**2.8 Bioassay of nano metal complex against** *Pythium ultimum*

Sterilized warm Potato Dextrose Agar (PDA) medium was prepared, and poured in sterile Petri dish supplemented with 100 μl of different concentration of nano metal complex (10, 20, 30 and 40 ppm). Agar discs (10 mm) were taken from the margins colony grown on water agar, and then placed in the center of each Petri dish. All petri-dishes were incubated in the dark at 26°C. Radial fungal mycelial growth was measured by determined colony diameters in each dishes, after 6 days of incubation. The percentage inhibition of fungal growth was calculated using the formula:

$$\text{Inhibition}\left(\emptyset\right) = \left(\left(\text{D} - \text{M}\right) \div \text{D}\right) \times \mathbf{100} \tag{1}$$

where D is the growth diameter of *P. ultimum* in the control (mm) and M is the growth diameter of *P. ultimum* in presence of Nano-metal complex (ppm) [26].

### **2.9 Microscopic analysis of the effect of nano metal complex on the mycelium growth**

The microscopic images were taken at the faculty of Agriculture, Cairo University, using a scanning electron microscope (JOEL brand model JSM6400) at 1000 and 3000 x. Samples were taken from mycelium treatment with tested nano metal complex at 30 ppm, then the mycelium was placed on a glass slide and using a metal coating equipment of samples (EDWARDS E306A), a copper bath was applied to the samples for 20 min for subsequent visualization. Finally, the general morphological and structural characteristics of the *P. ultimum* mycelium were evaluated**.**

*Biosynthesis of Zinc Nanocomplex Employing for Plant Growth Promotion and Bio-Control… DOI: http://dx.doi.org/10.5772/intechopen.100185*

#### **2.10 Evaluation of nano metal complex as antifungal activity and plant growth promotion in pot experiment**

The ability of nano metal complex in different concentrations (10, 20, 30 and 40 ppm), its antifungal and growth enhancement effects of Fenugreek seedlings in soil infested with *P. ultimum* were tested. For preparation *P. ultimum* infested soil, the propagule suspension of *P. ultimum* was added at thirty-propagules/g soil to the autoclaved soil. Thirty seeds were used for each treatment. The surface of the seeds were sterilized with 2% sodium hypochlorite for 3 min, and washed by sterile distilled H2O for 5 min and dried using sterile filter paper. Pre germination test was done to select viable seeds. The experiment was classified into five groups. Frist group: free *Pythium* soil were irrigated with sterile distilled water, while second groups: soil infested with *Pythium* and irrigated sterile distilled water. The remaining three groups: the soil infested with *Pythium* in each group and soil were irrigated with nano metal complex -solution with different concentrations (10, 20, 30 and 40 ppm). Pots were kept in a growth-illuminated cabinet (Precision, United States) at 25°C with 12 h photoperiod (91 μmol/ m2 s1 ) under humid conditions. Soil moisture content was preserved at 35%. Experiments were performed with ten replicate pots per treatment. Germination % was calculated after 48 and 72 hour by following formula:

$$\text{Germination percentage} = \frac{\text{Number of permuted seeds}}{\text{Total number of seconds}} \times 100\tag{2}$$

After 10 days of germination, seedlings growth were determined in terms of shoot and root length (mm).

#### **3. Results and discussion**

In the present study, the analytical data of the previously prepared Schiff base ligand and its Zn nano metal complex suggest the structures as in **Figure 1**. The Schiff base ligand and its Zn-nano complex were subjected to elemental analyses (C, H, N and O). The results of elemental analyses with molecular formula and the M.P. were in **Table 1**. The results obtained were in well agreement with those of

**Figure 1.** *Schematic route for preparation of the free ligand.*


#### **Table 1.**

*Elemental analysis and some physical measurements of the free ligand and its metal nano complex.*

#### **Figure 2.**

*1 H NMR data for (a) free ligand and (b) its Zn(II) nano complex.*


**Table 2.**

*1 H NMR data for the Schiff base ligand and its nano complex.*

the suggested formulae. The M.P. were sharp, indicating the purity of the prepared compounds. The results of the elemental analyses of the isolated complex are in agreement with those required by the proposed formula of the complex.

The NMR spectrum of the Schiff base ligand (**Figure 2**) (**Table 2**) showed a singlet peaks at 9.335 ppm and at 11.247 ppm due to the azomethine and phenolic protons, respectively. In addition to multiplet signals at 6.465–8.175 ppm attributed to the aromatic protons [27, 28]. The comparison of the NMR data of the ligand and its Zn (II)metal complex confirms the mode of coordination between the ligand and its metal ion. By complexation, the spectrum of the nano-Zn complex display

*Biosynthesis of Zinc Nanocomplex Employing for Plant Growth Promotion and Bio-Control… DOI: http://dx.doi.org/10.5772/intechopen.100185*


#### **Table 3.**

*Infra-red spectral data of the free ligand and its metal complex.*

a significant shift of the signals due to C=N group and OH protons indicating the involvement of both OH as well as C=N groups in coordination to the metal ions without their deprotonation suggesting that the ligand act as neutral tetradentate ligand, O2N2 coordination sphere.

The IR spectrum of the ligand was compared with those of its metal complex, in order to confirm the mode of bonding, **Table 3**.

The infrared spectrum of the schiff base ligand has a broad absorption band at 3456 cm−1 which were due to the phenolic group [29, 30]. The spectrum displays also medium band at 1290 cm−1 due to υC-O (phenolic group) [31]. Additionally, a strong band at1620 cm−1 was observed due to υC=N of azomethine group [32]. By careful comparison of the spectrum of the nano-metal complex with those of the Schiff base ligand it was found that: the band at 3456 cm−1 shifted to lower frequency region at 3401 cm−1 in the metal nano complex suggesting the involvement of OH group in complexation without its deprotonation. The strong bands at 1620 and 1290 cm−1 due to υC=N and υC-O (C=N and OH groups) are shifted to lower wave number (1532 cm−1) and (1240 cm−1), respectively in the metal complex indicating the coordination of nitrogen and oxygen to the metal ion. Also, the spectrum of the complex shows three bands at 1401 cm−1, 1372 cm−1 and 1025 cm−1 corresponding to unidentate coordination mode of NO3 group. All these results are consistent with the conductance data. Conclusive evidence of the bonding is also shown by observing new bands in the infrared spectrum of metal nano complex in low frequency region at 550 cm−1 and 515 cm−1 may be attributed to υM-O and υM-N, respectively that are not observed in the spectrum of the Schiff base ligand [33, 34].

The mass spectrum of the free ligand shows the parent peak at m/e = 474.36 (48.78%) that agree with the molecular formula C20H14Br2N2O2. Also, the spectrum shows numerous peaks corresponding to various fragments, their intensity indicates the stability of the fragments. **Figure 3** represent the proposed pathway for the decomposition steps for the ligand.

The conductivity Λm value of the Zn-nanocomplex can be calculated using the following relation Λm = K/C, where C is the molar concentration of the nano metal complex solution and K is the specific conductance. The complex was dissolved in (10−3 M) DMF and the molar conductivity of the solution at 25 ± 2°C were measured **Table 1**. It is concluded from the results that; the complex is found to has molar conductance value of 11.25 ohm−1mol−1cm2 indicating that this complex is non-electrolytic in nature. Also, the values indicate the bonding of the nitrate ions to metal cations [33, 35].

UV–vis spectra of the ligand and its metal complex was performed in DMF at room temperature at wavelength range 200–800 nm. The significant electronic absorption bands of the ligand, its nano complex and the magnetic moments of the complex are given in **Table 4**. 2 absorption peaks were observed in the spectrum of the ligand at 265 and 339 nm due to п-п\* and n-п\* transitions, respectively attributed to benzene and the azomethine groups [34, 36]. In the spectrum of metal complex, the absorption bands attributed to п-п\* and n-п\* transition were

#### **Figure 3.**

*Mass fragmentation pattern for the free ligand*


#### **Table 4.**

*Magnetic moment and electronic spectral data of the ligand and its Zn-nano complex.*

shifted to lower or higher frequency due to the coordination of the Schiff base ligand with the metal ions. Zn (II) complex is diamagnetic in nature with no *d-d* transition. So, according to all of this obtained data, the octahedral structure may be suggested.

The size and the morphology of the nano complex were studied by SEM (**Figure 4**). It was found that the particles are semispherical in nature with some agglomerations. The SEM images also revealed the stabilization of Zn(II) nanoparticles due to interaction with the Schiff base ligand [37].

*Biosynthesis of Zinc Nanocomplex Employing for Plant Growth Promotion and Bio-Control… DOI: http://dx.doi.org/10.5772/intechopen.100185*

#### **Figure 4.**

*SEM image of nanoparticle complex.*

**Figure 5.** *Suggested structure of Zn nano complex.*

Correlation of all this results, the structure of the complex can be suggested to be as in **Figure 5**.

Fungi are the most efficient microorganisms when it comes to the biological production of Mycogenic Metal Nanoparticles synthesis [38]. This is due to the fungi having enhanced processes, accumulating metals, have capacity for producing a high number of bioactive metabolites [39, 40], fungi do not require complex nutrients, these are easy to grow, easy to manipulate, also have high metabolites and production of biomass, and has high metal uptake and high wall-binding capability [41–43]. Our Experiment revealed that nano-metal complex had no obvious effect on the time required for Fenugreek seed germination, (**Figures 6** and **7**). Nanometal complex activated seeds germination, since seeds germinated 24 h prior to the germination of control. Results in **Figure 7** revealed that seedling elongation was retarded with different degrees in case of treated seeds compared with the controls. Nano-metal complex showed a slight retarding effect on Fenugreek seedling elongation compared with the control, especially after 7 days from seed germination.

**Table 5** shows the antifungal influence of different concentration of nano metal complex (10, 20, 30 and 40 ppm) on *P. ultimum* mycelial growth on PDA

#### **Figure 6.**

*Effect of nano-metal complex with different concentration on the time of seed germination.*

#### **Figure 7.**

*Pathogenicity tests of nano metal complex against Trigonella foenum-graecum germinating seeds grown in Erlenmeyer flasks containing 2% water agar. (A) Control, (B) treated seeds with nano-metal complex (10ppm), (C) treated seeds with nano-metal complex (20 ppm), (D) treated seeds with nano-metal complex (30 ppm), (E) treated seeds with nano-metal complex (40 ppm) after 7 days at 25°C.*

*Biosynthesis of Zinc Nanocomplex Employing for Plant Growth Promotion and Bio-Control… DOI: http://dx.doi.org/10.5772/intechopen.100185*


**Table 5.**

*Antifungal activity of nano metal complex against* P. ultimum *on PDA medium at 26°C for 6 days in the dark.*

medium after 6 days of incubation at 26°C. Results reveled that significantly reduced of mycelial growth of *P. ultimum* at different concentrations (10, 20, 30 and 40 ppm), especially in 30 and 40 ppm compared to control (p ≤ 0.001). As shown in **Figures 8** and **9** efficacy of nano metal complex at concentration 40 ppm, was the greatest one of all concentration tested *P. ultimum* growth inhibition, that observed no mycelium growth appear, follow by nano metal complex at concentration 30 ppm which mycelium growth were (32 mm). Percentage of inhibition of nano metal complex at different concentrations (10, 20, 30 and 40 ppm) were (23.52%, 29.4%, 62.4% and 100%) respectively. Biological control of damping-off diseases has been successfully. Applied using *T. harzianum* [44, 45]. El-Sayed [46] reported that *Trichoderma* spp. are known to control all pathogens either directly by inhibition of sporulation and growth of the pathogen mechanisms such as enzyme production and mycoparasitism, or indirectly by competing for nutrients and space, modifying the environmental conditions, or by promoting plant growth and enhancing plant defensive mechanisms and antibiosis. Microscopic examination of the mycelium showed coagulation of the protoplasm within the mycelium and lysis of cell wall of mycelium compared with the control (**Figure 10**).

Kamala and Indira [47], Evaluated the activity of three *Trichoderma* isolates (T73, T80 and T105) for their bio-control activity of *P. ultimum*. Also assayed the different bio control mechanisms such as protease, chitinase, β-1,3-glucanase

#### **Figure 8.**

*Inhibition of mycelial growth of P. ultimum. (A) Control, (B) Control containing Metalaxyl, (C) Treated containing Nano-metal complex (10 ppm), (D) Treated containing Nano-metal complex (20ppm), (E) Treated containing Nano-metal complex (30ppm) and F) Treated containing Nano-metal complex (40ppm) on PDA at 26°C for 6 days at the dark.*

#### **Figure 9.**

*Inhibition % of P. ultimum exposed to different concentration of nano metal complex, on PDA at 27°C for 6 days at the dark.*

#### **Figure 10.**

*Scanning electron micrograph showing the antagonistic effect of nano metal complex (30 ppm) against P. ultimum. (A and B) healthy mycelia of P. ultimum. (C) Abnormal structure, lysis and destruction of P. ultimum.*

activity, cellulase and production of volatile and non-volatile compounds. Their results show that, *Trichoderma* (T-105) reduced the.

Pre and post-emergence damping-off disease or Root rot disease were incidence in infested soil with *P. ultimum* and showed the highest disease control percentage under in vitro as well as the pot experiment [48–50]. In our study, we evaluated the biogenic Nano-metal complex in pot experiments, which reveal that data in **Table 6** have differences in the germination%, that It observed the beginning of germination *Biosynthesis of Zinc Nanocomplex Employing for Plant Growth Promotion and Bio-Control… DOI: http://dx.doi.org/10.5772/intechopen.100185*


#### **Table 6.**

*Effect of nano-metal complex treatments on germination (%) and root and shoot lengths (mm) of fenugreek.*

of seeds after 20 hour in Nano-metal complex 20, 30 and 40 ppm about 60%,compared with control 33.3%. gradullary, the germination % of seeds increase with increasing time until 48 h, all seeds were germinate 100%.

With observance to root and shoot lengths, results in **Table 6** show that Nanometal complex had effects in both concentration 20–40 ppm for shoot length about (180 mm), the Nano-metal complex (10 ppm) treatment recorded the lowest value for root length about 150 mm. In addition in the same concentration for root length about (95.2 mm, 96.5 mm and 96.5 mm), respectively. The Nano-metal complex (10 ppm) treatment recorded the lowest value for root length about 81.8 mm, **Figures 11** and **12**. From previous results, it can be concluded that Nano-metal complex (40 ppm) had the best record in both germination as well as shoot and root length.

The increased applications of Metal Nano Particles in several area required new biocompatible, safe, and active nanostructures with less dangerous by products of synthesis reactions [51]. Mycogenic Nano-Particles are observed as biocompatible,

#### **Figure 11.**

*The effect of bio-control agent and plant growth promotion of nano metal complex on Trigonella foenumgraecum seeds germination in pots containing sandy loam soil cultivated with P. ultimum. (1) Control seeds sown in free Pythium soil, (2–5) seeds sown in soil infested with P. ultimum and irrigated with nano-metal complex (40,30,20, 10 ppm), respectively after 4 weeks.*

#### **Figure 12.**

*Effect of nano-metal complex on Trigonella foenum-graecum plant biomass after 4 weeks of planting. (1) Shoot biomass. (2) Root biomass. (3) Total biomass.*

eco-friendly, less toxic, safe, and cheapest alternatives, with lowest consumption of energy and highest yields when compared with other physical or chemical synthesis [26, 52]. While well-known advantages of Metal Nano-Particles as the best alternatives against antimicrobial species of pathogenic fungi and other microflora, many

*Biosynthesis of Zinc Nanocomplex Employing for Plant Growth Promotion and Bio-Control… DOI: http://dx.doi.org/10.5772/intechopen.100185*

challenges and opportunities are ahead of us. Given that the effects of NPs result from a combination of multiple, the potential development of resistance against them is more difficulty and less likely [53]. In future should be focus on testing new isolates, discovering new Metal Nano-Particles, and clarifying their structures and mode of its action as antimicrobial agents. Novel discovery of mycogenic MNPs include the study of extremophilic and endophytic isolates. While the endophytic has increased its relevance during the last years, the extremophilic is still limited in studies and focused on other purpose rather than their use against antimicrobial species [54].
