Analysis of cell wall degrading enzymes from black spot pathogen and its pathogenicity

Abstract: 【Objective】The cell wall degrading enzymes produced by the black spot pathogen were studied, and their activities were determined. The role of cell wall degrading enzymes in the pathogenesis of the pathogens was discussed.【Methods】Jujube black spot disease was isolated from jujube in southern Xinjiang. Pathogen hyphae were separated from PDA culture medium. The hyphea were added into induction media containing sucrose, pectin, cellulose, filter paper powder, cotton powder, or jujube pulp as the inducers. The media were shaken in a shaker incubator set at 25 ℃ for 7 days, vacuum filtered to remove hyphae and spores, and centrifuged at 4 ℃ for 30 minutes at 10 000 r·min-1. The supernatant was used as the crude enzyme solution. The crude enzyme solution was vacuum filtered, and ethylenediamine tetraacetic acid was added to the filtrate. Then ammonium sulfate was added slowly with gentle stirring until 60% saturation. The mixture was allowed to stand at 4 ℃ for 5 hours and centrifuged at4 ℃ 15 000 r·min-1 for 20 minutes. The supernatant was discarded, and the precipitation was dissolved with acetic acid-sodium acetate buffer and placed in a dialysis bag. In the same buffer, dialysis was carried out at 4 ℃ for 48 hours and the dialysate was changed every 12 hours. The purified enzyme was used for enzyme activity determination. Crude enzymes were also extracted in vivo from different parts of jujube diseased fruit inoculated with spore suspension. The tissues were cut into slices and put into mortar, grinded with sodium chloride at 4 ℃, centrifuged at 4 ℃ and 5 000 r·min-1 after filtration, and the supernatant was collected as the crude enzyme for later use. Activities of six cell wall degrading enzymes were measured using the 3, 5-dinitrosalicylic acid (DNS) method and the Coomassie blue staining method. Among them, the activities of polygalacturonase, carboxymethylcellulase, β-glucosidase, xylanase and polygalacturonase were measured by the 3, 5-dinitrosalicylic acid method using pectin, carboxymethyl cellulose, salicin and xylan as substrate, respectively. Substrate solution was prepared with citric acid buffer. Using citric acid buffer solution as the blank, the enzymic hydrolysis was carried out at 50 ℃ for 30 to 60 minutes. The absorbance of the reaction mixture was measured by UV-Vis spectrophotometer, and the the enzyme activities were calculated according to the reducing sugar released. The pectin methyl trans-eliminating enzyme was determined by Coomassie Brilliant Blue method. The color density of the protein-Coomassie brilliant blue complex solution was proportional to protein concentration. The differences in color of the solution created by enzymic reactions was used to calculate the cell wall degrading enzyme activity.【Results】Jujube black spot bacteria produced six cell wall degrading enzymes under the induction of different carbon sources. They were polygalacturonase, carboxymethylcellulase, β-glucosidase, xylanase, polymethylgalacturonase and pectin methyl transeliminase. However, there were some differences in activity among enzymes under different inducers.When the jujube pulp was used as the inducer, the activity of β-glucosidase produced by the black spot pathogen was significantly higher than that of the other five enzymes, indicating that β-glucosidase played an important role in the pathogenesis of black spot pathogen. Filter paper as the inducer also produced the highest β-glucosidase activity. The activities of cell wall degrading enzymes produced by jujube black spot in infected jujube fruit was consistent with the results induced by different carbon sources in vitro, with the activity of β-glucosidase being higher than the other five enzymes. Moreover, the activity of β-glucosidase at the junction of diseased and healthy parts was the highest among different tissues sites.【Conclusion】Different inducers have different effects on the cell wall degrading enzymes secreted by the black spot pathogen, and the inducer most favorable for pathogen growth has an important effect on the enzyme production. Filter paper can be used as a good inducer of β-glucosidase. Combining the enzyme activity assay results in vitro and in vivo, it is speculated that β-glucosidase is the major cell wall degrading enzymes that play a role in the pathogenesis of the black spot pathogen. The key part of the impact is at the edge of the diseased tissue.