Genetic diversity of Pseudomonas syringae pv. lachrymans strains isolated from cucumber leaves...

Plant Pathology

(2007)

56

, 373–382 Doi: 10.1111/j.1365-3059.2006.01550.x

© 2006 The Authors Journal compilation © 2006 BSPP

373

Blackwell Publishing Ltd

Genetic diversity of

Pseudomonas syringae

pv.

lachrymans

strains isolated from cucumber leaves collected in Poland

H. Olczak-Woltman

a

*, A. Masny

b

, G. Bartoszewski

a

, A. P

l

ucienniczak

b

and K. Niemirowicz-Szczytt

a

a

Department of Plant Genetics, Breeding and Biotechnology, Warsaw Agricultural University, ul. Nowoursynowska 159, 02-776 Warszawa; and

b

Institute of Biotechnology and Antibiotics, ul. Staro

6

ci

5

ska 5, 02-516 Warszawa, Poland

Several strains of


pathovar (pv.)

lachrymans

and related bacterial pathogens were isolated fromcucumber (

Cucumis sativus

) leaves collected in central and southern Poland in 2001 and 2002. Twenty five originalstrains, together with five reference strains of

P. syringae

pv.

lachrymans

, pv.

syringae

and pv.

tomato

, were geneticallycharacterized by PCR-RFLP (polymerase chain reaction

−

restriction fragment length polymorphism), ADSRRS (ampli-fication of DNA fragments surrounding rare restriction sites), and PCR-MP (PCR

−

melting profiles) fingerprintingtechniques. Genetic similarity analyses of the PCR-RFLP and ADSRRS fingerprints showed that strains of

P. syringae

pv.

lachrymans

form distinct clusters. The results also indicated that the ADSRRS and the PCR-MP fingerprinting tech-niques may serve as more efficient tools for evaluating genetic similarity among pathovars and strains of

P. syringae

thanPCR-RFLP. The 25 strains showed diverse pathogenicity to cucumber seedlings and biochemical tests were varied. The

syrB

gene was identified in four cucumber strains, characterized as

P. syringae

pv.

syringae

.

Keywords

: ADSRRS, angular leaf spot, Cucurbitaceae, molecular characterization, PCR-melting profiles, PCR-RFLP

Introduction


pv.

lachrymans

is one of 50 patho-vars belonging to the heterogeneous species

Pseudomonassyringae

(Young

et al

., 1996) and is a pathogen of cucum-ber (

Cucumis sativus

). It causes angular leaf spot, thesymptoms of which include vein-limited, water-soakedlesions on the cucumber leaves, with or without a chlorotichalo, and water-soaked lesions on fruits, which may bemisshapen (Bradbury, 1986). Generally, pseudomonadsbelonging to

P. syringae

fluoresce on King’s medium B (KB)(King

et al

., 1954) when exposed to UV light, and possessneither a terminal cytochrome oxidase nor argininedihydrolase. The majority of

P. syringae

pathovarsexhibit no pectolytic activity.


produces smooth, mucoid colonies on nutrient agar with5% sucrose and causes a hypersensitive reaction ontobacco leaves (Bradbury, 1986; Lelliott & Stead, 1987).Several biochemical tests referred to as LOPAT tests(levan production on sucrose medium, oxidase reaction,pectolysis on potato slices or pectate gel, arginine

dihydrolase production, and hypersensitivity reaction ontobacco leaves) were developed to distinguish betweenpathogenic and saprophytic (e.g.

P. fluorescens

) fluorescentpseudomonads (Lelliott

et al

., 1966). According to theLOPAT tests,

P. syringae

pathovars belong to group Ia(Lelliott

et al

., 1966; Lelliott & Stead, 1987). However,the LOPAT tests cannot distinguish pathovars within

P. syringae

. Moreover, atypical strains occur which do notproduce fluorescent pigment on KB medium or exhibitpectolytic activity (Lelliott & Stead, 1987; Young & Triggs,1994). Hence, host pathogenicity tests remain the mostreliable method for pathovar differentiation. Although

P. syringae

pv.

lachrymans

is the casual agent of cucumberangular leaf spot (Lelliott & Stead, 1987), Bradbury (1986)reported that cucumber is also a host for

P. syringae

pv.

syringae

.DNA-based techniques form the basis of modern

microbial characterization and identification. Polymerasechain reaction

−

restriction fragment length polymorphism(PCR-RFLP) and repetitive sequence PCR (rep-PCR)have demonstrated considerable genetic diversity among

P. syringae

pathovars (Louws

et al

., 1994; Manceau &Horvais, 1996). Recent genetic investigations usingrep-PCR demonstrated that

P. syringae

pv.

lachrymans

and

P. s.

pv.

syringae

were heterogeneous (Stead

et al

.,

*E-mail: [email protected]

Accepted 13 September 2006

Plant Pathology

(2007)

56

, 373–382

374

H. Olczak-Woltman

et al.

2003). Three different rep-PCR clusters were identifiedamong strains of

P. s.

pv.

lachrymans

: i) a large, internallydiverse cluster with many strains showing similar bandingpatterns to

P. savastanoi

pv.

phaseolicola

, ii) two strainssimilar in banding patterns to

P. syringae

pv.

syringae

,and iii) a unique strain (Stead

et al

., 2003). This diversitywithin

P. syringae

pv.

lachrymans

strains was confirmedby AFLP analysis (Manceau & Brin, 2003), which revealedgroups of strains with no correlation to geographic originor phenotypic trait.

The aim of this study was to determine the geneticdiversity among strains of

P. syringae

isolated fromcucumber leaves with angular leaf spot symptomssampled in crops growing in Poland. Comparison wasmade to

P. syringae

reference strains using LOPAT andpathogenicity tests, PCR-RFLP analysis of the internaltranscribed spacer-1 (ITS1) region, and two novel finger-printing methods: amplification of DNA fragmentssurrounding rare restriction sites (ADSRRS), andPCR–melting profiles (PCR-MP) (Masny & Plucienniczak,2001, 2003). Both methods were originally described forevaluating differences in DNA sequence among clinical,human bacterial pathogens (Masny & Plucienniczak,2001; 2003). These two methods are described for thefirst time for identifying differences in plant pathogenicbacterial genomes.

Materials and methods

Bacterial strains

The bacterial collection included five reference strains and25 strains isolated from cucumber leaves showing angularleaf spot symptoms, that were collected in central (WW,WM) and southern (WK, WH) Poland in 2001 and 2002(Table 1). Strains WW and WM were isolated from leavescollected in one location, strains WK and WH from dif-ferent locations, in the southern Poland. In all locationseach leaf was collected from a different plant and eachstrain in the study was obtained from a different leaf.Exceptions were strains WH 1/01, WH 2/01 and WH 3/01,as well as WW 17/01 and WW 27/02, where each groupof strains was isolated from a single leaf. Cucumberleaves with symptoms of angular leaf spot were rinsed insterile water, ground in sterile water with a sterile porce-lain mortar and pestle, and 100

µ

L was spread onto KBplates. Several resulting single colonies with fluorescence(one per leaf, with two exceptions described above) weresubjected to the LOPAT tests. In addition, seven nonfluo-rescent colonies were included as negative control strains.Five reference strains were obtained from plant pathogenculture collections. Three reference strains belonged to

P. syringae

pv.

lachrymans

: YPG 1293, 814/98 (obtainedfrom the Institute of Plant Protection in Poznan, Poland) andLMG 5070 (obtained from the Microbiology Laboratoryin Gent, Belgium).


pv.

syringae

2905 and

P. syringae

pv.

tomato

, Pst 3 were obtained fromthe collection of the Research Institute of Pomology andFloriculture in Skierniewice, Poland. All reference strains

were subjected to the same LOPAT tests. All the strainswere stored on nutrient agar slopes at 4

°

C.

Cucumber pathogenicity tests

Seeds of cucumber accessions Wisconsin 18 SMR andinbred line B, selected from cv. Borszczagowski, and bothsusceptible to angular leaf spot, were individually sowninto plastic pots filled with a peat moss. The resultingplants were maintained in standard growth chamber con-ditions at 25

°

C day, 22

°

C night, with illumination for16 h from sodium lamps providing 50 W m

−

2

. Inoculumwas prepared by growing bacterial cells for 24 h on KBagar at 28

°

C. The resulting colonies were suspended insterile distilled water and adjusted to obtain an inoculumconcentration of 1

×

10

7

CFU (colony forming unit) mL

−

1

,estimated by the plate-count technique (Klement

et al

.,1990) to be equal to an optical density (OD

600

) of 0·050.Cucumber plants at the 2 to 3 leaf stage were inoculatedby spraying the lower side of each of three leaves per strain(Klement

et al

., 1990). Plants were kept in the dark at 100%relative humidity for 24 h, and then for 6 days in the cham-ber as above with 90% relative humidity. After 7 days, theleaves were scored for disease severity and the symptomswere described. The pathogenicity tests were performedusing six replicate plants of each cultivar per strain.

Bacterial DNA isolation

After growing the cultures for 24 h in Luria Broth liquidmedium on a rotary shaker at 28

°

C, total genomic DNAwas extracted from 1 mL using the Genomic DNA PrepPlus kit (A&A Biotechnology), following the manufac-turer’s instructions. The concentration of DNA wasestimated by electrophoresis of 2

µ

L DNA, together witha DNA marker, on a 1% agarose gel with 1

×

TEA bufferand stained with ethidium bromide.

Amplification of the ITS1 region

DNA amplification was performed using D21 and D22primers (Manceau & Horvais, 1996) according to theprotocol of Sorensen

et al

. (1998), with modifications. Theamount of DNA used was 20 ng, and 0·5 U of

Taq

DNApolymerase (Fermentas, Lithuania) was applied. The PCRamplifications were performed in a PTC-200 thermocycler(MJ Research) with the following parameters: templatedenaturation at 94

°

C for 1·5 min, primer annealing at 65

°

Cfor 1·5 min, and DNA extension for 3·0 min at 72

°

C. ThePCR was repeated for 35 cycles, with a final extensionstep of 10 min at 72

°

C. Amplicons were detected byelectrophoresis on a 1·5% agarose gel with 1

×

TEA buffer,stained with ethidium bromide and photographed using adigital camera and a UV trans-illuminator.

Amplification of the 752 bp fragment of

syrB

To identify

P. syringae

pv.

syringae

strains, PCR was doneusing

syrB

-based B1 and B2 primers (Sorensen

et al

.,

Plant Pathology

(2007)

56

, 373–382

Characterization of


pv

.

lachrymans 375

1998). The

syrB

gene encodes syringomycin toxin, and ischaracteristic for P. syringae pv. syringae. The reaction wascarried out for all strains in the collection, with the samePCR conditions as used for amplification of the ITS1 region.

PCR-RFLP analysis of the ITS1 region and the 752 bp syrB fragment

PCR-RFLP analysis of the ITS1 amplicons was performedusing enzymes BamHI, Bsu15I, BsuRI, Eco32I, EcoRI,Hin6I, HindIII, Hinf I, HpaII, PstI, RsaI, TasI, TaqI, XbaIand XhoI (Fermentas). The PCR-RFLP analysis of thesyrB 752 bp fragment was performed using HinfI, HpaIIand TaqI. The resulting target PCR band was excisedfrom the gel and purified using QIAquick Gel Extractionkit (Qiagen), following the manufacturer’s instructions.

The purified DNA (150 ng) was digested with 15 U ofenzyme for 2 h at the temperature and buffer recom-mended for each enzyme. Digested DNA fragments weredetected by electrophoresis on a 2% agarose gel with1× TEA buffer stained with ethidium bromide.

ADSRRS

Eighteen pathogenic and one saprophytic strain from thecollection were included. Genomic DNA (500 ng) wasdigested with two enzymes: XbaI (15 U µL−1; Eurx) andBglII (10 U µL−1; Fermentas) and the digested DNAprecipitated from the solution and dried, as describedby Masny & Plucienniczak (2001). Ligation was performedusing a short adapter, corresponding to the restriction siteleft by XbaI – POWIEBIS/XbaLIG, and a long adapter

Table 1 A list of bacterial strains isolated from cucumber leaves with angular leaf spot symptoms, collected in Poland in 2001 and 2002, and subjected to LOPAT and pathogenicity tests on cucumber, and the PCR reaction for syrB gene amplification product detection

Identification No. of strain

Place and Yearof isolationa

Detectionof syrBb

Fluorescenceon KBc

LOPAT tests resultsd LOPAT group

Pathogenicity on cucumber leaves

L O P A T Pe Symptoms

814/98 ref. strain − − + − + − − + Ia + water-soaked, necrotic lesionsLMG 5070 ref. strain − − + − − − + Ia + limited necrotic lesions, haloYPG 1293 ref. strain − + + − + − − + Ia + limited necrotic lesions, haloPss 2905 ref. strain + + + − − − + Ia + numerous, tiny chlorotic lesionsPst 3 ref. strain − + + − + − − + Ia − no symptomsWH 1/01 SP, 2001 − + + − − − + Ia + small lesionsWH 13/02 SP, 2002 − + − + + + − IVb − no symptomsWH 2/01 SP, 2001 − + + − − − + Ia + small lesionsWH 3/01 SP, 2001 − + + − − − + Ia + small lesionsWH 6/01 SP, 2001 − + + − + − − + Ia + small lesionsWH 7/02 SP, 2002 − − + − − − − − − no symptomsWK 1/02 SP, 2002 + + + − + − − + Ia + numerous, tiny chlorotic lesionsWK 2/02 SP, 2002 − − − − + − + II + a few large, dry necrotic lesionsWK 5/02 SP, 2002 − + − + + − + − IVb − no symptomsWK 8/02 SP, 2002 − + − − + − + II + a few large, dry necrotic lesionsWK 9/02 SP, 2002 − + − − + − + II + a few large, dry necrotic lesionsWK 10/02 SP, 2002 − + − − + − + II + a few large, dry necrotic lesionsWM 2/02 CP, 2002 − + + − + − − + Ia + limited necrotic lesions, haloWW 135/01 CP, 2001 − − + − + − + − − − no symptomsWW 17/01 CP, 2001 + + + − + − − + Ia + numerous, tiny chlorotic lesionsWW 18/02 CP, 2002 − + + − + + − + − IVa − no symptomsWW 19/02 CP, 2002 − + + − + − + − Vb − no symptomsWW 20/02 CP, 2002 − − − − + − − − − − no symptomsWW 254/02 CP, 2002 + + + − − − + Ia + numerous, tiny chlorotic lesionsWW 27/01 CP, 2001 + + + − − − + Ia + numerous, tiny chlorotic lesionsWW 3/02 CP, 2002 − − − − − − − − − no symptomsWW 4/02 CP, 2002 − + + − − − + Ia + limited chlorosisWW 5/02 CP, 2002 − − − − + − + II + few large dry necrotic lesionsWW 6/02 CP, 2002 − + − − + − − + II + limited chlorosisWW 7/02 CP, 2002 − − − − − − − − − no symptoms

aThe place and year of collection and isolation, where ref. strain = strain obtained from bacterial culture collection, SP = South Poland and CP = Central Poland.bStrains for which the syrB gene amplification product (752 bp) was detected. (+) = yes and (−) = no.cFluorescence on KB medium (King et al., 1954). (+) = yes and (−) = no.dResults of the LOPAT tests: (+) = positive, (−) = negative, and (+ −) = variable reaction, with the classification group of the LOPAT scheme determined in accordance with Lelliot et al. (1966).eP = the pathogenicity of the strains on cucumber accessions susceptible to angular leaf spot caused by Pseudomonas syringae pv. lachrymans. (+) = yes, and (−) = no; with specified symptoms.

Plant Pathology (2007) 56, 373–382

376 H. Olczak-Woltman et al.

BOXG/BOXD, corresponding to the restriction site leftby BglII (Masny & Plucienniczak, 2001). The DNA wasnot precipitated after ligation, since this process had noinfluence on the results (data not shown). PCR reactionswere performed using two primers: BOXPOWIE andPOWIEBIS (50 pmol each) (Masny & Plucienniczak,2001). PCR was performed in a PTC-200 thermocycler asfollows: 72°C for 2 min, and left constantly at 72°C untilTaq polymerase was added, additional 2 min at 72°C forfilling in the ends of the DNA fragments, followed bydenaturation at 94°C for 1 min. Thereafter, 25 cycleswere performed at: 94°C for 20 s, 65°C for 15 s, and68°C for 1 min, followed by an additional extension for2 min at 72°C. The amplified products were separated byelectrophoresis on a 6% native polyacrylamide gel with1× TAE buffer, and stained with ethidium bromide(0·5 mg L−1 aqueous solution) for 10 min. Images of thegels were taken using a digital camera and a UV trans-illuminator. Each amplification was carried out twice.

PCR-MP

The PCR-MP method described by Masny & Plucienniczak(2003) was used. Melting temperatures of genomic DNAfragments obtained after restriction digestion are dependenton their GC content and length. Lowering the denatura-tion temperature (Td) decreased the number of amplifiedfragments because only ssDNA molecules served astemplates (Masny & Plucienniczak, 2003). PCR-MP wasperformed for P. syringae pv. syringae 2905 and strainsWK 1/02, WW 17/01, WW 27/01 and WW 254/02.Genomic DNA (500 ng) was digested with HindIII (10 UµL−1; Fermentas) and ligation performed with an adapterassembled from two oligonucleotides POWIE and HIN-LIG at 20 pmol each (Masny & Plucienniczak, 2003). TheDNA phenol-chloroform-isoamyl extraction after diges-tion was omitted, as this process had no influence on theresults (data not shown). The PCR reaction was preparedwith the use of 50 pmol of POWAGCTT primer (Masny& Plucienniczak, 2003). Thermal cycling was performed ina PTC-200 thermocycler using two different denaturationtemperatures: 86°C and 85°C. The cycles were: 72°C for2 min to release the unligated oligonucleotides HINLIG,left constantly until 2·5 U of Taq polymerase was addedand mixed with a pipette tip, then left for an additional4 min at 72°C for filling in the ends of DNA fragments andcreating amplicons. Subsequently, the initial denaturationat 86°C or 85°C was performed for 2 min. Thereafter, 30cycles of denaturation at 86°C or 85°C for 80 s and anneal-ing with elongation at 68°C for 1 min were repeated,followed by 2 min at 68°C. The amplified products weredetected as described for the ADSRRS method. Repro-ducibility of the PCR-MP method was checked twice.

Data analysis

Polymorphisms generated by PCR-RFLP, ADSRRS andPCR-MP were analysed using the Gel Compare II v.2·5 software (Applied Maths). Genetic similarity (%) of

HpaII PCR-RFLP patterns of the bacterial strains wascalculated using the Dice coefficient and a dendrogramwas constructed with UPGMA. Genetic similarities of thestrains based on ADSRRS and PCR-MP patterns were basedon the Pearson correlation, and dendrograms producedusing UPGMA and neighbour-joining clustering methods.

Results

Phenotypic characterization of Pseudomonas syringae

Two of the three P. syringae pv. lachrymans reference strains(814/98 and YPG 1293) exhibited pectolytic activity, andtwo (814/98 and LMG 5070) did not fluoresce on KBmedium (Table 1). Pseudomonas syringae pv. tomato ref-erence strain Pst 3 exhibited limited pectolytic activity andproduced little levan from sucrose. Ten of the 25 Polishcucumber strains (40%) performed as expected forP. syringae LOPAT group Ia (+ − − − +), and six strains(24%) displayed results typical of the P. viridiflava groupII (− − + − +). All ten Polish cucumber strains from groupIa were fluorescent on KB medium, whereas two of the sixstrains belonging to LOPAT group II (WK 2/02 and WW5/02) were nonfluorescent on KB. Generally, strains fromLOPAT groups Ia and II produced blue-coloured fluores-cence on KB medium. All strains in LOPAT groups Ia andII produced a hypersensitivity reaction (HR) on tobaccoleaves within 24 h, confirming their pathogenicity (Table 1).Four Polish cucumber strains (16%) resulted in LOPATresults indicative of groups IVa, IVb or Vb. These strainsproduced intense, green fluorescent pigment on KB andwere nonpathogenic on tobacco leaves. Five nonfluores-cent strains (20%) could not be classified using theLOPAT tests, indicating they were unknown saprophyticspecies. Interpretation of the LOPAT tests results wasambiguous (‘+ −’ Table 1) for several cases, especially forthe pectolytic activity test.

Pathogenicity on cucumber leaves

The bacterial strains isolated from cucumber leaves andclassified by the LOPAT tests exhibited diverse patho-genicity results on susceptible cucumber accessions (Fig. 1).Pseudomonas syringae pv. lachrymans reference strain814/98 caused typical angular leaf spot symptoms oncucumber leaves (water-soaked, necrotic lesions eachwith a large chlorotic halo, and bacterial ooze on the under-side of the inoculated leaf) and was the most pathogenicof the strains tested (Fig. 1a). Pseudomonas syringae pv.lachrymans reference strains YPG 1293 and LMG 5070were only weakly pathogenic on cucumber leaves causingfast drying water-soaked lesions, and dry, light-coloured,papery lesions with or without a chlorotic halo (Fig. 1b).Strains WH 1/01, WH 2/01, WH 3/01, WH 6/01 and WM2/02, classified as LOPAT group Ia resulted in similarsymptoms to the P. s. pv. lachrymans YPG 1293 and LMG5070 reference strains, with additional limited chlorosis.This suggests that these strains belong to P. s. pv. lachry-mans. In contrast, strain WW 4/02, although classified


Characterization of Pseudomonas syringae pv. lachrymans 377

into LOPAT group Ia, produced only chlorotic lesions oncucumber leaves (Table 1).

The pathogenicity test results showed that strain 2905of P. syringae pv. syringae and four cucumber strains (WK1/02, WW 17/01, WW 27/01 and WW 254/02) causedsimilar symptoms when inoculated onto cucumber leaves(Table 1). These symptoms were numerous tiny chloroticlesions around dry, light-coloured, papery lesions on thecucumber leaves (Fig. 1 c), which were quite different fromthose caused by the P. syringae pv. lachrymans referencestrains. Strains that resembled P. viridiflava, according tothe LOPAT tests, caused large, round, papery, necroticlesions on cucumber leaves, similar to those caused by thereference strain LMG 5070, although without water-soaked lesions. The sole exception was strain WW 6/02,classified by the LOPAT tests as belonging to group II,which only produced chlorotic lesions on cucumber leaves.Strains classified into LOPAT groups IVa, IVb, Vb or notclassified into any group did not produce any symptomson cucumber leaves. These results confirmed that thesestrains were saprophytic.

Amplification of the ITS1 region

Primers D21 and D22 amplified a 560 bp fragment of the16S-23S rDNA spacer region from all of strains classifiedinto groups Ia and II by LOPAT tests (data not shown).Strains WK 1/02, WW 17/01, WW 27/01 and WW 254/02produced an amplicon of approximately 600 bp. Asmaller product, ranging in size from 540 to 560 bp, wasamplified from the saprophytic strains classified intoLOPAT groups IVa, IVb and Vb, and for those strains notclassified by the LOPAT scheme. Three strains from thisgroup had no amplification product (WH 13/02, WW 3/02, WW 20/02). Saprophytic strain WW 7/02 producedtwo amplicons, and saprophytic strain WK 5/02 pro-duced four closely spaced bands ranging in size from 270to 500 bp (data not shown).

Amplification of the syrB 752 bp fragment

Reference strain 2905 of P. syringae pv. syringae andstrains WK 1/02, WW 17/01, WW 27/01, and WW 254/02

isolated from cucumbers grown in Poland were all positive(Table 1). No amplification product was detected for theother strains. PCR-RFLP analysis of the syrB amplicon didnot reveal any polymorphisms among strains WK 1/02,WW 17/01, WW 27/01, WW 254/02 and the referencestrain 2905 (data not shown).

PCR-RFLP analysis of the ITS1 region

Only two of the 15 evaluated endonucleases, RsaI andHpaII, digested the PCR product well enough to yieldlegible, polymorphic patterns (data not shown). PCR-RFLPusing HpaII revealed more polymorphism than RsaI, andtyped the strains into six groups based on one to threepolymorphic bands (data not shown). Strains identified asP. syringae pv. syringae displayed a single banding patternafter PCR-RFLP analysis of the ITS1 fragment. StrainsWH 1/01, WH 2/01, WH 3/01 and WH 6/01, all isolatedfrom Polish cucumber plants collected in one location, insouthern Poland in 2001 and typed as pathovar lachrymansaccording to LOPAT and pathogenicity tests, exhibited asingle PCR-RFLP pattern. In addition, single banding pat-terns were obtained for strains classified as LOPAT groupII, i.e. WK 8/02, WK 9/02 and WK 10/02, all of whichcaused similar disease symptoms on cucumber (a fewlarge, dry necrotic lesions) and were isolated from leavescollected in southern Poland in 2002. The three P. syrin-gae pv. lachrymans reference strains had different bandingpatterns, which agrees with results of the pathogenicitytests (Table 1). However, the same banding patterns wereobtained for reference strains of P. syringae pv. lachrymans(LMG 5070) and P. syringae pv. tomato (Pst 3) as well as forYPG 1293 of P. s. pv. lachrymans and unknown WW 19/02(data not shown). These results indicate that PCR-RFLPanalysis of the ITS1 region does not fully differentiate thecucumber pathogenic P. syringae strains from the cucumbernon-pathogenic strains.

Strain differentiation by ADSRRS fingerprinting

ADSRRS fingerprinting showed significant diversity in thebanding patterns for the cucumber strains and referencestrains (Fig. 2). Each pattern consisted of approximately

Figure 1 Results of the pathogenicity tests on cucumber leaves of the accession ‘Line B’ susceptible to Pseudomonas syringae pv. lachrymans. Leaves were sprayed with inoculum containing 1 × 107 CFU mL−1 and results recorded 7 days after inoculation. (a) and (b): leaves inoculated with two reference strains of P. syringae pv. lachrymans (814/98 and LMG 5070, respectively); (c): leaf inoculated with original strain WW 17/01, isolated from cucumber leaves in Poland and later determined to be P. syringae pv. syringae.



25 bands, ranging in size from 100 to over 1200 bp. DNAbands larger than 1200 bp were not distinguishable due tosmearing. Identical results of ADSRRS were obtainedwhen the experiment was repeated.

Pseudomonas syringae pv. lachrymans reference strains814/98, LMG 5070 and YPG 1293 showed diverse band-ing patterns with only three common bands out of 20amplified (Fig. 2). The four strains identified as P. syringaepv. syringae, i.e. WK 1/02, WW 17/01, WW 27/01 andWW 254/02, had nine common bands, eight of whichwere the same size as for pathovar syringae referencestrain 2905. Furthermore, three additional commonbands were amplified for reference strain 2905, WW 17/01and WW 27/01, and strains 2905 and WW 254/02shared two other bands. The most different pattern wasobserved for the saprophytic strain WH 7/02 (Fig. 2).ADSRRS fingerprinting identified three homogenousgroups of strains with identical banding patterns: i) strains

WW 17/01 and WW 27/01; ii) strains WH 1/01, WH 2/01,WH 3/01; and iii) strains WK 8/02, WK 9/02, WK 10/02.The first two homogenous groups were composed ofstrains isolated from one leaf each, and the third groupfrom leaves collected in one field during the same year(2002).

Strain differentiation by PCR-MP fingerprinting

As expected, more DNA bands were obtained after PCRamplification at a Td of 86°C (16–23 bands) than at 85°C(8–20 bands) (Fig. 3). However, for reference strain 2905,a similar number of bands (19) was obtained for bothdenaturation temperatures. No differences in bandingpatterns were detectable for WW 17/01 and WW 27/01,two strains isolated from the same diseased leaf in 2001.Moreover, WW 254/02, which was isolated from a leafcollected in the same field in 2002, showed a very similar

Figure 2 The amplification of DNA fragments surrounding rare restriction sites (ADSRRS) fingerprinting patterns obtained from reference strains and original strains isolated from cucumber leaves in Poland in 2001 and 2002 (Table 1). Lane M, DNA molecular weight marker (fragment lengths in base pairs, bp). Lane 1, P. syringae pv. lachrymans 814/98; 2, P. syringae pv. lachrymans YPG 1293; 3, P. syringae pv. lachrymans LMG 5070; 4, P. syringae pv. tomato Pst 3; 5, P. syringae pv. syringae 2905; 6, WW 17/01; 7, WW 27/01; 8, WW 254/02; 9, WK 1/02; 10, WH 2/01; 11, WH 6/01; 12, WK 2/02; 13, WW 4/02; 14, WW 5/02; 15, WW 6/02; 16, WK 8/02; 17, WK 10/02; 18, WM 2/02; 19, WH 7/02.

Figure 3 The PCR–melting profile (PCR-MP) fingerprinting patterns of reference strain Pseudomonas syringae pv. syringae (2905) and original bacterial strains isolated from cucumber leaves in Poland and later determined as belonging to P. syringae pv. syringae (Table 1). Reactions were performed using two denaturation temperatures, 85°C and 86°C. Lane M, DNA molecular weight marker (fragment lengths in base pairs, bp). Remaining lanes are described in the figure.



banding pattern to WW 17/01 and WW 27/01. P. syringaepv. syringae reference strain 2905 and strain WK 1/02(isolated from a cherry tree and cucumber leaf collected insouthern Poland, respectively) were different in bandingpattern from WW 17/01, WW 27/01 and WW 254/02.Four common bands (900 bp, 800 bp, 400 bp, and 320 bp)were detected for all strains tested by PCR-MP (Fig. 3).

Genetic similarity analysis

Results of the dendrogram depicting genetic similarityamong bacterial strains, based on HpaII PCR-RFLPanalysis of the ITS1 region, was similar to results of thepathogenicity and LOPAT tests results (Fig. 4). Strain2905 and the four cucumber strains classified as P. syringaepv. syringae were clustered together. WW 4/02 and WW6/02, which were in groups Ia and II according to LOPAT,and both of which caused only chlorosis on cucumber

leaves, were also clustered together. Four strains classifiedas P. syringae pv. lachrymans, pathogenic on cucumberleaves (WH 1/01, WH 2/01, WH 3/01 and WH 6/01)were also grouped into one cluster. Within each of theseclusters, genetic similarity of the strains was 100%. How-ever, the pathovar lachrymans reference strain 814/98 andpathovar syringae strains were also grouped into one clusterwith 100% genetic similarity. P. syringae pv. lachrymansreference strain LMG 5070 and P. syringae pv. tomatoreference strain Pst 3 clustered together with 65% geneticsimilarity (Fig. 4).

Genetic similarities estimated by Pearson correlationcoefficients of ADSRRS fingerprints and calculated by theneighbour-joining clustering grouped the bacterial strainsinto three main clusters (Fig. 5). All reference strains,except pathovar syringae strain 2905, grouped into onecluster, along with WW 5/02 (LOPAT group II) and WH1/01, WH 2/01 and WH 3/01 (LOPAT group Ia, classified

Figure 4 Dendrogram of the genetic similarity (%) of bacterial reference strains and original strains isolated from cucumber leaves in Poland in 2001 and 2002. Bands obtained after digestion of the amplified product of the ITS1 region with HpaII (PCR-RFLP). Dendrogram constructed using Gel Compare software, based on the Dice coefficient and the UPGMA clustering method.

Figure 5 Dendrogram of the genetic similarity (%) of 18 pseudomonad strains pathogenic to cucumber and one saprophytic strain (WH 7/02). Strains were isolated from cucumber leaves in Poland in 2001 and 2002 and analysed in comparison to reference strains (see Table 1). Bands were obtained by amplification of DNA fragments surrounding rare restriction sites (ADSRRS) fingerprinting and analysed using Gel Compare software. The resulting dendrogram was based on the Pearson correlation coefficient and the neighbour-joining clustering method. The strains of Pseudomonas syringae pv. lachrymans, P. syringae pv. syringae and P. viridiflava grouped into different clusters indicating their diversity. Saprophytic strain WH 7/02 formed a separate group.



as pathovar lachrymans). The second cluster containedstrain 2905 and four cucumber strains of P. syringaepv. syringae, at a similarity of 60%, as well as the otherstrains from LOPAT groups Ia and II. WH 7/02 was theonly saprophytic strain evaluated by ADSRRS finger-printing and clustered separately (Fig. 5). However, theUPGMA clustering analysis (data not shown) did notindicate WH 7/02 as an outside group. Therefore, theneighbour-joining dendrogram agreed more with thepathogenicity results than did UPGMA. In the UPGMAdendrogram, pathovar lachrymans reference strains 814/98and YPG 1203, and strain Pst 3 of pathovar tomatoclustered together. P. syringae pv. lachrymans referencestrain LMG 5070 clustered with strain WH 2/01. Strainsof LOPAT group II were classified into different clusters(data not shown).

Genetic analysis based on PCR-MP fingerprinting,with a PCR Td of 86°C, and neighbour-joining (Fig. 6)or UPGMA (data not shown) clustering, suggested a highgenetic similarity (80%) of strains WK 1/02, WW 17/01,WW 27/01 and WW 254/02 to P. syringae pv. syringaereference strain 2905.

Discussion

Genetic characterization and fingerprinting of plantpathogenic bacteria by various molecular methods hasbeen the subject of a number of papers (Louws et al., 1994;Manceau & Horvais, 1996; Weingart & Volksch, 1997;Clerc et al., 1998; Manceau & Brin, 2003; Stead et al.,2003; Tian et al., 2003). Toxin-specific primers and rep-PCR have been shown to be useful for discrimination andidentification of certain pathovars, while techniques suchas AFLP or RAPD have been shown to be useful for deter-mining genomic differences among strains of P. syringae(Louws et al., 1994; Manceau & Horvais, 1996; Clercet al., 1998; Manceau & Brin, 2003).

The ADSRRS and PCR-MP fingerprinting methods arenew, powerful tools for differentiation of bacterial strains.ADSRRS fingerprinting identified clinical bacterial strains,including those of Enterococcus faecium, Escherichia colior Klebsiella pneumoniae (Masny & Plucienniczak, 2001;Krawczyk et al., 2002). The identical results with ADSRRSobtained in independent experiments performed fromDNA isolated from different cultures of the same strainshowed the method to be highly reproducible (Masny& Plucienniczak, 2001).

In this study, it was demonstrated that ADSRRS andPCR-MP clearly differentiated P. syringae pv. lachrymans,P. syringae pv. syringae and related strains isolated fromcucumber with angular leaf spot symptoms. Moreover,the accuracy of these methods at pathovar level was muchhigher than with PCR-RFLP analysis of the ITS1 region.Strain differentiation and identification based on thesemolecular methods agreed closely with traditional bio-chemical and pathogenicity tests.

The genetic diversity of plant microbes, even thosewithin a pathovar or species, is vast. Therefore, whendrawing conclusions about the differentiation of plantpathogens, it is necessary to compare results obtained fromdifferent techniques. Pathogenicity tests performed withthe strains from this collection resulted in various diseasesymptoms on cucumber leaves. The three P. syringae pv.lachrymans reference strains obtained from culture collec-tions caused a range of disease symptoms on cucumberleaves. Strain 814/98 produced the highest disease severityand caused typical symptoms on tested cucumber acces-sions, i.e. water-soaked lesions with a large chlorotic halo,and presence of bacterial exudates, whilst strains LMG5070 and YPG 1293 caused a few brown to black lesionswith limited chlorosis. Strains WH 1/01, WH 2/01, WH3/01, WH 6/01 and WM 2/02 caused similar diseasesymptoms to LMG 5070 and YPG 1293 but were evenless aggressive. This variation in disease symptoms reflectedthe diversity among strains shown by PCR-RFLP andADSRRS analysis, which revealed diverse DNA bandingpatterns of strains 814/98, LMG 5070, and YPG 1293.The same symptomatology and DNA patterns wereobserved for WH 1/01, WH 2/01, WH 3/01 and WH 6/01isolated from a single cucumber leaf collected in one field.In contrast, for strains isolated from different yearsor regions of Poland (e.g. WH 6/01, WM 2/02 and WW4/02) the DNA banding patterns and disease symptomswere different. These results agree with the rep-PCR andAFLP results of Stead et al. (2003) and Manceau & Brin(2003), respectively, demonstrating the genetic heteroge-neity in virulence in P. syringae pv. lachrymans.

Further diversity in P. syringae pv. lachrymans referencestrains of 814/98 and YPG 1293 was revealed by bothhaving pectolytic activity which is atypical for P. syringae(Lelliott et al., 1966). Similarly, two cucumber strains ofP. syringae pv. lachrymans, WH 6/01 and WM 2/02, alsoexhibited pectolytic activity: P. syringae pv. lachrymanshas been shown to be one of the few P. syringae pathovars

Figure 6 Dendrogram of the genetic similarity (%) of Pseudomonas syringae pv. syringae reference strain and four original bacterial strains (WK 1/02, WW 17/01, WW 27/01, and WW 254/02) isolated from cucumber leaves in Poland in 2001 and 2002 and determined to belong to P. syringae pv. syringae. Dendrogram was based on PCR-melting profiles fingerprint patterns after amplification at Td = 86°C, and constructed using Gel Compare software, based on the Pearson correlation coefficient and the neighbour-joining clustering method.



that frequently possess pectolytic activity (Riffaud et al.,2003). Both PCR-RFLP and ADSRRS revealed significantdiversity among P. syringae pv. lachrymans strains that wereclustered together with pathovars tomato and syringae orP. viridiflava. Finding that the strains of P. syringae pv.lachrymans grouped into different clusters was not un-expected. Such results have previously been demonstratedand it has been suggested that P. syringae pv. lachrymansis genetically heterogeneous (Manceau & Brin, 2003;Stead et al., 2003).

The pathogenicity tests showed that the reference strain2905 of pathovar syringae and the cucumber strainsWK 1/02, WW 17/01, WW 27/01 and WW 254/02 causedatypical symptoms that consisted of numerous tiny, chlo-rotic lesions, which differed from the water-soaked lesionscaused by P. syringae pv. lachrymans on cucumber leaves.Therefore, a 752 bp fragment was amplified from the syrBgene, which is responsible for syringomycin synthesis, acharacteristic typical of P. syringae pv. syringae (Quigley& Gross, 1994). The 752 bp product was present instrains 2905, WK 1/02, WW 17/01, WW 27/01 and WW254/02. No differences in the 752 bp amplicon were foundamong the strains by PCR-RFLP analysis. This furthersupports P. syringae pv. syringae being a natural pathogenof cucumber (Bradbury, 1986). Dendrograms depicting thegenetic similarity of the pathovar syringae strains, basedon PCR-RFLP analysis of the ITS1 region, confirmed thehomogeneity of these strains. However, a dendrogramdepicting the genetic similarity based on ADSRRS finger-printing indicated that strain WK 1/02 was the leastrelated genetically of these P. syringae pv. syringae strains,perhaps because strain WK 1/02 originated from southernPoland, whereas the other three cucumber strains wereisolated from central Poland.

Several strains of Pseudomonas isolated from cucumberleaves collected in Poland (WK 2/02, WK 8/02, WK 9/02,WK 10/02, WW 5/02, and WW 6/02) were classified asbelonging to group II according to LOPAT, indicating thatthey were P. viridiflava-like strains: P. viridiflava is anon-host specific pathogen and has been described as agenetically heterogeneous species (Stead et al., 2003; Tianet al., 2003; Moretti et al., 2005). This heterogeneity wasrevealed by the molecular tests; dendrograms groupedP. viridiflava-like strains into different clusters. Previously,P. viridiflava has been identified on Cucumis species(Goumans & Chatzaki, 1998; Riffaud et al., 2003; Morettiet al., 2005), and isolated from cucumber or melon leaveswith identification based on biochemical and physiologicaltests. In this study, the LOPAT results for cucumber strainsWK 2/02, WK 8/02, WK 9/02, WK 10/02, WW 5/02 andWW 6/02 indicated that these strains were P. viridiflava-like(Table 1). These isolates did not produce levan on sucrosemedium and clearly exhibited pectolytic activity. Onthe other hand, although pectolytic activity is typical forP. viridiflava strains, P. syringae pv. lachrymans have alsobeen reported to exhibit this property (Riffaud et al., 2003).The former has been shown to cause necrotic lesions onplant leaves without water-soaked symptoms and plantsreadily recover from the infection (Moretti et al., 2005)

and similar results were observed in this study for strainsclassified as P. viridiflava-like.

Overall, the results of this study indicate that path-ogenicity tests combined with both biochemical andmolecular analysis are accurate for differentiation anddiscrimination of plant pathogenic bacteria. Moreover,genetic clustering results should always be analysed takinginto account additional knowledge about the origin andproperties of the bacteria strains. It has been demonstratedthat P. syringae pv. lachrymans is genetically diverse, asexhibited by pathogenicity, biochemical and moleculartests. ADSRRS and PCR-MP fingerprinting methods suc-cessfully differentiated plant pathogens from saprophytes,and are reproducible and rapid techniques for determin-ing genetic differences among pathovars of P. syringae.

Acknowledgements

We thank Dr Malgorzata Schollenberger (Warsaw Agri-cultural University, Poland) and Professor Piotr Sobicze-wski (Research Institute of Pomology and Floriculture,Skierniewice, Poland) for supplying bacterial strains, andDr Anna Gzyl and Msc. Ewa Mosiej (National Institute ofHygiene, Warsaw, Poland) for help with genetic similarityanalysis. We thank also Dr Michael Havey (USDA andUniversity of Wisconsin, Madison, WI) for critical read-ing of the manuscript. This work was supported by grantNo 83/HiE/SK-114/2002 from the Agricultural PropertyAgency in Warsaw, Poland.

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