Enhancement Of Resistance Against Colletotrichum Gloeosporioides In Cassava Over-Expressing Rice Thaumatin-Like Protein (Pr-5) Gene

ABSTRACT

Cassava (Manihot esculenta Crantz), is an energy giving food and has a crucial role in many of the world‟s least developed countries by providing not only food but also economic security. However, its productivity has been hampered by constraints that reduce yields. One of the biotic constraints is cassava anthracnose disease (CAD) caused by pathogen Colletotrichum gloeosporioides, results into more than 30 per cent reduction of produce in the fields. The use of conventional methods like biological control, use of resistant cultivars and breeding for resistance to control CAD has offered limited success to the resource–poor farmers who depend on cassava. The objective of the study was to genetically engineer cassava resistant to CAD using Oryza sativa thaumatin-like protein gene (Ostlp), a member of the pathogenesis-related (PR) proteins in crop plants with potential for enhancing resistance against fungal attacks. Cassava (Manihot esculenta Crantz) model cultivar TMS 60444 over-expressing Ostlp under the control of CaMV-35S promoter were obtained through the Agrobacterium-mediated transformation of friable embryogenic calli (FEC), following a pre-screening susceptibility bioassay. The presence and integration of the transgene Ostlp in TMS 60444 genomic DNA was confirmed by PCR and Southern blot analyses. Subsequent Reverse transcriptase-PCR analysis revealed the expression of the transgene in the leaves tissues in the transformants. The in vivo antifungal activity of the transgene against the pathogen Colletotrichum gloeosporioides, the causative agent of cassava anthracnose disease (CAD) was evaluated using the leaf and stem in vitro bioassay. The results demonstrated that the Ostlp transgenic cassava leaves and stems significantly reduced the growth of the fungus and size of lesions compared to controls when leaves and stems were wound inoculated with C. gloeosporioides. Therefore, constitutive expression of rice thaumatin-like protein in cassava plants conferred enhanced tolerance against C. gloeosporioides compared to the non transformed cassava genotype. Since the evaluation of resistance was done in vitro, the study recommends field trials of the new germplasm and transformation of other farmers‟ preferred varieties using Ostlp gene.