摘要
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.16 SrRNA 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 16 S 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 WL168 HQ,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.WL168 HQ,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.
引文
[1] Zhang D.Isolation,identification and characteristics of endophytic bacteria from alfalfa.Harbin:Harbin Normal University,2016.张丹.紫花苜蓿内生菌的分离鉴定及特性分析.哈尔滨:哈尔滨师范大学,2016.
[2] Senthilkumar M,Anandham R,Madhaiyan M,et al.Endophytic bacteria:Perspectives and applications in agricultural crop production.Maheshwari(ed)Bacteria in Agrobiology Crop Ecosystems.New York,USA:Springer Publishing,2011:61-96.
[3] Tanuja B S C,Mishra P K.Ascending migration of endophytic Bacillus thuringiensis,and assessment of benefits to different legumes of D.W.Himalayas.European Journal of Soil Biology,2013,56(3):56-64.
[4] Beltrangarcia M J,White J F,Prado F M,et al.Nitrogen acquisition in Agave tequilanafrom degradation of endophytic bacteria.Scientific Reports,2014,4(2):6938.
[5] Mabood F,Zhou X M,Smith D L.Microbial signaling and plant growth promotion.Canadian Journal of Plant Science,2014,94:1051-1063.
[6] Pavlo A,Leonid O,Iryna Z,et al.Endophytic bacteria enhancing growth and disease resistance of potato(Solanum tuberosum L.).Biological Control,2011,56(1):43-49.
[7] Miliute I,Buzaite O,Baniulis D,et al.Bacterial endophytes in agricultural crops and their role in stress tolerance:A review.Zemdirbyste-Agriculture,2015,102(4):465-478.
[8] Ribeiro R A,Rogel M A,Lópezlópez A,et al.Reclassification of Rhizobium tropici type A strains as Rhizobium leucaenae sp.nov.International Journal of Systematic&Evolutionary Microbiology,2012,62(5):1179.
[9] Foxman B,Zhang L,Koopman J S,et al.Choosing an appropriate bacterial typing technique for epidemiologic studies.Epidemiologic Perspectives&Innovations,2005,2(1):1-8.
[10] Larsen M V,Cosentino S,Rasmussen S,et al.Multilocus sequence typing of total-genome-sequenced bacteria.Journal of Clinical Microbiology,2012,50(4):1355-1361.
[11] Berkum P V,Elia P,Eardly B D.Multilocus sequence typing as an approach for population analysis of Medicago-nodulating rhizobia.Journal of bacteriology,2006,188(15):5570-5577.
[12] Zhang Y M.Ecological characteristics and comparative genomics of soybean rhizobia with biogeographic feature.Beijing:China Agricultural University,2012.张延明.大豆根瘤菌生物地理分布的生态学特征及基因组学研究.北京:中国农业大学,2012.
[13] Martinez R E.Symbiovars in rhizobia reflect bacterial adaptation to legumes.Systematic&Applied Microbiology,2011,34(2):96.
[14] Rogel M A,Busto P,Santamaría R I,et al.Genomic basis of symbiovar mimosae in Rhizobium etli.BMC Genomics,2014,15(1):575.
[15] Ramírez-bahena M H,Chahboune R,Velázquez E,et al.Centrosemais a promiscuous legume nodulated by several new putative species and symbiovars of Bradyrhizobiumin various American countries.Systematic&Applied Microbiology,2013,36(6):392-400.
[16] Gubry-rangin C,Béna G,Cleyet-marel J C,et al.Definition and evolution of a new symbiovar,sv.rigiduloides,among Ensifer meliloti efficiently nodulating Medicago species.Systematic&Applied Microbiology,2013,36(7):490-496.
[17] Mnasri B,Mrabet M,Laguerre G,et al.Salt-tolerant rhizobia isolated from a Tunisian oasis that are highly effective for symbiotic N2.Archives of Microbiology,2007,187(1):79-85.
[18] Reiner R R,Lourdes L,Edith P,et al.Rhizobia with different symbiotic efficiencies nodulate Acaciellaangustissimain Mexico,including Sinorhizobium chiapanecumsp.nov.which has common symbiotic genes with Sinorhizobium mexicanum.FEMS Microbiology Ecology,2009,67(1):103-117.
[19] Ramírez-bahena M H,Hernández M,Peix A,et al.Mesorhizobial strains nodulating Anagyris latifolia and Lotus berthelotii in Tamadaya ravine(Tenerife,Canary Islands)are two symbiovars of the same species,Mesorhizobium tamadayense sp.nov.Systematic&Applied Microbiology,2012,35(5):334.
[20] Ulrich A,Zaspel I.Phylogenetic diversity of rhizobial strains nodulating Robinia pseudoacacia.Microbiology,2000,146(11):2997-3005.
[21] Laguerre G,Depret G,Bourion V,et al.Rhizobium leguminosarumbv.viciae genotypes interact with pea plants in developmental responses of nodules,roots and shoots.New Phytologist,2007,176(3):680-690.
[22] Qi J,Shi S L.Preliminary study on the ability of phosphorus-solubilizing and IAA-secreting of endogenous rhizobia in seeds of different alfalfa varieties.Grassland and Turf,2006,(5):18—20.祁娟,师尚礼.不同品种紫花苜蓿种子内生根瘤菌溶磷和分泌生长素能力.草原与草坪,2006,(5):18-20.
[23] Huo P H.Antimicrobial-resistant rhizobia screening and effect verification of undesired microbe control in the prepared rhizobia inoculant.Lanzhou:Gansu Agricultural University,2014.霍平慧.耐抑菌剂根瘤菌筛选及耐药菌株制备菌剂抑杂菌效果研究.兰州:甘肃农业大学,2014.
[24] Miao Y Y,Shi S L,Zhang J G,et al.Migration,colonization and seedling growth of rhizobia with matrine treatment in alfalfa(Medicago sativa L.).Acta Agriculturae Scandinavica,Section B-Soil&Plant Science,2017:1-13.
[25] Weisburg W G,Barna S M,Pelletier D A,et al.16Sribosomal DNA amplification for phylogenetic study.Journal of Bacteriology,1991,173(2):697-703.
[26] Vinuesa P,Silva C,Lorite M J,et al.Molecular systematics of rhizobia based on maximum likelihood and Bayesian phylogenies inferred fromrrs,atpD,recAand nifHsequences,and their use in the classification of Sesbania microsymbionts from Venezuelan wetlands.Systematic&Applied Microbiology,2005,28(8):702.
[27] Yoon S H,Ha S M,Kwon S,et al.Introducing ezbiocloud:A taxonomically united database of 16SrRNA gene sequences and whole-genome assemblies.International Journal of Systematic and Evolutionary Microbiology,2017,67(5):1613.
[28] Tamura K,Peterson D,Peterson N,et al.MEGA5:Molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods.Molecular Biology and Evolution,2011,28(10):2731-2739.
[29] Zhang X F,Shi S L,Nan L L,et al.Phenotype diversities of alfalfa rhizobium strains collected in different ecological regions in Gansu Province.Journal of Gansu Agricultural University,2009,6(3):106-111.张小甫,师尚礼,南丽丽,等.甘肃不同生态区域苜蓿根瘤菌表型多样性分析.甘肃农业大学学报,2009,6(3):106-111.
[30] Li J F,Zhang S Q,Shi S L,et al.Position and quantity of endogensis rhizobia in alfalfa plant.Chinese Journal of Eco-Agriculture,2009,17(6):1200-1205.李剑峰,张淑卿,师尚礼,等.苜蓿内生根瘤菌分布部位与数量变化动态.中国生态农业学报,2009,17(6):1200-1205.
[31] Zaied K A,Kosba Z A,Nassef M A,et al.Induction of Rhizobium inoculants harboring salicylic acid gene.Australian Journal of Basic&Applied Sciences,2009,(2):1386-1411.
[32] Shetta N D,Alshahranil T S.The symbiotic efficiency of legume tree rhizobia for host range legumes in central Saudi Arabia.International Journal of Agriculture&Biology,2016,18:851-857.
[33] Huo P H,Li J F,Shi S L,et al.Effect of seed ultra-drying storage on growth and resistance of the Medicago sativa seedlings affected by alkaline salt stress.Scientia Agricultura Sinica,2014,47(13):2643-2651.霍平慧,李剑峰,师尚礼,等.碱性盐胁迫对超干贮藏苜蓿种子幼苗生长及抗性的影响.中国农业科学,2014,47(13):2643-2651.
[34] Miao Y Y,Shi S L,Kang W J.Effects of gibberellin on migration and colonization of rhizobia and seedling growth of alfalfa.Scientia Agricultura Sinica,2017,(23):4545-4557.苗阳阳,师尚礼,康文娟.赤霉素对根瘤菌运移、定殖及苜蓿幼苗生长的影响.中国农业科学,2017,(23):4545-4557.
[35] Boukhatem Z F,Domerhue O,Bekki A,et al.Symbiotic characterization and diversity of rhizobia associated with native and introduced acacias in arid and semi-arid regions in Algeria.FEMS Microbiology Ecology,2012,80(3):534-547.
[36] Tang Z,An H,Deng L,et al.Effect of desertification on productivity in a desert steppe.Scientific Reports,2016,6(6):27839.
[37] Ke C L,Dai J X,Zhou D B,et al.Microbial community diversity in the soil fertilized with phosphate solubilizing bacteria by terminal restriction fragment length polymorphism analysis.Journal of Anhui Agricultural University,2017,44(3):471-477.柯春亮,戴嘉欣,周登博,等.利用T-RFLP技术在施用解磷菌剂土壤中微生物群落多样性分析.安徽农业大学学报,2017,44(3):471-477.
[38] Ren D W,Chen W F,Sui X H,et al.Rhizobium vignae sp.nov.a symbiotic bacterium isolated from multiple legume species.International Journal of Systematic and Evolutionary Microbiology,2011,61(3):580.
[39] Liu Y,Wang R P,Ren C,et al.Rhizobium marinum sp.nov.a malachite-green tolerant bacterium isolated from the sea water.International Journal of Systematic&Evolutionary Microbiology,2015,65(12):4449.
[40] Qi C M.The study on biodiversity and phylogenesis of rhizobia isolated fromTrifoliumand Medicago.Ya'an:Sichuan Agricultural University,2004.齐春梅.苜蓿、三叶草根瘤菌生物多样性和系统发育地位的研究.雅安:四川农业大学,2004.
[41] Fortuna M A,Zaman L,Ofria C,et al.The genotype-phenotype map of an evolving digital organism.PLoS Computational Biology,2017,13(2):e1005414.
[42] Salazar-ciudad I,Marín-riera M.Adaptive dynamics under development-based genotype-phenotype maps.Nature,2013,497(7449):361-364.