紫花苜蓿根瘤菌分子分型和生物型划分研究

Molecular typing and biotype classification of rhizobia of Medicago sativa

根据表型和共生效应,对种内根瘤菌划分生物型,为苜蓿与根瘤菌高效共生育种提供理论依据。以分离自甘肃省3个栽培区域5个苜蓿品种植株不同部位的根瘤菌株为材料,进行16SrRNA测序、多位点序列分型(Multilocus Sequence Typing,MLST)、表型数值分类和共生效应测定。结果表明,78株初筛菌经16SrRNA和MLST分析有20株属于Rhizobium radiobacter;表型特征数值分类将其聚为7个群。接种5个苜蓿品种,选择对共生效率贡献率最大的地上干重进行多重比较。当接种处理地上干重显著高于未接种处理CK时标记共生效应为A,与CK差异不显著时标记为B,显著低于CK时标记为C。以苜蓿品种为甘农3号、甘农9号、陇中、清水和WL168HQ的顺序,将各菌株共生效应结合,形成6种共生模式。结合表型和共生模式,20株R.radiobacter被划分为14种生物型,WL168HQ、甘农3号和陇中苜蓿品种内生物型多样性丰富,甘农9号和清水苜蓿品种内生物型单一。综上所述,根据表型特征和共生模型可以进行种内根瘤菌株的生物型划分,苜蓿品种内根瘤菌生物型多样性丰富。

This study aimed to classify biotypes in the same rhizobium species according to their phenotypic characteristics and the symbiotic efficiency between the alfalfa variety and rhizobium strain,and so to provide a knowledge base for breeding rhizobia that are effective symbiotic partners with alfalfa.Rhizobium strains were isolated from different plant parts of five Medicago sativa varieties in three cultivation regions of Gansu province.16S rRNA sequencing,multilocus sequence typing(MLST),numerical taxonomy analysis based on phenotypic characteristics and symbiotic efficiency determination were used to achieve rhizobia biotype classification.Of 78 rhizobia strains in a preliminary screening,20 were assigned to Rhizobium radiobacter by 16S rRNA sequencing and MLST analysis.Seven phenotypic clusters were formed based on numerical taxonomy analysis of phenotypic characteristics.Seedlings of the five M.sativa varieties were inoculated with the 20 R.radiobacter strains,and shoot dry weights,which contributed the most to the symbiotic efficiency between alfalfa and rhizobium,for varieties inoculated with each strain were compared using Duncan’s multiple range test.The symbiotic efficiency of strains was designated A,B,or C when the shoot dry weight of inoculated plants was,respectively,significantly higher than,not significantly different from,or significantly lower than that of non-inoculated control plants.The designated symbiotic efficiency of each rhizobia was combined according to the order of M.sativa cv.Gannong No.3,Gannong No.9,Longzhong,Qingshui,and WL168HQ,and six kinds of symbiotic combination patterns were formed.The 20 R.radiobacter strains were divided into 14 biotypes according to phenotypes and symbiotic patterns.For M.sativa cv.WL168HQ,Gannong No.3,and Longzhong many rhizobium biotypes were found,while the rhizobia of M.sativa cv.Gannong No.9,and Qingshui were monotypic.In conclusion,rhizobia of the same species(R.radiobacter)could be classified into biotypes using information on phenotypic characteristics and symbiotic patterns,and some alfalfa varieties were found to host a number of rhizobium biotypes.