ARTICLE IN PRESSJ. Pu"awska et al. / Systematic <strong>and</strong> Applied Microbiology 29 (2006) 470–479 471DNA:DNA hybridization [5], sequenceanalysis<strong>of</strong>16SrDNA [26,33] <strong>and</strong> 23S rDNA [23] indicate that biovaridentity <strong>of</strong> <strong>Agrobacterium</strong> is a reliable taxonomic trait.These studies also showed that the genus <strong>Agrobacterium</strong>is closely related <strong>and</strong> even entwined with bacteriabelonging to Rhizobium, Sinorhizobium <strong>and</strong> Mesorhizobium.Based on these results several proposals <strong>of</strong>nomenclature changes within <strong>Agrobacterium</strong> were presented[9,26] including the latest one suggesting rejection<strong>of</strong> this genus <strong>and</strong> incorporation <strong>of</strong> agrobacteria toRhizobium [34]. However, this proposal is not generallyaccepted [7] <strong>and</strong> therefore, we prefer to use the<strong>Agrobacterium</strong> nomenclature instead <strong>of</strong> the Rhizobium.Until now, no rapid, PCR-based system for <strong>identification</strong><strong>of</strong> taxa within the genus <strong>Agrobacterium</strong> has beenavailable to phytopathologists. However, such a methodwould be very useful in classification <strong>of</strong> isolated crowngall <strong>and</strong> hairy roots causal agents to biovar or species,because some species, e.g. A. rubi <strong>and</strong> A. vitis show hostspecialization. This kind <strong>of</strong> tool would be also helpful inecological studies on soil-borne bacteria. At present,classification <strong>of</strong> agrobacteria is performed most <strong>of</strong>ten onthe basis <strong>of</strong> several biochemical tests, which aregenerally time-consuming <strong>and</strong> labour intensive [18],even when modified into a microtiter system [2].The aim <strong>of</strong> our study was to develop a multiplex PCRsystem, based on the differences in sequences <strong>of</strong> 23SrRNA gene, for fast <strong>identification</strong> <strong>of</strong> <strong>Agrobacterium</strong>biovar 1, biovar 2, A. rubi, <strong>and</strong>A. vitis.1 mM EDTA pH 8.0). To lyse the cells, 200 ml <strong>of</strong> buffer(0.25% SDS, 0.2 M NaOH) was added <strong>and</strong> then themixture was incubated at 70 1C for 15 min. Next 200 ml<strong>of</strong> 5 M NaCl solution was added, the mixture wascarefully mixed <strong>and</strong> incubated on ice for 15 min.After centrifugation (10 min at 15 000g) DNA wasprecipitated with 2 volumes <strong>of</strong> cold EtOH at roomtemperature. The pellet was washed with 70% EtOH,dried <strong>and</strong> dissolved in 500 ml <strong>of</strong> 10 mM Tris-HCl buffer,pH 8.2.Primer designFive primers were designed on the basis <strong>of</strong> thenucleotide sequence <strong>of</strong> the 23S rDNA determined for12 <strong>Agrobacterium</strong>, 2 Sinorhizobium <strong>and</strong> 4 Rhizobiumstrains [23] <strong>and</strong> 23S rDNA sequences <strong>of</strong> related bacteriaobtained from GenBank (U45329, U28505, L39095,X88894, Z35330, X71840, X87283, X71839). One <strong>of</strong>them, UF, is a universal forward primer complementaryto 23S rDNA sequences <strong>of</strong> all agrobacteria. Four othersare reverse primers, each complementary to DNA <strong>of</strong> adifferent species/biovar <strong>of</strong> <strong>Agrobacterium</strong>: B1R – toDNA <strong>of</strong> biovar 1 strains, B2R – to biovar 2 strains, AvR–toA. vitis (ex biovar 3 strains) <strong>and</strong> ArR – to A. rubi(Table 3). All primers were checked for homology toother sequences in the GenBank <strong>and</strong> EMBL databasesusing the BLAST N program.Specificity <strong>of</strong> developed primersMaterials <strong>and</strong> methodsBacterial strainsAll bacterial strains used in this study are presented inTable 1. The <strong>Agrobacterium</strong>, Erwinia, Pseudomonas <strong>and</strong>Xanthomonas strains were grown on King’s B medium[14], strains <strong>of</strong> Allorhizobium, Mesorhizobium, Rhizobium<strong>and</strong> Sinorhizobium – on mannitol–yeast extractmedium (79 CA) [32] <strong>and</strong> Phyllobacterium – on nutrientagar with beef extract (Oxoid CM3). For the <strong>Agrobacterium</strong>strains with unknown identity, the biovar orspecies status was assessed on the basis <strong>of</strong> a set <strong>of</strong>biochemical tests: 3-ketolactose production, citrate <strong>and</strong>L-tyrosine utilization, growth <strong>and</strong> pigmentation in ferricammonium citrate broth, acid from erythritol <strong>and</strong>oxidase reaction [18] (Table 2).Total bacterial DNA preparationsTotal bacterial DNA was extracted using a methoddescribed by Sambrook et al. [24] in own modification.For DNA isolation bacteria taken from 1 colony wereresuspended in 100 ml <strong>of</strong> TE buffer (10 mM Tris-HCl,DNA <strong>of</strong> each bacterial strain (Table 1) was tested inseparate PCRs with the following sets <strong>of</strong> primers: (i)UF+B1R, (ii) UF+B2R, (iii) UF+AvR, (iv)UF+ArR <strong>and</strong> in a multiplex PCR with primersUF+B1R+B2R+AvR+ArR. DNA amplificationwas performed in total volume <strong>of</strong> 15 ml in ThermalCycler Trio – Thermoblock (Biometra, Germany). Allreactions were performed in PCR buffer (10 mM Tris-HCl, pH 9.0; 50 mM KCl; 0.1% Triton X-100) with1.5 mM MgCl 2 , 200 mM <strong>of</strong> each dNTP, 1 mM <strong>of</strong>eachprimer <strong>and</strong> 1 U <strong>of</strong> thermostable DNA polymerase(Promega, Madison, USA). The optimal amplificationconditions were determined experimentally: initial denaturationat 94 1C for 1 min, followed by 35 cycles <strong>of</strong>denaturation at 94 1C for 1 min, annealing at 67 1C for1 min, extension at 72 1C for 1.5 min <strong>and</strong> a finalextension step for 10 min. PCR products were analyzedby electrophoresis through 2% agarose gel. Each<strong>Agrobacterium</strong> strain was tested at least twice.Restriction analysisFor differentiation between strains <strong>of</strong> <strong>Agrobacterium</strong>biovar 2 <strong>and</strong> other representatives <strong>of</strong> family
472Table 1.Strains used in this studyARTICLE IN PRESSJ. Pu"awska et al. / Systematic <strong>and</strong> Applied Microbiology 29 (2006) 470–479Strain Source <strong>and</strong> 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 <strong>of</strong>UF+B2Rproduct aBiovar 1g-114-3-2 Unknown; Israel + nt32/1 Apple; Hungary + nt7/1 Raspberry; Hungary + + 365 Raspberry; Hungary + nt39/7 Plum; Hungary + + 3CFBP 2516, CFBP Poplar; France + nt2519,CFBP 2177B6 Tomato; France + ntC58 Sweet cherry; USA + ntT37 Unknown; France + ntACH5 Unknown; Belgium + ntAT4, 137 Cherry; Pol<strong>and</strong> + nt6 Cherry; Hungary + ntCh3, Ch6Chrysanthemum; + ntPol<strong>and</strong>13B Unknown; Israel + nt0 Grapevine; Hungary + + 3131a/b Apple; Pol<strong>and</strong> + ntBiovar 2K84 Peach; Australia + 2LMG 150 Apple; Unknown + 2K27 Poplar; Australia + 215b/94, 21/94, 307a, Apple; Pol<strong>and</strong> + 258031a/b, 32a, 32b, 39, Cherry; Pol<strong>and</strong> + 240, 88, 101, 107, 125,126, 129, 133, 301,387, 389, 392, 40046, 182, 192, 262 Pear; Pol<strong>and</strong> + 2122, 568 Plum; Pol<strong>and</strong> + 2103, 104 Peach; Pol<strong>and</strong> + 260, 67, 69, 71, 77, 81, Sweet cherry; Pol<strong>and</strong> + 283, 84, 89, 221b, 36530/2, 9 Peach; Hungary + 28/1, 51, 9/2 Raspberry; Hungary + 2P-Ag- 1, 2, 3, 4, 6 Pear; Japan + 2Pch-Ag- 2, 3, 4, 5, 6 Peach; Japan + 2A. vitisLMG 8750 Grapevine; Australia + ntAg162 Grapevine; Grecja + nt2/3 S5R, AB3, AB4, Grapevine; Hungary + ntTm4g-114-5-IV-1 Grapevine; Israel + ntCG49 Grapevine; USA + ntA. rubiLMG 156 Boysenberry; USA + ntLMG 159 Black raspberry; USA + ntLMG 294Eunonymus sp.;+ ntFranceA. larrymooreiLMG 21410Ficus benjaminant
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