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 475SDS-PAGE of whole-cell proteinsWhole-cell protein extracts were prepared and separatedby electrophoresis using slight modifications of theprocedure of Laemmli [16] as described by de Lajudieet al. [4].Results and discussionPresented study allowed to develop a rapid methodfor identification and differentiation of 4 taxa belongingto genus Agrobacterium using multiplex PCR withprimers complementary to the 23S rRNA gene. Up tonow, analysis of phenotypic features based on biochemicaltests has been the phytopathologist’s standardmethod to classify agrobacteria into biovar 1, biovar2, A. vitis and A. rubi [18]. However, unambiguousclassification of agrobacteria into a particular taxon islaborious and time-consuming, and the results are veryoften difficult to interpret. Modern systems of bacterialidentification exploit techniques based on analysis ofsequences of functional genes. One of the most oftenused for this purpose is the gene coding for 16S rRNA.Systems based on this gene were developed for rapididentification of bacteria belonging to genera Bifidobacterium[6], Mycobacterium, Nocardia and others (reviewedin [15]). However, among agrobacteria thedivergence in sequences of 16S rDNA is not highenough for this system to be effective. For example,dissimilarity between 16S rDNA sequences of A. rubiand Agrobacterium biovar 1 is only 1.5%, which limitsthe possibilities for developing primers or probesdifferentiating these 2 taxa [26,33]. Mougel et al. [19]designed probes complementary to 16S rDNA, whichallow the discrimination of biovar 2, but not biovar 1agrobacteria and A. rubi. The gene coding for 23SrRNA contains more information than that for 16SrRNA and has already been used for identification ofbacteria of other genera [17,30]. In the case ofAgrobacterium, higher dissimilarity between sequencesof all species/biovars was found for this gene. Forinstance, differences between 23S rDNA sequences ofbiovar 1 agrobacteria and A. rubi ranged from 2.37% to3.09% [23], offering more scope for the development ofspecific primers. Similar results were obtained also byPeplies et al. [20], who in study on oligonucleotide probedesign for some marine Alphaproteobacteria found that23S rRNA gene has up to 8 times more discriminatorysites than 16S rRNA.Based on BLAST N analysis, 4 of the developedprimers, UF, B2R, AvR and ArR showed 100%homology only to the 23S rDNA sequences of the taxathey were designed to identify. The sequence of primerB1R, in addition to homology to 23S rDNA sequence ofAgrobacterium biovar 1, also showed 100% homologyto gene sequences of A. rubi IAM 13569 (AY244376)and A. vitis IAM 14140 (AY244374). However, thesesequences may be misnamed, because on the basis ofcluster analysis of all known 23S rDNA sequences ofbacteria belonging to family Rhizobiaceae they are mostsimilar to sequences of biovar 1 strains of Agrobacterium,but not to other sequences of A. rubi (AF205373)and A. vitis (AF209071, AF209076, AY244370) strains(including the type strains) present in the publicdatabases.The specificity of the newly developed primers wastested in PCRs using DNA of 119 bacterial strainsincluding: 93 of Agrobacterium, 19 of rhizobia and 7strains of phytopathogenic bacteria representing variousgenera. Primers developed for identification of bacteriaclassified as A. vitis, A. rubi and Agrobacterium biovar 1,amplified DNA of strains belonging only to these taxaand gave products of the expected sizes: 478, 1006 and184 bp, respectively. However, primer B2R, combinedwith primer UF, in addition to strains of Agrobacteriumbiovar 2, also amplified DNA of some (though not all)strains of other, closely related genera such as:Allorhizobium undicola, Rhizobium etli, R. leguminosarumbv. trifolii, R. tropici and all tested strains of thegenus Sinorhizobium. None of tested primers amplifiedDNA of strains belonging to A. larrymoorei, Phyllobacterium,Arthrobacter, Erwinia, Pseudomonas or Xanthomonas(Table 1). The results obtained in separate PCRswith following sets of primers: UF+B1R, UF+B2R,UF+AvR and UF+ArR were identical with thoseobtained in the PCRs, using all 5 primers simultaneously.Four PCR products of expected sizes wereobtained as a result of multiplex PCR with DNAmixture of representative strains of all Agrobacteriumtaxa (Fig. 1).As a result of DNA amplification with primers UFand B2R, a characteristic product of 1066 bp wasobtained in PCRs with DNA of 3 strains of Agrobacteriumbiovar 1, 58 strains of biovar 2 and 10 rhizobiastrains (Table 1). Restriction analysis of these productsusing endonuclease Alw26I allowed differentiation ofbiovar 2 strains from the other strains. The followingDNA fragments were obtained after digestion of the1066 bp PCR product of agrobacteria belonging tobiovar 2: 1006 and 60 bp, whereas in case of the otherbacteria 3 products (830, 167 and 60 bp, as expected onthe basis of computer analysis) were obtained (Table 1,Fig. 2).In case of strain 47/7, a 184 bp product typical forbiovar 1 was obtained as a result of amplification withprimers UF+B1R. However, this strain could not beclearly classified in biovar 1 on the basis of phenotypicfeatures. It showed typical features for biovar 2, such asthe ability to produce an acid from erythritol and it was3-ketolactose negative but at the same time showed
476ARTICLE IN PRESSJ. Pu"awska et al. / Systematic and Applied Microbiology 29 (2006) 470–479M 1 2 3 4 5 6 7 8 9 10 11 12 13M 1 2 3 4 5 6 7 8 9500 bp→Fig. 1. Electrophoresis gel showing PCR products obtained inmultiplex PCR with DNA of following Agrobacterium strains:1 – B6 (bv 1); 2 – LMG 150 (bv 2); 3 – LMG 8750 (A. vitis);4–LMG 156 (A. rubi); 5 – LMG 21410 (A. larrymoorei); 6 –mixture of DNA of strains B6, LMG 150, LMG 8750, LMG156 and LMG 21410; 7 – 47/7; 8 – 0; 9 – 7/1; 10 – 39/7; 11 –Ch11; 12 – Ch12; 13 – negative control; M – molecular weightmarker 100 bp ladder (Fermentas, Lithuania).500 bp→features typical for biovar 1 such as presence of oxidaseC as well as growth and pigmentation in ferricammonium citrate broth (Table 2). We verified its 16SrDNA sequence and found that 47/7 was 100% similarto that of Agrobacterium biovar 1 strain IAM 13129(accession no. D12784) and thus we can conclude it isindeed a biovar 1 strain (data unpublished).DNA of 3 other Hungarian strains (0, 7/1, 39/7) wasamplified both with primers complementary to 23SrDNA sequence of Agrobacterium biovar 1 and biovar2. As a result of amplification with the 5-primer mixture2 PCR products (184 and 1066 bp) were obtained(Fig. 1). Analysis of phenotypic features showed, thatthese 3 strains were most similar to biovar 1, except intheir ability to grow and produce pigment in ferricammonium citrate broth (Table 2). On the other hand,restriction analysis with Alw26I of products obtainedwith primers UF+B2R gave the same 3 fragments (830,167 and 60 bp) as those found for rhizobial strains butnot the 2 fragments (1006 and 60 bp) characteristic forbiovar 2. SDS-PAGE of total cellular proteins was usedas a rapid tool to screen for overall similarities of theHungarian strains and other taxa in our protein profiledatabase. It was found that these 3 strains producedsimilar protein patterns distinct from type strains ofother biovars and species, but most similar to biovar 1strains (Fig. 3). They may represent a separate, hithertounrecognized taxon. The fact that none of the rhizobialstrains produced a product with the primer combinationUF+B1R as the Hungarian strains did, seems toconfirm that they may be a distinct group.Fig. 2. Restriction analysis with endonuclease Alw26I of PCRproducts obtained with primers UF+B2R. For comparisonnot digested product on lane 5 (1066 bp). Digestion fragmentsof UF+B2R PCR product of rhizobia (expected sizes: 830,167 and 60 bp): lane 1 – S. saheli LMG 7837; lane 2 – S.teranga LMG 7834; lane 3 – R. tropici LMG 9503; lane 4 – A.undicola LMG 11875. Digestion fragments of UF+B2R PCRproduct of some strains of agrobacteria biovar 2 (expectedsizes: 1006 and 60 bp): lane 6 – 39; lane 7 – LMG 150; lane 8 –K84; lane 9 – 129; lane 10 – 89. M – molecular weight marker100 bp ladder (Promega, USA).DNA of 2 Agrobacterium strains (Ch11 and Ch12)isolated from galls on chrysanthemum plants was notamplified with any set of primers, which suggests thatthey do not belong to any of the present biovars orspecies of Agrobacterium. Phenotypic characteristicsindicated that these strains are most similar to the A.rubi-type strain (Table 2), but FAME and RAPDanalysis did not confirm unambiguously their affinityto A. rubi [21]. Further studies should determine theirtaxonomic status.The method presented here is the first PCR-basedprotocol developed for identification and differentiationof bacteria belonging to biovars 1 and 2 of Agrobacterium,A. vitis and A. rubi. However, for discriminationbetween strains of biovar 2 and some rhizobia species
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