**4. Molecular based identification of fungi**

### **4.1 DNA extraction**

Before DNA extraction each purified *Aspergillus* spp. and *Fusarium* spp. was grown on PDA for 7–10 days at 28° C in an incubator. Then a 5 mm culture block was transferred on the conical flask containing PDA broth and the flasks were incubated at 28° C in an incubator for 7–10 days. Mycelium of each isolate was harvested and preserved at −80° C.

Genomic DNA was extracted from the fungal species isolated from maize grains following Wizard Genomic DNA extraction kit (Promega, USA) according to the manufacturer instructions from 100 mg fungal tissue ground with liquid nitrogen. Fungal tissue was processed by freezing with liquid nitrogen and grinding into a fine powder using a microcentrifuge tube pestle or a mortar and it was pestled. 0.04 g of this fungal tissues powder was added to a 1.5 ml microcentrifuge tube. 600 μl of Nuclei Lysis Solution was added and it was vortexed for 1–3 s to wet the tissue. The sample was incubated at 65° C for 15 min. 3 μl of RNase Solution was added to the cell lysate, and the sample was mixed by inverting the tube 2–5 times. The mixture was incubated at 37° C for 15 min. The sample was allowed to cool to room temperature for 5 min before proceeding. 200 μl of Protein Precipitation Solution was added, and it was vortexed vigorously at high speed for 20 s. The sample was centrifuged for 3 min at 13,000–16,000 × *g*. The precipitated proteins were formed into a tight pellet. The supernatant was carefully removed containing the DNA (leaving the protein pellet behind) and it was transferred to a clean 1.5 ml microcentrifuge tube containing 600 μl of room temperature isopropanol. The solution was gently mixed by inversion until thread-like strands of DNA form a visible mass. Then the sample was centrifuged at 13,000–16,000 × *g* for 1 min at room temperature. The supernatant carefully decanted. 600 μl of room temperature 70 % ethanol was added and was inverted gently into the tube several times to wash the DNA. It was centrifuged at 13,000–16,000 × *g* for 1 min at room temperature. The

ethanol was aspirated carefully using either a drawn Pasteur pipette or a sequencing pipette tip. The DNA pellet was very loose at this point and care must be used to avoid aspirating the pellet into the pipette. The tube was inverted onto clean absorbent paper and the pellet was air-dried for 15 min. 100 μl of DNA Rehydration Solution was added and the DNA was rehydrated by incubating at 65° C for 1 h. Periodically the solution was mixed by gently tapping the tube. Alternatively, the DNA was rehydrated by incubating the solution overnight at room temperature or at 4° C. The DNA was stored at 2–8° C.
