Root colonization of endophytic Trichoderma virens as a biological control for oil palm using SGFP reporter gene

Abstract Trichoderma virens 7b and 159c, are endophytes isolated from the roots of the oil palm. Both isolates are potential biological control agent that acts as a protective root shield against Ganoderma basal stem rot (BSR) disease. To investigate its mechanism in vivo, T. virens 7b and 159c were transformed via Agrobacterium tumefaciens, AGL1 strain carrying vector pCAMBgfp containing hygromycin (HygB) resistance cassette and synthetic green fluorescent protein (SGFP) gene. The putative transformed colonies were screened using SGFP-forward and SGFP-reverse primers that generated an amplicon of ~ 750 bp. Restriction analysis using SalI generated three fragments sized ~ 9700 bp, ~ 2865 bp and ~ 1435 bp, confirming the presence of the desired genes. Trichoderma virens 7b and 159c harbouring the SGFP gene were selected on PDA media supplemented with 175 µg/ml and 150 µg/ml Hygromycin B (HygB), respectively. Inoculation of transformed T. virens into the roots of 3-month-old oil palm tissue culture ramets showed positive colonization of the fungus examined under light microscope and confocal laser scanning microscopy (CLSM). These findings suggest that the Agrobacterium-mediated transformation of T. virens carrying the SGFP reporter gene can be used to monitor the colonization and detection of this beneficial biological control agent in the oil palm root system.