*2.2. Isolation and Identification of the Fungal Strain*

The fungal strain used for biosynthesis of MgO-NPs was isolated from Qalyubia Governorate, Egypt (31◦18'522.07" E, 30◦15'524.13" N), and has a code E3. The isolation procedure was achieved according to Fouda et al. [29] as follows: approximately 1.0 g of soil sample undergo diluted in sterilized dis. H2O. After that, 100 µL of the fourth dilution was inoculated onto MEA plates and incubated for 3–4 days at 30 ± 2 ◦C. The appeared colonies were picked up and re-inoculated onto the same media for purifications. The purified colony was preserved on an MEA slant for further use.

The identification was accomplished by cultural and microscopic characteristics and confirmed by molecular identification using internal transcribed spacer (ITS) sequence analysis. The ITS rDNA region was amplified using primers for ITS1 f (5-CTTG GTCATTTAGAGGAAGTAA-3) and ITS4 (5-TCCTCCGCTTATTGATATGC-3) [30]. The PCR mixture contained: 1X PCR buffer, 0.5 mM MgCl2, 2.5 U Taq DNA polymerase (QI-AGEN, Germantown, MD 20874, USA), 0.25 mM dNTP, 0.5 µL of each primer, and 1 µg of extracted genomic DNA. The PCR was performed in a DNA Engine Thermal Cycler (PTC-200, BIO-RAD, Hercules, CA, USA) with a program of 94 ◦C for 3 min, followed by 30 cycles of 94 ◦C for 30 s, 55 ◦C for 30 s, and 72 ◦C for 1 min, followed by a final extension performed at 72 ◦C for 10 min. The PCR product was checked for the expected sizes on 1% agarose gel and was sequenced by Sigma Company for Scientific Research, Egypt, with the two primers. The sequence was compared against the GenBank database using the NCBI BLAST tool. Multiple sequence alignment was done using the Clustal Omega software package (http://www.clustal.org/clustal2, accessed on 17 November 2010), and a phylogenetic tree was constructed using the neighbor-joining method with MEGA (v.6.1) software, with confidence tested by bootstrap analysis (1000 repeats).
