4.1.2. Dual Culture Antagonism Assay

Dual culture antagonism assays were also performed to evaluate the antagonistic capability of the *T. atroviride* strain I-1237 (Esquive) against the three *Lasiodiplodia* spp. isolates targeted for study (Bt105, LA-SOL3 and CBS124060), using dual culture assays [49,75]. *Trichoderma atroviride* strain I-1237 cultures used for this assay were obtained using the same method described for the mycelial growth inhibition assays. Mycelium plugs with 5 mm diameter of *T. atroviride* strain I-1237 and each *Lasiodiplodia* sp. isolate were cut from the actively growing margin of 3-day-old colonies, growing on PDA. Plugs were placed on opposite edges of 90 mm Petri dishes containing 15 mL of PDA. Plates were then incubated for 5 days in the dark at 22 ◦C. Each *Lasiodiplodia* sp. isolate was grown individually under the same conditions as control plates. Each combination *Trichoderma*/*Lasiodiplodia* spp. was replicated four times and the assay was performed twice. The percentage of mycelium growth inhibition was calculated using the formula, percent inhibition (PI) = [(B − A)/B] × 100 [42], where A is the radius of pathogen mycelium growth on the dual culture plates, and B is the radius of *Lasiodiplodia* spp. growth on the control plates.
