pH 7.2 to remove excess planktonic cells; then biofilms were fixed with 200 μl of methanol 95% for 10 min. Crystal violet (0.1%, w/v) was added and the plates were incubated for 15 min at room temperature. After the incubation, the crystal violet was removed, and the wells were gently washed with sterile distilled water to remove excess stain. Finally, the adhered biofilm bounded crystal violet was examined and photographed by an inverted microscope (Olympus Ck40 Japan) ×40 then, eluted in acetic acid (30%), and the absorbance was measured at 540 nm by ELIZA reader (Tecan, Elx800- USA). The treated wells were compared with that of the untreated control. All experiments were performed in triplicates.

In Vitro Antifungal Activity

The antifungal activity of ZnO-NPs and CuO-NPs was assessed by disc diffusion method versus phytopathogens fungi as Fusarium solani, Fusarium oxysporum, Sclerotium sclerotia, and Aspergillus terreus that obtained from the culture collection of Chemistry of Natural and Microbial Products, NRC, Egypt. Briefly, sterile discs of 0.7-mm diameter were placed into the potato dextrose agar (PDA) medium that contained the phytopathogenic strains [17]. The discs were loaded with 100 μl of ZnO-NPs and CuO-NPs at a concentration of 10 mg/mL and placed on the plates, separately. The plates were incubated for 5 days at 30 °C. Antifungal activity was evaluated by measuring the inhibition zone towards each strain being tested in triplicates.

Statistical Analysis

The means of three replications and standard error (SEr) were calculated for all the results obtained. The data were subjected to analysis of variance means by sigma plot 12.5 program.

Results and Discussions

Biosynthesis of ZnO-NPs and CuO-NPs Using P. chrysogenum

Recently, the green biosynthesis of oxides nanoparticles has gained great importance being suggested as a possible safe and eco-friendly alternative to chemical and physical synthetic methods. Proteins secreted by fungal strains have effective roles in the reduction of ZnO and CuO to ZnO-NPs and CuO-NPs. respectively, besides stabilizing the formed nanoparticles. In this context, P. chrysogenum filtrate was used as a bioreactor for green synthesis of ZnO-NPs and CuO-NPs through harnessing bioactive macromolecules such as proteins and enzymes secreted therefrom [11]. The appearance of white and green dark colors after contacting of biomass filtrate with zinc acetate and copper acetate at the end of reaction indicated the formation of ZnO-NPs and CuO-NPs, respectively. After calcination as described in “Materials and Method,” ZnO-NPs and CuO-NPs were obtained as a powder.

Characterizations of ZnO-NPs and CuO-NPs

Biosynthesis of ZnO-NPs and CuO-NPs was indicated by UV-visible spectroscopy as represented in Fig. 1. It was shown that the maximum absorption peak of the formed ZnO-NPs and CuO-NPs were occurred at 380 and 335 nm, respectively, due to their surface plasmon resonance. This observation is in agreement with the previous studies on the biosynthesis of ZnO-NPs and CuO-NPs, which indicates that the characterization peaks of ZnO-NPs and CuO-NPs were at 380 nm and 337 nm, respectively [37, 38]. Another report showed that the maximum absorption peaks of ZnO-NPs and CuO-NPs were 301 and 290 nm, respectively [39]. Moreover, the absorption bands for ZnO-NPs and CuO-NPs in Fig. 1 are slightly symmetrical with sharp in width at wavelength 335 and 380 nm, respectively. These could refer to the formation of ZnO-NPs and CuO-NPs with poly-dispersed narrow sizes particles.

Fig. 1
figure 1

Absorption peaks of the green synthesized ZnO-NPs and CuO-NPs using a UV-vis spectrophotometer

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TEM measurement was carried out to determine the approximate size and morphology of the biosynthesized ZnO-NPs and CuO-NPs. Figure 2 (A) and (B) indicates that ZnO-NPs and CuO-NPs with hexagonal and spherical shape were successfully formed with dispersed narrow sized particles surrounding with capping by active metabolites using P. chrysogenum filtrate. The average size of the formed ZnO-NPs and CuO-NPs are in the range of 9.0–35.0 nm and 10.5–59.7 nm, respectively. Figure 2 (A1) and (B1) shows the area selected electron diffraction (SAED) patterns of the ZnO-NPs and CuO-NPs, respectively. SAED pattern indicates good sharp rings that reveal the poly-crystalline nature of the ZnO-NPs and CuO-NPs. In previous literature, the size of ZnO-NPs and CuO-NPs biosynthesized by fungi is quite different. Gao et al. used Aspergillus niger for the mycosynthesis of ZnO-NPs with rod and cluster shapes, and the particle size distribution range was 80–130 nm [40]. Saravanakumar et al. reported that the particle size of CuO-NPs synthesized by Trichoderma asperellum ranged from 10 to 190 nm and it was almost spherical in shape [41]. Other reports showed various shapes of ZnO-NPs (hexagonal, nano-rod, and spherical) and CuO-NPs (spherical) with various sizes (10–42 nm, 8–38 nm, 10–45 nm, and 49.2 nm) that were synthesized by Fusarium keratoplasticum, Aspergillus niger and Aspergillus terreus, and Penicillium chrysogenum, respectively [6, 12, 17].

Fig. 2
figure 2

(A) TEM micrograph of ZnO-NPs, (B) TEM micrograph of CuO-NPs, (A1) SAED pattern of ZnO-NPs, and (B1) SAED pattern of CuO-NPs

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The SEM images of the myco-synthesized ZnO-NPs and CuO-NPs are shown in Fig. 3 (A) and (B). The SEM pictures showed randomly distributed ZnO-NPs and CuO-NPs with particle agglomeration. Other reported illustrated that the particles of metal oxide were predominantly spherical and aggregates into larger particles with no well-defined morphology [41, 42]. EDX examination was also investigated to confirm the purity and elemental composition of ZnO-NPs and CuO-NPs synthesized by P. chrysogenum as illustrated in Fig. 3 (A1) and (B1). Results showed the presence of zinc and oxygen elements. The highest peaks in the spectrum are related to the most concentrated elements. The ratio percentages of Zn and O were 58.3% and 20.0%, for ZnO-NPs, respectively, while CuO-NPs exhibited Cu and O with weight percentages 46.4% and 18.3%, respectively. The peaks for other elements such as C, Na, and K may appear from the filtrate of P. chrysogenum. This is in accordance with the earlier reports of the EDS analysis of ZnO-NPS and CuO NPs [23, 41, 43].

Fig. 3
figure 3

(A and A1) SEM-EDX spectrum of biosynthesized ZnO-NPs, (B and B1) SEM-EDX spectrum of biosynthesized CuO-NPs, and (C) FTIR spectra of fungal filtrate, ZnO-NPs and CuO-NPs biosynthesized by P. chrysogenum

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FTIR spectra obtained from the P. chrysogenum filtrate and green synthesized ZnO-NPs and CuO-NPs were shown in Fig. 3 (C). FTIR measurements were carried out to distinguish imaginable interactions between metal oxides (ZnO and CuO) and metabolites of P. chrysogenum, which may be accountable for capping, formation, stabilization, and dispersion of ZnO-NPs and CuO-NPs in colloidal solution. FT-IR analysis for P. chrysogenum filtrate showed the maximum bands at 1639.2 cm−1 that is a definite indicator of amide I and amide II linkages of peptides and band at 3238.86 cm−1 that corresponds to (N–H) stretching group from the previous results [11]. The recorded FT-IR spectra from the biosynthesized ZnO-NPs and CuO-NPs showed peaks at 602.64 and 564.07 cm−1, respectively, that are attributed to the ZnO and CuO stretching vibration mode. In addition, absorption bands in the range 400 to 700 cm−1 may be referred to the metal-oxygen (M−O) stretching mode [12, 44, 45]. While intense absorption peaks have appeared at 3319.85, 2927.41, 1696.08, 1533.13, 1364.39, 1013.40, and 868.77 cm−1. The peak at 3319.85 cm−1 corresponds to O–H stretching group of phenols and alcohol and may be due to the N–H asymmetric stretch mode of amines [6]. The band at 2927.41 cm−1 corresponds to the stretching of CH3 groups of the protein. The bands at 1696.08 and 1533.13 cm−1 correspond to the binding vibrations of amide I band of protein with N–H stretching’s [43]. The bands observed at 1364.39 and 1013.40 cm−1 are assigned to C–N stretching vibrations of aromatic and aliphatic amines [39]. While band at 868.77 cm−1 may be correlated with C–H and C=C alkene [32]. This refers to the coordinating bonds that occurred between metal oxides (ZnO and CuO) and metabolites secreted by P. chrysogenum. From the above results, it is evident that the metabolites secreted in biomass filtrate by P. chrysogenum have the main role in NP formation and size reduction of ZnO and CuO in well-stabilized nano-form.

The XRD patterns for ZnO-NPs and CuO-NPs are represented in Fig. 4. From XRD analysis, CuO-NPs exhibited a monoclinic structure. The typical diffraction peaks observed at 2θ = 31.72° (100), 34.47 (002), 36.25 (101), 47.71 (102), 56.59 (110), 62.98 (103), and 67.78 (112) confirmed the hexagonal phase crystals of ZnO [6], while the diffraction peaks of CuO-NPs observed at 2θ° of 36.44°, 42.37°, 61.41°, and 73.37° were ascribed to (111), (200), (220), and (311) planes of the CuO crystalline form, respectively [17]. The crystallite particle sizes of the biosynthesized ZnO-NPs and CuO-NPs were assessed using Scherrer’s equation [46]. In this regard, the average particle size of ZnO-NPs obtained from XRD analysis was ranged from 12.4 to 18.9 nm, while the average particle size of CuO-NPs was ranged from 12.7 to 42.6 nm.

Fig. 4
figure 4

XRD pattern of ZnO-NPs and CuO-NPs synthesized by P. chrysogenum

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In Vitro Antibacterial Activity of ZnO-NPs and CuO-NPs

One of the great importances of ZnO-NPs and CuO-NPs is their activities against pathogenic microbes. Therefore, ZnO-NPs and CuO-NPs biosynthesized using P. chrysogenum were firstly investigated for their antibacterial activity versus both Gram-positive and Gram-negative bacteria. In general, the inhibitory action for biosynthesized ZnO-NPs and CuO-NPs which represented by the diameter of the clear zone was more effective against Gram-positive than Gram-negative bacteria. The diameters of clear zones formed by ZnO-NPs (5 mg/mL) were 16.33 ± 0.88, 13.5 ± 0.28, 12.43 ± 0.23, 11.06 ± 0.34, and 11.13 ± 0.41 mm for Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli and Salmonella typhimurium, respectively. As well, the diameters of clear zones formed due to CuO-NPs (5 mg/mL) were 22 ± 0.57, 16.26 ± 0.63, 13.6 ± 0.4, 11.93 ± 0.52, and 11.66 ± 0.33 mm, respectively (Fig. 5). The results indicated that CuO-NPs have better inhibitory effects versus Gram-positive and Gram-negative pathogenic-bacteria than those caused by ZnO-NPs. The inhibitory action for biosynthesized ZnO-NPs and CuO-NPs may be due to the reaction of nanoparticles with bacterial protein by combining the thiol (-SH) group and hence leads to inactivation of proteins and bacterial growth. Therefore, it is compulsory to detect the MIC to ZnO-NPs and CuO-NPs for each bacterial strain (Table 1). To this end, the inhibitory effect of different concentrations of ZnO-NPs and CuO-NPs (0.009–5.0 mg/mL) were investigated. Results showed that the MIC for ZnO-NPs were 2 mg/mL against Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and 3 mg/mL against Bacillus subtilis and Salmonella typhimurium, while the MIC for CuO-NPs were 1.0, 1.5, 2, and 3 mg/mL against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, and Salmonella typhimurium, respectively. Thus, CuO-NPs are more effective than ZnO-NPs against Gram-positive bacteria being exhibited the lowest inhibitory concentration at 1.0 and 1.5 mg/mL against Staphylococcus aureus and Bacillus subtilis, respectively. Salem and Fouda, explained the mechanism of nanoparticles in bacterial growth reduction through the interaction of NPs with phosphorous moieties in DNA, which lead to inactivation of DNA replication and hence inhibition of enzymes functions [8]. Also, it can inhibit respiratory enzymes of bacterial cells and stop ATP production which leads to cell death [47]. Besides, electrostatic attractions between positive charge of NPs and negative charge of bacterial surface lead to several changes as membrane detachment, cytoplasmic shrinkage, and ultimately membrane of cell rupture [24]. Hydrogen peroxide (H2O2) produced by metal oxide in the presence of water atmosphere plays an important role in the decomposition and death of the bacterial cell when it interacts with the surface of the bacterial cell [48].

Fig. 5
figure 5

Antibacterial activity for ZnO-NPs and CuO-NPs at (5 mg/mL) against different pathogenic bacteria

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Table 1 Minimum inhibitory concentration (MIC) of ZnO-NPs and CuO-NPs (mg/mL)
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In Vitro Anti-Biofilm Activity of ZnO-NPs and CuO-NPs

In this work, the antibiofilm activity of nanoparticles exhibited varied effects against different microorganisms (Fig. 6). Accordingly, CuO-NPs exhibited the highest effect against biofilm formation of Staphylococcus aureus without affecting the bacterial growth when performed at concentrations under MIC value. CuO-NPs at 0.3, 0.15, 0.07, 0.03, and 0.01 mg/mL had reduced the biofilm formation by 95, 94.1, 94.4, 85.9, and 68.8%, respectively, without affecting bacterial growth (Fig. 6a). On the other hand, CuO-NPs did not affect biofilm formations by a Gram-negative bacterium, Pseudomonas aeruginosa (Fig. 6c). Inverted microscopic analysis was carried out to evaluate the effect of nanoparticles on the surface topology of the biofilm matrix. The results showed the presence of biofilm matrix in the positive-control sample, while CuO-NPs-treated wells exhibited reduced surface colonization and biofilm matrix in Staphylococcus aureus. The images of light microscopic analysis confirmed that the CuO-NPs had achieved the whole dispersion of biofilm style by loosening the micro-colonies in the sample treated at 3.0 and 1.5 mg/mL (Fig. 7). Both the results from quantitative and qualitative analyses confirm that the CuO-NPs inhibited the biofilm fashioning of Staphylococcus aureus at the first stage. On the other hand, ZnO-NPs had exhibited no considered effect against biofilm formation of both Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) strains (Fig. 6b and d). Another report showed that biologically synthesized metal oxides inhibited the activities of biofilm figuration at its irreversible adherence stage. Interestingly, inhibition of primary stage biofilm activity was observed at the MIC values [49]. Kaweeteerawat et al. evaluated that the mechanism of metal oxide NPs in biofilm inhibition may consequence from NPs interacting with cell membranes of bacteria and stimulating oxidative stress [50]. Previous study explained the anti-bioflim activity according SEM images which showed deformation, external cell roughness, and cell wall’s shrinkage of bacterial cells. Moreover, biofilm formation and viable cells were reduced [51].

Fig. 6
figure 6

Anti-biofilm activity of the ZnO-NPs and CuO-NPs against Gram positive and Gram negative strains. a CuO-NPs against Staphylococcus aureus, b ZnO-NPs against Staphylococcus aureus, c CuO-NPs against Pseudomonas aeruginosa, and d ZnO-NPs against Pseudomonas aeruginosa

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Fig. 7
figure 7

Light inverted microscopic images of S. aureus biofilms grown with various concentrations of CuO-NPs: a 0.0 mg/mL, represent the positive control; b negative control; c, d 3.0 and 1.5 mg/mL above the MIC value; e 0.7 mg/mL; f 0.3 mg/mL; g 0.15 mg/mL; h 0.07 mg/mL; i 0.03 mg/mL; and j 0.01 mg/mL. At concentrations from 0.01 to 0.3 mg/mL (f–j) bacteria have appeared as scattered cells and cannot aggregated together to perform normal biofilm

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In Vitro Antifungal Activity of ZnO-NPs and CuO-NPs

The antifungal activity of the biosynthesized ZnO-NP and CuO-NPs against four phytopathogenic fungi (Fusarium solani, Fusarium oxysporum, Sclerotium sclerotia, and Aspergillus terreus) were investigated. ZnO-NP and CuO-NPs were proved to have antifungal activity against all the phytopathogenic fungi at 10 mg/mL (Fig. 8). The diameters of clear zones formed by the effect of ZnO-NPs were 12.33 ± 0.88, 11.83 ± 1.36, 23 ± 1.15, and 14 ± 0.5 mm for Fusarium solani, Fusarium oxysporum, Sclerotium sclerotia, and Aspergillus terreus, respectively, while CuO-NPs exhibit stronger antifungal activity where the inhibition zone diameters were 31.66 ± 0.88, 22.66 ± 0.66, 26.83 ± 0.60, and 28.66 ± 1.76 mm for Fusarium solani, Fusarium oxysporum, Sclerotium sclerotia, and Aspergillus terreus, respectively. ZnO-NP and CuO-NPs have also showed activities against phytopathogenic fungal strains Alternaria solani, Aspergillus niger, Fusarium oxysporum, Pythium ultimum, and Alternaria alternate [32, 52]. Nanomaterial inhibited the fungal growth by affecting cellular functions, which caused distortion in fungal hyphae and prevented the expansion of conidia and conidiophores and consequently lead to cell death [53].

Fig. 8
figure 8

Antifungal activity for ZnO-NPs and CuO-NPs at 10 mg/mL against different phytopathogenic fungal strains

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Conclusion

In this study, a promising biogenic ZnO and CuO nanoparticles were produced by P. chrysogenum. They showed obvious ability to inhibit several pathogens including Gram-positive and Gram-negative bacteria, as well as some phytopathogenic fungi. CuO-NPs at 0.3, 0.15, 0.07, 0.03, and 0.01 mg/mL had reduced the biofilm formation by 95, 94.1, 94.4, 85.9, and 68.8%, respectively, without affecting bacterial growth. The obtained biosynthesized ZnO-NPs and CuO-NPs are smart nano-materials that exhibited the potential to be applied in the medical field and limit the spread of some pathogenic microbes.