Pathogenicity Of Cell Wall Degrading Enzymes Produced By Rhizoctonia Cerealis And Isolation, Purification,Characterization Of The Pathogen Polygalacturonase

Wheat sharp eyespot (Rhizoctonia cerealis) is a serious disease in China and the world. In order to further reveal the functions of cell wall degrading enzymes (CWDEs), especially polygalacturonase (PG), in the pathogenesis of the pathogen and provide a new theoretical basis for controlling the disease, the kinds and the pathogenicity of the CWDEs produced by R. cerealis were preliminarily studied, and the purification and characterization of the pathogen PG was first made. The results showed as follews.Five CWDEs such as polygalacturonase (PG), polymethylgalacturonase (PMG), cellulase (Cx), polygalacturonic acid trans-eliminase (PGTE) and pectin methyltrans-eliminase (PMTE) could be produced in the improved Marcus’s medium by R. cerealis. Among them, PG had the highest activity with722.22U/mL; PMG and Cx had the higher activity with444.44U/mL and245.79U/mL, respectively; PGTE and PMTE had much low activity. In the lesion on the wheat sheath, the activity of PG, PMG and Cx was significantly higher tthan that of PGTE and PMTE. Among three parts of the lesion, the activity of CWDEs in the fading part (the outer) was the highest; the next was that in the brown; the lowest was in withered (the inner). It suggested that the CWDEs such as PG, PMG and Cx produced by the pathogen played an important role during the infection of the pathogen.After wheat sheath was inoculated with PG, Cx and their mixtuer (1:1), respectively, the yellowing and watery lesions occurred and the permeability of cell membrane was changd, which showed that these enzymes could damage the sheath tissue and the cell membrane to some extent. The crude PG in the culture filtrate could be precipitated with acetone and obtained by centrifugation (8000r/min) at4℃for15min. It was successively purified by DEAE Sepharose Fast Flow ion exchange column, Phenyl-Sepharose6Fast Flow hydrophobic column and Sephadex G-75gel column. The molecular weight of the purified PG was41.78kD by SDS-PAGE and the pI value was5.34by IEF-PAGE. The PG was demonstrated as a kind of glycoprotein that contained9.1%saccharide and a-amino acids, but no lipid and aromatic amino acids. The PG activity was observed in the range of pH value from4to12, and the strongest one was at pH6. The PG was not stable in the higher temperature (≥40℃) and lost its activity in the treatment of100℃for20min. The PG was also sensitive to ultraviolet radiation, chloroform, trypsin and proteinase K, with a decrease in the activity by81.4%,85.1%,84.3%and89.8%, respectively after the treatment.