*2.7. Production of Secondary Metabolites from Trichoderma spp.*

The agar plugs of *Trichoderma* spp., measuring 7 mm in diameter, were taken from actively growing margins of *T. harzianum* (PGT4), *T. reesei* (PGT5) and *T. reesei* (PGT13) cultures grown on PDA media and were inoculated individually: dual cultures (*T. harzianum* (PGT4) and *T. reesei* (PGT5); *T. reesei* (PGT5) and *T. reesei* (PGT13) and *T. reesei* (PGT13) and *T. harzianum* (PGT4)) and co-culture sample PGTA (*T. harzianum*, *T. reesei* and *T. reesei*) were inoculated into 250 mL Erlenmayer flasks containing 100 mL of potato dextrose broth medium (PDB, HIMEDIA) supplemented with chloramphenicol antibiotics, incubated in static condition for 9 days at 25 ◦C [42]. After the incubation period, to avoid fragmentation of the mycelium, it was removed using a microbial loop and the cultures were filtered under vacuum through filter paper (Whatman No. 4). The final filtrate was called a crude extract of secondary metabolites. A control assay was performed to ensure an optimum filtration (to check for the absence of conidia and mycelia) procedure by spreading 30 µL of the final filtrate on petri plates under sterile conditions containing PDA medium. The plates were examined for fungal growth [41].
