Differences of defensive enzyme activities and metabolites between resistant and susceptible tobacco cultivars infected by Ralstonia solanacearum
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摘要:目的
为烟草抗病育种和抗性鉴定提供生化代谢指标。
方法采用茎部注射法对抗病品种粤烟97和感病品种长脖黄接种青枯雷尔菌Ralstonia solanacearum，并采用比色法和GC-MS法分别进行相关防御性酶活性及代谢物质的测定。
结果抗病品种粤烟97的PAL、SOD、POD、PPO活性，接菌组与对照组均分别高于感病品种长脖黄的接菌组与对照组；抗病品种粤烟97和感病品种长脖黄分别在接菌后第7天和第5天，PAL活性高于对照组，其余时间酶活性接菌组均低于对照组；抗病品种粤烟97和感病品种长脖黄在受青枯雷尔菌侵染前期，SOD、POD、PPO酶活性均提高，随着时间延长酶活性均低于对照组。POD和PPO同工酶凝胶电泳表明：抗病品种粤烟97同工酶类型多于感病品种长脖黄；接菌后第3天，粤烟97的同工酶谱带宽度与色度增强，而长脖黄无增强现象。说明抗病品种能快速应对外界刺激，加速相关抗病物质的形成。代谢物检测表明：抗病品种粤烟97接菌组中肌肉肌醇、烟碱、苹果酸、L-苏氨酸等物质的相对质量分数高于对照组，而感病品种长脖黄接菌组中这些物质均低于对照组，反映品种间抗青枯病能力的强弱可能与上述物质有关。
结论烟草不同抗病品种叶片中PAL、SOD、POD、PPO酶活性的高低，以及上述代谢物质含量的变化，可作为反映烟草抗青枯病的生化指标。
Abstract:ObjectiveIn order to provide the biochemical and metabolic basis of tobacco breeding for disease resistance and resistance identification.
MethodResistant (Yueyan 97) and susceptible (Changbohuang) tobacco cultivars were inoculated with Ralstonia solanacearum by trunk injection. Defensive enzyme activities and metabolites were measured by using colorimetric method and GC-MS.
ResultThe activities of phenylanine ammonia lyase (PAL), superoxide dismutase (SOD), peroxidase (POD) and polyphenol oxidase (PPO) in the resistant cultivar 'Yueyan 97' were higher than those in the susceptible cultivar 'Changbohuang' with the same inoculation treatment. The PAL activities of Yueyan 97 and Changbohuang were higher than that in control on the seventh and fifth days after inoculation, respectively. The PAL activities of both cultivars were lower compared to control at any other time. For both cultivars, SOD, POD and PPO activities increased compared to control in the earlier days, but were lower compared to control during the later time period. The types of POD and PPO isozymes in the resistant cultivar 'Yueyan 97' were higher than those in the susceptible cultivar 'Changbohuang'. The width and color of isozyme bands of Yueyan 97 were enhanced at 3 d after inoculation, while those of Changbohuang were not enhanced. It suggested that the resistant cultivar could quickly respond to external stimuli and accelerate the formation of defense-related substances. Detection of metabolites showed that the relative contents of some substances in Yueyan 97 such as myo-inositol, nicotine, malic acid, and L-threonine were higher compared to control, but in Changbohuang were lower compared to control, suggesting that cultivar differences in resistance against R. solanacearum might be related to these substances.
ConclusionThese four defensive enzyme activities and the contents of the above metabolites could be used as biochemical indexes for evaluating tobacco resistance against R. solanacearum.
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Keywords:
- tobacco /
- Ralstonia solanacearum /
- defensive enzyme /
- enzyme activity /
- isozyme /
- metabolite
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图 1 不同抗病烟草品种接种青枯雷尔菌后叶片PAL活性(以鲜质量计)的变化
相同时间同一烟草品种不同柱子上方凡是有一个相同小写字母者，表示该品种对照组和接菌组间差异不显著(P>0.05，Duncan's法)。
Figure 1. Changes of PAL activities (based on fresh mass) in leaves of resistant and susceptible tobacco cultivars after inoculation of Ralstonia solanacearum
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图 2 不同抗病烟草品种接种青枯雷尔菌后SOD活性(以鲜质量计)的变化
相同时间同一烟草品种不同柱子上方凡是有一个相同小写字母者，表示该品种对照组和接菌组间差异不显著(P>0.05，Duncan's法)。
Figure 2. Changes of SOD activities (based on fresh mass) in leaves of resistant and susceptible tobacco cultivars after inoculation of Ralstonia solanacearum
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图 3 不同抗病烟草品种接种青枯雷尔菌后POD活性(以鲜质量计)的变化
相同时间同一烟草品种不同柱子上方凡是有一个相同小写字母者，表示该品种对照组和接菌组间差异不显著(P>0.05，Duncan's法)。
Figure 3. Changes of POD activities (based on fresh mass) in leaves of resistant and susceptible tobacco cultivars after inoculation of Ralstonia solanacearum
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图 4 不同抗病烟草品种接种青枯雷尔菌后PPO活性(以鲜质量计)的变化
相同时间同一烟草品种不同柱子上方凡是有一个相同小写字母者，表示该品种对照组和接菌组间差异不显著(P>0.05，Duncan's法)。
Figure 4. Changes of PPO activities (based on fresh mass) in leaves of resistant and susceptible tobacco cultivars after inoculation of Ralstonia solanacearum
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图 5 不同抗病烟草品种接种青枯雷尔菌后叶片POD同工酶谱带
CK：对照组，TR：接菌组；A：抗病品种粤烟97，B：感病品种长脖黄。
Figure 5. Electrophoretic isoenzyme patterns of POD in leaves of resistant and susceptible tobacco cultivars after inoculation of Ralstonia solanacearum
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图 6 不同抗病烟草品种接种青枯雷尔菌后叶片PPO同工酶谱带
CK:对照组, TR：接菌组；A：抗病品种粤烟97, B：感病品种长脖黄。
Figure 6. Electrophoretic isoenzyme patterns of PPO in leaves of resistant and susceptible tobacco cultivars after inoculation of Ralstonia solanacearum
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图 7 不同抗病烟草品种接种青枯雷尔菌后代谢组分的GS-MS总离子流图
1：感病品种长脖黄对照组；2：感病品种长脖黄接菌组；3：抗病品种粤烟97对照组；4：抗病品种粤烟97接菌组。
Figure 7. Total ion chromatograms of metabolites of resistant and susceptible tobacco cultivars after inoculation of Ralstonia solanacearum
表 1 不同抗病烟草品种接种青枯雷尔菌后品种间代谢物差异比较1)
Table 1 Comparison of metabolites from resistant and susceptible tobacco cultivars after inoculation of Ralstonia solanacearum
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