Materials and methods
Using ordinary laboratory method, the pathogen fungus A. alternata was isolated from transitional part between healthy tissue and brown spots on tobacco leaves. T. harzianum was isolated from the root zone of rhizosphere, by a method of dilution, using Czapek agar.
In-vitro biological activity of T. harzianum on A. alternata was investigated by double cultures on the potato dextrose agar.
Trials were set up in three replications, with five Petri dishes by each variant. They were incubated at 25oC and the diameter of the colonies was measured for 10 days.
In diffusible metabolites, investigations were maintained with the variants:
- Placing the fragments of test fungus and pathogen on the separated half of a media
- Placing the fragments at the same time, with near-contact
- Temporal and spatial advantage of pathogen growth (up to filing the half of a medium) and then place the T. harzianum fragment.
In volatile metabolites investigations, fragments of test fungus and pathogen were placed in the center of two separate PDA plates sealed with parafilm, so, the test fungus was in the under plate and, the pathogen was in upper plate. There were two controls (T. harzianum and A. alternata ) in an inverted position.
After the period of incubation, micro observations were made of the pathogen in the two kinds of double cultures.