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

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

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476ARTICLE IN PRESSJ. Pu"awska et al. / Systematic <strong>and</strong> 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 <strong>of</strong> following <strong>Agrobacterium</strong> 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 <strong>of</strong> DNA <strong>of</strong> strains B6, LMG 150, LMG 8750, LMG156 <strong>and</strong> 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 <strong>of</strong> oxidaseC as well as growth <strong>and</strong> pigmentation in ferricammonium citrate broth (Table 2). We verified its 16SrDNA sequence <strong>and</strong> found that 47/7 was 100% similarto that <strong>of</strong> <strong>Agrobacterium</strong> biovar 1 strain IAM 13129(accession no. D12784) <strong>and</strong> thus we can conclude it isindeed a biovar 1 strain (data unpublished).DNA <strong>of</strong> 3 other Hungarian strains (0, 7/1, 39/7) wasamplified both with primers complementary to 23SrDNA sequence <strong>of</strong> <strong>Agrobacterium</strong> biovar 1 <strong>and</strong> biovar2. As a result <strong>of</strong> amplification with the 5-primer mixture2 PCR products (184 <strong>and</strong> 1066 bp) were obtained(Fig. 1). Analysis <strong>of</strong> phenotypic features showed, thatthese 3 strains were most similar to biovar 1, except intheir ability to grow <strong>and</strong> produce pigment in ferricammonium citrate broth (Table 2). On the other h<strong>and</strong>,restriction analysis with Alw26I <strong>of</strong> products obtainedwith primers UF+B2R gave the same 3 fragments (830,167 <strong>and</strong> 60 bp) as those found for rhizobial strains butnot the 2 fragments (1006 <strong>and</strong> 60 bp) characteristic forbiovar 2. SDS-PAGE <strong>of</strong> total cellular proteins was usedas a rapid tool to screen for overall similarities <strong>of</strong> theHungarian strains <strong>and</strong> other taxa in our protein pr<strong>of</strong>iledatabase. It was found that these 3 strains producedsimilar protein patterns distinct from type strains <strong>of</strong>other biovars <strong>and</strong> species, but most similar to biovar 1strains (Fig. 3). They may represent a separate, hithertounrecognized taxon. The fact that none <strong>of</strong> 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 <strong>of</strong> PCRproducts obtained with primers UF+B2R. For comparisonnot digested product on lane 5 (1066 bp). Digestion fragments<strong>of</strong> UF+B2R PCR product <strong>of</strong> rhizobia (expected sizes: 830,167 <strong>and</strong> 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 <strong>of</strong> UF+B2R PCRproduct <strong>of</strong> some strains <strong>of</strong> agrobacteria biovar 2 (expectedsizes: 1006 <strong>and</strong> 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 <strong>of</strong> 2 <strong>Agrobacterium</strong> strains (Ch11 <strong>and</strong> Ch12)isolated from galls on chrysanthemum plants was notamplified with any set <strong>of</strong> primers, which suggests thatthey do not belong to any <strong>of</strong> the present biovars orspecies <strong>of</strong> <strong>Agrobacterium</strong>. Phenotypic characteristicsindicated that these strains are most similar to the A.rubi-type strain (Table 2), but FAME <strong>and</strong> 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 <strong>identification</strong> <strong>and</strong> differentiation<strong>of</strong> bacteria belonging to biovars 1 <strong>and</strong> 2 <strong>of</strong> <strong>Agrobacterium</strong>,A. vitis <strong>and</strong> A. rubi. However, for discriminationbetween strains <strong>of</strong> biovar 2 <strong>and</strong> some rhizobia species

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