3 − 1. The effect of copper (Cu) and Trichoderma harzianum fungi application on biochemical parameters of guar
Based on variance analysis, interaction effects of copper (Cu) and Trichoderma harzianum fungi were significant in enzymatic antioxidants activities (Table 1). As shown in the mean comparison table, the highest enzymatic antioxidants activities were obtained when 100 µlit Cu and T. harzianum fungi were use. As, non-inoculation with T. harzianum and non-application of Cu-NPs decreased the catalase (63.59%), peroxidase (44.02%) and poly phenol oxidase (46.83%) compared to inoculation with T. harzianum and Cu application (Table 2). In fact, it can be said that copper as abiotic stress can produce more ROS in the cell and cause secondary oxidative stress to plants. On the other hand, in this paper, it is demonstrated that enzymatic antioxidant activities such as catalase, peroxidase and poly phenol oxidase were implicated in the tolerance of Trichoderma harzianum to oxidative the stress caused by exposure to Cu. So, previous studies have reported that Trichoderma inoculation and copper application have the greatest effect on enzymatic antioxidants activities (Ernesto Juniors et al., 2020).
On the other hand, the results analysis of variance showed that main effect of copper and the interaction of copper (Cu) and Trichoderma harzianum fungi significantly affected proline (Table 1). As can be seen in Table 2, the highest content of proline was 9.35 µg/g.Fw− 1 that obtained from 100 µlit Cu application and inoculation with T. harzianum fungi and the lowest content of proline was in non-inoculation with T. harzianum fungi and no application of copper (3.08 µg/g.Fw− 1). In fact, the absence of fungi and copper reduced the content of proline by approximately 67.05% when compared to the application of 100 µlit Cu and T. harzianum fungi. Of many plants, the use of organic solute such as proline, mineral ions, particularly Ca and K, for osmotic regulation. In fact, when cells are under stress, increased proline content protects cell membranes, proteins, cytoplasmic enzymes, and reactive oxygen species, as well as scavenges free radicals. Plants, in fact, can withstand the stress by increasing proline, polyamine, and protein production (Hosseinzadeh et al., 2018).
Table 1
Analysis of variance of biochemical traits as affected by copper and Trichoderma harzianum
S.O.V
|
df
|
Mean square
|
|
Catalase
|
Peroxidase
|
Poly phenol oxidase
|
Proline
|
Replication
|
2
|
28.16
|
1.602
|
0.0015
|
1.44
|
Copper (Cu)
|
1
|
3909.53**
|
658.97**
|
485.19**
|
67.60**
|
Trichoderma harzianum (TH(
|
1
|
298.55*
|
4.407ns
|
65.56*
|
0.688ns
|
Cu*TH
|
1
|
420.48*
|
127.76**
|
83.57*
|
6.95*
|
Error
|
6
|
48.49
|
3.033
|
11.02
|
0.925
|
C.V (%)
|
-
|
15.00
|
4.53
|
12.25
|
16.08
|
ns, * and ** indicating non-significant and significant at 5 and 1 % level, respectively
|
Table 2
Means comparison of biochemical traits as affected by copper and Trichoderma harzianum
Treatments
|
Catalase
|
Peroxidase
|
Poly phenol oxidase
|
Proline
|
(OD µg Protein min− 1)
|
(µg g Fw− 1)
|
Cu0TH0
|
27.44
|
27.14
|
20.43
|
3.08
|
Cu0TH1
|
29.30
|
34.88
|
21.04
|
4.13
|
Cu1TH0
|
53.56
|
43.17
|
28.48
|
7.35
|
Cu1TH1
|
75.37
|
48.49
|
38.43
|
9.35
|
LSD
|
14.95
|
5.77
|
6.90
|
1.71
|
TH0 and TH1: Non-inoculated, Inoculated with Trichoderma harzianum, Inoculation with Trichoderma harzianum
Cu0 and Cu1: 0, 100 µlit Copper
|
3-2. Scanning Electron Microscopy (SEM)
The Scanning Electron Microscopy (SEM) pictures of the synthesized copper nanoparticles are showed in Fig. 1 (A and B). This shape is used to confirm the size of the nanoparticles. In fact, SEM provided further insight into the surface morphology of the Cu-NPs. The experimental results showed that the diameter of the prepared copper nanoparticle, according to the image of SEM at 300 nm magnification, was about 15–30 nm and the shape is found to be spherical as shown in the Figs. 1A and 1B. The above results are in agreement with the findings of Hassanien et al. (2018).
3-3. Transmission Electron Microscopy (TEM)
Through transmission electron microscopy (TEM), it was easy to observe the shape and particle size of Cu-NPs. Therefore, the TEM analysis was used to observe the size and shape of nanoparticles. The TEM image of the synthesized copper nanoparticles is showed in Fig. 2 (A and B). This figure shows the spherical-sized particles of Cu-NPs in nano-dimensions. In fact, the TEM image (Figs. 2A and 2B), also confirms the spherical shape of Cu-NPs which the average particles are in the size range of 10–30 nm. The above results are in agreement with the findings of Ismail (2019).
3-4. X-Ray Diffraction
XRD analysis is a very useful tool for identifying the structure of metal nanoparticles. Therefore, the XRD analysis was used to evaluate the crystallinity of green synthesized Cu-NPs, type and crystal phase. The XRD pattern of Cu-NPs as shown in Fig. 3 that demonstrates the diffraction peaks of Cu-NPs exhibiting three peaks of \(2\theta\) at 43.6 (111), 50.80 (200) and 74.4 (220). The peaks match with the literature values of metallic copper (File No. 04-0836) (Rajesh et al., 2018), which further proves the formation of crystals of the copper nanoparticles. In fact, it can be said that the material in question was the copper nanoparticles and the sharpness indicates the crystalline nature of the as-prepared Cu nanostructure. The peaks observed in the XRD spectrum of copper nanoparticles synthesized in this study are consistent with the above (Hasheminya et al., 2018). Moreover, the average crystallite size of copper nanoparticles was analyzed using Scherrer’s formula.
(Eq. 1)
Where k = 0.94, the Scherrer‘s constant, D is the mean crystallite size, λ is the wavelength of the copper target, β is the full width half maximum value (FWHM) of the diffraction peaks and θ is the diffraction angle. Thus, XRD is commonly used to determine the chemical composition and crystal structure, type and crystal phase of a material. (Caroling et al., 2015).
3-5. Evaluation of factor groups by FT-IR
The FT-IR technique was used to confirm the synthesis of nanoparticles as well as to investigate the interactions between the different species and changes in chemical compositions of the mixtures during bio-synthesis. The FT-IR spectra of the synthesized Cu-NPs are shown in Fig. 4. As can be seen in the Figure, the peaks observed in the range of 3410 and 2920 are associated with the O–H and H-bonded functional groups in the copper nanoparticles, respectively. Also, the band at the 2848 was ascribed to C–H stretching vibrations. Furthermore, the carbonyl group, C–OH stretching vibrations, and C–O stretching were represented by the peaks at 1620, 1504, 1224, and 1320, respectively. This result also confirms that water soluble compound such as saponins which are present in the aqueous extract of guar leaf that have the ability to perform the stabilization of copper nanoparticles. A similar observation has been reported by the several works (e.g. Gopalakrishnan & Muniraj, 2019).
3–6. Antibacterial properties of copper nanoparticles, aqueous extract and essential oil extracted from the guar plant
The results of the MIC and MBC analysis of the effect of extracted nanoparticles, aqueous extract and essential oil (Control, application of Cu, application of Trichoderma harzianum and combined application of T. harzianum and Cu) on bacteria Escherichia coli and Staphylococcus aureus treated guar showed that, the essential oils treated with T. harzianum and copper at a concentration of 80 µl/ml inhibited the growth of Escherichia coli and Staphylococcus aureus. Also, the results of the MBC study showed that the concentration of 100 µl/ml in this treatment is effective in killing these two bacteria. E. coli and S. aureus bacteria were both inhibited by extracts treated with T. harzianum and copper at concentrations of 80 and 40 mg/ml, respectively. The concentration of 100 mg/ml in this treatment is effective in killing these two bacteria. The other factors investigated in this study included the minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC) of copper nanoparticles extracted from guar.
According to the findings of this study, the extracted copper nanoparticles of plant inhibited the growth of E. coli and S. aureus bacterial in all of the treatments. Also, according to MBC results, the copper nanoparticles at a concentration of 0.25 mg/ml are able to kill this bacterium. Besides that, the extracted nanoparticles in the fourth treatment inhibit the growth of S. aureus bacteria at a concentration of 0.5 mg/ml, and a concentration of 0.25 mg/ml is sufficient to kill this bacterium. Generally, by comparing MIC and MBC obtained from nanoparticles, the aqueous extract and essential oils of the studied treatments it can be concluded that, the copper nanoparticles and aqueous extract of the fourth treatment (combined application of fungus and copper) have better antibacterial properties compared to the essential oils of this plant. So, the essential oil of this plant in concentrations of 120 µl/ml inhibited the growth of E. coli and S. aureus. Sources revealed that no research on the synthesis of copper nanoparticles with aqueous extract of guar plant has been published to date, and this is the first. However, the similar studies on other plants have been conducted (Amer & Awwad, 2021; Dlamini et al., 2020; Hasheminya et al., 2018). The results of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Cu-NPs on the aforementioned bacteria are presented in Fig. 5 and Table 3.
Table 3
Antibacterial activity of copper nanoparticles, aqueous extract and essential oil extracted from the guar
Bacteria
|
Extracted
|
Antibacteria
|
Control
|
Cu
|
TH
|
TH*Cu
|
E.coli
|
Essential oil
|
MIC (µl/ml)
|
120
|
-
|
120
|
80
|
MBC (µl/ml)
|
-
|
-
|
-
|
100
|
Aqueous extract
|
MIC (mg/ml)
|
120
|
120
|
100
|
80
|
MBC (mg/ml)
|
-
|
-
|
120
|
100
|
Cu-NPs
|
MIC (mg/ml)
|
2
|
1
|
1
|
0.5
|
MBC (mg/ml)
|
0.5
|
0.25
|
0.5
|
0.25
|
S.aureus
|
Essential oil
|
MIC (µl/ml)
|
100
|
120
|
100
|
80
|
MBC (µl/ml)
|
-
|
-
|
120
|
100
|
Aqueous extract
|
MIC (mg/ml)
|
100
|
100
|
100
|
40
|
MBC (mg/ml)
|
120
|
120
|
120
|
100
|
Cu-NPs
|
MIC (mg/ml)
|
1
|
1
|
1
|
0.5
|
MBC (mg/ml)
|
0.25
|
0.25
|
0.5
|
0.25
|
3-5. Evaluation of factor groups by FT-IR
The results of studying the diameter and area of non-growth halo by image j software on the antibacterial properties of guar aqueous extract, essential oil and copper nanoparticles on Escherichia coli and Staphylococcus aureus by disk diffusion test showed that the nanoparticles extracted from the plant have the antibacterial properties on these two bacteria. In fact, by applying a concentration of 100 µl of copper with Trichoderma harzianum, each of these three extracted compounds (extract, essential oil and nanoparticles) increased the diameter and area of non-growth halo in Escherichia coli and Staphylococcus aureus bacteria.
Copper nanoparticles extracted from guar in all of the four treatments showed the high antibacterial properties on E. coli and S. aureus. So that, in the concentration of 1 mg of copper nanoparticles, the highest diameter and area of non-growth halo (3.268 mm) was observed in S. aureus bacteria and in the fourth treatment (combined application of fungus and copper). Furthermore, a 50 µl aqueous extracts concentration significantly increased the diameter and area of the non-growth halo in E. coli bacteria. So that, the fourth treatment (combined application of fungus and copper) had the largest diameter and area of non-growth halo in S. aureus (2.382 mm), while the control treatment had the smallest diameter and area of non-growth halo (0.931 mm). In S. aureus bacteria, therefore, a concentration of 50 µl of guar essential oil increased the diameter and area of non-growth halo. The results show that in comparison with the essential oil of this plant, the nanoparticles and aqueous extract from the fourth treatment (combined application of fungus and copper) had stronger antibacterial properties. In summary, most of the diameter and area of non-growth halo in the two studied bacteria in copper nanoparticles and aqueous extract was related to the fourth treatment. The above results are in agreement with the findings of Amer & Awwad (2021).