APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUly 1994, p. 2278-2285 Vol. 60, No. 70099-2240/94/$04.00+0Copyright ©) 1994, American Society for Microbiology

Derivation of Mutants of Erwinia carotovora subsp. betavasculorumDeficient in Export of Pectolytic Enzymes with Potential

for Biological Control of Potato Soft RotJOSE M. COSTA AND JOYCE E. LOPER*

Horticultural Crops Research Laboratory, U.S. Department of Agriculture, Agricultural Research Service, andDepartment of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97330

Received 13 January 1994/Accepted 19 April 1994

Erwinia carotovora subsp. betavasculorum Ecbl68 produces an antibiotic(s) that suppresses growth of therelated bacterium Erwinia carotovora subsp. carotovora in culture and in wounds of potato tubers. StrainEcbl68 also produces and secretes pectolytic enzymes and causes a vascular necrosis and root rot of sugar beet.Genes (out) involved in secretion of pectolytic enzymes by Ecbl68 were localized to two HindlIl fragments (8.5and 10.5 kb) of Ecbl68 genomic DNA by hybridization to the cloned out region ofE. carotovora subsp. carotovoraand by complementation of Out- mutants of E. carotovora subsp. carotovora. Out- mutants of Ecbl68, whichdid not secrete pectate lyase into the culture medium, were obtained when deletions internal to either Hindlllfragment were introduced into the genome of Ecbl68 through marker exchange mutagenesis. Out- mutants ofEcbl68 were complemented to the Out' phenotype by introduction of the corresponding cloned HindlIlfragment. Out- mutants of Ecbl68 were less virulent than the Out' parental strain on potato tubers. StrainEcbl68 and Out- derivatives inhibited the growth of E. carotovora subsp. carotovora in culture, indicating thatthe uncharacterized antibiotic(s) responsible for antagonism was exported through an out-independentmechanism. Strain Ecbl68 and Out- derivatives reduced the establishment of large populations of E.carotovora subsp. carotovora in wounds of potato tubers and suppressed tuber soft rot caused by E. carotovorasubsp. carotovora.

Erwinia carotovora subsp. carotovora is a major pathogen ofpotato, causing seed piece decay, black leg, and aerial soft rotin the field and soft rot of tubers in storage (29). Managementof diseases caused by this pathogen relies on cultural practicessuch as the use of certified seed, sanitation, and maintenanceof controlled environmental conditions for tuber storage.Biological control of seed piece decay by inoculation of seedpieces with antagonistic bacteria prior to planting has beensuccessful in field experiments (9, 19, 33, 44) and offerspromise for the management of the disease in commercialagriculture. Fluorescent pseudomonads are the predominantgroup of bacteria that have been evaluated for biologicalcontrol of seed piece decay and tuber soft rot (9, 14, 19, 33, 43).Fluorescent pseudomonads may not be ideally suited as bio-logical control agents under all conditions favoring tuber softrot or seed piece decay because environmental conditionsmaximizing populations of Pseudomonas spp. (16, 25) are notidentical to those most conducive for disease development (1,6, 22, 26). In certain environments, an avirulent strain of E.carotovora that proliferates under the same conditions as thetarget pathogen could be a superior antagonist.

Competition among strains of E. carotovora occurs in planttissues, including potato tubers (11). Erwinia carotovora subsp.betavasculorum Ecbl68, a pathogen that causes a vascularnecrosis and root rot of sugar beets (40), suppresses growth ofE. carotovora subsp. carotovora in culture and in wounds ofpotato tubers. The antagonistic activity of Ecbl68 is partially

* Corresponding author. Mailing address: Horticultural Crops Re-search Laboratory, Agricultural Research Service, U.S. Department ofAgriculture, 3420 N.W. Orchard Ave., Corvallis, OR 97330. Phone:(503) 750-8771; Fax: (503) 750-8764. Electronic mail addrcss: LoperJ@bcc.orst.edu.

due to its production of an antibiotic(s) with activity against abroad spectrum of E. carotovora subsp. carotovora strains (4).Although E. carotovora subsp. betavasculorum Ecbl68 reducespopulation densities of E. carotovora subsp. carotovora in softrot tissue, its potential to decrease the severity of tuber soft rothas not been evaluated because Ecbl68 itself macerates potatotubers (4). An avirulent mutant of Ecbl68 which no longermacerates potato tubers may be an effective agent for thebiological control of tuber soft rot.

Soft rot erwinias produce several cell wall-degrading en-zymes such as pectate lyases, polygalacturonases, and cellu-lases that contribute to their virulence (7). Attempts to derivean avirulent strain of E. carotovora subsp. betavasculorumcould target genes involved in pectolytic enzyme production,enzyme export, enzyme regulation, or host recognition, all ofwhich are virulence factors (7). Derivation of an avirulentstrain through sequential disruption of structural genes encod-ing pectolytic enzymes could be extremely laborious because asfew as one gene encoding a functional pectate lyase conferspathogenicity to Escherichia coli (18, 35). For example, a strainof Erwinia chrysanthemi with deletions in four structural genesencoding pectate lyases still macerates potato tuber tissue(34a). In contrast, strains with a single mutation in an out gene,required for secretion of these pectolytic enzymes, are aviru-lent (2) or have greatly reduced virulence (27). The purpose ofthis study was to identify out genes of E. carotovora subsp.betavasculorum and derive Out- mutants of E. carotovorasubsp. betavasculorum to test as agents for the biologicalcontrol of tuber soft rot caused by E. carotovora subsp.carotovora. Out- mutants had a greatly reduced capacity tomacerate potato tubers but inhibited the growth of E. caroto-vora in culture and in potato tissue, indicating that a functionalout region was not required for antibiotic secretion.

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Out- MUTANTS OF E. CAROTOVORA SUBSP. BETAVASCULORUM 2279

TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Description Relevant characteristics' Source or reference

E. coliDH5ot

S17-1

E. carotovora subsp.carotovora

AC5008AC5018Ecc7Ecc7b

E. carotovora subsp.betavasculorum

Ecbl 68Ecb 168c-6Ecb 168bLA290

LA291

LA292LA294

F- endAl hsdRJ7(rK- MK+) supE44thi-l recAI gyrA96 relAl 480dlacZAM15, A-

RP4 2-Tc::Mu-Km::Tn7 mobilizingstrain

out::Tn5out::TnlO-lacZWild typeDerivative of Ecc7

Wild typeDerivative of Ecbl68Derivative of Ecbl68Derivative of Ecbl68b, out::nptI-sacB-sacR in Sau3AI site

Derivative of Ecbl68b, out::nptl-sacB-sacR in BamHI site

Derivative of LA290, A(out)Derivative of LA291, A(out)

Out- Gibco BRL Life Technologies

Tra+, Tpr, Smr

Out-, KmrOut-, TcrOut+Out+, Nalr

Out+Acc+, Out+, RiftOut+, SmrOut+, Kmr, sucrose sensitive, Smr

Out-, Kmr, sucrose sensitive, Smr

Out-, SmrOut-, Smr

out genes of E. carotovora subsp.carotovora on 15.7-kb Sall fragmentcloned in pDSK519

cos, IncPl replicon, polylinker ofpUC8

Mobilizing plasmidMobilizing plasmidpUC4K derivative containing nptI-sacB-sacR cartridge

pVS1 and pACYC184 replicons,polylinker of pUC8

10.5-kb HindlIl fragment containingout gene(s) from Ecbl68 clonedinto pVSP61

10.5-kb HindIll fragment containingout gene(s) from Ecbl68 clonedinto pLAFR3

8.5-kb HindIII fragment containingout gene(s) from Ecbl68 clonedinto pLAFR3

8.5-kb HindIlI fragment containingout gene(s) from Ecbl68 clonedinto pVSP61

nptI-sacB-sacR cartridge cloned intoBamHI site of 10.5-kb HindIIlfragment of pJEL1898

nptl-sacB-sacR cartridge cloned intoSau3AI site of 8.5-kb Hindlllfragment of pJEL1899

pJEL1899 with internal CIaI deletionpJEL1898 with internal NsiI deletion

Out-, KMr 27

Tra-, Mob', Tcr

Tra+, KmrTra', AprKmr, Apr, sucrose sensitive

Mob+, KMr

Out+, Kmr

Out+, Tcr

Out+, Tcr

Out+, KMr

Out+, Tcr, KMr

Out+, Tcr, KMr

Out, TcrOut, Tcr

38

123934

W. Tucker, DNA PlantTechnologies, Oakland, Calif.

This study

This study

This study

This study

This study

This study

This studyThis study

" Acc+, accepts foreign DNA at a higher frequency than the wild-type strain; Mob', mobilizable plasmid; Out' and Out-, export proficient and export deficient,respectively; Tra+, self-transmissible plasmid or strain; Apr, ampicillin resistant; Kmr, kanamycin resistant; Nalr, nalidixic acid resistant; Rifr, rifampin resistant; Smr,streptomycin resistant; TCr, tetracycline resistant; Tpr, trimethoprim resistant.

(An abstract of this research has been published previously cultured on Luria-Bertani medium (LB) (36) at 35°C. E.[10].) carotovora subsp. carotovora and E. carotovora subsp. betavas-

MATERIALS AND METHODS

Bacterial strains and media. Bacterial strains and plasmidsused in this study are listed in Table 1. Escherichia coli was

culorum were cultured on LB at 27°C. Growth rates of E.carotovora subsp. betavasculorum strains were determined bymeasuring the change in optical density at 600 nm of culturesgrown with shaking (200 rpm) at 27°C in LB broth. In some

37

27274This study

4324This study

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This studyThis study

PlasmidspAKC601

pLAFR3

pRK2013pSa322pUM24

pVSP61

pJEL1888

pJEL 1898

pJEL1899

pJEL1903

pJELI915

pJEL1921

pJELl959pJEL1960

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cloning experiments, LB agar was supplemented with 5-bromo-4-chloro-3-indolyl-,-D-galactopyranoside (X-Gal) (40 ,g/ml;International Biotechnologies, Inc., New Haven, Conn.) andisopropyl-p-D-thiogalactopyranoside (IPTG) (100 ,ug/ml;Sigma Chemical Co., St. Louis, Mo.) for screening transfor-mants of E. coli. Antibiotics (Sigma) were used at the followingconcentrations: ampicillin, 100 ,ug/ml; kanamycin, 50 p.g/ml;rifampin, 100 jig/ml; and tetracycline, 20 jig/ml (except wheredifferent concentrations are specified).

Nucleic acid methods. Plasmids were isolated by an alkalinelysis procedure (36) and purified by ethidium bromide-cesiumchloride density gradient centrifugation. For isolation ofgenomic DNA, cells were lysed with sodium dodecyl sulfate,treated with proteinase K, and extracted with chloroform inthe presence of hexadecyltrimethylammonium bromide (3)prior to standard phenol-chloroform extraction and ethanolprecipitation. Electrophoresis was carried out in 0.5 to 1.0%agarose gels with Tris-phosphate-EDTA (TPE) buffer (36).Ligations, alkaline phosphatase treatments, restriction endo-nuclease digestions, and transformation procedures were stan-dard (36).

Southern hybridizations. Probes for Southern blots were (i)out region of E. carotovora subsp. carotovora, a 15.7-kb Sallfragment of pAKC601 (27); (ii) pJEL1921, which contained a8.5-kb genomic region of E. carotovora subsp. betavasculorumand the nptI-sacB-sacR cartridge cloned in pLAFR3; and (iii)pJEL1915, which contained a 10.5-kb genomic region of E.carotovora subsp. betavasculorum and the nptI-sacB-sacR car-tridge cloned in pLAFR3. Restriction fragments used asprobes were purified from agarose gels (SeaKem GTG; FMCBioProducts, Rockland, Maine) by adsorption and elutionfrom NA-45 DEAE membranes as recommended by themanufacturer (Schleicher & Schuell, Keene, N.H.). DNAprobes were labeled by random-hexamer-primer labeling with[32P]dCTP as recommended by the manufacturer (Gibco BRLLife Technologies, Inc., Gaithersburg, Md.). The 32P-labeledprobes were purified on a Nu-clean D-50 column (Internation-al Biotechnologies, Inc., New Haven, Conn.). Hybridizationconditions were moderately stringent (42°C, 50% formamide-0.16X SSC [lx SSC is 0.15 M NaCl plus 0.015 M sodiumcitrate]) and were followed by washes at 55°C in O.1X SSC(36).

Mobilization of plasmids. Plasmids pVSP61 and pLAFR3and their derivatives were mobilized from E. coli DH5o intothe rifampin-resistant strain Ecbl68c-6 of E. carotovora subsp.betavasculorum in triparental matings with pRK2013 as ahelper plasmid or from E. coli S17-1 in biparental matings.Transconjugants of strain Ecbl68c-6 harboring plasmids weretypically obtained at a frequency of ca. 10-' transconjugant perdonor. Plasmids were mobilized from Ecbl68c-6 into thestreptomycin-resistant strain Ecbl68b of E. carotovora subsp.betavasculorum by triparental matings with pRK2013 orpSa322 as the helper plasmid. Transconjugants of E. carotovorasubsp. betavasculorum harboring pLAFR3 derivatives wereselected on LB agar amended with tetracycline (60 ,ug/ml).

Cloning of out genes of E. carotovora subsp. betavasculorum.Genomic DNA of strain Ecbl68 was cut with HindlIl; frag-ments between 7 and 14 kb were isolated from agarose gels andcloned into pUC8 (42); and recombinant plasmids were intro-duced into E. coli DH5ot by transformation. We probed 764ampicillin-resistant, LacZ- colonies with the out region fromE. carotovora subsp. carotovora by colony hybridization. Insertsof hybridizing plasmids were cloned into pLAFR3 and pVSP61and tested for complementation of the out mutations of E.carotovora subsp. carotovora mutants AC5008 and AC5018

(27). Secretion of pectolytic enzymes was evaluated on pectate-yeast extract-citrus pectin (PYCP) agar (27).

Derivation of Out- derivatives of E. carotovora subsp.betavasculorum. The marker exchange eviction mutagenesistechnique of Ried and Collmer (34) was used to constructdirected, unmarked mutations in Ecbl68b, a streptomycin-resistant derivative of E. carotovora subsp. betavasculorumEcbl68. The nptI-sacB-sacR cartridge, which is carried on a3.8-kb BamHI fragment of pUM24 (34), confers kanamycinresistance to E. carotovora subsp. betavasculorum as a result ofnptI and confers sucrose sensitivity as a result of the productionof levan sucrase encoded by sacB. E. carotovora subsp. betavas-culorum grew on minimal 925 agar medium (21) amended with10% sucrose as the sole carbon source, whereas cells thatcontained the sacB gene did not grow on this medium.Exchange recombination events between unstable recombi-nant plasmids and the chromosome that resulted in insertionof the cartridge into the bacterial genome were selected on LBamended with kanamycin; those resulting in eviction of thecartridge from the genome were selected on minimal 925 agarmedium containing 10% sucrose. Cultures of Ecbl68b contain-ing pLAFR3 derivatives were grown at 27°C with shaking in200 ml of LB broth in the absence of tetracycline. After 10 to24 h, 0.1 ml of culture was transferred to 200 ml of fresh LBbroth. After four or five successive transfers, pLAFR3 deriva-tives were lost from more than 90% of bacterial cells.

Pectate lyase assay. Cultures of E. carotovora subsp. betavas-culorum were grown with shaking for approximately 12 h inPYCP medium at 27°C, and 1 ml was centrifuged at 6,000 x g.The cell pellets were washed once in cold fresh medium andsonicated in 1 ml of cold PYCP medium with three pulses of 30s each. Culture supernatant and sonicates were incubated at30°C for 30 min with a 2.5-ml aliquot of 0.1 M Tris-HCI buffer(pH 8.5)-1.5 M calcium chloride-0.1% polygalacturonic acid(8). The reaction was stopped by addition of 160 ,ul of ZnSO4and 80 ,ul of 1 M NaOH. The mixture was centrifuged (6,000x g), and 650 KIl of the resulting supernatant was mixed with anequal volume of thiobarbituric acid reagent; the mixture wasboiled for 30 min, and theA550 was measured (23). The proteincontent was determined by using a protein assay kit (Bio-RadLaboratories, Richmond, Calif.). One unit of enzyme activitywas defined as an increase of 1 A550 unit per hour permilligram of protein (23).

Protease assay. Protease activity was determined by usingthe gelatin plate assays described by Hankin and Anagnostakis(15). Samples (5 p,l) of cultures grown overnight were spottedonto the plate and incubated at 27°C for 48 h.

Antibiotic assay. Overnight bacterial cultures were spottedon nutrient agar (Difco) containing 1% glycerol (NAG),incubated for 48 h, and then oversprayed with a culture of thetest strain E. carotovora subsp. carotovora Ecc7 (ca. 108 CFUml ') (4).Tuber soft rot severity and competition among strains in

wounds of potato tubers. The severity of tuber soft rot causedby E. carotovora subsp. betavasculorum was evaluated by usingthe method of Axelrood et al. (4). Medium-size potato tubersof the cultivar Chieftain were washed, surface disinfested bybeing submerged in 0.5% sodium hypochlorite, and rinsedthoroughly with tap water. Tubers were wounded midwaybetween the apical and stem ends by being punctured with aflamed steel rod (diameter, 3 mm) to a depth of 15 mm. Potatojuice released by the wounding was removed by wicking with apaper tissue, leaving an air cavity in the wound. An inoculumwas prepared from 48-h-old cultures of individual strainsgrown at 27°C on NAG by washing and suspending cells in 0.1M potassium phosphate buffer (pH 7.0). The inoculum was

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H CE E Sau Ss C HI,

B nptU-sacB-sacR B

H CE E. Sau-' Ss C Hl,' ,L

H C C H

pJEL 1899

pJEL 1921

Restoration ofOut phenotype

pJEL 1959

1 kb

FIG. 1. Restriction map of insertion and deletion derivatives of the cloned 8.5-kb HinzdIIl out fragment of E. carotovora subsp. betaviasculorunmEcbl68 used to construct an Out- mutant and complementation of the out mutation of E. carotovora subsp. carotovora AC5008. Dashed linesdenote the insertion of the nptl-sacB-sacR cartridge into a Sau3AI site of pJEL1899 to create pJEL1921. Plasmid pJEL1959 was derived frompJEL1899 by deletion of an internal C/al fragment (deletion is indicated by the bar with diagonal lines). Restoration of the Out' phenotype wasdetermined on PYCP agar (27) and by the pectate lyase assay (8, 23). Abbreviations: B, BamnHI; C, Clal; E, EcoRI; H, HindIll; Sau, Sau3AI; Ss,SstI.

adjusted to 108 CFU/ml for strains of E. carotovora subsp.betavasculorumn, and 107 CFU/ml for E. carotovora subsp.carotovora. Cell densities were confirmed by dilution plating onNAG medium. Equal-volume suspensions of competing strainswere mixed immediately prior to inoculation. Suspensions ofeach individual strain were mixed with an equal volume of 0.1M potassium phosphate buffer (pH 7.0) for single-strain inoc-ulations. Then 20 ,ul of the inoculum was introduced into eachwound, giving a density of 106 CFU of E. carotovora subsp.betavasculonim per wound and a density of 105 CFU of E.carotovora subsp. carotovora per wound. Tubers were incu-bated in a moist chamber at 24°C for 48 h. Macerated tissuewas removed, weighed, and suspended in 0.1 M potassiumphosphate buffer (pH 7.0). Macerated tissue suspensions wereagitated by vortexing for 1 min, and the population size wasestimated by dilution plating on minimal 925 medium supple-mented with glucose (0.5%), cycloheximide (100 ,Lg/ml), andnalidixic acid (100 Lg/ml) or streptomycin (100 Lg/ml) toenumerate colonies of E. carotovora subsp. carotovora or E.carotovora subsp. betavasculorum, respectively. The detectionlimits of the dilution plating were approximately 100 CFU perwound. Densities of strains that were not detected were givenan arbitrary value of 99 CFU per wound.Data analysis. Tuber soft rot and bacterial population data

were analyzed by the analysis of variance procedure (SASInstitute, Inc., Cary, N.C.). To achieve homogeneity of vari-ances in the tuber soft rot data, the control (nontreated) data,which were more uniform than the data for bacterial treat-ments, were removed from the data set prior to analysis. Thelogarithmic (base 10) transformation was applied to individualestimations of bacterial population size prior to analysis.Fisher's protected least significant difference was used formean separation for the soft rot and bacterial population data(13).

RESULTSIdentification and cloning of out genes of E. carotovora

subsp. betavasculorum. Genes (olut) involved in secretion ofpectolytic enzymes by Ecbl68 were identified by hybridizationto the cloned out region of E. carotovora subsp. carotovora andby complementation of Out- mutants of E. carotovora subsp.carotovora. In Southern blots, a 10.5-kb HindlIl fragment of

genomic DNA of E. carotovora subsp. betavasculorurn Ecb168hybridized to the out region of E. carotovora subsp. carotovora.A second 8.5-kb HindIll fragment hybridized weakly to the outgene probe. From a library of genomic HindlIl fragments ofstrain Ecbl68 cloned in pUC8, plasmids containing the 8.5- or10.5-kb Hindlll fragments were identified by colony hybridiza-tion to the out gene probe of E. carotovora subsp. carotovora.Plasmids containing the 8.5- or 10.5-kb HindIlI fragmentrestored the Out' phenotype to the E. carotovora subsp.carotovora Out- mutant AC5008 or AC5018, respectively.

Derivation of Out- derivatives of E. carotovora subsp.betavasculorum. Sequences within the two out loci were deletedfrom the genome of E. carotovora subsp. betavasculorum bymarker exchange eviction mutagenesis (34). A 3.8-kb BamHIfragment containing the nptI-sacB-sacR cartridge was clonedinto a Sau3AI site of pJELI 899, which contained the 8.5-kb outHindlIl fragment, to obtain pJEL1921, a derivative of theunstable plasmid pLAFR3 (Fig. 1). From plasmid pJEL1921,which contained a functional out' gene, a Clal fragment wasdeleted to derive pJEL1959, which did not complement the outmutation of AC5008 (Fig. 1). The cartridge-containing regionof pJEL1921 was exchanged into Ecbl68b to produce LA290.The cartridge was then evicted from LA290 by exchangesubstitution of the sequences in pJEL1959 to produce theOut- strain LA292, which contained a genomic Clal deletion(Fig. 1).The nptI-sacB-sacR cartridge was also cloned into the

BamHI site of the insert in pJEL1898, which contained the10.5-kb out HindIll fragment, to obtain pJEL1915, a derivativeof the unstable plasmid pLAFR3 (Fig. 2). From plasmidpJEL1915, which contained a functional olut gene, a NsiIfragment was deleted to derive pJEL1960, which did notcomplement the out mutation of strain AC5018 (Fig. 2). Thecartridge-containing region of pJEL1915 was exchanged intoEcbl68b to produce LA291. The cartridge was then evictedfrom LA291 by exchange substitution of the sequences inpJEL1960 to produce the Out- strain LA294, which containeda genomic NsiI deletion (Fig. 2). Southern analysis of restric-tion enzyme-digested DNA probed with pJEL1921 or

pJEL1915 confirmed that insertions and deletions into thegenomic DNA of LA290, LA291, LA292, and LA294 were asdescribed in Fig. 1 and 2 (data not shown).

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H NN B S N HI I

B nptl-sacB-sacR B

H N"N B S N HI I " I

H N N H_mm

_f,I

pJEL 1898

pJEL 1915

Restoration ofOut phenotype

pJEL 1960

FIG. 2. Restriction map of insertion and deletion derivatives of the cloned 10.5-kb Hindlll out fragment from E. carotovora subsp.betavasculorum Ecbl68 used to construct an Out- mutant and complementation of the out mutation of E. carotovora subsp. carotovora AC5018.Dashed lines denote the insertion of the nptI-sacB-sacR cartridge into a BamHI site of pJEL1898 to create pJEL1915. Plasmid pJEL1960 was

derived from pJEL1898 by deletion of an internal NsiI fragment (deletion is indicated by the bar with diagonal lines). Restoration of the Out'phenotype was determined on PYCP agar (27) and by the pectate lyase assay (8, 23). Inserts in pJEL1898 and pJEL1960 were cloned in pVSP61to test for complementation of AC5018. Abbreviations: B, BamHI; H, HindIll; N, NsiI; S, Sall).

Characterization of Out- derivatives ofE. carotovora subsp.betavasculorum. Out- mutants of E. carotovora subsp. caroto-vora do not degrade pectate or secrete pectate lyases butexport proteases (27). The Out phenotype of E. carotovorasubsp. betavasculorum strains was evaluated by three charac-teristics: (i) pectate degradation on PYCP plates, (ii) secretionof proteases, and (iii) secretion of pectate lyase. Strains LA292and LA294, which contained genomic deletions in out regions,did not degrade pectate on PYCP plates. These strains pro-duced pectate lyase at levels equivalent to or in excess of thatproduced by the parental strain Ecbl68b, but the Out-mutants failed to export the enzyme (Table 2). PlasmidpJEL1888, which contained the cloned 10.5-kb out Hindlllfragment, and pJEL1903, which contained the cloned 8.5-kbout HindIlI fragment, restored pectate lyase secretion toLA294 and LA292, respectively. Therefore, a role of thecloned regions in pectate lyase export by strain Ecbl68b wasconfirmed. Out- mutant strain LA294 grew at a rate compa-rable to that of the parental strain Ecbl68 in LB, whereasLA292 grew more slowly than Ecbl68 (Table 2). Out- deriv-atives of E. carotovora subsp. betavasculorum utilized galactose,mannitol, maltose, or sorbitol as the sole carbon source, as didthe Out' parental strain. The Out' parental strain Ecbl68 andthe Out- derivative strains LA292 and LA294 secreted pro-tease, as observed on gelatin nutrient plates, indicating that theout region was not required for protease secretion. Out-

mutants of E. carotovora subsp. betavasculorum were notimpaired in antibiotic secretion in culture: zones of inhibitionagainst E. carotovora subsp. carotovora that surrounded colo-nies of Out' and Out- strains were similar in size (diameter,20 mm).

Virulence of Out- derivatives of E. carotovora subsp. be-tavasculorum. The Out- mutants LA292 and LA294 of E.carotovora subsp. betavasculorum caused less maceration ofpotato tuber tissue than did the Out' parental strain Ecbl68b;therefore, the Out- phenotype was associated with reducedvirulence of the bacterium (Table 3). Out- strains of E.carotovora subsp. betavasculorum established large populationsizes in wounds of potato tubers, although these populationswere smaller than those established by the Out' parentalstrain (Table 3).

Suppression of E. carotovora subsp. carotovora by E. caroto-vora subsp. betavasculorum in wounds of potato tubers. E.carotovora subsp. betavasculorum Ecbl68b reduced the popu-lation size of E. carotovora subsp. carotovora Ecc7b and theamount of tissue maceration caused by E. carotovora subsp.carotovora in wounds of potato tubers (Table 3). In addition,the establishment of Ecc7b was reduced by 6.6 logl0 CFU perwound when Ecbl68 and Ecc7b were coinoculated into potatowounds (Table 3). E. carotovora subsp. betavasculorum Out-strains caused reductions in populations of Ecc7b that rangedfrom 5.8 to 6.2 loglo CFU per wound. Strain Ecc7b had no

TABLE 2. Pectate lyase activity and generation times of E. carotovora subsp. betavasculorum and Out- derivatives'

Pectate lyase activityStrain Phenotype (U/mg of protein) in: % of pectate lyase Generation time

in supernatant (min)bCell lysate Supernatant

Ecbl68b Out+ 6.5 341.0 98.1 76.ObLA292 Out- 455.6 5.0 1.1 97.6aLA292(pJEL1903) Out+ 3.7 201.5 98.2 NDcLA294 Out- 435.0 4.4 1.0 72.2bLA294(pJEL1888) Out+ 4.0 93.3 95.9 ND

a Bacterial strains were grown in PYCP medium (27) for about 12 h at 28°C. Cell lysates were prepared by sonication. Values represent means of two replicatecultures.

b Values followed by a common letter are not different statistically (P = 0.05; Fisher protected least significant difference test).c ND, not determined.

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TABLE 3. Competition between E. carotovora subsp.betavasculorum Out- strains and an antibiotic-sensitive

E. carotovora subsp. carotovora strain in wounds of potato tubers

Log,,( CFU/woundb Amt ofmacerated

Strain(s)" tissue/E. carotovora subsp. E. carotovora subsp. wound

carotovora betavasculorum (g)h

Ecc7b 10.3a 1.22aEcbl68b 9.6a 0.56bEcc7b + Ecbl68b 3.7c 9.4ab 0.56bEcc7b + LA292 4.5b 9.1bc 0.34cLA294 8.9c 0.23cdLA292 8.9c 0.17dEcc7b + LA294 4.1b 9.Oc 0.17d

a Ecbl68b is the Out' E. carotovora subsp. betavasculorum parental strain;LA292 and LA294 are Out- derivative strains. Ecc7b is a nalidixic acid-resistantderivative of E. carotovora subsp. carotovora Ecc7 (4).

b Results represent mean values of three experiments involving 10 replicationseach. Means in the same column that are followed by a common letter are notdifferent statistically (P = 0.05; Fisher protected least significant difference test).Initial cell densities were approximately 1io' CFU of E. carotovora subsp.betavasculorum and 105 CFU of E. carotovora subsp. carotovora Ecc7b perwound. Inoculated potatoes were incubated for 48 h at 24°C in a moist chamber.

significant effect on the establishment of Ecbl68b in wounds;population sizes of Out' and Out- strains of E. carotovorasubsp. betavasculorum resulting from mixed-strain inoculationswith Ecc7b did not differ from those resulting from corre-sponding single-strain inoculations. The weight of maceratedtissue surrounding wounds that were coinoculated with E.carotovora subsp. betavasculorum and E. carotovora subsp.carotovora was similar to that caused by the correspondingOut' or Out- strain of E. carotovora subsp. betavasculorum insingle-strain infections (Table 3). Wounds coinoculated with E.carotovora subsp. carotovora Ecc7b and Out- derivatives of E.carotovora subsp. betavasculorum developed less soft rot thandid those coinoculated with Ecc7b and the Out' strainEcbl68b, probably because the Out- derivatives caused lesstissue maceration than did the Out' parental strain of E.carotovora subsp. betavasculorum.

DISCUSSION

The presence of out genes in the genome of E. carotovorasubsp. betavasculorum Ecbl68 was established by several linesof evidence. (i) The putative out genes of Ecb168 hybridized tothe cloned out region of E. carotovora subsp. carotovora (27).We attempted unsuccessfully to introduce an out mutation intothe genome of Ecbl68 by homologous recombination with amutagenized form of the out region of E. carotovora subsp.carotovora (data not shown). Although out genes from the twosubspecies hybridized to one another, we presume that se-quence similarity was not sufficient to allow for detectablelevels of homologous recombination between the out regionsof E. carotovora subsp. carotovora and E. carotovora subsp.betavasculorum. (ii) The putative out genes of E. carotovorasubsp. betavasculorum complemented two Out- mutants of E.carotovora subsp. carotovora (Fig. 1 and 2). Thus, products ofout genes function interchangeably between E. carotovorasubsp. betavasculorum and E. carotovora subsp. carotovora,indicating that these subspecies share a common mechanism ofpectate lyase export. (iii) Derivatives of E. carotovora subsp.betavasculorum Ecb168 with deletions in putative out genesexpressed a phenotype typical of Out-- mutants of E. chrysan-themi and E. carotovora subsp. carotovora (2, 27, 41); pectate

lyase remained inside the cell, whereas protease was exported.(iv) Cloned out genes of E. carotovora subsp. betavasculorumEcbl68 complemented the corresponding Out- mutants de-rived from this strain (Table 2). Although linkage of the twoHindIll genomic fragments containing the out genes of Ecbl68was not determined in this study, it is possible that the outgenes of E. carotovora subsp. betavasculorum are linked, as arethose of E. carotovora subsp. carotovora and E. chrysanthemi,which span a 12.6- to 12.7-kb region of genomic DNA (24, 31).Out- derivatives of E. carotovora subsp. betavasculorum

macerated potato tuber tissue but to a lesser extent than didthe Out' parental strain Ecbl68b. Similarly, Out- mutants ofE. carotovora subsp. carotovora cause tuber soft rot but are lessvirulent than the Out' parental strain (27). In contrast, Out-mutants of E. chrysanthemi are avirulent on their host, Saint-paulia ionantha (2). A possible explanation for the differencebetween the virulence of Out- derivatives of these species maybe that E. carotovora subsp. carotovora and E. carotovora subsp.betavasculorum produce greater concentrations of pectatelyase (27) than does E. chrysanthemi (2). Release of enzymes asbacterial cells die and lyse may explain the small amount oftissue maceration caused by Out- strains of E. carotovorasubsp. betavasculonrm and E. carotovora subsp. carotovora.The Out- derivatives of E. carotovora subsp. betavasculorum

had several properties of an effective biological control agent.(i) They suppressed populations of E. carotovora subsp. caro-tovora in wounds of potato and suppressed tuber soft rotcaused by E. carotovora subsp. carotovora (Table 3). (ii) Theyinhibited E. carotovora subsp. carotovora in culture and onpotato tubers at a level comparable to the parental Out'strain, indicating that an antibiotic(s) required for inhibition ofE. carotovora subsp. carotovora (4) was secreted by an out-independent process. (iii) They established large populationsin wounds of potato tubers (Table 3), indicating that the Out-phenotype was not associated with loss of ecological fitness. Itshould be noted, however, that the generation time of one ofthe Out- mutants of E. carotovora subsp. betavasculorum(LA292) was longer than that of the Out' parental strainEcbl68b in culture (Table 2). Because LA292 has a largedeletion of genomic DNA, it is possible that neighboring geneslinked to out were deleted, causing a deleterious effect ongrowth. Populations of both Out- mutants in the wounds ofpotato tubers were smaller than that of the Out' parentalstrain (Table 3). It is not surprising that Out- strains did notestablish equivalent population sizes, because Out- strainscaused less rot; compounds released upon maceration of tubertissue serve as substrates for growth of Ervinia spp. (7, 20). Onthe basis of the population sizes established in wounds ofpotato tubers in this study, Out- mutants of E. carotovorasubsp. betavasculorum are sufficiently fit to be successful an-tagonists of E. carotovora subsp. carotovora.The success and reliability of biological control may be

optimized if a biological control agent is active under the fullspectrum of conditions favoring proliferation of the pathogenand subsequent infection of a plant host. In this study, weattempted to derive an avirulent strain of E. carotovora subsp.betavasculorum that proliferates under the environmental con-ditions favoring the target pathogen E. carotovora subsp.carotovora. Mutagenesis of the out genes of E. carotovorasubsp. betavasculorum resulted in reduced virulence. Theseefforts could serve as the first step in the derivation of anavirulent strain. A technique by which directed unmarkeddeletions can be introduced into the bacterial genome (34) wasselected for this study because these Out- mutants can now besubjected to further mutagenesis. For example, mutagenesis ofhrp genes (hypersensitivity reaction and pathogenicity) or

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2284 COSTA AND LOPER

regulatory genes that effect pectolytic enzyme productionresults in greatly reduced virulence of E. chrysanthemi (5) or E.carotovora subsp. carotovora (17, 28, 30), respectively. Mu-tagenesis of corresponding genes in E. carotovora subsp.betavasculorum is a promising strategy to generate avirulentmutants with potential as biological control agents, if suchmutations do not affect antibiotic production or ecologicalfitness of the bacterium.

ACKNOWLEDGMENTS

We thank Paige Axelrood and Arun Chatterjee for providing strains,valuable advice, and encouragement; Jennifer Kron for assistance inthe derivation of mutant strains and biological control assays; MarcellaHenkels for drawing the figures; and Mike Boehm, Jennifer Kraus,Daniel Roberts, and Virginia Stockwell for critical reviews of themanuscript.

This research was supported in part by grant 193-5358-095 from theU.S. Department of Agriculture, Cooperative Research Service.

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