Vol. 56, No. 11

Identification of Different Agrobacterium Strains Isolated from theSame Forest Nursery

MARIE-FRANCE MICHEL,' ANA CRISTINA MIRANDA BRASILEIRO,2 CHRISTIANE DEPIERREUX,3LEON OTTEN,4 FRANCIS DELMOTTE,3 AND LISE JOUANIN2*

Station d'Amelioration des Arbres Forestiers, Institut National de la Recherche Agronomique, Ardon, F45160 Olivet,'Laboratoire de Biologie Cellulaire, Institut National de la Recherche Agronomique, Route de Saint-Cyr, F-78026

Versailles Cedex,2 Centre de Biophysique Moleculaire, Departement de Biochimie des Glycoconjugues etLectines Endogenes, Centre National de la Recherche Scientifique et Universite d'Orleans,

F45071 Orleans Cedex 2,3 and Institut de Biologie Moleculaire des Plantes, CentreNational de la Recherche Scientifique, F-67084 Strasbourg, France

Received 3 May 1990/Accepted 30 August 1990

Several Agrobacterium strains isolated from the same forest nursery from 1982 to 1988 were compared byserological, biochemical, and DNA-DNA hybridization methods. Similarities among strains belonging to biovar2 were observed by indirect immunofluorescence, whereas biovar 1 strains showed serological heterogeneity.Electrophoretic analysis of bacterial envelope-associated proteins showed that few bands appeared in thestrains belonging to biovar 1, whereas many proteins appeared in the case of biovar 2 strains. ChromosomalDNA was analyzed with six random C58 chromosomal fragments. None of the six probes hybridized to the DNAof the two biovar 2 strains. One of the probes gave the same hybridization pattern with all biovar 1 strains,whereas the other probes yielded different patterns. The vir regions were closely related in the differentpathogenic strains. The T-DNA and replication regions were less conserved and showed some variations amongthe strains.

Agrobacterium tumefaciens is a soil bacterium which isthe causative agent of the plant disease crown gall. Thisdisease is the result of a form of genetic parasitism by thebacterium on plants and affects most of the dicotyledonousplants, particularly fruit trees, ornamental plants, grape-

vines, and some forest trees such as poplars, wild cherrytrees, and birches (6, 19). We have studied this disease in a

surveyed nursery located at the National Agronomic Re-search Institute (INRA) in Orleans, France. Several forestspecies, including coniferous, wild cherry, walnut, and pop-

lar trees belonging to the Tacamahaca, Aigeros, and Leucesections have been cultivated at INRA since 1973. In thisnursery, crown gall disease was first detected in poplarplants belonging to the Leuce section (essentially Populusalba, Populus tremula, and Populus tremuloides) and in wildcherry trees (Prunus avium L.) (40). From 1982 to 1988,nearly 700 Agrobacterium isolates were identified accordingto biochemical (45) and serological characteristics by indi-rect immunofluorescence. Recently, the disease has spreadto several nurseries, probably through the planting of in-fected forest plants, and this spread has been enhanced bythe fact that poplars from the Leuce section were propagatedby root cuttings. These root cuttings could have beencontaminated by agrobacteria living in the rhizosphere;therefore, the evolution of this Agrobacterium populationwas explored in this study. From a phytopathological pointof view, the identification of Agrobacterium strains bychromosomally determined traits is of little use, becausepathogenicity is controlled by the Ti plasmids. It is wellknown that Ti plasmids can be exchanged between strainsbelonging to different biovars under laboratory conditions(27).

In this work, we identified the most frequently occurring

* Corresponding author.

agrobacterial strains according to their biochemical or sero-

logical patterns with the aim of characterizing more com-pletely the strains isolated from a single nursery. The se-

lected strains were also characterized by molecularhybridization using probes derived from essential regions ofthe Ti plasmid from strain C58 isolated from Prunus avium(50).

MATERIALS AND METHODS

Agrobacterium strains. A. tumefaciens C58 was chosen as

the reference strain for bacteriological, biochemical, immu-nological, and molecular studies. Strain 82.143 was isolatedfrom a grapevine (Danam grapevine cultivar, Herault,France) and used as a reference strain from an independentlocation. The other strains came from a single nurserylocated at the INRA Forest Institute, Orleans, France.Bacterial isolations were performed with nonnecrotic gallsharvested from 1982 to 1988. The first number of each straincorresponds to the year of harvest. Crushed gall sectionswere spread on selective biovar media or on YPGA medium(distilled water containing 5 g of yeast extract per liter, 5 g ofBacto Peptone per liter, 10 g of glucose per liter, and 15 g ofBacto Agar per liter) (40). Isolations from soil were donewith 10 g of nursery soil mixed with 95 ml of distilled waterand homogenized with a Waring blender for 2 min. Approx-imately 250 ,ul was spread on selective biovar media. Onemedium was selective for biovar 1 isolates with dulcitol at2.73 g/liter (5) as the carbon source. The other medium was

selective for biovar 2 isolates and contained erythritol at 5.0g/liter (41) as the carbon source. Genus and biovar statuswere determined according to the method of Kerr andPanagopoulos (29) as modified by Nesme et al. (40). Theisolates were routinely grown at 25°C on MYA medium(distilled water containing 5 g of yeast extract per liter, 0.5 gof Casamino Acids per liter, 8 g of mannitol per liter, 2 g of

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ammonium sulfate per liter, 5 g of sodium chloride per liter,and 15 g of Bacto Agar per liter) (52). The bacteria werestored in 40% glycerol at -50°C.

Pathogenicity tests. Each isolate was tested for pathoge-nicity by inoculation on carrot disks and on explants of aLeuce poplar hybrid clone, P. tremula x P. alba (INRA no.717.1B4), cultivated in vitro on a modified Murashige andSkoog (MS) medium containing half-strength macronutrientsand micronutrients as described by Murashige and Skoog(39). This medium was supplemented with the followingingredients (per liter): 1 mg of nicotinic acid, 1 mg ofpyridoxine hydrochloride, 1 mg of thiamine hydrochloride,1 mg of calcium pantothenate, 1 mg of cystine dihydrochlo-ride, 0.01 mg of biotin, 100 mg of myoinositol, and 200mg ofglutamine. Poplar plantlets were maintained on this MSmedium without a growth regulator and with 16 h of light perday. The plantlet stems were examined 3 weeks after inoc-ulation for the presence or absence of tumors.

Agrocin sensitivity. In vitro sensitivity tests to agrocinproduced by Agrobacterium radiobacter K84 were doneaccording to the method described by Stonier (51).

Determination of opine products. (i) Opine extraction.Poplar tumors were cultured in vitro on growth regulator-free modified MS medium. Tumor tissues were ground withan Ultra-Turrax homogenizer (400 to 800 mg of fresh tissueper ml of ethanol). The extract was concentrated to 100 ,ulwith a Speed Vacuum concentrator for 2 h. Aliquots (50 RI)of arginine, nopaline, and octopine at 2 mg/ml each indistilled water as standards, tumor extract (100 ,ul), andcontrol plant extract (100 pl) were applied onto Whatman3MM filter paper as spots. The paper was wetted withpyridine-acetic acid-water (3:10:387; pH 3.9) as an electro-phoretic buffer (37). Vertical electrophoresis was conductedfor 5 h at 400 V (10 V/cm) in a roof electrophoresis tankwhich allowed a rheoelectrophoretic effect.

(ii) Detection of opines. The electrophoretograms weredried overnight under a vacuum hood. Opines were detectedwith modified Sakaguchi reagent (47). The electrophoreto-grams were dipped into a freshly prepared solution of 0.1%8-hydroxy-quinoline and 0.1% urea in distilled acetone.After being dried under a vacuum hood, the electrophoreto-grams were sprayed with a freshly prepared solution ofsodium hypobromite (0.2% Br2 in 0.5 N sodium hydroxide).Red-purple spots appeared and were identified by theirposition on the electrophoretogram by using arginine, no-paline, and octopine as standards.

Serological survey. Six antisera were prepared from six A.tumefaciens strains. Antisera against strains C58 and 84.7were prepared as described by Digat (11), while the otherantisera were prepared according to the method of Depier-reux et al. (9).

(i) Antigen preparation. Antisera were prepared fromwhole cells from a 48-h-old culture grown on Roberts andKerr medium (45) (distilled water containing 10 g of mannitolper liter, 20 g of L-glutamic acid per liter, and 2 ,ug of biotinper liter) (pH 7.1). After being washed in phosphate-bufferedsaline (distilled water containing 8.5 g of NaCl per liter, 8.62g of Na2HPO4 .2H20 per liter, and 2.48 g of KH2PO4 perliter) (pH 7.2), the bacteria were heated at 100°C for 2 h.After three washes with buffer, the bacterial concentrationwas adjusted to 108 cells per ml.

(ii) Immunization schedule. Antisera against Agrobacte-rium cells were raised in rabbits (Fauve de Bourgogne) bythree multiple subcutaneous injections of the antigen suspen-sion in incomplete Freund adjuvant on days 0, 7, and 14.Each rabbit received 1 ml of an emulsion containing 0.5 ml of

bacterial cells in saline and 0.5 ml of incomplete Freundadjuvant. Booster doses of this antigen in incomplete Freundadjuvant were given after 3 weeks, and rabbits were bled (50ml) from the marginal ear vein 7 days later. Crude sera werestored at -20°C.

(iii) Immunofluorescence tests. Indirect immunofluores-cence was carried out with heat-fixed bacteria on microprintslides (Flow Laboratories S.A., Asnieres, France). Rabbitantisera were diluted at 1/500 and incubated with fixedbacteria for 30 min. Fluorescein-labeled goat anti-rabbitserum (Hoechst-Behring, Paris, France) was diluted at 1/100for indirect immunofluorescence assays. Positive resultswere scored on a scale of + to + + +, with +++ indicatingthat the cell walls were strongly fluorescent and + indicatingthat the cell walls were only slightly fluorescent.

Protein extraction from bacterial envelope. Bacteria from200-ml cell cultures grown in liquid mannitol medium (45)were harvested by centrifugation at 6,000 x g for 20 min.The supernatant was discarded, and proteins from the cellenvelope were extracted with 5 ml of 0.05 M Tris-HCl (pH9.0) in the presence of 20 mM benzamidine chloride as aprotease inhibitor with gentle stirring at 23°C for 1 h accord-ing to the method of Depierreux et al. (C. Depierreux, M.-F.Michel, M. Monsigny, and F. Delmotte, submitted forpublication). The bacterial suspension was centrifuged at12,000 x g for 15 min, and the supernatant was filtered on an0.22-,um-pore-size filter (Amicon, Epernon, France) and wasfurther desalted on a Trisacryl GF 05 column (IBF-Biotech-nics) (18 by 2 cm), equilibrated, and eluted with 0.05 Mammonium acetate buffer (pH 7.2). The protein fractionseluted in the void volume of this column were pooled andfreeze-dried. The salt-free residue was dissolved in distilledwater for further analysis by polyacrylamide gel electropho-resis at a final concentration of 3 mg of protein per ml.

Polyacrylamide gel electrophoresis in SDS. Gel slabs (16 cmwide, 18 cm long, and 1.5 mm thick) were made of 12.5%acrylamide in the presence of 0.1% sodium dodecyl sulfate(SDS) according to the method of Laemmli (33). The gelswere stained with Coomassie blue G 250 and then scannedby absorption densitometry at 550 nm (Camag TLC ScannerII). Apparent molecular weight values (Mr) for the polypep-tides were calculated from a graph of relative mobilitiesversus log Mr. The Mr standards (Sigma) used were phos-phorylase A (Mr, 92,500), bovine serum albumin (Mr,66,200), ovalbumin (Mr, 45,000), carbonic anhydrase (Mr,31,000), soybean trypsin inhibitor (Mr, 21,500) and lysozyme(Mr, 14,400).

Plasmids. The plasmids used as probes in this study (Table1) were isolated from Escherichia coli strains and weregrown at 37°C in liquid LB medium (38) supplemented withthe appropriate antibiotics.

Cloning of C58 chromosomal regions. Total DNA extractedfrom Agrobacterium strain GV3101(pTiTm-4) (C58 cured ofits pTi and carrying pTiTm-4 [23]) was partially digested withHindlIl and cloned in pPM1016, a modified version ofpUC18 (43). The chromosomal origin of the clones wasverified by the absence of hybridization to pTiTm-4.DNA isolation and digestion. Plasmid DNA was isolated

from E. coli strains by the method of Birnboim and Doly (3)and purified on a CsCl gradient. Total Agrobacterium DNAwas isolated by the method of Dhaese et al. (10). The cellswere grown overnight in 3 ml of MYA to an optical densityof 0.8 at 600 nm. The total DNA pellet was suspended in 100pJ of buffer (10 mM Tris-HCl [pH 8.0], 0.1 mM EDTA).One-third of the DNA solution was digested with variousrestriction enzymes and then loaded on a horizontal 0.8%

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~~~~~~IDENTIFICATION OF AGROBACTERIUM STRAINS 3539

TABLE 1. Plasmids used as probes in this studyPlasmid Characteristics Origin Reference

pGV0345 pTiC58 Hindlll fragments 22, 31b, 23, 33, 2, and 30b T-DNA 7pGV0319 pTiC58 Hindlll fragments 14b, 19, 41, 22, and 31b T-DNA 7pGV0342 pTiC58 HindIll fragments 10, 15, and 14b T-DNA 7pGV0361 pTiC58 Hindlll fragments 27, 16, 37a, and 7 Virulence region 7pGV0340 pTiC58 Hindlll fragments 18, 14a, 27, and 16 Virulence region 7pGV0348 pTiC58 HindlIl fragments 38, 17, 34, 39, 12, 9, and 18 Virulence region 7pUCD500 pTiC58 EcoRI fragments 27 and 25 Replication region 16pT-10 Random C58 Hindlll fragments Chromosome This studypT-25 Random C58 Hindlll fragments Chromosome This studypT-62 Random C58 HindIII fragments Chromosome This studypT-70 Random C58 HindIII fragments Chromosome This studypT-173 Random C58 Hindlll fragments Chromosome This studypT-179 Random C58 HindllI fragments Chromosome This study

agarose gel in TEA buffer (40 mM Tris acetate [pH 8.0], 2

mM EDTA) and run at 3.5 V/cm for 10 h.

DNA-DNA hybridizations. The gels were blotted onto

Hybond N' membranes in 0.4 N NaGH by the protocol of

the manufacturer (Amersham). Prehybridization was per-

formed for 3 h at 430C in 50% formamide-4 x SSPE (40 mM

NaH2PO4 2H2O, 72 mM NaCl, 4 mM EDTA)-l% SDS-500

p.g of denatured, fragmented salmon sperm DNA per ml-

0.5% nonfat dried milk. The probe DNA was labeled with

32P by using random oligonucleotide primers (48). The

denatured radioactive probe was added to the prehybridiza-tion solution and hybridized overnight at 430C. Membranes

were washed three times (for 20 min each time at 430C) with

2x SSC (300 mM NaCl, 30 mM tri-sodium citrate)-0.1%

SDS and three times with 0.2x SSC-0.1% SDS, dried, and

exposed to Kodak X-Omat autoradiography film with inten-

sifying screens at -80'C for various periods.

RESULTS

Characterization of bacterial population diversity. The bac-

terial strains were chosen according to their frequency of

isolation from nearly 700 isolates obtained from 1982 to 1988

at the INRA nursery located in Orldans. The isolates were

characterized by bacteriological and serological tests. Most

of the isolates belonged to biovar 1 (70 to 80%), and among

biovar 1 isolates, strain 84.5 was the most frequent (nearly

50%) (40). We established that the frequency of different

strains did not vary from year to year (unpublished data),

and we selected some strains for further studies. Their

biochemical and chromosomal backgrounds were compared

TABLE 2. Origins and characteristics of Agrobacterium strains

Strain Origin Bio- Patho- Agrocin Opine~var genicitya sensitivity detected

C58 Prunus avium (50) 1 P + Nopaline82.143 Vitis vinifera 1 P NDb Nopaline82.139 Prunus avium 2 P + Nopaline84.5 Populus Sp. 1 P + Nopaline84.7 Populus Sp. 1 P + Nopaline85.122 Populus sp. 2 P + Nopaline86.15 Populus sp. 1 NP None

87.170 Populus sp. 1 P Nopaline87.240 Populus sp. 1 P Nopaline

88.10 Nursery soil 1 NP None

P, Pathogenic (tumor formation on poplar plantlets and carrot disks); NP,

nonpathogenic.b ND, Not determined.

with those of strains 82.143, isolated from a grapevine, andC58, which were chosen as references. Most of the isolatedbacteria were pathogenic and were sensitive to agrocin.Some strains isolated since 1986 were resistant to agrocin;this resistance could be related to a treatment of the cuttingswith A. radiobacter K84 in 1984. Only nopaline-positiveagrobacteria were present in galls (Table 2).Immunofluorescence study. The antiserum titers varied

from 1/800 to 1/6,400, depending on the strain. A routineserological study with a 1/500 dilution was done by indirectimmunofluorescence on pure culture isolates. The cross-reactions occurring between antisera and various strains areshown in Table 3. Antiserum against strain 84.7 was highlyspecific for this strain, and cross-reactions against otherstrains did not occur. Almost complete cross-reaction tookplace between strains C58 and 86.15. The antisera raisedagainst biovar 1 were unable to recognize biovar 2 strains,and those raised against biovar 2 were unable to recognizebiovar 1 strains (Table 3).

Electrophoresis protein patterns. The reference strain,C58, showed two major bands at Mr5 of 32,400 and 33,500.Biovar 2 strains 82.139 and 85.122 showed a dense proteinpattern between Ms5 of 12,000 and 62,000 (Fig. 1). Strain82.139 had an intense protein band at an Mr of 15,200 whichmight be specific to this strain. All biovar 1 strains had alimited number of proteins. Moreover, strains C58, 84.5,

TABLE 3. Indirect immunofluorescence cross-reactions

Reaction' with antiserum to indicated strain of biovar:Biovarand 12

strainsC58 84.5 84.7 86.15 82.139 85.122

C58 +++ +++ - + - -

82.143 - - - - -84.5 +++ +++ - + - -84.7 - + - -86.15 +++ - --87.170 - - - - - -87.240 - - - - - -88.10 - - - - - -

282.139 - - - - +++ +++85.122 - - - - +++ +++

a+++, Strong fluorescence reaction; +, weak fluorescence reaction; -no fluorescence.

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3540 MICHEL ET AL.

FIG. 1. SDS-polyacrylamide gel electrophoresis protein patterns. The patterns of strains 82.143, 84.7, 87.240, and 88.10 are not shown.

86.15, 87.170 (Fig. 1), 87.240, and 88.10 (data not shown) hada major common band around an Mr of 34,000.

Molecular analysis. The nine Agrobacterium isolates (Ta-ble 2) were characterized on the basis of total DNA hybrid-ization against selected C58 probes. These probes includesome fragments of chromosomal DNA and the essential Tiplasmid regions involved in T-DNA transfer (vir region andT-DNA) and in plasmid maintenance (replication region).Strain C58 was used as the reference strain.

(i) Chromosomal DNA analysis. Hybridization results areschematically summarized in Fig. 2A. In order to confirmand to further support the data which indicated that strainsisolated from the same soil have difference chromosomalbackgrounds, we used six random C58 chromosomal frag-ments of about 15 kb each for probing (pT-10, pT-25, pT-62,pT-70, pT-173, and pT-179). There was no hybridization

against the two biovar 2 strains (85.122 and 82.139). Allbiovar 1 strains displayed the same hybridization patternwith pT-179, but different hybridization patterns were ob-tained with pT-10 and pT-70 (Fig. 3A). No hybridization wasobserved with probes pT-25 and pT-62. Probe pT-173 hy-bridized at a different position only with strains 84.5 and88.10 and did not hybridize with the other strains.

(ii) Ti plasmid analysis. The results of this analysis areschematically summarized in Fig. 2B.

(a) vir region. Three overlapping fragments from thepTiC58 virulence (vir) region (pGV0348, pGV0340, andpGV0361 [Fig. 3B]) were used to probe the various strains.With each of these probes, all isolates revealed the sameHindlIl hybridization profile except strains 88.10 and 86.15.The Southern blot analysis, with pGV0361 as a probe, isshown in Fig. 3B as an example. Moreover, the BamHI and

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IDENTIFICATION OF AGROBACTERIUM STRAINS 3541

A

B

Strain

85.12284.5

87.24088.1086.1582.14384.7

87.17082.139

Biovar

2

1111

2

vir T-DNA OriStrain_ region pGV0342 pGV0319 pGV0345 pUCD50085.122 jj EZ/84.5 11 -1 1 Zl

87.240 1V82.143 I84.7 11

87.170 ..I......X.l82.139 M-

FIG. 2. Molecular hybridization analysis. The comparisonsamong strains are valid for each probe separately, i.e., within eachcolumn. Symbols: FII, no hybridization; E, different hybridiza-tion patterns against C58 strain but homologouis between strains infigure; E]i, different hybridization patterns: . same hybridiza-tion patterns. (A) Chromosomal analysis. Probes pT-25 and pT-62are omitted in this figure because no hybridization was observedwith any strains. (B) Ti plasmid analysis. Strains 88.10 and 86.15 didnot hybridize with these probes, and the results for those strains are

not shown in this figure. The three probes from the vir region(pGV0348, pGV0340, and pGV0361) are combined in a column,since they gave identical results. Ori, Region of origin of replication.

EcoRI digestion patterns were also identical (data notshown). These results indicated that the vir regions of thedifferent strains are closely related. The two nonpathogenicstrains (Table 2), 88.10 and 86.15, did not hybridize witheither vir region probes or with the other pTi plasmid probes(T-DNA and replication region) described below.

(b) T-DNA. To identify regions homologous to the T-DNAof pTiC58, recombinant pBR322 plasmids covering its entireT-DNA (pGV0342, pGV0319, and pGV0345) were used as

probes. Restriction patterns were conserved for the left partof the region with four of the strains (82.139, 84.5, 85.122,and 87.240), but 82.143, 84.7, and 87.170 showed some slightmodifications. With the right half of the C58 T-DNA (probespGV0319 and pGV0345), we also observed a conservedprofile, except with two strains (82.139 and 87.170) whichshowed very different patterns (Fig. 3C). For these twostrains, more precise hybridizations with subfragments H19,H41, H22, H31, and H23 were performed to determine theexact location of their nonhomologous regions. Strain 82.139showed no hybridization with fragments H22 and H31,which carry the 3' end of the iaaM (tms-1) gene, the ipt (tmr)and the ons (opine secretion) genes, and the 5' end of the 6b(tml) gene (Fig. 3C). Strain 87.170 did not hybridize with theH22 subfragment probe. For strains 82.139 and 87.170 hy-bridizations with the other subfragments (H19, H41, andH23) revealed bands of sizes differing from these of the C58strain.

(c) Replication region. Hybridization analysis was per-formed with pUCD500, which is maintained in a Ti-plasmid-less Agrobacterium strain (16). pUCD500 includes a kana-mycin resistance gene, EcoRI fragment 27, and an

incomplete, deleted EcoRI fragment 25. These two frag-

ments contain the replication and maintenance functions ofpTiC58 (restriction map in Fig. 3D). Fragment 27 yieldedidentical hybridization profiles (replication and incompatibil-ity functions), except for the 87.170 and 82.139 isolates (Fig.2B and 3D). The copy number locus (fragment 25) showeddifferent hybridization profiles with all strains; however, aband of the same size was observed in the profiles of strains84.5, 85.122, and 87.240.

DISCUSSION

In this work, we have characterized at different levelssome Agrobacterium isolates obtained from the same nurs-ery during a period of 7 years (1982 to 1988). Most of theisolates belong to biovar 1 (70 to 80%) (40). These data aresimilar to those obtained by Ophel and Kerr (42), whoobserved that biovar 1 strains are ubiquitous soil bacteriawith pathogenic and nonpathogenic forms found in a widerange of dicotyledonous hosts.

Despite the fact that in natural conditions the agrobacteriawere found only on tumors of Leuce poplars or wild cherrytrees, they could induce tumors on coniferous species,walnut trees, and poplars of other sections in laboratoryconditions (unpublished data). Moreover, they were patho-genic on carrot disks and on tomato, tobacco, cabbage, andrapeseed plants (unpublished results). Thus, they can beconsidered wide-host-range strains like strain C58. All thestrains are of the nopaline type, which seems to be morepathogenic on forest trees than octopine strains are (1).The chosen strains were characterized by bacteriological

and serological tests. The antisera made with boiled cellswere in most cases too specific to recognize all the sero-groups of agrobacteria which can be present in a definedbiotope. Reactions with these antisera occurred only withsome strains, e.g., C58 antiserum reacted with homologousstrains and some heterologous strains such as 84.5 and 86.15(Table 3).However, the two antisera raised against biovar 2 strains

cross-react and can be extended to all the strains of biovar 2(unpublished data). These results confirmed the studiesperformed by Alarcon et al. (2).

In order to prepare specific antisera able to recognize alarge number of Agrobacterium isolates, proteins associatedwith the bacterial envelope were prepared and analyzed bySDS-polyacrylamide gel electrophoresis. With the strainsbelonging to biovar 1, we observed very few protein bands.This is in contrast to previous results obtained by Engstromet al. (15) and Alarcon et al. (2), who studied the pattern oftotal proteins extracted after ultrasonication. Moreover, amajor band at an Mr of around 34,000 was conserved amongall strains. Biovar 1 and biovar 2 protein patterns were verydifferent (Fig. 1), and the biovar 2 strains showed a denseprotein pattern comparable to the result of Alarcon et al. (2).The molecular analysis of the chromosome of the isolates

further supports the bacteriological, serological, and bio-chemical studies. The two biovar 2 strains (82.139 and85.122) did not hybridize with any of the six chromosomalC58 probes (Fig. 2A), which suggests that the chromosomesof these two strains are distantly related to the chromosomeof C58 (a biovar 1 strain). On the other hand, all biovar 1strains hybridized with at least three C58 probes. pT-179 isan interesting probe because it had a uniform hybridizationprofile with all biovar 1 strains. This probe could cover aconserved biovar 1 chromosomal region. Chromosomalanalysis also allowed the detection of differences in thebiovar 1 group. Strains 84.5, 86.15, 87.240, and 88.10 were

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FIG. 3. Chromosomal and plasmid DNA-DNA hybridization. All four panels show Southern blot hybridization of Agrobacterium strainsusing C58 probes. Total DNA was prepared and hybridized under stringent conditions as detailed in Materials and Methods. (A) HindlIldigests hybridized with the pT-70 chromosomal probe. On the left, the fragment sizes are shown in kilobases (Kb). (B) HindIII digestshybridized with pGV0361 (right part of pTiC58 virulence region). The transcriptional organization of vir loci is from Rogowsky et al. (46). (C)HindIlI digests hybridized with pGV0345 (right part of pTiC58 T-DNA region). The transcriptional organizations of acs (agrocinopinesynthase), nos (nopaline synthase), and transcript 5 are from Joos et al. (24). tmr (tumor morphology rooty) and tms (tumor morphologyshooty) are from Koukolikova-Nicola et al. (32). tml (tumor morphology large) is from Tinland et al. (54), and ons (opine secretion) is fromMessens et al. (36). Strain 84.5 is not shown in this autoradiograph. Strains 84.7 and 87.240 show an additional band between HindlIlfragments 2 and 22 (H2 and H22), probably due to partial digestion. (D) EcoRI digests hybridized with pUCD500 that covers the replicationregion. The locations ofpar (stable plasmid inheritance), ori (origin of replication), inc (incompatibility with other incRh-1 members), and cop

(copy number control) are from Gallie et al. (16). Restriction maps are from Depicker et al. (7, 8). In panels B through D, the restrictionfragments corresponding to the bands are represented on the left of the autoradiographs and are designated by the first letter of the restrictionenzyme (H for HindIll and E for EcoRI) followed by the fragment number.

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distinguished by their restriction fragment length polymor-phism when hybridized with probes pT-10, pT-70, andpT-173 (Fig. 2A). In these cases, no additional test isrequired. However, this analysis was insufficient to distin-guish strains 82.143, 84.7, and 87.170 (Fig. 2A). Theirhybridization patterns were identical with each of the probesbut different from the C58 pattern. These strains are proba-bly very closely related, and additional serological or bio-chemical tests, or both, are required to differentiate them.Finally, probes pT-25 and pT-62 did not hybridize with anyof the nine strains studied, indicating that they cover veryspecific C58 regions.

Previous studies have analyzed the chromosomal proper-ties of Agrobacterium isolates; there have been, however,few studies of the variability of the Ti plasmid harbored bystrains isolated from the same biotope (31, 43). Therefore weperformed Ti plasmid hybridization analysis to determinethe potential variability in the samples. The absence of a Tiplasmid in strains 86.15 and 88.10, which neither produceopine nor form tumors on poplar trees and carrots, wasexamined by hybridization. They did not hybridize with anyof the Ti plasmid probes, confirming that they are probablyplasmidless Agrobacterium strains (i.e., A. radiobacter).

vir region hybridizations performed with the remainingstrains showed a highly conserved profile that correspondsto about 50 kb of DNA (Fig. 2B and 3B). Considering thatmost of the Ti plasmids isolated so far have sizes rangingbetween 190 and 240 kb (35), this homologous vir regioncould represent about 25% of the total Ti plasmid size. Thisis in agreement with previous work showing functional andDNA sequence homology within the vir region of Ti plas-mids (12, 14, 17, 21) and also that of Ri plasmids (4, 25, 44,55).

Although the T-DNA is also considered a highly con-served region (14), we observed some divergence in theT-DNA of the analyzed strains, showing that in these strainsthe T-DNA is less conserved than the vir region (Fig. 2B).This is true in particular for 82.139 and 87.170, which lackhomology in the right part of their T-DNA (Fig. 3C). Thisregion comprises the H22 and H31 pTiC58 subfragmentscarrying the 3' ends of tms, tmr, and ons and the 5' end oftml (restriction map in Fig. 3C) and belong to the so-called"common DNA" fragment (8). The unexpected absence ofhomology in the common DNA may indicate that functionsencoded by these oncogenes are modified in these strains. Amore detailed study of their T-DNA regions is needed todefine these modifications.

Replication region hybridization showed that five strains(82.143, 87.240, 84.5, 84.7, and 85.122) probably belong tothe same incompatibility group as C58 because fragment 27,which covers its incompatibility function, yields the samepattern (Fig. 3D). Hence, this study supports earlier reportswhich classified nopaline strains as members of the incRh-1incompatibility group and therefore as containing plasmidsincompatible among them (20, 30, 34). For strains 82.139 and87.170, only weak homology was found, perhaps indicatingthat they belong to another incompatibility group. The copynumber locus (fragment 25) hybridization revealed a partialhomology between C58 and the other strains.On the basis of the hybridization pattern with functionally

important Ti plasmid regions (plasmid maintenance andT-DNA transfer), the seven plasmids are closely related toone another, especially the 84.5, 85.122, and 87.240 plasmidsidentical with each of the pTi probes. Sciaky et al. (50) andHooykaas et al. (20) found that nopaline strains form aheterogenous group. In our case, the Ti plasmid seemed well

conserved, and this result suggests that the strains isolatedfrom poplars harbored plasmids which could be derived froma common ancestor with some minor changes during theirrespective evolution. These sequences may be conservedbecause they contain essential genetic information for agro-bacteria. The partial homology detected in the T-DNA and inthe replication origin region for strains 82.139 and 87.170indicates that either the corresponding regions have sufferedsome modifications or these regions did not arise from acommon ancestor. For strains 82.139 and 87.170, the T-DNAdifference could be explained by the concept proposed byThomashow et al. (53) and Knauf et al. (31), according towhich the common DNA is found in many Ti plasmidsbecause these DNA sequences are required for a wide hostrange. During Agrobacterium evolution, modification of thecommon DNA would lead to a reduction in host range. Thesame authors have shown that limited and wide host rangesare correlated with the absence or presence of a highlyconserved common DNA. The biovar 2 strains, such as82.139, are associated with specific hosts (42) and may,according to this concept, increase or retain their virulenceby modifications in the T-DNA. Interestingly, strain 82.139is more virulent on poplar plantlets than the other strainsanalyzed here (A. C. M. Brasileiro, J. C. Leple, J. Muzzin,D. Onnought, M.-F. Michel, and L. Jouanin, unpublisheddata).

This work focused on the comparison between differenttechniques to identify agrobacteria in their natural environ-ment. Bacteriological and serological techniques are so farthe most frequently employed methods to check plants andsoil for bacteria. These techniques are time consuming andare not very successful. This work shows that it is possibleto distinguish biovar 1 and biovar 2 strains by hybridizationwith a single probe such as pT-179. Moreover, it is possibleto identify virulent Agrobacterium strains with a hybridiza-tion test using a vir region probe which is well conserved.These probes could be used directly against purified bacte-rial DNA from the bacterial soil community, as proposed byHolben et al. (18).By comparing chromosomes and Ti plasmids of the same

strains, we have shown that identical Ti plasmids can befound in different chromosomal backgrounds. This suggeststhat these Ti plasmids can be exchanged in the soil popula-tion by conjugative transfer from virulent strains to avirulentstrains. Transfer of virulence was earlier demonstrated invivo (26) and in vitro (13, 22, 28) for Agrobacterium spp. andin soil populations for species of the genus Rhizobium (49),another genus of the family Rhizobiaceae.

ACKNOWLEDGMENTS

We thank A. Depicker and C. I. Kado for providing bacterialstrains, B. Digat for providing antisera, P. Charest for discussionsand critical reading of the manuscript, and J.-F. Muller, B. Guerin-Dauphin, V. Combes, and N. Millet for helpful technical assistance.A.C.M.B. is supported by a doctoral grant from the Brazilian

Ministry of Science and Technology (CNPq), and C.D. was sup-ported by a fellowship from the French Ministry of Research andTechnology. This research was supported by grants from theEuropean Economic Community to M.-F.M. and AIP/INRA (grant88/4617) and Arbocentre to F.D. and M.-F.M. We thank A.-M.Camus for typing the manuscript.

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