Rapid and specific identification of four Agrobacterium ... - Cost 873

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ARTICLE IN PRESSJ. Pu"awska et al. / Systematic and Applied Microbiology 29 (2006) 470–479 4775060708090100M. huakuii LMG 14107M. plurifarium LMG11892 TA. rhizogenes LMG 150M. ciceri LMG 14989 TR. huautlense LMG 18254Agrobacterium sp. 39/7Agrobacterium sp. 7/1Agrobacterium sp. 0A. radiobacter (bv. 1) LMG140 TA. tumefaciens (bv. 1) LMG 232A. tumefaciens (bv. 1) LMG 243A. tumefaciens (bv. 1) LMG 52A. larrymoorei LMG 21410 TA. tumefaciens (bv. 1) LMG 187 TS. terangae LMG 7834 TB. japonicum LMG 6138 TAz. caulinodans LMG6465 TS. arboris LMG15624S. saheli LMG 7837 TR. galegae LMG 6214 TR. leguminosarum LMG 8817 TR. tropici LMG 9503 TR. tropici LMG 9517M. loti LMG 6125 TS. kostiense LMG 15610S. meliloti LMG 6130S. meliloti LMG 6133 TR. etli CFN42 TS. medicae LMG 16580S. fredii LMG 6217 TR. mongolense LMG 19141M. mediterraneum LMG17148 TM. tianshanense LMG15767 TA. tumefaciens (bv. 1) LMG 37R. hainanense LMG 18075A. vitis LMG8750 TAl. undicola LMG 11874Fig. 3. Dendrogram showing the similarities between the SDS-PAGE protein profiles of Hungarian strains 39/7, 7/1, 0 and aselection of reference strains. Using the program GelCompar v. 4.2, the Pearson product moment correlation coefficient wascalculated and converted to percentage values. The similarities are represented as a UPGMA (unweighted pair grouping methodusing averages) dendrogram.(Allorhizobium undicola, Rhizobium etli, R. leguminosarumbv. trifolii, R. tropici and tested Sinorhizobium sp.) itis necessary to carry out RFLP analysis of the obtained1066 bp PCR product. Although as inferred fromtheoretical analysis, the primer B2R was expected tobe 100% complementary only to 23S rDNA of biovar 2strains, it also allowed amplification in some strains ofrhizobia. This may be due to the fact that all rhizobialstrains producing the typical 1066 bp amplicon withprimers UF+B2R, are phylogeneticaly closely relatedto agrobacteria, as is apparent from 16S rDNAphylogeny [3,34]. Similarity of 23S rDNA of Agrobacteriumbiovar 2 and some rhizobia is very high. In somecases, 23S rDNA sequences of biovar 2 strains are evenmore similar to the same gene sequence of rhizobia (e.g.R. tropici) than to those of other Agrobacterium biovar/species [23].The presented method we use for fast identificationand classification of isolated agrobacteria from plantsand soil. Simultaneous application of primers complementaryto tms2 gene which amplify DNA of onlytumour inducing strains [22,29] or pathogenicity tests onplants [28] allows for complete characterization ofstudied agrobacteria. The developed method should behelpful in phytopathological and ecological studies.AcknowledgmentsThe authors wish to thank J.H. Haas, S. Su¨le,X.Nesme,M. Maes, H. Sawada, S. Martyniuk, H. Bagin´ska for
478ARTICLE IN PRESSJ. Pu"awska et al. / Systematic and Applied Microbiology 29 (2006) 470–479providing bacterial strains. J. Pu"awska is deeply indebtedto the Foundation for Polish Science for NationalScholarship for Young Scientist (Edition 2001/2002).A. Willems is grateful to the Fund for ScientificResearch – Flanders for a postdoctoral research fellowship.This work was supported by Polish Scientific Committee(KBN) Grant 5P06A02119.References[1] J. Brosius, T.J. Dull, D.D. Sleeter, H.F. Noller, Geneorganization and primary structure of a ribosomal RNAoperon from Escherichia coli, J. Mol. Biol. 148 (1981)107–127.[2] J. Cubero, M.M. Lopez, An efficient microtiter system todetermine Agrobacterium biovar, Eur. J. Plant Pathol. 107(2001) 757–760.[3] P. de Lajudie, E. Laurent-Fulele, A. Willems, U. Torck,R. Coopman, M.D. Collins, K. Kersters, B. Dreyfus, M.Gillis, Allorhizobium undicola gen. nov., sp. nov., nitrogen-fixingbacteria that efficiently nodulate Neptunianatans in Senegal, Int. 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Ludwig,Comparative sequence analysis and oligonucleotide probedesign based on 23S rRNA genes of Alphaproteobacteriafrom North Sea bacterioplankton, Syst. Appl. Microbiol.27 (2004) 573–580.[21] J. Pu"awska, Z. Piotrowska-Seget, Characterization ofAgrobacterium isolates from chrysanthemum on the basisof biochemical tests, FAME analysis and RAPD,Phytopathol. Pol. 30 (2003) 9–17.[22] J. Pu"awska, P. Sobiczewski, Development of a seminestedPCR based method for sensitive detection oftumorigenic Agrobacterium in soil, J. Appl. Microbiol. 98(2005) 710–721.[23] J. Pu"awska, M. Maes, A. Willems, P. Sobiczewski,Phylogenetic analysis of 23S rRNA gene sequences ofAgrobacterium, Rhizobium and Sinorhizobium strains,Syst. Appl. Microbiol. 23 (2000) 238–244.[24] J. Sambrook, E.F. Fritsch, T. Maniatis, MolecularCloning, A Laboratory Manual, second ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY,1989.[25] H. Sawada, H. Ieki, Phenotypic characteristics of thegenus Agrobacterium, Ann. 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