APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1994, p. 1905-1913 Vol. 60, No. 60099-2240/94/$04.00+ 0Copyright © 1994, American Society for Microbiology

Identification and Grouping of Mycoplasmalike OrganismsAssociated with Grapevine Yellows and Clover Phyllody

Diseases Based on Immunological and Molecular AnalysesK. H. CHEN,' R. CREDI,2 N. LOI,3 M. MAIXNER,4 AND T. A. CHEN"*

Department of Plant Pathology, Rutgers University, New Brunswick, New Jersey 08903'; Istituto di Patologia vegetale,Universita degli Studi, Bologna,2 and Universita di Udine, Udine, Italy; and Federal Biological Research Center for

Agriculture and Forestry, Institute for Plant Protection in Viticulture, Bemkastel-Kues, Germany4

Received 30 August 1993/Accepted 15 March 1994

Immunofluorescent staining, dot blot hybridization, PCR, random amplified polymorphic DNA (RAPD)markers, and restriction fragment length polymorphism were used to study the genetic relatedness amongmycoplasmalike organisms (MLOs) associated with several geographically diverse grapevine yellows diseases(CA1, CH1, SA1, and SA2 from Bologna, Italy; GYU from Udine, Italy; GYR from Rome, Italy; and GYG fromGermany). The relationship between these and MLOs associated with clover phyllody diseases in Italy (CPhBand CPhC) and Canada (CPhCa) was also examined. Two monoclonal antibodies reacted with MLOs of GYU-,CPhB-, and CPhC-infected periwinkles. Dot blot hybridization with two cloned GYU DNA fragments, GYD-1and GYD-2 inserts, showed that both hybridized with DNAs of GYU-, CPhB-, and CPhC-infected periwinklesbut not with those of GYR and CPhCa. In addition, GYD-1 insert hybridized with DNAs of CA1, CH1, SA1,SA2, and GYG. Three primer pairs were developed in PCR experiments for this study. By using primer setGYD2P1F and GYD2P1R, a 600-bp DNA fragment was amplified only when DNAs from GYU-, CPhB-, andCPhC-infected plants were used as templates. With the primer pair GYD2P1F and GYD2P2R, a 550-bp DNAfragment was amplified from GYU, CPhB, CPhC, and GYG. The primer pair GYDlPlF and GYD1P2R, on theother hand, could amplify all isolates, although the patterns ofPCR products were not identical for all isolates.Six different primers used in RAPD analysis have resulted in 13 RAPD markers which were either specific forone isolate or shared by two, three, or four isolates and thus could be used for the identification of MLOisolates. On the basis of these results, all isolates studied except GYR and CPhCa could be distinguished fromone another, and they could be broadly classified into five subgroups: subgroup I, CAl and CH1; subgroup II,SAl and SA2; subgroup III, GYU, CPhB, and CPhC; subgroup IV, GYG; and subgroup V, GYR and CPhCa.Restriction fragment length polymorphism analyses with cloned GYD-1 insert were in good agreement withresults obtained from other approaches used in this study.

Grapevine flavescence doree (FD), an economically impor-tant yellows-type disease occurring worldwide, was first discov-ered in vineyards of southern France (11). The etiologicalagent is believed to be a mycoplasmalike organism (MLO) (9)which is naturally transmitted to grapevines (Vitis vinifera L.)by a leafhopper, Scaphoideus titanus Ball (Scaphoideus littoralisBall) (7, 33). Since the first report of FD in France in 1957,similar grapevine yellows (GY) diseases have been reported inAustralia, northern France, Germany, Italy, and the UnitedStates (4, 8, 19, 26, 30). They are also termed Vergilbungs-krankheit in Germany and bois noir in northern France.Symptoms of these diseases are, in general, similar to that ofFD, although minor differences have been noted in variousregions. In Australia and the United States MLOs have beenshown to be associated with the diseases, whereas in Italy andGermany no pathogens or vectors have been identified to date.Although S. titanus has been reported to occur in northernItaly (29), the occurrence and spread of GY do not seem to becorrelated with the presence of S. titanus (28a).

In France, FD MLOs were transmitted from diseased grape-vines to broad beans (Vicia faba L.) by using S. titanus collectedfrom FD-affected vineyards (10). Experimental transmissionfrom diseased broad beans to periwinkles [Catharanthus roseus(L.) G. Don] was carried out by another leafhopper, Eusce-

* Corresponding author. Phone: (908) 932-9199 or (908) 932-9425.Fax: (908) 932-9377.

lidius variegatus Kbm, which is not a natural vector of FD. InItaly, GY diseases on different grape varieties have beenreported in several regions, and minor differences in symptomshave also been observed. MLOs have been transmitted directlyfrom infected grapevines to periwinkles by dodder either in thegreenhouse or under controlled conditions in the vineyards(15). In Germany, the Vergilbungskrankheit MLO also hasbeen transmitted to periwinkle plants by dodder. At least twodifferent types of symptoms have been observed on the peri-winkles infected with Italian and German GY MLOs: oneinvolves stunting, proliferation of lateral shoots, and phyllody,and the other involves general yellowing of the leaves. To date,there is no direct evidence to indicate whether these GYdiseases are actually the same disease with slightly variedsymptoms as a result of cultivar and geographic differences orwhether more than one type or one strain of MLO are involvedin the disease etiology.

In the course of studying GY, two monoclonal antibodies toGY MLO (Udine, Italy) had been developed in our laboratory(12). They were tested for specificity against nine other MLO-associated yellows diseases. Both monoclonal antibodies re-acted strongly with MLOs in periwinkles infected with isolatesof clover phyllody (CPh) MLO originally collected from theFriuli region of Italy (unpublished data). The CPh MLOs weretransmitted from diseased clovers (Trifolium repens L.) toperiwinkles by the vector Euscelis plebejus Fall (6). The factthat clover plants showing typical phyllody symptoms are

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frequently found in northern Italy, where most of the Italianvineyards are also located, raises some questions regarding thepossible relatedness between GY MLOs and CPh MLOs. Itwould also be interesting to know whether clover plants play animportant role in the epidemiology of GY disease.The objectives of this study were to identify the differences

among various GY MLO isolates and to compare these MLOswith those associated with CPh diseases. Comparison studiesincluded extensive immunological and molecular analyses byusing monoclonal antibodies, DNA probes, specific PCR,restriction fragment length polymorphism (RFLP) analyses ofchromosomal DNA, and random amplified polymorphic DNA(RAPD) markers. This work has resulted in a better under-standing of GY disease in general and helped to clarify thepresence of GY MLO strains. In addition, it suggests theimportance of CPh MLOs in the complex etiology of GYdisease.

MATERIALS AND METHODS

Plant material. Periwinkle plants (Catharanthus roseus) withsymptoms of phyllody proliferation from Udine, Italy, werekindly provided by L. Carraro. The MLO believed to be thecausal agent, designated GYU, had been transmitted by dod-der (Cuscuta campestris L.) from diseased grapevine cultivarChardonnay grown in the Friuli region of Italy. Four periwin-kles infected with MLOs denoted as CA1, CH1, SA1, and SA2were kindly provided by R. Credi. These MLOs were originallytransmitted by dodder (C. campestris) from infected grapevinecultivars Caveccia, Chardonnay, and Sangiovese grown in theBologna region of Italy (15). The sample from Germany,designated GYG, was kindly provided by M. Maixner, who hadsuccessfully transmitted the GYG MLO from grapevine culti-var Riesling to periwinkle in Bernkastel-Kues by using thedodder C. odorata. Diseased periwinkle infected with theRoma isolate of GY MLO (GYR) was kindly provided by M.Barba. The Italian isolates of CPh MLOs (CPh-Bergamo[CPhB] and CPh-Cavazzo [CPhC]) in periwinkle were kindlyprovided by L. Carraro, Udine, Italy. These MLOs wereoriginally collected from naturally infected clover (T. repens L.)plants grown in vineyards in the Friuli region of Italy andshowing typical phyllody symptoms. The vector Euscelis incisus(Euscelis plebejus) Fall (6) was used to transmit the MLOsfrom clover to periwinkle. A Canadian strain of the CPh MLO(CPhCa) in periwinkle was kindly provided by I.-M. Lee, U.S.Department of Agriculture, Beltsville, Md. All diseased peri-winkles were maintained by grafting and kept in an insect-controlled greenhouse.

Immunofluorescent staining. Cross sections of leaf midribsof both diseased and healthy periwinkles were prepared byfreehand sectioning and fixed in cold acetone for 10 min. Thesections were air dried and incubated in monoclonal antibodies(culture supernatants) specific to GYU MLO for 30 min at37°C. After being washed in phosphate-buffered saline (PBS),the sections were then stained with anti-mouse immunoglobu-lins G and M conjugated with fluorescein isothiocyanate (KPL;Kirkegaard and Perry Lab., Inc., Gaithersburg, Md.) at adilution of 1:1,000 (vol/vol) for 30 min at 37°C. The sectionswere thoroughly rinsed with several changes of PBS andobserved under a Nikon epifluorescence microscope (13).DNA isolation. Total DNA from healthy or diseased plants

was isolated by using the cetyltrimethylammonium bromide(CTAB) method as described by Doyle and Doyle (18) withminor modifications. Midribs of leaves were cut longitudinallywith sharp forceps from infected or healthy periwinkle leavesand ground to a fine powder in liquid nitrogen. The pulverized

tissue was added to preheated (65°C) CTAB buffer (100 mMTris-HCl [pH 8.0], 1.4 M NaCl, 50 mM EDTA, 1% polyvi-nylpyrrolidone [PVP], 0.2% [vol/vol] 3-mercaptoethanol, 2.5%hexadecyltrimethylammonium) and incubated at 65°C for 30min with occasional shaking. Next, a two-thirds volume ofchloroform-isoamyl alcohol (24:1, vol/vol) was added to themixture, and the solution was mixed by inversion beforecentrifugation at 4,300 x g for 10 min. The aqueous phase wastransferred to a clean tube, and a two-thirds volume of coldisopropanol was added. Total nucleic acids were precipitatedby keeping the mixture at -70°C for 30 min. After centrifuga-tion at 12,000 x g for 10 min, the precipitated nucleic acidswere resuspended in TE buffer (10 mM Tris-HCl, 1 mMEDTA [pH 8.0]) and extracted once with an equal volume ofTE buffer-saturated phenol and twice with chloroform-isoamylalcohol (24:1, vol/vol). Nucleic acids were precipitated againwith ethanol, collected by centrifugation as described above,rinsed with 70% ethanol, air dried, and resuspended in TEbuffer. These crude extracted nucleic acids were used in dotblot hybridization, Southern hybridization, and PCR experi-ments.Dot blot hybridization. Crude nucleic acid samples extracted

from healthy and MLO-infected periwinkle plants were dilutedin 6x SSC (lx SSC is 150 mM NaCl and 15 mM sodiumcitrate [pH 7.0]), denatured by boiling for 10 min, and imme-diately chilled on ice for 5 min. Each denatured DNA sample(2 pLg per spot) was blotted onto a nylon membrane (BiotraceRP; Gelman Science Inc., Ann Arbor, Mich.) by using the S &S Minifold apparatus (Schleicher & Schuell Inc., Keene, N.H.).Membranes were air dried for 30 min, baked at 80°C for 2 h,prehybridized at 65°C for 3 h or overnight, and then hybridizedwith DNA probes for 24 h at 65°C. The probes were labeledwith [32P]dCTP by using the random-primer DNA-labelingsystem (Bethesda Research Laboratories [BRL], Life Technol-ogies Inc., Gaithersburg, Md.). The inserts of GYD-] andGYD-2 (GYD-1 contained a 9.0-kb insert, and GYD-2 con-tained a 1.6-kb insert), derived from GYU and previouslydeveloped in our laboratory (12), were recovered from gels byusing the Geneclean kit (Bio 101 Inc., La Jolla, Calif.) and usedas probes in tests to study the genetic interrelatedness amongthese MLOs. After hybridization, blots were subjected tohigh-stringency wash conditions consisting of two washes in 2xSSC-0.5% sodium dodecyl sulfate (SDS) at room temperaturefor 20 min each and two washes in 0.1X SSC-0.5% SDS at65°C for 30 min each. Filters were then wrapped in plastic wrapand exposed to Fuji X-Omat AR diagnostic film for a mini-mum of 12 h at -70°C.

Primers for PCR. Three oligonucleotides, GYD2P1F, GYD2P1R, and GYD2P2R, previously derived from the cloned GYD-2fragment (12) were used in PCR to analyze sequence homologyamong MLOs used in this study. The sequences of these primerswere as follows: 5'-TACTGCTGCGCGACGTCTAATT-3' (GYD21P1F), 5'-GACATTCGATTAACAT'fIA7TfTGATAAATT-3'(GYD2P1R), and 5'-CCAAGCTGTGACTGTC(TTA-3' (GYD2P2R). In addition, two primers (one forward [GYDIPIF] and onereversed [GYDIP2R]), used as one primer set, were designed andsynthesized (DNA synthesizer; Applied Biosystems, Foster City,Calif.); they were based on the partial sequence data of the DNAfragment from GYD-1. The sequences were as follows: 5'-TTTCG'1'1TCAATTAAGGC'1'1'1`TV-3' (GYD1P1F) and 5'-CGAAAG'f'TAGC(1'CAACTGC(1'AT-3' (GYDlP2R).PCR amplification. The PCR amplification was performed

in a 25-pLl PCR mixture containing 200 ng of template DNA;1.0 ,uM each primer of a pair; 250 pLM each dATP, dCTP,dGTP, and dTTP; 25 mM Tris (pH 8.0); 5 mM MgCl2; 50 mMNaCl; 2.5 ,ug of bovine serum albumin (BRL, Life Technolo-

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gies Inc.); and 1.25 U of Taq DNA polymerase (PromegaCorp., Madison, Wis.). A 25-[lI volume of mineral oil was

added to each reaction mixture, and PCR was carried out for30 or 35 cycles in a Hybaid Thermal Reactor (National LabnetCo., South Plainfield, N.J.) under the following conditions:initial denaturation for 2 min at 94°C and then 30 or 35 cyclesconsisting of denaturation for 1 min at 94°C, annealing for 1

min at 50 or 55°C, and extension for 3 min at 72°C (the finalextension step continued for 20 min at 37°C). PCR productswere analyzed by electrophoresis in a 0.8% agarose gel, stainedwith ethidium bromide, and visualized under UV light.RFLP-Southern blot hybridization. Nucleic acid samples

(about 2.5 pLg of total nucleic acid per sample) from healthy or

MLO-infected periwinkle plants were singly digested withEcoRV or HinidlIl or doubly digested with BglII and Hindlll or

with EcoRV and HindlIl at 37°C for 3 h. Digested DNAsamples were electrophoresed in a 0.8% agarose gel, alkalidenatured, and transferred to nylon membrane by the methodof Sambrook et al. (32). The membranes were air dried, baked,prehybridized, hybridized with 32P-labeled cloned DNA probeGYD-1 insert, and washed as described for dot blot hybridiza-tion.RAPD markers. A total of eight random primers, five that

were 10 nucleotides long, two that were 14 nucleotides long,and one that was 12 nucleotides long, were used. The arbitrarydecamer primers obtained from Operon Technologies Inc.,Alameda, Calif., have been designated by an OP prefix andthen the kit letter and primer number. They were OPAl(CAGGCCCTTC), OPA5 (AGGGGTCTTG), OPA12 (TCGGCGATAG), OPA16 (AGCCAGCGAA), and OPA17 (GACCGCTTGT). Primers RAPD1 (AATGAAAGTCGA7TF) with71%o A+T, RAPD2 (ATAGTTGGGAAT) with 67% A+T,and RAPD3 (AGAGAAAAAGGAGG) with 57% A+T were

synthesized by using a DNA synthesizer (Applied Biosystems).Amplification reactions were carried out in volumes of 12.5 plcontaining 25 mM Tris-HCI (pH 8.0); 5 mM MgCl2; 50 mMNaCl; 1.25 jig of bovine serum albumin (BRL, Life Technol-ogies Inc.); 250 FM each dATP, dCTP, dGTP, and dTTP(Promega Corp.); 0.36 ,uM primer (OPAl, OPA5, OPA12,OPA16, or OPA17) or 0.5 VLM primer (RAPDI, RAPD2, or

RAPD3); 1.25 U of Taq DNA polymerase (Promega Corp.);and 25 ng of template DNA. Amplification was performed in a

Hybaid Thermal Reactor programmed for one cycle of 5 minat 94°C; 45 cycles of 1 min at 94°C, 1 min at 28°C (OPAl,OPA5, OPA12, OPA 16, OPA17, RAPD1 or RAPD2) or 36°C(RAPD3), and 2 min at 72°C; followed by 10 min at 72°C.One-half of the reaction product was taken from each of thetubes and separated by polyacrylamide gel electrophoresis witha Mini-Protean II slab cell (Bio-Rad Laboratories, Richmond,Calif.). Separation was performed with 0.75-mm-thick slab gelsof 7.5c acrylamide. The ratio of acrylamide to N'N'-bis-methylene-acrylamide was 29:1. Gels and running buffer were

prepared by the method of Laemmli (23), except that SDS was

omitted. Samples were mixed with 9 ,ul of sample buffer (0.05M Tris-HCl [pH 6.8], 20Ccl glycerol, 0.004% bromophenolblue) and loaded into the wells. A 123-bp DNA ladder (BRL,Grand Island, N.Y.) was used to determine the size of theRAPD marker. Electrophoresis was carried out at 200 V untilthe dye front reached the end of the gel (approximately 45min). DNA bands were visualized by silver staining with a

silver staining kit (Bio-Rad Laboratories), following the pro-tocols suggested by the manufacturer with slight modifications.Briefly, gels were fixed with 10% acetic acid for 30 min withgentle agitation, soaked in oxidizer for 5 min with shaking,rinsed with water until they became clear, impregnated withsilver reagent for 20 min with gentle shaking, rinsed with

TABLE 1. Immunofluorescence reactions of monoclonal antibodiesderived from the GY MLO Udine isolate with sections of healthy or

MLO-infected periwinkles

Origin of leaf midrib Immunofluorescence of:sections GYM-1 GYM-2

GY CAI - -

GY CHI - -

GY SAI - -

GY SA2 - -

GY Udine + +GY RomaGY GermanyCPh Bergamo + +CPh Cavazzo + +CPh CanadaHealthy periwinkle

" +, typical apple-green fluorescence in the phloem area; -, no fluorescencein the phloem area. GYM-1 and GYM-2 are the monoclonal antibodies used inthis study.

deionized water for 1 min, and then developed at roomtemperature in freshly prepared developer until the bandsreached the desired intensity in relation to background. De-velopment was stopped by adding 5% acetic acid, and the gelswere preserved for permanent record by being soaked in 5%glycerol for at least 20 min and dried between two sheets ofcellophane at room temperature.

RESULTSImmunofluorescent staining. Two anti-GYU MLO mono-

clonal antibodies, GYM-1 and GYM-2, previously developedin our laboratory (12) were used in this study. Both monoclo-nal antibodies reacted with MLOs present in the phloemelements of GYU-, CPhB-, and CPhC-infected periwinkleplants, showing typical apple-green fluorescence. No specificfluorescence was observed from cross sections of healthyperiwinkles, other GY-MLO isolate-infected periwinkles, orCPhCa-infected periwinkles. Results of the immunofluores-cence study are summarized in Table 1.

Dot blot hybridizations. Results of dot blot hybridizations ofcloned DNA probes against nucleic acids from healthy orMLO-infected periwinkle plants are shown in Table 2. Neitherprobe (GYD-1 insert and GYD-2 insert) derived from GYUhybridized with total DNA extracted from healthy periwinkle

TABLE 2. Dot blot hybridization analyses of -3P-labeled DNAprobes against GY MLO Udine isolate to DNAs extracted from

healthy and MLO-infected periwinkles

Hvbridization of DNA probe':MLO in periwinkles

GYD-1 GYD-2

GY CAI + -

GY CHI + -

GY SAI + -

GY SA2 + -

GY Udine + +GY RomaGY Germany +CPh Bergamo + +

CPh Cavazzo + +

CPh CanadaHealthy periwinkle

' +, positive hybridization, -, no hybridization signal.

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plants or with that from GYR- or CPhCa-infected periwinkleplants under stringent conditions. Both probes hybridizedstrongly with nucleic acid samples from plants infected withCPhB, CPhC, or GYU. The GYD-1 insert also hybridized withDNA of CA1, CH1, SAl, SA2, and GYG, indicating thatGYD-1 was a broader probe to this group of MLOs than wasGYD-2.PCRs. Three oligonucleotides, GYD2P1F, GYD2P1R, and

GYD2P2R, previously derived from the GYD-2 sequence,were used in combinations of two as primer pairs in PCR totest sequence homology among MLO isolates examined. Withprimer pair GYD2P1F and GYD2P1R, a 600-bp DNA frag-ment was amplified from GYU-, CPhB-, and CPhC-infectedperiwinkle plants, whereas this specific fragment was notobserved in the remaining isolates (Fig. 1A). When GYD2P1Fand GYD2P2R were used as primers, a 550-bp DNA fragmentwas amplified not only from those three MLO-affected plantsbut also from plants infected with GYG (Fig. iB). No specificPCR products were obtained when nucleic acids isolated fromhealthy periwinkle plants or plants infected with CA1, CH1,SAl, SA2, or CPhCa were used as templates (Fig. 1B). Forcomparison, two primers, GYDlPlF and GYD1P2R, based onthe partial sequence of the broad-range DNA probe GYD-1were designed and used for the same purpose. With the primerpair, fragments from all isolates could be amplified but thepatterns of DNA bands were not identical for all isolates. Atleast four DNA fragments were produced from CAl and CHIMLOs, and the size and pattern of their PCR products werethe same. However, the pattern was different from that of theremaining isolates, which produced only a single DNA bandcorresponding to the 500-bp DNA fragment present in CAland CHI PCR products (Fig. 1C).RAPD markers. Since MLOs cannot be cultured in vitro, the

nucleic acids used for PCR templates were always contami-nated with healthy plant DNA. In fact, only a minute portionof the DNA preparations were derived from the MLOs. Thus,most of the bands that appeared in amplified PCR products ofhealthy or infected periwinkles were present in both samples.To identify prominent RAPD markers that could be used toseparate MLO isolates, we used several primers to amplifyDNAs from various isolates. Two primers (RAPD2 andOPA16) did not produce specific discernible bands and werediscarded. The rest of the primers produced RAPD markersthat were specific for one isolate or were shared between twoto four isolates (Fig. 2). With these markers, between one andfour isolates could be distinguished. Although some minor,faint bands were also present in some but not all MLO isolates,they were discounted because they lacked definitive clarity.Only bands that appeared regularly from different DNApreparations and repeated PCR experiments were selected asreproducible RAPD markers and used for analyses.A total of 13 prominent RAPD markers were chosen, of

which 5 were unique to a single isolate: marker 9 from primerRAPD1 was unique to CPhC, marker 13 from primer RAPD3was unique to SA2, marker 6 from primer OPA17 was uniqueto GYG, and markers 1 and 2 from primer OPAl were uniqueto CAl and CPhB, respectively. Four other markers wereshared by two isolates: marker 8 from primer RAPD1 waspresent in GYU and CPhB, marker 3 from primer OPA5 waspresent in SAl and SA2, and markers 11 and 12 from primerRAPD3 were common in SAl and SA2. One marker wasshared by three isolates, i.e., marker 4 from primer OPA5 inGYU, CPhB, and CPhC. Three markers were present in eachof four isolates, for example, marker 7 from primer RAPD1 inCA1, CH1, SAl, and SA2. The results are summarized inTable 3 and shown in Fig. 2A to F.

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FIG. 1. PCR analyses of DNA extracted from healthy and MLO-infected periwinkles. Primer pairs used in PCR amplification areGYD2P1F and GYD2P1R (A), GYD2P1F and GYD2P2R (B), andGYDlPlF and GYD1P2R (C). The primers in panels A and B werederived from GYD-2 sequence, and that in panel C was derived fromGYD-1 sequence. See Materials and Methods for the definition ofstrain abbreviations. In panels A and B, all samples were amplified inthe same reaction and run on three gels. The figure shows results fromelectrophoresis of PCR products in a 0.8% agarose gel and stainingwith ethidium bromide. The marker was lambda DNA digested withHindlIl; from top to bottom, the bands are 23.1, 9.4, 6.6, 4.4, 2.3, 2.0,and 0.56 kb. H-P, healthy periwinkle. In panel A, a 600-bp DNAfragment was amplified from GYU-, CPhB-, and CPhC-infectedperiwinkles. In panel B, a 550-bp DNA fragment was amplified fromGYU-, CPhB-, CPhC-, and GYG-infected periwinkles. Numbers 1, 2,and 3 indicate different isolations of the same samples.

RFLP/Southern blot hybridizations. RFLP patterns of en-donuclease-digested DNAs in Southern hybridizations probedwith GYD-1 from GYU MLO DNA showed genetic similari-ties between the MLOs. Results of analyses with probe GYD-1insert against EcoRV-, HindIII-, BglII-HindIII-, or EcoRV-HindlIl-digested DNAs from healthy plants and from plantsinfected with different isolates of GY-MLOs and CPh-MLOsare shown in Fig. 3. In all cases, no hybridization signal wasobserved when DNA from healthy periwinkles was probed(Fig. 3). The probe hybridized with all DNA samples extractedfrom MLO-infected plants except those from CPhCa- and

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FIG. 2. RAPD amplification product analysis of genomic DNA isolated from healthy and MLO-infected periwinkles. Strain abbreviations arethe same as in Fig. 1. PCR was performed under the conditions outlined in Materials and Methods with the primers OPAl (A), OPA5 (B), OPA12(C), OPA17 (D), RAPD1 (E), and RAPD3 (F). Amplification products were separated on 7.5% polyacrylamide gels and visualized by silverstaining. Lane M, molecular marker (BRL 123-bp DNA marker). Lane C, control with no genomic DNA. Lane H-P, healthy periwinkle. The linesand numbers indicate the RAPD markers. The molecular sizes of the internal standards marked a, b, and c in lane M are (from a to c) 492, 984,and 1,476 bp.

GYR-infected periwinkles. These results were consistent withthose obtained from dot blot hybridizations described above.Multiple-banding patterns were common in all of the MLORFLPs and could be broadly divided into four groups. GYMLO isolates CAl and CH1 were moderately related to eachother. Although some DNA bands observed in RFLP patternsof CAl MLO corresponded to those of the same size fromCH1 MLO, each has its own distinctive pattern. RFLP patternsof SAl and SA2 MLOs were identical when EcoRV-, BglII-HindIll-, or EcoRV-HindIII-digested DNA was probed. WhenDNA was digested with Hindlll and hybridized with the sameprobe, SAl MLO could be distinguished from SA2 MLO bythe absence of one band that was present in the pattern of SA2(Fig. 3B). Nevertheless, these two patterns were markedlydifferent from those of other isolates. The RFLP patterns for

GYU, CPhB, and CPhC exhibited very high similarities to oneanother (Fig. 3), but they were not identical because each hadits own unique pattern. On the other hand, GYG MLO showedan RFLP pattern that was different from those of all the otherMLOs examined (Fig. 3).

DISCUSSION

Although MLOs have been implicated in more than 300plant yellows diseases around the world (28), the number ofgenetically distinct MLOs involved in these diseases has re-mained a mystery. The principal impediment to their identifi-cation and classification has been the inability to isolate themin pure culture in vitro. For this reason, traditional identifica-tion and classification of MLOs has relied on biological criteria

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TABLE 3. Summary of RAPD markers obtained by using sevendifferent primers for genomic DNAs extracted from MLO-

infected periwinkles

RAPD No. of RAPD Marker(s) present inprimer marker MLOs

OPAl 1 CAl2 CPhB

OPA5 3 SAl, SA24 GYU, CPhB, CPhC

OPA12 5 CA1, CH1, SAl, SA2

OPA17 6 GYG

RAPD1 7 CA1, CHI, SAI, SA28 GYU, CPhB9 CPhC

RAPD3 10 CA1, CH1, SAl, SA211 SAl, SA212 SAl, SA213 SA2

such as disease symptoms expressed from infected plants, hostplant range, and characteristics of vector transmission. Al-though these criteria are helpful in the study of MLO-relateddiseases, the use of biological properties alone may not distin-guish among different MLOs that induce similar symptoms ina plant species or may not reveal different diseases caused by

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the same MLO. Recent development of hybridoma and recom-binant DNA technologies has made it possible to identify andto study the genetic relatedness of these uncultured MLOs (17,21, 22, 24, 25). By using cloned DNA fragments of MLOs asprobes in dot blot hybridization and RFLP analyses, it hasbeen possible to differentiate among MLO strains (17, 22, 24,25). Lee et al. have used the DNA probes derived from severalMLOs to classify the complex aster yellows (AY) MLO clusterinto three genomic subclusters (types) (25). On the basis ofthese genetic analyses, some MLOs previously not designatedas AY strains have also been classified into the AY MLOcluster (25). In this study, we have used immunological andgenomic DNA analyses to successfully identify the similarityand differences among various GY MLO isolates. In addition,we have provided evidence that there is genetic relatednessbetween GY MLO isolates and MLOs associated with CPh inItaly. On the basis of our results, we propose to classify theisolates into at least five subgroups.Using the GYU MLO monoclonal antibodies GYM-1 and

GYM-2 in immunofluorescent staining and the DNA probesGYD-1 insert and GYD-2 insert from the same MLO in dotblot hybridization analyses, we found that there were epitopesand homologous DNA sequences commonly present in GYUMLO and Italian CPh MLOs, which suggested that theseMLOs are closely related at the genetic level. Moreover, theGYU MLO and the Italian CPh MLOs that caused seeminglydifferent types of diseases in different natural hosts in northernItaly gave rise to very similar RFLP patterns of their DNAs.This has further confirmed our earlier presumption that thesetwo MLOs shared very high genetic homology and should be

N *I4 r.1 Pl X So N '-4 .4,-'

,< x g x 0XA C) CC Co ".W iw A 't

B

0. 0.) M 0iX S A A P N .4 P4'I P: 0V S S P 44 41

04A >4 4 x4 Nx4S C U

0 00 0 00 0 a 00a00a0 0 00

FIG. 3. RFLP-Southern blot hybridization analyses of chromosomal DNAs extracted from healthy and MLO-infected periwinkles. Nucleic acidsamples were digested with restriction endonucleases EcoRV (A), HindIII (B), HindlIl and BglII (C), and HindIII and EcoRV (D) and probedwith a 9.0-kb GYU MLO chromosomal DNA fragment from GYD-1 plasmid. The identity of strain abbreviations is the same as in Fig. 2. H-P,healthy periwinkle. In panel B, the arrow indicates the band which is absent in SAl.

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GRAPEVINE YELLOWS AND CLOVER PHYLLODY MLOs 1911

placed within a group of related MLOs, although they wereoriginally obtained from two divergent host plants. In addition,by using the primer pairs GYD2P1F and GYD2P2R orGYD2P1F and GYD2P1R in PCR experiments, the same sizesof DNA fragments were amplified from both Italian CPhMLO- and GYU MLO-infected plants but not from any otherMLO-infected plants tested. These results have further indi-cated the close phylogenetic relatedness between GYU andItalian CPh MLOs. Interestingly, neither the monoclonalantibodies nor the DNA probes reacted with a CPh MLOisolate from Canada (CPhCa), which produced symptoms inperiwinkles and clovers almost identical to those produced bythe isolates of Italian CPh MLO. Thus, at least two geneticallydistant groups of MLOs that are geographically isolated canproduce the same phyllody symptoms in clover plants.The discovery of phylogenetic relatedness between GYU

and Italian CPh MLOs has led us to speculate that, in nature,Italian CPh disease might have originated from GY-infectedgrapevines in the field in Europe or vice versa. In Turin, Italy,transmission of GY MLOs from grapevines to clovers (T.repens L.) by the leafhopper Euscelidius vaniegatus Kbm hasbeen reported (1). Thus, clover plants must play an importantrole as an alternative host in the epidemiology of GY. Furtherevidence is needed to show whether Italian CPh MLO caninfect grapevines and produce GY symptoms. In an earlierreport (12), we demonstrated the common occurrence of GYMLO in symptomless wild Vitis riparia in the Finger Lakesregion of New York. Thus, it is possible that GY MLO isendemic in eastern North America and was transferred toEurope via the introduction of American grapevine rootstocks.If such were the case, we should anticipate the presence ofItalian CPh MLOs in clovers in North America. Therefore, itwas surprising when our results indicated there are majordifferences between Italian CPh MLO and Canadian CPhMLO (CPhCa).On the basis of immunofluorescence tests and dot blot

hybridization analyses, the 10 MLOs (7 GY MLO isolates fromItaly and Germany and 3 CPh MLO isolates from Italy andCanada) could be broadly classified into three subgroups.Subgroup I consisted of five GY MLO isolates that reactedonly with the DNA probe GYD-1 (i.e., CA1, CH1, SAl, SA2,and GYG). Subgroup II consisted of three MLOs that reactedwith both DNA probes (i.e., GYU, CPhB, and CPhC). Sub-group III was represented by the two MLOs that reacted withneither probe (i.e., GYR and CPhCa). Since GYR and CPhCadid not hybridize with the two GYU MLO DNA probes, it wasnot possible to determine whether there are genetic relation-ships between them and with other isolates.To further distinguish isolates within subgroups I and II, we

needed other approaches to differentiate these isolates. Byusing primer pair GYDlPlF and GYD1P2R in PCR, sub-group I could be further divided into another two subgroups.One consisted of CAl and CH1, and the other consisted ofSAl, SA2, and GYG. Additional discrepancies between thePCR results of the two primer sets, i.e., GYD2PlF-GYD2PlRand GYD2PlF-GYD2P2R, have made it possible to separateGYG from SAl and SA2. MLOs in subgroup II, however, stillcould not be separated from one another.The PCR technique with arbitrary primers (5, 34, 35) has

been applied to distinguish isolates by using RAPD markers (3,20, 34, 35). Unfortunately, the nucleic acids we used as PCRtemplates were not pure MLO DNA. We could not takeadvantage of RAPD markers for studying genetic similaritieswithin these MLO isolates. RAPD markers from this study,however, could still be used for the identification of isolates. Byusing RAPD marker 9, specific for CPhC MLO, and RAPD

CMl, CR1, SA1, SA2, GYU, CPhB,CPhC, GYG, GYR, CPhCa

Immunofluorescent stainingwith GDI-1 and GD(-2

.1(-) I

CAM, CR1, SAM, SA2,GYG, GYR, CPhCa

GYD-1GYD-2

CSAM

GYU, CPhB, CPhCDot blot hybridizationusing GYD-1 and GYD-2

inserts

Ct) I I GYD-1 (-) GYD-1 (+)-) | GYD-2 (-) GYD-2 Ct)MAl, CR1, GYR GYC, CPhB, CPhC

SA2, GYG CPhCa RAPD marker

PCR with the No. 9primer pairGYD2P1F a GYD2P2R (+) (-)

(+ I(-)GYG CMl, CH1, SA1, SA2

PCR with theprimr pairGMDIPIF a G3D1P2R

F I

CPhC GYU, CPhBRARD marker

No. 2

(+I) (-)

CPhB GYU

CMi, Cal SMl, SA2

RAID marker RAPD mrkerNo. 1 No. 13

(+) (-) (-)r (+)CAM CR1 SM SA2

FIG. 4. Schematic procedures for the identification of variousisolates of GY MLOs and CPh MLOs.

marker 2, specific for CPhB MLO, isolates within subgroup IIcould be distinguished from one another (Fig. 2A and E).Similarly, CAl and CH1 MLOs could be separated from eachother by using RAPD marker 1, and SAl and SA2 MLOs couldbe distinguished by using marker 13 (Fig. 2A and F). Thus,except for the isolates in subgroup III, all the MLO isolatesused in this study could be differentiated from one another. Aflow chart for identifying these isolates is presented in Fig. 4.RFLPs have been used to identify genetic similarity among

the culturable mollicutes (31). RFLP analysis was also used asan alternative means of characterizing the genetic relationshipsamong the various MLO isolates. All MLO isolates thathybridized with the probe GYD-1 insert could be distinguishedfrom one another by RFLP analysis of chromosomal DNAs.On the basis of hybridization patterns, these isolates could bebroadly classified into five subgroups which are in goodagreement with results obtained from other genetic analysesused in this study. Thus, the methods used in this study wouldbe very useful for investigating the genetic relatedness amongother different geographical isolates of the GY MLO groupsuch as FD and NB MLOs in France. They could also facilitatethe search for insect vectors responsible for field transmissionof GY-MLO isolates and help in controlling the spread of GYdisease. Multiple hybridization patterns were observed inRFLP-Southern blot hybridizations, which suggested the pres-ence of similar or identical sequences in several regions of theMLO genome.

Recently, a paper for comparing MLOs associated with twoGY diseases and Italian periwinkle virescence disease waspublished by Davis et al. (16). In this paper, comparisonstudies included only RFLP analyses and dot hybridizationswith six DNA probes from three different MLOs. The authorsconcluded that at least two distinct MLOs are associated with

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GY in Italy. Our studies, however, included immunologicaland several molecular analyses. In addition to the methodsused by Davis et al. (16), we used immunofluorescence tests,PCR, and RAPD markers amplified with arbitrary primers tocompare seven GY MLO isolates from different geographicalareas and three CPh MLO isolates from Italy and Canada.Thus, the information obtained in this paper has expanded ourunderstanding of the relationships among these MLO isolatesbeyond that reported by Davis et al. It would also be interest-ing to know if the strains used in their study were the same asany of the strains used in our study.On the basis of the symptoms expressed by plants infected by

various MLOs, plant-pathogenic MLOs can be broadly dividedinto two categories: those that cause symptoms of phyllodyand/or virescence (virescence MLOs), and those which do notproduce virescence and phyllody but, rather, produce a generaldecline symptom (decline MLOs) (14). In most cases, MLOsthat infect woody plants seldom cause the symptoms of vires-cence and phyllody on herbaceous plants and virescence MLOsrarely induce diseases in woody plants. It is interesting, how-ever, that GYU transmitted directly from diseased grapevinesto periwinkles by dodder induced phyllody and virescencesymptoms on periwinkles. In Bologna, Italy, MLOs associatedwith GY were also transmitted to periwinkles by dodder, buttwo types of symptoms were observed (15). SAl and SA2produced yellowing of the foliage, flower abortion, or smallflowers, whereas plants affected by CAl and CH1 exhibitedpartial yellowing of leaves, phyllody, and virescence (15). GYMLOs transmitted by dodders from symptomatic vines toperiwinkles in Germany and Latium, Italy (2, 27), also exhib-ited phyllody and virescence symptoms. On the basis of thisinformation and results from RFLP-Southern blot hybridiza-tions (Fig. 3), at least five different GY MLO strains, CA1,CH1, GYG, GYU, and GYR, belong to the group of vires-cence MLOs while the remaining two, SAl and SA2, can beclassified as the decline MLOs.

ACKNOWLEDGMENTS

We thank Irvin S. Y. Chen, Department of Microbiology andImmunology, School of Medicine, University of California, Los Ange-les, for helping with oligonucleotide syntheses. We thank Donald Y.Kobayashi, Department of Plant Pathology, Rutgers University, forreviewing the paper.

This research was supported in part by the New Jersey AgriculturalExperiment Station.

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