Molecular marker development from ISSR for fungal pathogens of Mangifera indica L.

Abstract

Pathogen causes severe loss of crop plants worldwide, the study presents isolation, identification and molecular marker development of six fungal pathogens from the different varieties of the mango from Rajkot and nearby areas. These include two strains of Colletotrichum gloeosporioides (SUF24) and C. gloeosporioides (SUF29), Fusarium fujikuroi, Phaeosphaeria nodorum, Botryosphaeria dothidea, and Pseudofusicoccum ardesiacum. They were cultured and their DNA was isolated. PCR amplification was performed using ISSR primers. Amplification was checked by agarose gel electrophoresis and band pattern was scored. PCR amplification showed monomorphic and polymorphic bands with the studied primers. ISSR primer 880 showed monomorphic band in all studied fungi. Each monomorphic band was cloned, sequenced and selected for further bioinformatics analysis. From the obtained sequences, multiple sequence analysis, sequence similarity matrix, frequency distribution plot, pairwise identity using heat map, restriction site analysis, polymorphism and haplotype identification was performed. It was observed that the monomorphic band of these fungi have nearly similar base pairs length but the nucleotide patterns were found to be different. Even two strains of Colletotrichum gloeosporioides showed distinct patterns. Each one of the sequences showed unique nucleotide sequence composition and RE cutting sites, thus monomorphic band can be converted to the polymorphic band pattern. This approach is used to design new DNA markers for the fungi studied and its applicability to discriminate fungi even at a strain level is discussed.