| Botrytis cinerea is an important pathogenic fungi of plant and could produce many kinds of enzymes in disease development. Study the cell-wall degrading enzymes (CWDEs) and cutinase by Botrytis cinerea and their pathogenicity to plant could make us best understanding about pathogenic mechanisms of plant pathogens.The activities of cutinase and pectinases could be detected in the cultured filtrate from the conidia of Botrytis cinerea, and polygalacturonase (PG), pectin methylgalacturonase (PMG), pectin methylesterase (PME), pectin methyl trans-eliminase (PMTE) and polygalacturonic acid trans-eliminase (PGTE) were involved in the cultured filtrate except for cellulase. Among these pectinases from conidia, PG's activity was the highest with 1.604 U/mg. Besides cutinase and above pectinases, the mycelium of Botrytis cinerea could produce cellulase. Among the pectinase of mycelium, PMG's activity was the highest with 48.539 U/mg. There were some differences of the enzymes activity by conidia in vitro and in vivo. In vitro, the activity peak of cutinase with 2.238×10-6 U/mg appeared at the 24th hours after conidia germination, while in vivo the activity peak with 1.345×10-5 U/mg appeared at the 12th hours after inoculation using conidial suspension. At the same time the activity peaks of pectic hydrolases produced by conidia appeared at the 9th hours after conidia germination in vitro and at the 12th hours after inoculation using conidia in vivo.Treated by the crude enzymes of pectinase, cellulase and mixed enzymes, the tomato leaves were yellow brown and presented macerated while the leaves treated by the inactivity enzymes didn't appear any lesion. The symptoms of leaves treated by enzymes were similar with the lesions of the leaves inoculated with Botrytis cinerea. The symptoms of leaves treated with pectinase and mixed enzymes were more serious than that of leaves treated by cellulase. Otherwise, when the leaves were wounded the lesions which caused by CWDEs was more serious than that of leaves unwounded. It indicated that the CWDEs of Botrytis cinerea were primary pathogenic factors.The observation the process of conidial invasion by scanning electron microscope show that the penetration peg of pathogen could penetrate tomato leaves through the space of epidermis cells, nevertheless the event of the penetration peg penetrating through the cell-wall of epidermis was not discovered. These results, just as the conidial invasion mode and characteristics of secretion pectinase by conidia, implied that the pectinases, especially PG and PMG, were favorable to the pathogen in the process of penetration.Treated tomato leaves with the crude enzymes the polysaccharide of cell walls could be degraded, the cytomembrane be damaged obviously, the osmosis of cytomembrane was changed, and finally the electrolyte substances of cell leaked out. In general, the mixed enzymes could produce the more serious lesions to cytomembrane which lesion rate was 53.2%, and the next was pectinase with 47.1%, and lower lesions were produce by cellulase which lesion rate was about 31.1%. Observations by transmission electricity microscope found that the crude enzymes of Botrytis cinerea had prominent damage effect on the cell ultrastructure of the tomato leaf. The crude enzymes could cause cell plasmolysis, cell-wall broken fractionally and even decomposition, the stroma lamella of chloroplast and the ridge of mitochondria unclearly, and other organelle damaged and even disappearance. By the way, after treated by mixed enzymes for 6 hours the cells exhibited damaged phenomenon, and the cells treated for 36 hours were damaged so seriously that the cells were breakdown.Detection the activities of CWDEs with different parts of tomato plant showed that the activity of PMG in infected flowers was the highest with 11.475 OD/g.min among the all samples. The next was in infected younger stem, and the activity in infected leaves was similar with that in older stem. The PMG'activity in infected fruit was the lowest with 2.036OD/g.min. Otherwise, PMG's activity in brown region of necrosis was higher with 4.139 OD/g.min, which PMG's activity were lower in yellow region of necrosis with 0.583 OD/g.min and healthy region of necrosis periphery with 0.649 OD/g.min. Whereas,The cellulase activity could be detected in brown region of necrosis, but in other regions of necrosis.Basing on above experiments we concluded that the CWEDs of Botrytis cinerea palyed an important role as pathogenetic factors in penetration of the penetration peg and disease development. The pathogenic effect of pectinase on tomato plant was stronger than that of cellulase.
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