The kinds and activities of main enzymes in the pathogenic process of shot hole disease caused by Wilsonomyces carpophilus in Prunus armeniaca

Abstract:【Objective】Prunus armeniaca is the origin species of apricot widely cultivated in the world, and an important germplasm resource of characteristic fruit trees in Xinjiang, China. The occurrence of perforation disease of drupes has become an important factor threatening the healthy growth of the wild fruit forest. Wilsonomyces carpophilus is the most serious pathogen causing perforation disease, which can not only damage the leaves of stone fruit plants, but also damage the fruits and shoots. The study aimed to clarify the kinds and activities of main enzymes in the pathogenic process of shot hole disease caused by W. carpophilus in P. armeniaca.【Methods】W. carpophilus were cultured for 10 days on PDA medium, the hyphae were added into induction media containing sucrose, pectin, CMC, cellulose particle, filter paper powder, and cotton pectin or P. armeniaca leaves respectively as 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 healthy leaves of P. armeniaca were inoculated with W. carpophilus, and the blank culture medium was used as control. Samples were taken on 2, 4, 6, 8, 10, 12, 14 and 16 d after inoculation. The samples 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. The 3,5-dinitrosalicylic acid (DNS) and the Coomassie blue staining method was used to detect the kinds and activies of cell wall degrading enzymes. 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 degradation of Apricot leaves by cell wall degrading enzymes produced by W. carpophilus. The obtained enzyme solutions induced by 7 inducers (20 mL) were inoculated into the leaves of P. armeniaca by in vitro needling method, and the inactivated enzyme solution was boiled as the control treatment. The leaves were cultured at about 25 ℃ under light conditions, and the changes of leaves were observed after 7 days. The extraction and activity determination of the antioxidant enzymes from the leaves of P. armeniaca. 1 g of P. armeniaca leaves were weighed, cut into pieces and put into a mortar. An appropriate amount of phosphate buffer was added and ground into a homogenate in an ice bath. The mixture was frozen and centrifuged at 12 000 r·min-1 for 30 min at 4 ℃, and the supernatant was used as the crude enzyme extract. The activity of peroxidase (POD) was determined by guaiacol method, and the activity of superoxide (SOD) was determined by tetrazolium azolium reduction method. The enzyme activity was calculated according to the formula.【Results】The W. carpophilus produced six CWDES, including xylanase, PG, and PMG) under the induction of different carbon sources. However, there were some differences in the activity of enzymes under different inducers. The CWDES was produced in the leaves of P. armeniaca after infection by W. carpophilus. The activities of the PG and PMG were the highest, while the activities of the carboxymethyl cellulase were the lowest. The PMG and PG of pectinase were secreted the earliest in the pathogenic process of W. carpophilus, and the pectinase had more pathogenic effect on P. armeniaca leaves than cellulase. The cell wall degradation enzyme solution added with different inducers showed different degrees of degradation after inoculation on P. armeniaca leaves, and the control leaves showed no symptoms. Among them, the enzyme solution induced by sucrose, pectin and P. armeniaca leaves presented yellowish brown spots 2 days after inoculation on the leaves, and black brown spots 7 days after inoculation, and the degradation degree was severe. The enzyme solution induced by the microcrystalline cellulose, carboxymethyl cellulose (CMC), filter paper powder and degreased cotton bran on the leaves presented smaller lesions. The activities of the peroxidase (POD), superoxide enzyme (SOD) and catalase (CAT) increased firstly and then decreased with the extension of infection time.【Conclusion】In conclusion, the pectinase would play an important role in the pathogenic process of shot hole disease caused by W. carpophilus in P. armeniaca. The leaves of P. armeniaca also produced the antioxidant enzymes to resist the pathogen, indicating that the leaves of P. armeniaca could reduce the damage of reactive oxygen species production in the plants by regulating their own antioxidant enzyme system after being infected by W. carpophilus. The results would provide a theoretical basis for the molecular pathogenic mechanism of the pathogen and be of great significance for the prevention and control of perforation disease.