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

474ARTICLE IN PRESSJ. Pu"awska et al. / Systematic and Applied Microbiology 29 (2006) 470–479Table 1. (continued )Strain Source and location Fragments produced in multiplex PCR with primersUF+B1R+B2R+AvR+ArRBiovar 1(184 bp)Biovar 2(1066 bp)A.vitis(478 bp)A. rubi(1006 bp)PCR-RFLP ofUF+B2Rproduct aPseudomonas syringaepv. tomatoXanthomonas arboricolapv. pruniUnknown; PolandUnknown; Unknownntnta 2, two DNA fragments of 1006 bp and 60 bp were obtained as a result of RFLP with Alw26I; 3, three DNA fragments of 830 bp, 167 bp and 60 bpwere obtained; nt, not tested.Table 2.Characteristic of phenotypic features of some Agrobacterium strains testedStrain 3-ketolactoseproductionCitrateutilizationL-tyrosineutilizationFerricammoniumcitrateAcid fromerythritolNaCltolerance(%)Oxidase cAgrobacterium B6 T (bv.1) + + 4 +Agrobacterium Ch 3, 131a/b + + 3 +Agrobacterium Ch-6, 137, 6, + + 4 +CFBP 2519Agrobacterium 0, 39/7, 7/1 + 4 +Agrobacterium LMG 150 T+ + + 1(bv.2)Agrobacterium 400 + + + 1 +/Agrobacterium 77 + + + 1A. vitis LMG 8750 T + 3 +A. rubi LMG 156 T nt 4 +Agrobacterium Ch-11 5 +Agrobacterium Ch-12 3 +/Agrobacterium 47/7 + + 3 +T , type strain; nt, not tested.Table 3.Agrobacterium species/biovar-specific primers based on 23S rDNA sequencesName of primer Target position a Sequence (5 0 –3 0 )UF f 171–193 GTAAGAAGCGAACGCAGGGAACTB1R r 338–360 GACAATGACTGTTCTACGCGTAAB2R r 1207–1230 TCCGATACCTCCAGGGCCCCTCACAAvR r 640–662 AACTAACTCAATCGCGCTATTAACArR r 1150–1173 AAAACAGCCACTACGACTGTCTTf, forward; r, reverse.a E. coli position numbering [1].Rhizobiaceae which produced a 1066 bp amplificationproduct with primers UF+B2R (Table 1),the restriction analysis of this product was applied.On the basis of computer analysis, endonucleaseAlw26I was chosen as the most appropriate for thispurpose. For each reaction 50 ng of PCR product wasused. Restriction fragments were separated in 2%agarose gels.