**2. Methodology**

### **2.1 Materials**

In this study, all chemicals used were of highest purity. Organic solvents used included C2H5OH and (DMF) were spectroscopic pure from British Drug House (BDH). Distilled water collected from glass equipment. The other materials such as, 5-bromo-salicylaldehyde (Sigma), *o*-phenelendiamine (Aldrich), Zn(NO3)2 (Merck) were also used.

#### **2.2 Instrumentation**

Microanalytical determinations were carried out in the Cairo University (Microanalytical center). The IR spectra were recorded on a Perkinelmer spectrophotometer (400 – 4000 cm−1) (KBr technique). proton-NMR spectra (DMSO-*d*6) were measured at a Pruker spectrophotometer, using TMS as an internal standard. The uv–vis spectra were recorded on a Perkin-Elmer spectrophotometer. Mass spectra were recorded with the aid of a Shimadzu using a direct insertion probe (DIP) at temperature range 50–800°C. The nano-sized complex was characterized with (SEM) a scanning electron microscope.

#### **2.3 Synthesis of the Schiff base ligand**

A solution of 5-bromo-salicylaldehyde (4.02 g, 20 mmol.) in absolute ethanol (50 ml) was added to a hot stirred solution of *o*-phenylenediamine (1.08 g, 10 mmol.). This mixture was refluxed for 3 hr. in water bath and cooling, then, the products formed were collected by filtration. Yield (87.4%, 90.2%); M. P; 240 and 200°C; respectively (the ligand was synthesized in our previous work [24].

#### **2.4 Studied micro-organisms**

Isolates of *Pythium ultimum* and *Trichderma harzianum* were obtained from biology department –Jouf University, KSA. Precultures of *Trichderma harzianum* (MW459195) and *Pythium ultimum* (MW830915) were made by grown on potato dextrose agar (PDA) for 7 days at 26°C in dark.

#### **2.5 Preparation of biomass of** *Trichderma harzianum* **(MW459195)**

Two discs (5 mm in diameter) of *T. harzianum* (MW459195) was inoculated onto 100 ml PD broth in a 250 Erlenmeyer flask and incubated at 26°C for 7 days in the dark. Mycelium mats were collected by using filter paper (Whatman No. 1) and washed three times with sterilized distilled water to eliminate any adhering media may present. Mycelium mats (3 g) were dried for bio-synthesis of nanoparticles.

#### **2.6 Pathogenicity tests of nano metal complex against seedling and germination of seeds**

In order to use Nano metal complex as bio-control agent, it must not produce metabolic toxins that effect on seed germination and plant growth. Seeds of Fenugreek were soaked in the solution of sodium hypochlorite (2% w/v) for 3 min and washed with sterile distilled H2O several times. Ten ml of different concentration of nano metal complex (10, 20, 30 and 40 ppm) were prepared and mixed with 0.6 g of carboxymethyl cellulose (CMC). Fifty of Fenugreek seeds were added to the mixture and stirred thoroughly and allowed to dry overnight in a laminar flow cabinet at room temperature, and incubated at 20°C until seeds germinated. Ten ml of sterile distilled H2O were used as control. After Fenugreek seeds were germinated and beginning to form radicles and plumules, the time of seed germination were determined. Choose the vital seeds and 3 seeds were planted in Erlenmeyer flasks, which containing sterilized 100 ml of WA (3%) was decanted in conical flasks with 10 ml of different concentration of nano-metal complex (10, 20, 30 and 40 ppm). All flasks were incubated in growth chamber at 25°C with 12 h photoperiod (91 μmol m−2 s−1) for 4 week. All seeds were then used in study the pathogenicity of nano metal complex on Fenugreek seed germination and seedling elongation.

#### **2.7 Biosynthesis of the nano metal complex**

The Zn nano complex was prepared by mixing the Schiff base ligand (1 mmol) and 1 mmol of Zn (II) nitrate in the presence of *T. harzianum* powder as a reducing agent as a novel method for the preparation of Zn-nanocomplex. The mixture was ground with a pestle in an open mortar at room temperature. The melted mixture was then allowed to solidify. The solid was filtered off and crystallized twice-using C2H5OH to give Zn-nanocomplex as a pale brown crystal [25].
