Protein profiling of Colletotrichum falcatum

Abstract

Red rot caused by Colletotrichum falcatum is a deleterious disease of sugarcane, responsible for 29.07 % loss in cane yield and 31 % loss in sugar production in Pakistan. Disease detection, at premature stage, will help to protect the crop from detrimental effects of red rot. Proteins extracted from C. falcatum can be used as an antigen source to design an immunological detection assay for the early detection of disease. Aim of present study was to identify C. falcatum and its protein profiling. Identification was done using morphological features and by amplification of Internal Transcribed Spacer (ITS) region. Total DNA was isolated from frozen mycelia by small-scale extraction method and PCR amplification was performed using primers (ITS1 and ITS4) that produced an amplification product of approximately 600bp. The purified PCR product was cloned and sequenced. Obtained DNA sequence showed the maximum sequence identity (99%) to Gibberella moniliformis (accession no. GU982311). Phylogenetic analysis also confirmed the similarity of this sequences with Gibberella moniliformis. Two types of conidia were observed under scanning electron micrograph in vicinity of mycelial culture, i.e. falcate shaped conidia as primary spores and oval shaped conidia as secondary spores. Total protein was extracted from frozen mycelia of C. falcatum through Trichloroacetic acid (TCA)/ acetone extraction method. Total protein was run on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) but a fainted band was observed. There may be two probable reasons for this: 1) due to less concentration of mycelial sample, which in turn resulted in a lesser amount of protein content, 2) C. falcatum has tough and sturdy cell wall which was unable to disrupt at room temperature.