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APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/99/$04.0010

Sept. 1999, p. 4197–4206 Vol. 65, No. 9

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Ti Plasmids from Agrobacterium Characterize Rootstock ClonesThat Initiated a Spread of Crown Gall Disease in

Mediterranean CountriesSANDRINE PIONNAT,1 HARALD KELLER,1* DELPHINE HERICHER,1 ANDREE BETTACHINI,1

YVES DESSAUX,2 XAVIER NESME,3 AND CHRISTINE PONCET1

Institut National de la Recherche Agronomique (INRA), Phytopathologie et Botanique, Unite Sante Vegetale etEnvironnement, 06606 Antibes Cedex,1 Institut des Sciences Vegetales, Centre National de la Recherche

Scientifique (CNRS), 91198 Gif-sur-Yvette Cedex,2 Laboratoire d’Ecologie Microbienne du Sol,Unite Mixte de Recherche CNRS 5557 and INRA, Universite Claude Bernard-Lyon 1,

69622 Villeurbanne Cedex,3 France

Received 31 March 1999/Accepted 21 June 1999

Crown gall caused by Agrobacterium is one of the predominant diseases encountered in rose cultures.However, our current knowledge of the bacterial strains that invade rose plants and the way in which theyspread is limited. Here, we describe the integrated physiological and molecular analyses of 30 Agrobacteriumisolates obtained from crown gall tumors and of several reference strains. Characterization was based on thedetermination of the biovar, analysis of 16S ribosomal DNA restriction fragment length polymorphisms byPCR (PCR-RFLP), elucidation of the opine type, and PCR-RFLP analysis of genes involved in virulence andoncogenesis. This study led to the classification of rose isolates into seven groups with common chromosomecharacteristics and seven groups with common Ti plasmid characteristics. Altogether, the rose isolates formed14 independent groups, with no specific association of plasmid- and chromosome-encoded traits. The predom-inant Ti plasmid characteristic was that 16 of the isolates induced the production of the uncommon opinesuccinamopine, while the other 14 were nopaline-producing isolates. With the exception of one, all succinamo-pine Ti plasmids belonged to the same plasmid group. Conversely, the nopaline Ti plasmids belonged to fivegroups, one of these containing seven isolates. We showed that outbreaks of disease provoked by the succi-namopine-producing isolates in different countries and nurseries concurred with a common origin of specificrootstock clones. Similarly, groups of nopaline-producing isolates were associated with particular rootstockclones. These results strongly suggest that the causal agent of crown gall disease in rose plants is transmittedvia rootstock material.

The soilborne, gram-negative bacterium Agrobacterium tu-mefaciens infects dicotyledonous plants from almost 100 dif-ferent families, causing crown gall disease throughout theworld (11). This disease is characterized by the formation oftumors at wound sites, an event resulting from a natural in-terkingdom DNA transfer. Approximately 15 genes from a200-kb tumor-inducing (Ti) plasmid of the bacterium are trans-ferred to the plant cells, where they become integrated into thehost genome (7; for reviews, see references 10, 22, and 42) andexpressed. The transfer requires both the products of othergenes located in the nontransferred virulence (vir) region ofthe Ti plasmid and proteins that are encoded by the chromo-some (4). The transferred DNA (T-DNA) portion of the Tiplasmid carries the genes tms and tmr, which encode proteinsinvolved in the synthesis of the plant hormones auxin andcytokinin, respectively, which are responsible for uncontrolledcell proliferation during crown gall tumorigenesis (23, 26, 29).The T-DNA also encodes enzymes used for the synthesis oftumor-specific compounds, called opines. Opines are releasedby the plant and can be used by the bacterium as the solecarbon and/or nitrogen sources (14) and perceived as signalsfor the conjugal transfer of the Ti plasmid between strains ofAgrobacterium (1, 32, 43). The opines are mostly amino acid or

sugar derivatives. The metabolism of opines is encoded bynontransferred genes on the Ti plasmid (17). The presence ofthe opine molecules in crown galls therefore provides an eco-logical niche favoring pathogen development and Ti plasmiddissemination (2).

The extent of crown gall disease depends largely on thephysiological conditions of the host plants. When the plants arein a good state of health, tumors are limited and do not influ-ence the viability of the hosts. In contrast, the disease becomessevere when preinfections, wounding, or other environmentalfactors weaken the hosts (37). At present, crown gall is thepredominant disease encountered on rose cultures in the Med-iterranean region, reducing both the vigor of the plants and theyields of marketable flowers (33). The severity of the diseaseon rose plants can be related to the development and use ofnew production methods in nurseries, such as vegetative mul-tiplication of plant material. The wounds induced by cutting,grafting, and root pruning generate additional infection sitesfor Agrobacterium (27). In nurseries, transmission of the bac-teria occurs via soil or via water (24). Furthermore, growthconditions encountered in nurseries (temperature and humid-ity) favor the development of the pathogen. Additionally, theincrease in commercial exchanges of contaminated plant ma-terial must be taken into account when one is investigating theepidemic spread of the disease. Due to the stability of geneticcolonization by Agrobacterium (2), current curative methodsare not effective for controlling the disease. In the absence ofrose varieties naturally resistant to crown gall disease, further

* Corresponding author. Mailing address: INRA, Phytopathologieet Botanique, Unite Sante Vegetale et Environnement, BP 2078,F-06606 Antibes Cedex, France. Phone: 33-4 93 67 88 67. Fax: 33-4 9367 88 88. E-mail: [email protected].

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propagation of the disease can be avoided only through pre-vention and selection of healthy plants before vegetative mul-tiplication. Therefore, methods that allow detection of thepathogen in contaminated plant material must be developed.Better knowledge of the Agrobacterium strains that invade roseplants and the way in which they spread is therefore needed.

Here, we present the establishment of a collection ofAgrobacterium isolates that were obtained from diseased roseplants from France, Spain, and Morocco. The physiologicaland molecular characteristics of these isolates were analyzed,with particular emphasis on the characteristics affecting viru-lence and tumorigenesis. We collected information about theorigins of plant material that was used for flower productionand the horticulture conditions used for generating the roseplants. We present data indicating a correlation between themolecular characteristics of Agrobacterium and rose plant ori-gins and discuss the possible implications of our results for abetter understanding of the epidemiology of crown gall dis-ease.

MATERIALS AND METHODS

Plant samples. The majority of rose plant samples harboring crown gall tu-mors were graftings obtained from flower producers. Two samples were root-stocks. The 28 samples were collected from 23 different growers in France, Spain,and Morocco between 1991 and 1997. For confidentiality, floriculturists weredesignated by numbers, and professional grafters, multipliers, and breeders ofrootstocks were designated by letters.

Agrobacterium reference strains. Strains with the prefix CFBP and Agrobacte-rium sp. strain C58 were obtained from the Collection Francaise de BacteriesPhytopathogenes (CFBP; Institut National de la Recherche Agronomique[INRA], Angers, France). Strains A6, Bo542, and EU6 were gifts from W. S.Chilton (North Carolina State University, Raleigh). Strains ACH5 and T37 wereobtained from the Centre National de la Recherche Scientifique (CNRS), Vil-leurbanne, France. Strains 287-7 and 282-1 were obtained from M. M. Lopez(Instituto Valenciano de Investigaciones Agrarias, Moncada, Spain). StrainANT4 was isolated at INRA, Antibes, France.

Isolation of A. tumefaciens from crown galls. Tumors were cut from rose plants,washed with water, ground in a mortar, and extracted in sterile water. Theinsoluble residues were allowed to settle, and 1-ml aliquots of the supernatantswere spread on petri dishes containing YPGA medium (5 g of yeast extract, 5 gof Bacto Peptone, 10 g of glucose, and 15 g of agar liter21) and incubated for 4days at 25°C. Typical Agrobacterium colonies (16) were picked and dispersed in1 ml of water at 25°C under continuous agitation. Aliquots from overnightcultures were streaked on YPGA medium. The procedure was repeated untilhomogeneous bacterial cultures were obtained.

Isolate reference numbers. The isolates RiM9, RiM10, RiM12, RiM15,RiM19, RiM20, RC21, RiM23, RiM26, RiM27, RiM30, RiM42, RiM45, RiM50,RiM57, RiM60, RiM66, RiM67, RM71, RiM74, and RiM76 were deposited atCFBP and appear in the catalog of phytopathogenic bacterial strains under thenumbers CFBP4418, CFBP4419, CFBP4421, CFBP4423, CFBP4424, CFBP4425,CFBP4426, CFBP4427, CFBP4430, CFBP4431, CFBP4434, CFBP4440, CFBP4442,CFBP4443, CFBP4444, CFBP4445, CFBP4447, CFBP4449, CFBP4451, CFBP4453,and CFBP4454, respectively.

Biochemical analysis of the isolates. The recovered bacteria were assayed forthe presence of b-glucosidase, b-galactosidase, and urease activities; the Gramstrain response was also investigated (20). Isolates that were identified as be-longing to the genus Agrobacterium were stored at room temperature on YPGAmedium, as well as under liquid nitrogen in an aqueous solution containing 15%glycerol and 15% dimethyl sulfoxide. The biovars of the isolates were determinedaccording to their growth characteristics on selective medium (3), on 2% NaCl,and on ferric ammonium citrate; production of 3-ketolactose; utilization ofcitrate; and medium alkalization in the presence of malonic acid, L-tartaric acid,and mucic acid (25). The pathogenicity of the bacteria was assessed by evaluatingtheir ability to induce crown gall formation 3 weeks after the inoculation of rosecuttings. All growth assays and the investigation of tumor induction ability wereperformed at 25°C.

Opine analyses. To obtain large tumors and to avoid the extraction of rosecompounds interfering with opine analyses, opine production by tumors on gallsdeveloping 4 weeks after stem inoculation of tobacco (Nicotiana tabacum cv.Xanthi-nc) with bacteria was analyzed. Opines were extracted from 1 g (freshweight) of tumors by homogenization of the galls in 10 ml of methanol. Theextracts were clarified by filtration through Whatman GF/C filters, dried undervacuum, resuspended in 10 ml of ethyl acetate, and extracted with 10 ml of water.The aqueous phase was reextracted with 10 ml of ethyl acetate and concentratedto 1 ml. For separation of opines, 20 ml of the extracts was spotted on Whatman3MM paper (38 by 20 cm). Paper electrophoresis was performed at a constant

voltage (34 V cm21) for 1 h in 1 M formic acid–0.8 M acetic acid at pH 1.8 forseparating agropine, nopaline, mannopine, chrysopine, and octopine and in 0.15M formate buffer at pH 2.8 for separating succinamopine and leucinopine (8,13). Opine spots were revealed as described by Dessaux et al. (13). Authenticnopaline, octopine, and mannopine standards were purchased from Sigma.Leucinopine and succinampine were gifts from W. S. Chilton and P. Guyon(CNRS, Gif sur Yvette, France), respectively. Agropine was synthesized as de-scribed previously (12).

Isolation of DNA and PCR protocols. DNA was extracted as described byChen and Kuo (6) from 1.5 ml of A. tumefaciens cultures grown for 2 days at 25°Cin YPG medium (YPGA medium without agar). All PCR experiments wereperformed with 50-ml reaction mixtures containing 13 PCR buffer (10 mMTris-HCl [pH 9.0], 0.1% Triton X-100, 1.5 mM MgCl2, 0.2 mg of bovine serumalbumin ml21), 200 mM each nucleotide (Promega), 0.1 mM each primer, 0.25 Uof Taq polymerase (Appligene-Oncor, Illkitvh, France), and 25 ng of templateDNA. The temperature profile for the amplification of tmr(171), vir(246), andvir(418) was as follows: initial denaturation at 94°C for 3 min, 35 cycles ofdenaturation at 94°C for 5 s, annealing at 57°C for 15 s, and elongation at 71°Cfor 30 s, and final extension at 71°C for 3 min. For the amplification of the 16Sfragment, the annealing temperature was increased to 59°C. For the amplifica-tion of tms(587) and vir(1673), the duration of all steps was doubled. The primersused to amplify Ti plasmid fragments were as follows: FGPtmr530 and FG-Ptmr7019 for amplifying tmr(171), FGPtms21949 and FGPtms1469 for amplifyingtms(587), FGPvirA2275 and FGPvirB21649 for amplifying vir(1673), FGP-virB11121 and FGPvirG159 for amplifying vir(246), and ANTvirB11887 (59GGTGAGACAATAGGCGATCT39) and FGPvirG159 for amplifying vir(418). Chro-mosomal DNA corresponding to the 16S rRNA sequence was amplified withprimers FGPS6 and FGPS15099. All primers designated FGP were describedpreviously (28). PCR products were analyzed on 1.2% agarose gels by coelec-trophoresis with a 123-bp ladder (Gibco BRL). Electrophoresis and staining ofgels with ethidium bromide were carried out by standard procedures (35).

PCR-RFLP. In a total volume of 15 ml, 5 ml of a PCR product was digestedwith 8 U of the enzyme CfoI (Boehringer GmbH, Mannheim, Germany), DdeI orMspI (Appligene-Oncor), HaeIII (Amersham, Buckinghamshire, United King-dom), or MseI (New England Biolabs) in 13 reaction buffer as indicated by thesuppliers. Restriction fragments obtained after 1 h of digestion were separatedon 8% acrylamide gels and stained with ethidium bromide. Similarity assign-ments were made by the Dollop software program included in the PHYLIPpackage (15). Characteristics considered were opine synthesis (succinamopine,nopaline, octopine, agropine, chrysopine, and null); amplification of vir(247),vir(416), and vir(1672); and the restriction fragment length polymorphism(RFLP) banding pattern after digestion of vir(416), vir(1672), and tms(587).

RESULTS

Combined physiological and molecular analyses of Agrobac-terium. The analysis that was established to identify and dis-tinguish A. tumefaciens isolates involved characteristics deter-mined by chromosomal genes and by genes of the Ti plasmid,as indicated in Fig. 1. For simplicity reasons, species ofAgrobacterium were designated according to phytopathogeniccharacteristics (9). In other words, tumor- and root-inducingagrobacteria were termed A. tumefaciens and A. rhizogenes,respectively. Physiological and molecular characteristics thatwere determined by the chromosome were biovars (25) and theRFLP of a PCR fragment of the 16S rRNA gene, respectively.Ti plasmid-determined characteristics were opine synthesis intumors, amplification by specific primers (28) of regions withinthe tumorigenesis genes tmr and tms and within some virulencegenes (Fig. 1), and RFLPs of the tms fragment and the twolongest vir gene fragments. The analyses were applied to 17reference strains of A. tumefaciens from different geographicorigins and isolated from different hosts. Four reference strainsof A. vitis and two strains of A. rhizogenes were also included(data not shown).

16S rDNA RFLP analysis of reference strains. A 1,479-bpfragment of the 16S ribosomal DNA (rDNA) was amplifiedfrom all Agrobacterium reference strains with the universalprimers FGPS6 and FGPS15099. The PCR products were di-gested with the enzymes MspI and HaeIII. A. tumefaciens gaverise to the profiles Ms1 (four restriction sites), Ms2 (threesites), and Ms3 (three sites), and the profiles H1 (seven sites),H2 (six sites), and H3 (seven sites), respectively (Fig. 2). Onthe basis of the PCR-RFLP profiles, the reference strains were

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classified into seven chromosome groups belonging to biovars1 and 2 (Table 1). The two A. rhizogenes strains were frombiovar 2. A. rhizogenes A4 showed 16S rDNA RFLP patternsidentical to those of A. tumefaciens CFBP1317 and CFBP1935,whereas A. rhizogenes CFBP3001 and A. tumefaciens CFBP296and CFBP1904 exhibited similar RFLP patterns. All A. vitisstrains were from biovar 3. Their 16S rDNA RFLP profileswere not found within the analyzed A. tumefaciens strains.

Opine analyses and PCR of plasmid-encoded pathogenicitygenes of reference strains. Of 17 A. tumefaciens referencestrains, 11 caused the synthesis of the opine nopaline in tu-mors. Five other reference strains caused octopine, agropine,chrysopine, or succinamopine synthesis in tumors. StrainCFBP2746 did not produce these opines, leucinopine, or man-nopine. The A. vitis strains caused the production of nopaline,vitopine, cucumopine, and octopine (31). The two strains of A.

rhizogenes caused the production of mikimopine (40) and agro-pine (39).

tmr(171) and tms(587) were amplified from all A. tumefaci-ens reference strains with the specific primer pairs FGPtmr7019–FGPtmr530 and FGPtms21949–FGPtms1469 (28), respec-tively. Independent of the opine type, tmr(171) was also am-plified from the A. vitis strains. tms(587) was obtained onlyfrom the nopaline-type strains of A. vitis. Neither the tmr northe tms fragment was amplified from the A. rhizogenes strains.With primer pair FGPvirB11121–FGPvirG159, a 247-bp frag-ment spanning the intergenic region between virB11 and virGfrom 198 bp 59 to 49 bp 39 of the virG start codon was obtainedfrom all nopaline-type strains of A. tumefaciens as well as fromthe succinamopine-type strain EU6 and strain CFBP2746. Noamplification occurred with DNA from strains harboring oc-topine-, agropine-, and chrysopine-type Ti plasmids (Table 1).The combination of primers ANTvirB11887 and FGPvirG159allowed amplification of vir(416) from all nopaline-type strains,the succinamopine-type strain, and strain CFBP2746. Thisfragment spanned the region from 149 bp 59 of the stop codonof virB11 to 49 bp 39 of the start codon of virG. The size of thisfragment was reduced to 370 bp when PCR was performedwith template DNA isolated from the octopine-, agropine-,and chrysopine-type strains (Table 1). With primer pair FGP-virB21649–FGPvirA2275, vir(1673), spanning the region from217 bp 59 of the stop codon of virA to 164 bp 39 of the startcodon of virB2, was amplified from DNA preparations of no-paline- and succinamopine-type A. tumefaciens strains as wellas from strain CFBP2746. The fragment size reached 2,400 bpwhen PCR was performed with DNA isolated from the octo-pine-, agropine-, and chrysopine-type strains (Table 1). Noneof the primer combinations mentioned above allowed amplifi-cation of vir fragments from A. vitis or A. rhizogenes DNA.

RFLP analysis of amplified tms and vir genes of referencestrains. Digestion of the PCR product tms(587) from A. tume-faciens with the enzymes CfoI and DdeI gave rise to the profilesCt1 (three restriction sites), Ct2 (three sites), and Ct3 (twosites), and the profiles D1 (one site), D2 (two sites), D3 (twosites), D4 (two sites), and D5 (no site), respectively (Fig. 3).Digestion of vir(418) with the enzymes MspI and MseI led tothe profiles M1 (no site) and M2 (one site) and the profiles S1(two sites) and S2 (one site), respectively (Fig. 4). The PCRproduct vir(1673) was digested with CfoI and gave rise to theprofiles C1 (eight sites) and C2 (seven sites) (Fig. 4). Due tothe size differences of the PCR fragments, polymorphismswithin the vir region of the octopine-, agropine-, and chryso-pine-type strains were not determined. On the basis of theopine type of the Ti plasmids and the RFLP profiles of thePCR products, the Ti plasmids of the 17 A. tumefaciens refer-ence strains fell into 12 plasmid groups. All results of RFLPanalyses of PCR products are summarized in Table 1.

Assembling a collection of A. tumefaciens crown gall isolatesfrom rose plants. Rose samples that presented symptoms ofcrown gall disease were collected from 23 different flowerproducers, rootstock multipliers, or breeders in France, Spain,and Morocco. With the exception of one Rosa canina and threeRosa manetti rootstocks, all others were of the species Rosaindica Major. Twenty-four samples were obtained from flow-ering, grafted plants. Two samples were obtained from un-grafted rootstocks, and two samples were obtained fromgrafted rootstocks to be sold to flower producers. Among the28 different plant samples, 22 had massive crown gall tumorsonly on the rootstocks (Fig. 5A to C). Two plants had crowngall tumors only on the roots (Fig. 5D to E), and two plants hadtumors on both the rootstocks and the roots but not on scions.Galls from roots and rootstocks of the same plant were treated

FIG. 1. Schematic and simplified maps of the Ti plasmid and the chromo-some from A. tumefaciens. Regions that were used for PCR amplification andphysiological characterization of isolates are indicated. For primer assignments,see Materials and Methods. lb and rb, left and right T-DNA borders, respec-tively.

FIG. 2. Restriction patterns of the amplified 1,500-bp 16S rDNA fragmentsafter digestion with MspI (profiles Ms1 to Ms4) and HaeIII (profiles H1 to H4).A 123-bp ladder (lanes L) was used as a DNA size marker.

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separately. Two samples had the rarely observed galls on scions(Fig. 5F). Altogether, bacteria were obtained from 30 indepen-dent galls. We obtained pure cultures of these 30 isolates,which all belonged to the genus Agrobacterium. All isolateswere pathogenic and induced the formation of galls when in-oculated on rose and tobacco plants.

16S rDNA RFLP analysis of rose isolates. The 1,479-bpfragment of the 16S rDNA was amplified from all rose isolates.Digestion of the PCR products from 27 isolates with MspI gaverise to profile Ms1 or Ms2. No profile corresponding to Ms3was observed. MspI digestion of PCR products from three roseisolates yielded a new profile, Ms4 (three restriction sites; Fig.2). Digestion of the 16S rDNA fragment with HaeIII gave riseto the profiles H1, H2, and H3 for PCR products from 26isolates. A new profile, H4 (six sites), was obtained after HaeIIIdigestion of the PCR products from four isolates. Three iso-lates gave rise to the novel profile Ms4-H4, and one isolate hadthe novel profile Ms2-H4. However, 21 of the isolates had themost represented profiles Ms1-H1 and Ms2-H2 (Table 2), asalready observed with Agrobacterium reference strains. On thebasis of the PCR-RFLP profiles, the rose isolates were classi-

fied into seven chromosome groups belonging to biovars 1 and2. No isolate was classified as biovar 3.

Ti plasmid characteristics of rose isolates. Among the A.tumefaciens rose isolates, 16 caused the synthesis of succinamo-pine in tumors. All other rose isolates induced the productionof nopaline in crown gall tumors (Table 2), and no other opinesynthesis trait was found. tmr(171) and tms(587) as well asvir(247), vir(416), and vir(1673) were amplified by PCR fromall rose isolates (Table 2).

Restriction digestion of the rose isolate PCR producttms(587) with CfoI and DdeI gave rise to the simple profile Ct1and Ct2 and the profile D1 and D2, respectively (Fig. 3).Digestion of vir(418) with MspI and MseI led to the profile M1and M2 and the profile S1 and S2, respectively, as was observedfor A. tumefaciens reference strains. Similarly, restriction di-gestion of vir(1673) with CfoI engendered the profiles C1 andC2 (Fig. 4 and Table 2). On the basis of the opine type of theTi plasmids and the RFLP profiles of the PCR products, the Tiplasmids of the 30 rose isolates of A. tumefaciens fell into sevenplasmid groups (Table 3). Groups II, V, VI, and VII had newcharacteristics that were not encountered during the analysis ofthe reference strains. The predominant groups II and III rep-resented 22 of the 30 isolates. With the exception of one isolate(RiM45) that defined a specific plasmid group, all succinamo-pine-type isolates were from plasmid group II. However, the 15isolates of this group could be dispatched over five of the seven

FIG. 3. Restriction profiles after digestion of amplified tms(587) with theenzymes CfoI (profiles Ct1 to Ct3) and DdeI (profiles D1 to D5). A 123-bp ladder(lanes L) was used as a DNA size marker.

FIG. 4. RFLP analysis of amplified vir(418) and vir(1673). vir(418) was re-striction digested with the enzymes MspI (profiles M1 and M2) and MseI (profilesS1 and S2). Digestion of vir(1673) was performed with the enzyme CfoI (profilesC1 and C2). A 123-bp ladder (lanes L) was used as a DNA size marker.

FIG. 5. Symptoms of crown gall disease on rose plants. Galls developedfrequently on rootstocks (A, B, and C) and roots (D and E) but rarely on scions(F).



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chromosome groups. Among the nopaline-type isolates, sevenbelonged to plasmid group III. Although showing the same Tiplasmid characteristics, they were from three different chro-mosome groups. Thus, among the rose isolates of A. tumefa-ciens, no specific correlation between plasmid type and chro-mosome characteristics was found.

Relationship between A. tumefaciens genotypes and the ori-gins of rose plant samples. To draw conclusions regarding thepropagation of A. tumefaciens in rose cultures, we collectedinformation on the origins of rootstocks, the conditions forrootstock propagation, and the culture conditions used forflower production. This information was compared with theexperimental data that we obtained by molecular characteriza-tion of the bacterial isolates. We did not find any apparentcorrelation between the chromosome characteristics of the A.tumefaciens isolates and the origin of the rose plants or theculture conditions. In contrast, a strong correlation betweenthe plasmid characteristics of the bacterial isolates and theorigin of rootstock clones was evident (Fig. 6). A similarityanalysis with equal weights of Ti plasmid characteristics con-firmed the homogeneity among the isolates that clustered indefined plasmid groups (Fig. 6). With the exception of RM77,all succinamopine-type isolates were from rose plants thatwere multiplied and cultured under conditions that we termedgraft/A (Fig. 6). A further common feature identified was thatall rootstocks which gave rise to rose plants contaminated bysuccinamopine-type isolates were processed by breeder C or D(with the exception of RM77). Additionally, breeder D fre-quently obtained plant material from breeder C (Fig. 6). RM77

was the only isolate originating from a plant that was cultivatedby the graft/C method and harboring a group II plasmid. Thisisolate had the same chromosomal background as the nopa-line-type isolate RM78, which was from a plant produced bymultiplier-grafter E. The group II Ti plasmid of RM77 mighthave originated in rootstocks obtained from breeder C or D,but in this case it was impossible to trace the contaminationback to its origin (Fig. 6).

All isolates that clustered in the well-represented plasmidgroup III were isolated from rose plants grafted on rootstocksobtained from breeder B. Isolate RiM57 (chromosome groupII) was recovered from a rootstock obtained directly from thisbreeder. Additionally, the culture conditions that gave rise tothe different gall-diseased samples were heterogeneous(graft/A, graft/B, and graft/C), making contamination throughsoil or water improbable. The only A. tumefaciens referencestrain harboring a group III Ti plasmid (strain 287-7) wasisolated in Spain from a rose plant propagated by multiplier/grafter E. In general, multiplier/grafter E acquired rootstocksfrom breeder B.

Two A. tumefaciens isolates in our collection were from roseplant scions, on which crown galls rarely developed. Both iso-lates belonged to independent plasmid groups, and no corre-lation could be established between disease and rootstock or-igin (Fig. 6). We believe that the contamination happenedduring the grafting process or was due to wounding occurringbetween grafting and flower production. In general, the occa-sional infection of scions did not contribute to the propagationof the disease.

TABLE 3. Classification of the analyzed A. tumefaciens isolates into Ti plasmid groupsa

Profiletms(587)

vir(1673) profile C1 vir(1673) profile C2

Profile vir(418)M1S1 Profile vir(418)M2S1 Profile vir(418)M1S1 Profile vir(418)M1S2 Profile vir(418)M2S2

Opine Isolates Group Opine Isolates Group Opine Isolates Group Opine Isolates Group Opine Isolates Group

Ct1D2 Succ. RiM45 INop. CFBP1904 Nop. CFBP296 Nop. RiM10 VNop. C58 Nop. RiM10r VNop. CFBP1932Nop. CFBP2516

Ct2D1 Nop. 282-1 Nop. RiM97s VII Nop. RiM89 IIINop. RiM12 IIINop. RiM15 IIINop. RiM30 IIINop. RiM57 IIINop. RiM60 IIINop. RiM66r IIINop. 282-7 III

Ct2D2 Nop. RiM67r IV Succ. RiM9 II Nop. RiM96s VINop. RM71 IV Succ. RiM18.1 IINop. RM78 IV Succ. RiM18.2 IINop. CFBP2177 IV Succ. RiM18.2r IINop. T37 IV Succ. RiM19 II

Succ. RiM20 IISucc. RC21 IISucc. RiM23 IISucc. RiM26 IISucc. RiM27 IISucc. RiM42 IISucc. RiM50 IISucc. RiM74 IISucc. RiM76 IISucc. RM77 II

a Opines were succinamopine (Succ.) and nopaline (Nop.). Rose isolates are shown in plain text, and reference strains are shown in italic type.



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DISCUSSION

We performed an integrated analysis of the physiologicaland molecular characteristics of 30 A. tumefaciens isolates thatwere recovered from diseased rose plants. In comparison tothe reference bacterial strains used in this study, the roseisolates showed strong homogeneity, whatever the geographicorigin of the samples.

This homogeneity was characterized in particular by Ti plas-mids encoding only the opines succinamopine and nopaline,although more than 20 different opines are known (14, 40).One surprising finding was that 16 of 30 isolates harboredsuccinamopine-type Ti plasmids. Plasmids encoding this opineare relatively rare, and until now only three A. tumefaciensstrains that induce succinamopine production in galls weredescribed (8). The virulence traits of isolates harboring succi-namopine-type Ti plasmids were more stable than those ofnopaline-type isolates. As already reported in a previous study(38), nopaline-type isolates lost virulence when kept at tem-peratures above 30°C. We found that this loss of virulence wascorrelated with an absence of amplification by PCR of vir, tms,or tmr gene fragments from the DNA of the isolates and wasthus most probably due to a loss of the Ti plasmid (data notshown). In contrast, none of the succinamopine-type isolateslost virulence and appeared to be well adapted to an extendedexposure to the high temperatures that commonly occur in thecountries where the rootstocks were selected and propagated.The opine type of a Ti plasmid also influences the effectivenessof conjugal transfer between bacteria (30 and references there-in). Although the effect of succinamopine on this transfer hasnot yet been analyzed, our results suggest that succinamopine-type Ti plasmids have been transferred frequently to differentchromosomal backgrounds and that recipient strains are betteradapted for the infection of rose cultures.

Despite the fact that rose isolates constitute a rather homo-geneous group, their specific characteristics allowed differen-tiation from our reference strains. The plasmid characteristicsof most of the rose isolates thus defined groups that were notrepresented among the reference strains. Furthermore, thisspecificity was also found at the chromosome level. Usually,the analysis of 16S rDNA provides good information for theidentification of Agrobacterium species (36, 41) and biovarswithin A. tumefaciens. Ponsonnet and Nesme (34) amplified a1,500-bp 16S rDNA fragment from 41 different Agrobacteriumstrains by using primers FGPS6 and FGPS15099. They digestedthe fragments with HaeIII and found that the profiles H1 andH3 always correlated with strains from biovar 1, while biovar 2strains gave rise to profile H2. In the present study, we foundthe same strict correlation when analyzing the referencestrains. In contrast, 5 of the 30 rose isolates did not fit into theabove scheme or gave rise to the new profile H4 upon analysisof their 16S rRNA genes. The 16S rDNA PCR-RFLP profilesthat we encountered most frequently for 21 of the 30 isolatesfrom all seven plasmid groups were Ms1-H1 (biovar 1) andMs2-H2 (biovar 2). It seems likely that bacteria with thesechromosomal backgrounds are common in rose cultivation ar-eas and are good recipients for Ti plasmids.

To our knowledge, the present study represents the firstdemonstration of a close correlation between the Ti plasmidtype of A. tumefaciens isolates found in diseased rose plantsfrom different countries and a common origin of the plantsamples that were used for isolation of the bacteria. The resultsindicate that rootstocks from breeder C or D were the sourcefor the dissemination of the succinamopine-type isolates thatwe found in rose plants from 14 independent flower producers.We believe that the succinamopine-type group II Ti plasmidoriginated in a rootstock from one of these breeders and thatthis plasmid was further disseminated to other A. tumefaciensstrains through conjugal transfer. Even more clearly, our find-ings strongly suggest that rootstocks obtained from breeder Bwere the origin of the dissemination of the group III Ti plasmidto eight independent nurseries in three different Mediterra-nean countries.

To obtain grafted plants, three culture methods were used:

FIG. 6. Similarity analysis of discrete Ti plasmid characteristics of A. tume-faciens reference strains and rose isolates, classification of chromosome groups ofrose isolates, and origin and culture conditions of the plants that served forbacterial isolation. Characteristics analyzed were opine type, amplification of virgene fragments, and PCR-RFLP banding patterns for the long vir and tmsfragments. Values indicating the confidence of branch point assignments werecreated in a bootstrap analysis from 1,000 trials. Rose isolates of A. tumefaciensare shown in shaded boxes. Sample types were either rootstocks or graftedplants. In graft/A, grafting and planting into pots occurred at the same time.Plants were grown under defined culture conditions and were sold for flowerproduction 6 to 8 weeks later. In graft/B, cuttings of the rootstock were plantedinto pots and cultured under defined conditions. Graftings were performed afterroot formation, and plants were sold for flower production 10 to 12 weeks aftercutting. In graft/C, cuttings of the rootstocks were planted in the ground, andgraftings were performed 6 month later. Plants were sold for flower productionafter 1 year. Nd, neither the breeder of R. manetti rootstocks for samples RM77,RM71, and RM78 nor the breeder of the rootstock for sample RiM97 could bedetermined. Grafter F propagated rootstocks in Morocco but performed graft-ings in France on either his own rootstocks (RiM10 and RiM89) or rootstocksthat were obtained from grafter/multiplier E (RiM66 and RiM67).



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graft/A, graft/B, and graft/C. In terms of phytopathology, theprocedures involving graft/A and graft/B almost eliminate therisk of new contamination of plants by soil bacteria, as all stepsare performed with a soil-free substrate. However, the rapidturnovers and exchanges between rootstocks and scions favorthe propagation of disease through contaminated material. Incontrast, the classical graft/C method restricts this risk butfavors new contamination by soil bacteria. For our collection,only the three samples isolated from R. manetti rootstocks andsamples RiM12 and RiM15 were cultivated by the graft/Cmethod by multiplier/grafter E. The five A. tumefaciens isolatesfrom these samples could be classified into four distinct chro-mosome groups. In contrast, the 14 isolates from graft/A sam-ples, which were all obtained from breeder C, were from 5chromosome groups. Thus, plants in soil cultures acquire bac-teria with different chromosomal backgrounds more frequentlythan do those in cultures with soil-free substrates.

In grapevine, A. tumefaciens can persist in the roots but inthe spring becomes mobilized throughout the plant with thevessel sap (5, 18, 19). In tobacco, persisting Agrobacterium canbe detected preferentially in the basal parts of the plants (21).These authors stressed the risks of dissemination through veg-etative propagation, which involves stem bases and roots. Wedeveloped an A. tumefaciens detection method based on PCRof vir(418). With this method, we were able to detect thebacteria in rose plants even in the absence of disease symp-toms. Furthermore, in diseased plants the bacteria could belocalized in organs distant from crown galls (data not shown).Movement of Agrobacterium within the plants would accountfor the identification of the same isolates in cuttings and roots(RiM10 and RiM10r; RiM18.2 and RiM18.2r [Table 2]). Ourresults indicate that Agrobacterium can persist in rose plantsand that it is able to move systemically in the plants. Thesefactors increase the risks for dissemination of the microorgan-ism through vegetative propagation of rose plants.

In summary, we have shown that the exponential spread ofcrown gall disease in Mediterranean rose cultures is due to thevegetative propagation of rootstocks; to the frequent exchangeof plant material between professional breeders, multipliers,and grafters; and to the increasing turnover rates for flowerproduction. As efficient chemical or genetic control of thedisease will not be applicable in the near future and as it willnot be possible to restrict commercial exchanges and to de-crease turnover rates, further propagation of the disease canbe reduced only through selection of healthy rootstocks. Thus,sensitive methods for the detection and characterization of thebacteria are required. In this study, we presented putativetargets for detection by PCR (vir, tms, and tmr regions) and asubset of molecular markers that will be valuable tools for suchpurposes.

ACKNOWLEDGMENTS

We thank all growers who kindly provided us with samples of crowngall-diseased rose plants. We are grateful to William Scott Chilton forthe gifts of opines and of A. tumefaciens A6, Bo542, and EU6 and toMaria Lopez for strains 287-7 and 282-1. We thank Claude Antonini,Louis Simonini, and Jean-Marie Drapier for plant care. We thank NeilLedger for editorial assistance.

This work was supported by grant 639/92 from the Association Natio-nale de la Recherche Technique and Comite National Interprofessionnelde l’Horticulture to S.P. and by EEC contract ERBIC18CT970198 toX.N. and Y.D.

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14. Dessaux, Y., A. Petit, and J. Tempe. 1993. Chemistry and biochemistry ofopines, chemical mediators of parasitism. Phytochemistry 34:31–38.

15. Felsenstein, J. 1993. PHYLIP: phylogenetic inference package (version3.572). Department of Genetics, University of Washington, Seattle.

16. Kersters, K., and J. De Ley. 1984. Agrobacterium Conn 1942, p. 244–254. InN. R. Krieg and J. G. Holt (ed.), Bergey’s manual of systematic bacteriology,vol. 1. Williams & Wilkins, Baltimore, Md.

17. Kim, H., and S. K. Farrand. 1998. Opine catabolic loci from Agrobacteriumplasmids confer chemotaxis to their cognate substrates. Mol. Plant MicrobeInteract. 11:131–143.

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