【作者】 吴洁云；

【导师】 童蕴慧；

【作者基本信息】 扬州大学 ， 植物病理学， 2007， 硕士

【摘要】 灰葡萄孢(Botrytis cinerea)是重要的植物病原菌,在致病过程中可以产生多种酶类。研究病菌角质酶和胞壁降解酶种类及其在致病中的作用,可以为进一步阐述病菌的致病机理提供理论依据。在灰葡萄孢分生孢子的萌发液中,可以检测到角质酶和果胶酶,果胶酶包括多聚半乳糖醛酸酶(PG)、果胶甲基半乳糖醛酸酶(PMG)、果胶甲基酯酶(PME)、果胶甲基反式消除酶(PMTE)和多聚半乳糖醛酸反式消除酶(PGTE),其中以PG活性最高,为1.604 U/mg。在分生孢子的萌发液中没有纤维素酶(Cx)的活性。病菌菌丝除了可以产生角质酶和以上5种果胶酶外,还可以产生Cx。菌丝的果胶酶中以PMG活性最高,达48.539 U/mg。分生孢子在离体和活体条件下的产酶动态有一定差异。离体条件下,角质酶最大活性高峰出现在培养后的24h,活性为2.238×10-6 U/mg,但在活体下,只要12h就能达到高峰值,活性可以达到1.345×10-5 U/mg。分生孢子的果胶水解酶在离体培养时只要9h就能达到活性高峰,但活体植物体表,PMG高峰出现在分生孢子接种后的第12h。分别用果胶酶、纤维素酶以及两者的混合酶液处理番茄叶片,所有处理的叶片均出现不同程度的褐色、黄色病变以及水渍状病斑。症状表现形式与病菌接种较为相似。三种不同酶液处理中,以果胶酶和混合酶对叶片的致病症状严重。另外,胞壁降解酶在叶片有伤口时的症状要比无伤口时严重得多。番茄叶片用失活的酶液处理不出现任何病变症状。因此,病菌的胞壁降解酶能够对番茄叶片造成明显的致病作用。用扫描电镜观察分生孢子侵入时发现,分生孢子的侵染丝可以通过番茄叶片表皮细胞间隙侵入,但未见直接从表皮细胞壁侵入的现象。从分生孢子产生果胶酶的特点和侵染丝从细胞间隙侵入的现象可以得知,果胶酶特别是PG和PMG在病菌侵入阶段起着重要作用。酶液处理番茄叶片,可以使叶片细胞壁多糖降解,细胞膜明显受到损伤,膜透性改变,最终导致细胞内电解质渗漏。酶液对细胞膜的损伤,以混合酶最严重,损伤率达52.3%,其次是果胶酶,为47.1%,Cx损伤作用最小,为31.1%。通过投射电镜观察,粗酶液对番茄叶片细胞超微结构也有显著的破坏作用,主要表现为使叶片细胞质壁分离、细胞壁断裂甚至消解、叶绿体片层结构模糊不清,线粒体嵴模糊甚至消失、细胞核等细胞器受损并分解等现象。另外,混合酶液处理后6h即出现细胞被破坏的现象,36h时严重受害的细胞完全崩解。在番茄植株不同发病部位检测胞壁降解酶活性,发现发病花朵中PMG活性最高,为11.475 OD/g.min,其次为幼嫩茎杆中,叶片与成株期茎杆中活性相似,果实中活性最低,仅2.036OD/g.min。在病斑中心的褐色部位,也是PMG活性最高,为4.139 OD/g.min,病斑外圈黄色部位和病健交界处的健康部位PMG均较低,分别为0.583 OD/g.min与0.649 OD/g.min。另外,在病斑中心的褐色部位可以检测到纤维素酶活性,但病斑其它部位均没有活性。综上所述,灰葡萄孢胞壁降解酶在病菌侵入和致病过程中都发挥了重要作用,果胶酶作用又强于纤维素酶。更多还原

【Abstract】 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.更多还原