中国农业科学2018，Vol.51Issue(2)：268-278,11.DOI:10.3864/j.issn.0578-1752.2018.02.007
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
Heterogeneity Analysis of Ralstonia solanacearum Mutants by Tn5 Transposon Using High Performance Ion-Exchange Chromatography
摘要
Abstract
[Objective] The objective of this study is to analyze the heterogeneity ofRalstonia 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 (CTIi)was expressed as CTIi=Si/ (S1+S2+S3)× 100％ (i=l,2 or 3;S1,S2 and S3 represent peak areas of P1,P2 and P3,respectively),and the relationship of CTIi 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 ofR.solanacearum.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Key words
high performance ion-exchange chromatography/Ralstonia solanacearum/bacterial wilt disease/Tn5 transposon/mutant strain引用本文复制引用
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):268-278,11.基金项目
国家重点研发计划(2017YFD0201100)、国家自然科学基金(31701835)、福建省农业科学院青年英才基金(YC2016-15)、福建省农业科学院科技创新团队(STIT2017-1-11) （2017YFD0201100）
