Construction of new GFP-tagged fusants for Trichoderma harzianum with enhanced biocontrol activity

Abstract

Trichoderma is one of the most exploited biocontrol agents for the management of plant diseases. In biocontrol ecology, the critical factors are detection, and the monitoring and recovery of specific biocontrol agents either naturally present or deliberately released into the environment. Protoplast fusion is an appropriate tool for the improvement of biocontrol Trichoderma strains. Protoplast isolation from Trichoderma harzianum was achieved using 24 h culture age, 6.6 mg/ml Novazym L 1412 at 30°C which resulted the maximum protoplast yield of 5 × 108/ml. The self-fused protoplasts were regenerated and 12 fusants were selected based on their growth rate on 2% colloidal chitin medium. Next, a comparison was done for chitinase and antagonistic activity. Transcriptomic analysis based on quantitative real-time RT-PCR, demonstrated that T8-05 fusant expressed 1.5 fold of chit42 transcript as compared with the parental line. This fusant with 7.02±0.15U chitinase activity showed a higher growth inhibition rate (100%) than the parent strain, against Rhizoctonia solani. To obtain a genetically marked fusant that can be used as a biomonitor, the fusant was cotransformed with the gfp and amdS genes. The morphology and viability of selected cotransformant (FT8-7MK-05-2) was similar to the parent. Green fluorescing conidia were observed within the first 2 days of incubation in the soil, and this was followed by the formation of chlamydopores after 60 days. The colonisation of the gfp-tagged fusant was also monitored visually on R. solani sclerotia by scanning electron microscopy. Production of gfp-tagged fusant of Trichoderma spp. provides a potentially useful tool for monitoring hyphal growth patterns and the population of biocontrol agent isolates introduced into environmental systems.

Keywords

• biomonitor
• fusant
• green fluorescent protein
• real-time RT-PCR
• Trichoderma