TRANSFORMATION OF Trichoderma harzianum T8 WITH GFP GENE TO FACILITATE ITS MONITORING IN SOIL AND USE OF SEM TO STUDY INTERACTION OF T. Harzianum WITH Rhizoctonia solani SCLEROTIA

Monitoring of the behavior of biocontrol agents in situ is a powerful tool for successful management of diseases. In this research a Trichoderma isolate was identified as T. harzianum by the analysis of rDNA-ITS regions using the TrichOKEY and TrichoBLAST sequence identification programs. For gfp transformation the maximum protoplast yield (5 × 108/ml) was obtained using the optimized conditions of protoplast isolation. The highest transformation efficiency (96%) was obtained with 10 µg DNA and 30 µl protoplast (5 × 108/ml). To obtain a genetically marked Trichoderma that can be used as a biomonitor, T. harzianum was cotransformed with p3SR2 and pDL2 plasmids containing amdS and gfp genes, respectively. Transformation of gfp gene was confirmed by PCR pattern. The morphology and viability of selected cotransformant (T. harzianum T8-7MK) was similar to that of the wild type. The biocontrol activity of transformant was confirmed by Dual culture with Rhizoctonia solani and microscopic analysis. The monitoring of gfp tagged T. harzianum was followed in agricultural soil (in 60 days after inoculation). Also the colonization of T. harzianum T8-7MK was monitored visually on R. solani sclerotia by scanning electron microscopy. The treated sclerotia showed no detectable growth on PDA after six days, while the untreated sclerotia were able to grow after two days on the same medium. These observations confirmed the biomonitor and biocontrol activity of T. harzianum T8-7MK transformant.