Initial extensive screen for copper metal resistance fungi, it was discovered that Penicillium olsonii outperformed other fungi with regard to fungal growth with copper different metal concentration levels up to 800 ppm. The identification of P. olsonii funguswas confirmed using molecular characterization, which is based on ITS rDNA (Fig. 1) and applied for CuPNs synthesis. Identification of fungi to the species level by molecular manner is principally based on the variable nature of the DNA’s ITS regions [29]. The 18S rRNA sequence of the P. olsonii isolate was searched on a database (Basic Local Alignment Search Tool (BLAST) using multiple sequence alignment (Fig. 1b) with theMEGA6 software. The results of alignment profile, concluded that the P. olsonii 18SrRNA amplicon closely matched other P. olsonii strains at more than 99%). Figure 1 illustrated the structured phylogenetic relatedness of the full sequence of P. olsonii 18S rRNA that was compared to the closely related strains from the database (blast.ncbi.nlm.nih.gov/Blast.cgi).
During the present study, the capacity of P. olsonii MT635310.1 to synthesize CuNPs was observed where the aqueous Cu2+ ions were reduced during exposure to the culture supernatant of P. olsonii MT635310.1. The color of this solution changes from blue to green color after 72 h of mixing fungus biomass with copper sulfate solution which indicated the formation of CuNPs extracellularly (Fig. 2). The present findings are in agreement with the recent findings [30]. The aforementioned studies reported that the appearance of green color color is due to reduction of copper sulfate and its bioconversion to CuNPs.
The finding appeared that the reaction solution has an absorption maximum around 565 nm attributed to the surface plasmon resonance band (SPR) of the CuNPs. In general, it has been reported that CuNPs display a surface plasmon peak at 580-59031. Although Sudhir et al.[32] demonstrated that the biogenic CuNPs were characterized by UV-Vis spectrophotometer showing a typical resonance (SPR) at about 631 nm which is specific for CuNPs. CuNPs were studied recently by [30] at 200–1000 nm wavelength range and it has absorbance at 550 to 650 nm.
The size distributions of CuNPs in the aqueous solution was evaluated by SEM images (Fig. 3). The synthesized CuNPs by P. olsonii were observed by SEM with almost of spherical shape and the particle sizes varied from 6–26 nm. According to Ponnusamy et al. [33] CuNPs size were varied from 5 to 50 nm in diameter. SEM measurements by Chalandar et al.[34] estimated that extracellular biosynthesis of CuNPs with the diameters 40 nm. It is also reported that Penicillium citrinum, P. waksmanii and P. aurantiogriseum produced spherical CuNPs of size 90–295 nm6. The size variation may be related to culture conditions and producing microorganism.
FTIR analyses of the nanoparticles biosynthesized using P. olsonii MT635310.1 was recorded (Fig. 4). The FTIR spectra of the CuNPs biosynthesized revealed the presence of different functional groups. The strong and broad band observed at 3,471 cm− 1 indicates the presence of polyphenolic O-H group and primary amine O-H band. A narrow band at 1639.73 cm− 1, indicates the presence of amide I. While band at 1448.19 cm− 1 corresponding to C-C stretching aromatic ring, C-O stretching carboxylic acid group assigned at 1,243 cm − 1, ring, C-O stretching carboxylic acid group assigned at 1242 cm− 1. Cuevas et al. [35] mentioned that bands at 1243 and 1244 cm− 1 are designated for bending vibration movements in amides I and amides III. The weak bands at 657 cm − 1 may correspond to alkyl halides. These main bands indicate the presence of protein on the surface of the CuNPs and copper oxide nanoparticles. Ore results agreement with previous studies[36, 37].
It has been reported that CuNPs act as a fungicide against different species of phytopathogenic fungi such as Fusarium solani, F. oxysporum, Neofusicoccum sp.[38]. CuNPs showed various levels of inhibition on colony growth of F. oxysporum, F. solani and C. curvulata. CuNPs at concentrations of 50, 1000 and 200 ppm. Inhibition % of fungal growth increased with increasing CuNPs concentrations (Table 1 & Fig. 5). These results were agreement with recently result of Chalandar et al.[34] who show that antifungal properties of CuPNs increased by increasing the concentration of nanoparticles to 15%. The obtained results showed that the sensitivity of tested fungi to CuNPs depend on species, since F. oxysporum was more sensitive to different levels of CuNPs than Fusarium solani; for example the inhibition % was 86.25 and 32.92, respectively at 200 ppm. Similar results have been reported for the antifungal activity of CuNPs against different fungal species [38]. Furthermore, similar to present findings, Sudhir et al.[32] reported that Fusarium culmorum was found to be most sensitive to CuPNs followed by F. oxysporum and F. Graminearum. The best chariteria of copper was observed by Ramesh et al.[39] where, the application of CuPNs against plant pathogenic fungi exhibited improvement to plant growth. The previous author estimated the antifungal activity of CuPNs against the pathogenic fungi F.culmorum, F. oxysporum, F. graminearum and Phytophthora infestans beside CuPNs at concentrations below 100 ppm have been reported to enhance germination and growth of some plants. CuNPs not only repress the fungal growth but also inhibit the sporulation process. This may help in elucidating the antifungal mechanisms of CuNPs .The increment of CuNPs concentration was accompanied by reduction of sporulation rate that reached to 10.5, 20.2 and 26.5% at 200 ppm for F. oxysporum, F. solani and C. curvulata, respectively compared to the control (100%). Sporogenisis of Aspergillus niger has been recently described as sensitive to CuNPs [40]
Table 1
Antifungal activity of different concentrations of CuNPs
CuNPs concentration ppm
|
F. oxysporum
|
F. solani
|
C. curvulata
|
Colony radius (cm)
|
Growth Inhibition %
|
Sporulation
%
|
Colony radius
(cm)
|
Growth Inhibition
%
|
Sporulation
%
|
Colony radius
(cm)
|
Growth Inhibition %
|
Sporulation
%
|
Control
|
8.0 ± 0.1
|
0.0
|
100
|
8.2 ± 0.2
|
0.0
|
100
|
3.8 ± 0.2
|
0.0
|
100
|
50
|
3.4 ± 0.2
|
57.50
|
82.0
|
8.0 ± 0.1
|
2.44
|
95
|
3.6 ± 0.1
|
5.26
|
88.0
|
100
|
1.8 ± 0.2
|
77.50
|
39.4
|
7.2 ± 0.2
|
12.10
|
25.5
|
2.2 ± 0.1
|
42.10
|
50.6
|
200
|
1.1 ± 0.5
|
86.25
|
10.5
|
5.5 ± 0.4
|
32.92
|
20.2
|
1.2 ± 0.5
|
68.42
|
26.5
|
Morphological of the tested fungi hyphae after exposure to CuNPs was examined in an attempt to understand its inhibition mechanism. Therefore, the underlying mechanism by which CuNPs kill fungi is reflected by changes of fungal morphology (Fig. 6). From the observed micrographs, the hyphae had a bigger and swollen appearance particularly at 200 ppm of CuNPs treatment. Hyphae vaculation of F. oxysporum could be clearly observed at 200 ppm of CuNPs. On the other hand, spore size and shape of F. solani were dramtecaly changed at high concentration. Chlamydospores were observed in C.curvulata, beside disruption of hyphae. Abd El-Mongy and Abd El-Ghany [41] reported that chlamydospores were formed in fungi under stress conditions. Microscopic observation revealed that the CuNPs clearly damaged the hyphae of Alternaria alternata and Botrytis cinerea [42]. Effect of CuNPs Transmission electron microscopy revealed that CuSO4 and CuNPs treatments encouraged the deformed appearance of the A. niger at 200 ppm and 300 ppm, particularly CuNPs[40].