Use of RAPD-PCR and Phage display techniques to study variation in Colleto trichum gloeosporioides isolated from Papaya (Carica papaya L)

Abstract

ScFv monoclonal antibodies were raised against Colletotrichum gloeosporioides (Penz.) Penz. and Sacc. isolates of papaya using phage display technology. Phages obtained after fourth round of biopanning were used to generate monoclonal antibodies against the pathogen. Four monoclonals were identified having the highest binding affinity to C. gloeosporioides and were selected to differentiate isolates of C. gloeosporioides along with isolates of C. capsici, Fusarium oxysporum f.sp. cubense and Alternaria spp. Cetyle trimethyl ammonium bromide (CTAB) method with some modifications was followed to isolate total genomic DNA in another experiment. Initially, DNA samples were amplified with 20 different OPA random primers and two of them were selected to amplify individual DNA samples from the eight papaya isolates and C. capsici, Fusarium oxysporum f.sp. cubense and Alternaria spp . All monoclones found to have high binding affinity towards different isolates of C. gloeosporioides compared to three other species, indicating their specificity towards C. gloeosporioides. There was a greater variation ob served among isolates according to band patterns when amplified with OPA 3 and OPA 14 primers se lected. However, with both these primers, there were common bands(1200 bp with OPA 3 and 965 bp with OPA 14) that may be specific to C. gloeosporioides.