高效离子交换色谱法分析青枯雷尔氏菌Tn5转座子突变菌株的异质性

doi:10.3864/j.issn.0578-1752.2018.02.007

摘要/Abstract

摘要： 【目的】研究青枯雷尔氏菌（Ralstonia solanacearum）Tn5转座子突变菌株的异质性，筛选出无致病力高效生防菌株。【方法】利用已构建的青枯雷尔氏菌致病力参考指标“弱化指数（attenuation index，AI）”和接种番茄植株的生物测定法对60株供试的青枯雷尔氏菌Tn5转座子突变菌株进行致病力测定；利用高效离子交换色谱法研究不同致病力突变菌株的色谱行为异质性；构建色谱效价指数（chromatography titer index, CTI_i），CTI_i=S_i/（S₁+S₂+S₃）×100%（i=1、2或3；S₁、S₂和S₃分别对应P₁、P₂和P₃的峰面积），测定不同致病力青枯雷尔氏菌突变菌株的CTI_i值，用皮尔逊相关系数（Pearson correlation coefficient，PCC）分析色谱效价指数与突变菌株的致病力和青枯病害防治效果的相互关系。【结果】基于AI值和生物测定结果，青枯雷尔氏菌Tn5转座子突变菌株具有3种不同致病力类型：强致病力、无致病力和中间型。强致病力菌株共33株，其AI值介于0.49—0.63，接种番茄15 d，植株发病率为66.33%—100%；无致病力菌株共有20株，其AI值介于0.78—0.89，接种番茄15 d，植株发病率为0；中间型菌株共有7株，其AI值介于0.68—0.73，接种番茄15 d，植株发病率为24.17%—45.92%。20株无致病力突变菌株对番茄青枯病的防治效果测定结果显示，防治效果介于16.68%—92.45%，菌株T831防治效果最好，达91.74%，其次是菌株T780，防治效果为87.51%，菌株T497防治效果最差，为16.68%。不同致病力青枯雷尔氏菌的高效离子交换色谱分离结果显示，青枯雷尔氏菌经高效离子交换色谱可以分离出P₁（出峰时间为0.6 min）、P₂（出峰时间为4.4或4.5 min）和P₃峰（出峰时间为5.9或6.0 min）；强致病力菌株和无致病力菌株均具有3种不同峰类型：单峰、双峰和3个峰，强致病力菌株的主峰为P₃峰，无致病力菌株的主峰为P₁峰，中间型菌株有双峰和3个色谱峰两种类型；强致病力菌株中CTI₃为100%的菌株，致病力最强，诱导番茄植株发病率均达85%以上，CTI₃＜100%的菌株，接种后番茄植株发病率介于66.33%—84.14%；无致病力菌株中CTI₁达100%的菌株，其防治效果高，均达到80%以上，CTI₁＜90%的菌株，其防治效果低，介于16.68%—63.62%；CTI₃与突变菌株致病力呈极显著正相关，相关系数PCC值为0.62；CTI₁与无致病力突变菌株的防治效果呈极显著正相关，相关系数PCC值为0.80。【结论】青枯雷尔氏菌Tn5转座子突变菌株具有3种致病力类型，不同致病力菌株的色谱行为不同。构建的色谱效价指数CTI₁可用于快速筛选出无致病力高效生防菌株，CTI₃可作为青枯雷尔氏菌致病力的参考指标。

关键词: 高效离子交换色谱, 青枯雷尔氏菌, 青枯病, Tn5转座子, 突变菌株

Abstract: 【Objective】The objective of this study is to analyze the heterogeneity of Ralstonia solanacearum mutants by Tn5 transposon and screen out an avirulent mutant with high control efficiency. 【Method】 The pathogenicity of sixty R. solanacearum Tn5 transposon mutants was identified by using the attenuation index (AI, a quantitative index for calculating the pathogenicity of R. solanacearum) and tomato plant inoculation bioassay. The chromatographic behavior heterogeneity of different pathogenic mutants of R. solanacearum was analyzed by using high performance ion-exchange chromatography. A chromatography titer index (CTI_i) was expressed as CTI_i=S_i/ (S₁+S₂+S₃)×100% (i=1, 2 or 3; S₁, S₂ and S₃ represent peak areas of P₁, P₂ and P₃, respectively), and the relationship of CTI_i with the pathogenicity and control efficiency of tested mutants was also analyzed based on Pearson correlation coefficient (PCC). 【Result】 Based on the value of AI and the results of bioassay, sixty R. solanacearum mutants were divided into three pathogenic types: virulent, avirulent and interim. Thirty-three mutants belonged to virulent mutant with the value of AI ranged from 0.49 to 0.63 and disease incidence ranged from 66.33% to 100% (15 days after inoculation). Twenty mutants were avirulent with the value of AI ranged from 0.78 to 0.89 and the disease incidence was 0. There were only seven interim mutants with the value of AI ranged from 0.68 to 0.73 and disease incidence ranged from 24.17% to 45.92%. The control efficiency of twenty avirulent mutants against tomato bacterial wilt disease ranged from 16.68% to 92.45%. The best control efficiency was obtained by T831 of 91.74%, followed by T780 (87.51%). The mutant of T497 had the worst control efficiency of 16.68%. Using high performance ion-exchange chromatography, R. solanacearum mutants were successfully separated into three chromatographic peaks: P₁ (retention time of 0.6 min), P₂ (retention time of 4.4 or 4.5 min) and P₃ (retention time of 5.9 or 6.0 min). Both virulent and avirulent mutants had three different peaks: single peak, double and three peaks. However, the interim mutants only had double peaks and three peaks types. For virulent mutants, the intense peak is P₃, while for avirulent mutants, P₁ is the majority. The virulent mutants with CTI₃ of 100% had high pathogenicity, which could induce tomato plant bacterial wilt with disease incidence up to 85%. The virulent mutants with CTI₃ less than 100% had the disease incidence ranged from 66.33% to 84.14%. The avirulent mutants with CTI₁ of 100% had high control efficiency which reached up to 80%, and the avirulent mutants with CTI₁ less than 90% had low control efficiency which ranged from 16.68% to 63.62%. The correlations of CTI₃ and the pathogenicity of virulent mutants were significantly positive (P＜0.01), with PCC of 0.62. The relationships between CTI₁ and control efficiency against bacterial wilt disease of the avirulent mutants were also significantly positive (P＜0.01), with PCC of 0.80. 【Conclusion】 R. solanacearum mutants by Tn5 transposon have three pathogenic types, which resulted in different chromatographic behaviors. CTI₁ constructed in this study can be used for rapidly selecting an avirulent mutant with high control efficiency and CTI₃ can be used for calculating the pathogenicity of R. solanacearum.

Key words: high performance ion-exchange chromatography; Ralstonia solanacearum')">

high performance ion-exchange chromatography; Ralstonia solanacearum, bacterial wilt disease, Tn5 transposon, mutant strain

郑雪芳，刘波，朱育菁，陈德局，陈小强. 高效离子交换色谱法分析青枯雷尔氏菌Tn5转座子突变菌株的异质性[J]. 中国农业科学, 2018, 51(2): 268-278.

ZHENG XueFang, LIU Bo, ZHU YuJing, CHEN DeJu, CHEN XiaoQiang. Heterogeneity Analysis of Ralstonia solanacearum Mutants by Tn5 Transposon Using High Performance Ion-Exchange Chromatography[J]. Scientia Agricultura Sinica, 2018, 51(2): 268-278.

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