油菜抗菌核病品种的筛选和抗性机理分析
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摘要
核盘菌Sclerotinia sclerotiorum导致的菌核病是我国油菜上危害最严重的病害。本文应用苗期、成株期病菌接种和草酸处理等4种方法,测定了来自江苏省的37个甘蓝型油菜品种对菌核病的抗性,并比较了不同鉴定方法之间的差异。结果表明,4种鉴定方法均显示出不同品种间的抗(耐)性存在显著性差异,但不同鉴定方法的吻合度不高。根据油菜菌核病的发病过程,提出成株期叶柄菌丝块接种方法,该方法操作简便、发病率高。利用此方法对83个油菜品种(系)抗菌核病性的抗性进行了鉴定。
在品种抗性比较的基础上,选取4个抗感不同的油菜品种,比较在Sclerotiniasclerotiorum侵染后,不同品种组织内活性氧产生情况。通过DAB和伊文思兰染色,分别检测接种病菌后不同抗感品种的氧进发(Reactive Oxygen Intermediares,ROIs)的产生及其诱导的细胞死亡情况。结果表明,抗性品种中双9号、皖油14产生ROIs及细胞死亡的程度均轻于感病品种沪油15、史力丰。使用抗氧化剂如抗坏血酸、H_2O_2酶预处理叶片后,病斑比对照减小。H_2O_2预处理则能显著增大病斑,忍受ROIs可能是油菜抗S.sclerotiorum的机理之一。通过甲苯胺蓝染色检测细胞壁修复情况的结果表明,抗性品种叶片在菌丝定殖早期的细胞壁中多酚化合物过氧化结合物的产生量大于感病品种。
本文通过RT-PCR的方法,检测了不同抗感品种在接种病原菌后不同时间段PDF1.2、PR-1、EDS1、Cu/ZnSOD、FeSOD、PAL、nsLTP、GLP等8个基因的表达。结果表明,核盘菌的侵染激活了油菜的JA/ET途径,抗病品种组织内的标记基因PDF1.2上调表达。病菌接种后,抗性品种的EDS1的表达量下调,Cu/ZnSOD、FeSOD的表达水平提高,从而抑制细胞死亡。抗性品种在接种病原菌后,与具有细胞壁修复等功能的GLP和nsLTP表达也被上调。参与产生次生代谢物质的PAL表达在接种后24h被明显上调。首次在油菜上扩得EDS1基因的部分序列。
The stem rot caused by Sclerotinia sclerotiorum is a devastating disease of rapeseed in China.In this study,to determine resistance of 37 rapeseed varieties(Brassica napus L.) from Jiangsu province to S.sclerotiorum,the methods including mycelium inoculation at seedling and flowering stage and oxalic acid infiltration were employed.The consistency of methods was analyzed.Results indicated that tested varieties showed significant difference in resistance to S.sclerotiorum with 4 identification methods,but the similarity level among methods was low.A petiole inoculation method at flowing stage was proposed because it was easy to implement and had higher disease incidence.Eight-three rapeseed varieties(stains) were identified for resistance to S.sclerotiorum by this method.
For comparison of ROIs(Reactive Oxygen Intermediates) production and cell death in rapeseed leaves after inoculated with S.sclerotiorum at first,DAB and Evans blue staining were used.Results indicated that the resistant variety Zhongshuang 9 and Wanyou 14 produced less ROIs and cell death than the susceptible variety Huyou 15 and Shilifeng after inoculation with S.sclerotiorum.The pretreatment of rapeseed leaves with antioxidants such as ascorbic acid、catalase made all varieties much more resistant than controls,and H_2O_2 pretreatment increased lesions significantly.It suggested ROIs tolerance is likely one of the resistance mechanisms of rapeseed against S.sclerotiorum.In addition,toluidine bule staining was used to detect the peroxidative incorporation of phenolic compounds into the cell wall.Results showed that the resistant varieties had more peroxidative incorporation of phenolic compounds than the susceptible varieties during the early S. sclerotiorum infection stage.
In this study,the leaves of different rapeseed varieties were inoculated with S. sclerotiorum,and the expression of defense genes such as PDF1.2、PR-1、EDS1、Cu/ZnSOD、FeSOD、PAL、nsLTP、GLP was respectively detected by RT-PCR from 0-24 h after inoculation.Results indicated that the interaction of rapeseed and S.sclerotiorum induced the jasmonic acid and ethylene(JA/ET)-dependent pathway,and PDF1.2 was up-regulated.Upregulated expression of EDS1 and downregulted expression of Cu/ZnSOD and FeSOD of resistance varieties after inoculation meant that the hypersensitive(HP) cell death could be decreased.Expression of nsLTP and GLP that were related to cell wall modification was up-regulated after inoculation in the resistant varieties.PAL expression of resistance varieties also increased at 24h after inoculation.EDS1 partial mRNA sequence on rapeseed varieties was obtained.
在品种抗性比较的基础上,选取4个抗感不同的油菜品种,比较在Sclerotiniasclerotiorum侵染后,不同品种组织内活性氧产生情况。通过DAB和伊文思兰染色,分别检测接种病菌后不同抗感品种的氧进发(Reactive Oxygen Intermediares,ROIs)的产生及其诱导的细胞死亡情况。结果表明,抗性品种中双9号、皖油14产生ROIs及细胞死亡的程度均轻于感病品种沪油15、史力丰。使用抗氧化剂如抗坏血酸、H_2O_2酶预处理叶片后,病斑比对照减小。H_2O_2预处理则能显著增大病斑,忍受ROIs可能是油菜抗S.sclerotiorum的机理之一。通过甲苯胺蓝染色检测细胞壁修复情况的结果表明,抗性品种叶片在菌丝定殖早期的细胞壁中多酚化合物过氧化结合物的产生量大于感病品种。
本文通过RT-PCR的方法,检测了不同抗感品种在接种病原菌后不同时间段PDF1.2、PR-1、EDS1、Cu/ZnSOD、FeSOD、PAL、nsLTP、GLP等8个基因的表达。结果表明,核盘菌的侵染激活了油菜的JA/ET途径,抗病品种组织内的标记基因PDF1.2上调表达。病菌接种后,抗性品种的EDS1的表达量下调,Cu/ZnSOD、FeSOD的表达水平提高,从而抑制细胞死亡。抗性品种在接种病原菌后,与具有细胞壁修复等功能的GLP和nsLTP表达也被上调。参与产生次生代谢物质的PAL表达在接种后24h被明显上调。首次在油菜上扩得EDS1基因的部分序列。
The stem rot caused by Sclerotinia sclerotiorum is a devastating disease of rapeseed in China.In this study,to determine resistance of 37 rapeseed varieties(Brassica napus L.) from Jiangsu province to S.sclerotiorum,the methods including mycelium inoculation at seedling and flowering stage and oxalic acid infiltration were employed.The consistency of methods was analyzed.Results indicated that tested varieties showed significant difference in resistance to S.sclerotiorum with 4 identification methods,but the similarity level among methods was low.A petiole inoculation method at flowing stage was proposed because it was easy to implement and had higher disease incidence.Eight-three rapeseed varieties(stains) were identified for resistance to S.sclerotiorum by this method.
For comparison of ROIs(Reactive Oxygen Intermediates) production and cell death in rapeseed leaves after inoculated with S.sclerotiorum at first,DAB and Evans blue staining were used.Results indicated that the resistant variety Zhongshuang 9 and Wanyou 14 produced less ROIs and cell death than the susceptible variety Huyou 15 and Shilifeng after inoculation with S.sclerotiorum.The pretreatment of rapeseed leaves with antioxidants such as ascorbic acid、catalase made all varieties much more resistant than controls,and H_2O_2 pretreatment increased lesions significantly.It suggested ROIs tolerance is likely one of the resistance mechanisms of rapeseed against S.sclerotiorum.In addition,toluidine bule staining was used to detect the peroxidative incorporation of phenolic compounds into the cell wall.Results showed that the resistant varieties had more peroxidative incorporation of phenolic compounds than the susceptible varieties during the early S. sclerotiorum infection stage.
In this study,the leaves of different rapeseed varieties were inoculated with S. sclerotiorum,and the expression of defense genes such as PDF1.2、PR-1、EDS1、Cu/ZnSOD、FeSOD、PAL、nsLTP、GLP was respectively detected by RT-PCR from 0-24 h after inoculation.Results indicated that the interaction of rapeseed and S.sclerotiorum induced the jasmonic acid and ethylene(JA/ET)-dependent pathway,and PDF1.2 was up-regulated.Upregulated expression of EDS1 and downregulted expression of Cu/ZnSOD and FeSOD of resistance varieties after inoculation meant that the hypersensitive(HP) cell death could be decreased.Expression of nsLTP and GLP that were related to cell wall modification was up-regulated after inoculation in the resistant varieties.PAL expression of resistance varieties also increased at 24h after inoculation.EDS1 partial mRNA sequence on rapeseed varieties was obtained.
引文
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