Purification and Partial Characterization of Polygalacturonase From Virulent Isolate Of Ascochyta rabiei (IK06): Causal Agent of Ascochyta Blight in Chickpea

Ascochyta blight caused by Ascochyta rabiei is one of the major diseases of chickpea (Cicer arientinum) in Iran. Many phytopathogenic microorganisms, incuding A .rabiei, attack their host plant by secreting pectic enzymes including polygalacturonase (PG) which causes modification of cell-wall structure, increasing accessibility of cell-wall components for degradation by other enzymes. Polygalacturonase is the major factor in the initiation of Ascochyta blight disease, therefore in this study, the enzyme was purified from a virulent isolate of A .rabiei (IK06). Fungi were cultured in PZ medium culture media were harvested and after dialysis used for purification. Purification was achieved by Carboxy Methyl Sepharose Fast Flow ion exchange column equilibrated to pH= 5.5. Zero to one molar NaCl gradient was used for elution of the proteins from the column. Determination of protein content and enzyme activity of each fraction showed that PG was eluted from the column in 0.3 to 0.4 M salt. The purity of the protein and the MW of the enzyme were determined using SDS-PAGE technique. The MW was found to be around 27 KDa. The activity of the purified protein was also evaluated using polyacrylamide gel containing pectin as substrate (zymogram gel). Optimum pH for the purified enzyme was 7.5.