杧果细菌性角斑病菌细胞壁降解酶的致病作用Pathogenic effect of cell wall degrading enzymes produced by pathogen causing mango bacterial leaf spot
张大智;詹儒林;柳凤;李国平;赵艳龙;常金梅;
摘要(Abstract):
【目的】从细胞壁降解酶的角度开展其致病机制的研究,探讨杧果细菌性角斑病在发病过程中产生细胞壁降解酶的种类及其在致病过程中的作用,以期为对该病害的有效监测和研发防控新技术提供理论依据。【方法】采用活体外诱导培养和病原菌接种处理,利用3,5-二硝基水杨酸法(DNS法)和紫外分光光度计法检测病原菌侵染过程中细胞壁降解酶活性变化,DDS-ⅡA型电导率仪测定叶片组织浸出液的电导率,并观测细胞壁降解酶对杧果叶片的致病作用。【结果】病原菌在改良的Marcus培养液和罹病组织中均能产生6种主要细胞壁降解酶,其中羧甲基纤维素酶(Cx)活性较高,多聚半乳糖醛酸酶(PG)、果胶甲基半乳糖醛酸酶(PMG)和β-葡萄糖苷酶次之,果胶甲基反式消除酶(PMTE)和多聚半乳糖醛酸反式消除酶(PGTE)较低。病原菌接种杧果叶片后,6种酶活性显著升高,在病菌侵染前期(0~4 d),PMG接种后2 d达到最大活性高峰,酶活值为8.470 U?mg-1,以未含致病菌株的培养基接种杧果叶片作为对照,此时接种处理酶活值是对照的3.805倍;β-葡萄糖苷酶接种后4 d酶活性值最大,为15.190 U?mg-1,是对照的4.388倍。在病菌侵染后期(4~14 d),Cx、PG、PMTE和PGTE均在接种后的10 d达到最高峰,酶活性值分别为25.941、14.605、0.009、0.014 U?mg-1,是对照的2.672、14.634、12.571和4.121倍,在整个测定过程中,对照始终处于较低水平。此外,细胞壁降解酶对杧果叶片具有浸解作用,且浸解损伤程度与酶浓度成正比。【结论】细胞壁降解酶在病原菌的入侵和致病过程中起重要作用。果胶酶在病原菌侵染初期最早分泌并起作用,纤维素酶主要降解次生壁,致病作用发生在后期,并且果胶酶比纤维素酶对杧果叶片的致病作用明显。
关键词(KeyWords): 杧果细菌性角斑病;细胞壁降解酶;酶活性;致病作用
基金项目(Foundation): 海南自然科学基金(20163109);; 中央级非盈利性研究机构基本科研业务费研究专项(1630062014007);; 农业行业科研专项(201203092-3)
作者(Author): 张大智;詹儒林;柳凤;李国平;赵艳龙;常金梅;
Email:
DOI: 10.13925/j.cnki.gsxb.20150038
参考文献(References):
- [1]文衍堂,黄圣明.杧果细菌性黑斑病症状与病原菌鉴定[J].热带作物学报,1994,15(1):79-85.WEN Yantang,HUANG Shengming.Symptoms and pathogen identification of bacterial black spot of mango(Mangifera indica L.)[J].Chinese Journal of Tropical Crops,1994,15(1):79-85.
- [2]PRUVOST O,COUTEAU A,PERRIER X,LUISETTI J.Phenotypic diversity of Xanthomonas sp.mangiferaeindicae[J].Journal of Applied Microbiology,1998,84:115-124.
- [3]漆艳香,谢艺贤,张贺,张欣,陆英,喻群芳,蒲金基.海南省杧果细菌性黑斑病原菌生理生化特性初步研究[J].中国农学通报,2014,30(28):301-305.Qi Yanxiang,XIE Yixian,ZHANG He,ZHANG Xin,LU Ying,YU Qunfang,PU Jinji.Preliminary study on physiological and biochemical features of the pathogen of mango bacterial black spot in Hainan province[J].Chinese Agricultural Science Bulletin,2014,30(28):301-305.
- [4]董春,何汉生.杧果细菌性黑斑病研究进展[J].果树科学,1999,16(S1):47-51.DONG Chun,HE Hansheng.Advance in research on mango bacterial black spot[J].Journal of Fruit Science,1999,16(S1):47-51.
- [5]GAGNEVIN L,PRUVOST O.Epidemiology and control of mango bacterial black spot[J].Plant Disease,2001,85(9):928-935.
- [6]MIDHA S,RANJAN M,SHARMA V,PINNAKA A K,PATIL P B.Genome sequence of Xanthomonas citri pv.mangiferaeindicae strain LMG,941[J].Journal of Bacteriology,2012,194(11):30-31.
- [7]漆艳香,蒲金基,张欣,陆英,张贺,张辉强,谢艺贤.杧果细菌性黑斑病菌Xcm R基因的克隆与序列分析[J].热带作物学报,2011,34(4):663-667.QI Yanxiang,PU Jinji,ZHANG Xin,LU Ying,ZHANG He,ZHANG Huiqiang,XIE Yixian.Cloning and sequence analysis of Xcm R from Xanthomonas campestris pv.mangiferaeindicae[J].Chinese Journal of Tropical Crops,2011,34(4):663-667.
- [8]詹儒林.杧果主要病虫害诊断与防治原色图谱[M].北京:中国农业出版社,2011:10-12.ZHAN Rulin.Diagnosis and treatment of the primary mango diseases and insect pests[M].Beijing:China Agricultural Press,2011:10-12.
- [9]漆艳香,张贺,喻群芳,谢艺贤,张欣,陆英,张辉强,蒲金基.杧果细菌性黑斑病菌在不同场所的存活期[J].植物保护,2015,41(5):140-144.QI Yanxiang,ZHANG He,YU Qunfang,XIE Yixian,ZHANG Xin,LU Ying,ZHANG Huiqiang,PU Jinji.Survival period of Xanthomonas campestris pv.mangiferaeindicae in different places[J].Plant Protection,2015,41(5):140-144.
- [10]韦运谢,张贺,蒲金基,刘晓妹,谢艺贤,漆艳香.杧果细菌性黑斑病病菌抗铜基因cop A克隆与序列分析[J].中国植保导刊,2015,35(7):18-22.WEI Yunxie,ZHANG He,PU Jinji,LIU Xiaomei,XIE Yixian,QI Yanxiang.Cloning and sequence analysis of cop A from Xanthomonas campestris pv.mangiferaeindicae[J].China Plant Protection,2015,35(7):18-22.
- [11]王金生.植物病原细菌学[M].北京:中国农业出版社,2000:173-191.WANG Jinsheng.Pathogenic bacteriology[M].Beijing:China Agriculture Press,2000:173-191.
- [12]GUMMADI S N,PANDA T.Purification and biochemical properties of microbial pectinases-a review[J].Process Biochemistry,2003,38(7):987-996.
- [13]李壁鹰,贺伟.果树采后病害病原菌胞外酶的启动及其调控[J].河北林果研究,2005,20(2):174-182.LI Biying,HE Wei.Activation and regulation of pathogen ectoenzymes in postharvest diseases[J].Hebei Journal of Forestry and Orchard Research,2005,20(2):174-182.
- [14]余兴莲,王丽,徐伟民.纤维素酶降解纤维素机理的研究进展[J].宁波大学学报(理工版),2007,20(1):78-82.YU Xinglian,WANG Li,XU Weimin.Progress in the studies of cellulose degradation by cellulase[J].Journal of Ningbo University(Natural Science&Engineering Edition),2007,20(1):78-82.
- [15]周婷.受条锈菌侵染后小麦叶片中细胞壁降解酶活性变化的研究[D].杨凌:西北农林科技大学,2007.ZHOU Ting.Studies on the changes of cell wall degrading enzymes in wheat leaves infected by Puccinia striiformis F.sp.tritici[D].Yangling:Northwest A&F University,2007.
- [16]LALAOUI F,HALAMA P,DUMORTIER V,PAUL B.Cell walldegrading enzymes produced in vitro by isolates of Phaeosphaeria nodorum differing in aggressiveness[J].Plant Pathology,2000,49(6):727-733.
- [17]RUIZ M C,PIETRO A D,RONCERO M I G.Purification and characterization of anacidic endo-β-1,4-xylanase from the tomato vascular pathogen Fusarium oxysporum f.sp.Lycopersici[J].FEME Microbiology Letters,2006,148(1):75-82.
- [18]MARCUS L,BARASH B,SNEH Y.Purification and characterization of pectolytic enzymes produced by virulent and hypovirulent isolates of Rhizoctonia solani Kuhn[J].Physiological and Molecular Plant Pathology,1986,29:325-336.
- [19]刘志恒,亓烨,黄欣阳,杨红,侯悦,张睿.黄瓜棒孢叶斑病(褐斑病)病菌细胞壁降解酶产生条件及活性分析[J].中国蔬菜,2011(8):76-80.LIU Zhiheng,QI Ye,HUANG Xinyang,YANG Hong,HOU Yue,ZHANG Rui.Conditions and activity analysis of cell wall degrading enzymes produced from Corynespora cassiicola of brown spot of cucumber[J].China Vegetagle,2011(8):76-80.
- [20]李宝聚,周长力,赵奎华.黄瓜黑星病菌致病机理的研究Ⅱ细胞壁降解酶及其在致病中的作用[J].植物病理学报,2000,30(1):13-18.LI Baoju,ZHOU Changli,ZHAO Kuihua.Pathogenic mechanism of scab of cucumber caused by Cladosporium cucumerinumⅡthe cell wall degrading enzymes and its pathogenic action[J].Acta Paytopathologica Sinica,2000,30(1):13-18.
- [21]方中达.植病研究方法[M].北京:中国农业出版社,1998:65.FANG Zhongda.Research methods of plant diseases[M].Beijing:China Agricultural Press,1998:65.
- [22]赵亚华.生物化学实验技术教程[M].广州:华南理工大学出版社,2000:15-17.ZHAO Yahua.Tutorial experiment technology in biochemistry[M].Guangzhou:South China Uuiversity of Technology Press,2000:15-17.
- [23]赵艳琴,吴元华,伏颖,赵秀香,陈建关.烟草靶斑病菌(Rhizoctonia solani)细胞壁降解酶活性分析及其致病作用[J].烟草科技,2014(11):84-88.ZHAO Yanqin,WU Yuanhua,FU Ying,ZHAO Xiuxiang,CHEN Jianguan.Activity pathogenic effect of cell wall degrading enzyme in tobacco target spot pathogen Rhizoctonia solani[J].Tobacco Science&Technology,2014(11):84-88.
- [24]伏颖.烟草靶斑病菌遗传分化、侵染特性及致病机理研究[D].沈阳:沈阳农业大学,2011.FU Ying.Studies on the genetic differentiation,infection characteristics and pathogenic mechanism of Rhizoctonia solani[D].Shenyang:Shenyang Agriculture University,2011.
- [25]BRADFORD M.A rapid and sensitive of method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72:248-254.
- [26]EUGENE R,HONG J P,MYEONG O K,YOUNG R C,CHANG W L,JAE W K.Expression of exo-polygalacturonases in Botrytis cinerea[J].FEMS Microbiology Letters,2001:105-109.
- [27]VERHOEFF K,WARREN J M.In vitro and in vivo production of cell wall degrading enzymes by Botrytis cinerea from tomato[J].European Journal of Plant Pathology,1972,78(4):179-185.
- [28]FONSECA M J V,SAID S.Sequential production of pectinases by Penicillium frequentans[J].Journal of Microbiology and Biotechnology,1995,11(2):174-177.
- [29]高增贵,陈捷,高洪敏,唐朝荣,宋佐衡,薛春生.玉米茎腐病菌产生的细胞壁降解酶种类及其活性分析[J].植物病理学报,2000,30(2):148-152.GAO Zenggui,CHEN Jie,GAO Hongmin,TANG Chaorong,SONG Zuoheng,XUE Chunsheng.The kinds and activity of cell wall degrading enzymes produced from corn stalk rot pathogens[J].Acta Paytopathologica Sinica,2000,30(2):148-152.
- [30]陈晓林,牛程旺,李保华,李桂舫,王彩霞.苹果树腐烂病菌产生细胞壁降解酶的种类及其活性分析[J].华北农学报,2012,27(2):207-212.CHEN Xiaolin,NIU Chengwang,LI Baohua,LI Guifang,WANG Caixia.The kinds and activities of cell wall-degrading enzymes produced by Valsa ceratosperma[J].Acta Agriculturae Boreali-Sinica,2012,27(2):207-212.
- [31]石延霞,李宝聚,刘学敏.黄瓜霜霉病菌致病作用与两种细胞壁降解酶关系初探[J].园艺学报,2003,30(4):465-466.SHI Yanxia,LI Baoju,LIU Xuemin.The relation of pathogenesis action by Pseudoperonospora cubensis and two sort of zymin in cucumber[J].Acta Horticulturae Sinica,2003,30(4):465-466.
- [32]齐凤坤,康立功,许向阳,李景富.番茄芝麻斑病原菌产生的细胞壁降解酶种类及其活性变化[J].植物保护,2010,36(5):47-51.QI Fengkun,KANG Ligong,XU Xiangyang,LI Jingfu.The kinds and activity of cell wall degrading enzymes produced from tomato Helminthosporium carposaprum pathogens[J].Acta Horticulturae Sinica,2010,36(5):47-51.
- [33]陈夕军,张红,徐敬友,童蕴慧,纪兆林.水稻纹枯病菌胞壁降解酶的产生及致病作用[J].江苏农业学报,2006,22(1):24-28.CHEN Xijun,ZHANG Hong,XU Jingyou,TONG Yunhui,JI Zhaolin.Cell wall degrading enzymes produced by Rhizoctonia solani and their pathogenicity to rice plants[J].Jiangsu Journal of Agriculture Sciences,2006,22(1):24-28.
- [34]杨媚,杨迎青,郑丽,李明海,周而勋.水稻纹枯病菌细胞壁降解酶组分分析、活性测定及其致病作用[J].中国水稻科学,2012,26(5):600-606.YANG Mei,YANG Yingqing,ZHENG Li,LI Minghai,ZHOU Erxun.Component analysis,activity measurement and pathogenic effect of cell wall degrading enzymes from rice sheath blight pathogen Rhizoctonia solani[J].Chinese Journal of Rice Science,2012,26(5):600-606.