APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/98/$04.0010

May 1998, p. 1967–1971 Vol. 64, No. 5

Copyright © 1998, American Society for Microbiology

Endopolygalacturonase PG1 in Different FormaeSpeciales of Fusarium oxysporum

ANTONIO DI PIETRO,* FE I. GARCIA-MACEIRA, M. DOLORES HUERTAS-GONZALEZ,M. CARMEN RUIZ-ROLDAN, ZAIRA CARACUEL, ANDREA S. BARBIERI,†

AND M. ISABEL G. RONCERO

Departamento de Genetica, Facultad de Ciencias, Universidad de Cordoba, 14071 Cordoba, Spain

Received 21 October 1997/Accepted 19 February 1998

PG1, the major endopolygalacturonase of the vascular wilt pathogen Fusarium oxysporum, was secretedduring growth on pectin by 10 of 12 isolates belonging to seven formae speciales, as determined with isoelectricfocusing zymograms and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. A Southern analysisof genomic DNA and PCR performed with gene-specific primers revealed that the pg1 locus was highlyconserved structurally in most isolates. Two PG1-deficient isolates were identified; one lacked the encodinggene, and the other carried a pg1 allele disrupted by a 3.2-kb insertion with sequence homology to hATtransposases. The virulence for muskmelon of different F. oxysporum f. sp. melonis isolates was not correlatedwith PG1 production in vitro. We concluded that PG1 is widely distributed in F. oxysporum and that it is notessential for pathogenicity.

It has been proposed that endopolygalacturonases (en-doPGs) (poly-a-1,4-galacturonide glycanohydrolases; EC3.2.1.15) play a key role in fungal pathogenicity for plants bydepolymerizing homogalacturonan, a major component of theplant cell wall (4). They may also function as avirulence deter-minants by releasing oligogalacturonide inducers of plant de-fense mechanisms (6) and interacting with plant proteins thatmodulate polygalacturonase (PG) activity (3). Fusarium oxys-porum Schlecht. is an economically important soilborne plantpathogen that has a worldwide distribution and causes vascularwilt disease in a wide variety of crops. This species includesmore than 120 described formae speciales that are defined onthe basis of specificity for host species (1). The mechanisms ofpathogenicity and wilt symptom induction by this fungus arepoorly understood, although it has been suggested that en-doPGs may be involved (2). Until now, no information con-cerning the occurrence and distribution of specific pectinolyticisozymes in this species has been available. Recently, PG1, themajor endoPG produced by F. oxysporum f. sp. lycopersici dur-ing in vitro growth on pectin, was purified and characterized(7), and the corresponding gene was cloned (10). In thepresent study, we compared 12 F. oxysporum isolates belongingto seven different formae speciales to determine the occur-rence and diversity of PG1 and the corresponding gene in thespecies and determined if PG1 production by F. oxysporum f.sp. melonis was correlated with virulence for muskmelon.

The F. oxysporum isolates used in the study were stored asmicroconidial suspensions in 30% glycerol at 280°C (Table 1).PG1 is the major pectinolytic enzyme secreted by F. oxysporumduring early phases of growth on pectin (7, 10). Thus, to ana-lyze PG1 production, 108 microconidia obtained by filteringcultures grown for 4 days in potato dextrose broth (PDB)(Difco) were germinated by incubating them for 12 h in 50 mlof fresh PDB. The resulting germlings were washed twice in

sterile water, transferred to 20 ml of synthetic medium (7)supplemented with 1% (wt/vol) citrus pectin (Sigma ChemicalCo., St. Louis, Mo.), and incubated on a rotary shaker for 24 hat 150 rpm and 28°C. Total PG activity was determined indialyzed culture filtrates by measuring the release of reducinggroups from polygalacturonic acid by the method of Nelson-Somogyi. Appropriate control experiments without either en-zyme or substrate were performed simultaneously. The quan-tity of reducing sugar released was calculated by usingD-galacturonic acid standards (Sigma). Medium to high extra-cellular PG activity was detected in most filtrates; the onlyexceptions were the isolate 218 and 58110 filtrates, which con-tained low activity, and the 18M, A34, and 275 filtrates, whichcontained extremely low or no PG activity (Table 2). In orderto check degradation of citrus pectin in the cultures, 1.2 ml ofeach culture filtrate was lyophilized in an Eppendorf tube.Total or partial degradation of the pectin polymers due to theaction of endoPG was observed with all isolates except 18M,A34, and 275 (Fig. 1A). To visualize pectinolytic isozymes,filtrates from pectin cultures grown for 48 h were concentratedby acetone precipitation and were analyzed by using zymo-grams in isoelectric focusing gels (pH 3 to 9) (16). Two activitybands (pI 6.85 and 7.0) corresponding to PG1 isoforms (7, 10)were detected in all isolates except 18M and 275 (Fig. 1B). Afaint activity band with a pI of 7.0 produced by these twoisolates probably corresponded to an exopolygalacturonase(exoPG) (PG3) having the same pI as the main PG1 isoformthat was masked in other isolates by the more active PG1 (11).After 48 h of growth, some isolates also produced significantamounts of other pectinolytic isozymes with pIs lower than thePG1 pIs.

Cultures grown for 24 h on pectin were analyzed by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 11% (wt/vol) discontinuous acrylamide gels, fol-lowed by staining with AgNO3, which revealed two major pro-tein bands (35 and 37.5 kDa) (Fig. 1C) that corresponded todifferentially glycosylated isoforms of PG1 (7). After filtrateswere concentrated up to 100-fold by acetone precipitation, thePG1 protein bands were detected in all isolates except 18Mand 275 (results not shown). Three of the PG1 producers, 281(F. oxysporum f. sp. lycopersici), 58110 (F. oxysporum f. sp.

* Corresponding author. Mailing address: Departamento de Ge-netica, Facultad de Ciencias, Avda. de S. Alberto Magno s/n, 14071Cordoba, Spain. Phone: 34-57218601. Fax: 34-57218606. E-mail:ge2dipia@uco.es.

† Present address: Dipartimento di Patologia Vegetale, Universitadegli Studi di Pisa, 56100 Pisa, Italy.

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conglutinans), and A34 (F. oxysporum f. sp. melonis), secretedonly small amounts of the enzyme during this growth phase(growth for 24 h) (Fig. 1C), although the zymogram resultssuggested that larger amounts of PG1 accumulated in filtratesof these isolates at later stages of growth (after growth for 48 h)(Fig. 1B). In summary, the combined results obtained from PGactivity assays, pectin degradation studies, zymograms, andSDS-PAGE gels indicate that PG1 was the major pectinolyticenzyme secreted by F. oxysporum during the early phases ofgrowth on pectin (growth for 24 h) and, due to its endo modeof action, was the main enzyme responsible for rapid degrada-tion of the pectin polymer. Nevertheless, at later stages ofgrowth (Fig. 1B) or on different pectic substrates, additionalpectinolytic isozymes were produced. Thus, when the 12 iso-lates were grown on solid medium containing polygalacturonicacid (18), even PG1-deficient isolate 275 produced a clearhalo, indicating that pectinolytic isozymes other than PG1 werepresent (data not shown). In fact, two additional exopolygalac-turonases and one pectate lyase from F. oxysporum f. sp. lyco-persici have been characterized previously (8, 9, 11).

The presence of the pg1 gene in the different F. oxysporumisolates was studied by performing a Southern hybridization

analysis (17) of total genomic DNA extracted from mycelium(14) grown for 5 days in PDB on a rotary shaker at 150 rpm and28°C. Two micrograms of DNA was digested with restrictionenzymes EcoRV and HindIII and hybridized with a 735-bp

FIG. 1. Analysis of pectin degradation by lyophilization (A), analytical iso-electric focusing followed by pectinolytic activity staining (B), and SDS-PAGEfollowed by silver staining (C) of culture filtrates of F. oxysporum isolates grownfor 24 h (A and C) or 48 h (B) on synthetic medium supplemented with 1%(wt/vol) citrus pectin. Lane 1, strain 42-87; lane 2, strain 281; lane 3, strain 77r;lane 4, strain 58110; lane 5, strain 15651; lane 6, strain 8503; lane 7, strain 18M;lane 8, strain 1127; lane 9, strain Fo-8; lane 10, strain A34; lane 11, strain 275;lane 12, strain G-60301. In panel A the white residue that was visible afterlyophilization was undegraded pectin. In panels B and C culture filtrates wereconcentrated 25-fold by acetone precipitation. The positions of isoelectric focus-ing and molecular weight markers are indicated on the left.

TABLE 1. F. oxysporum isolates used in the present study

Isolate Taxon Race Host plant Sourcea

42-87b F. oxysporum f. sp. lycopersici 2 Tomato I.N.I.A.281b F. oxysporum f. sp. lycopersici 2 Tomato I.N.I.A.77rb F. oxysporum f. sp. radicis-lycopersici NDc Tomato I.N.I.A.58110 F. oxysporum f. sp. conglutinans 2 Radish ATCC15651 F. oxysporum f. sp. tuberosi ND Potato ATCC8503d F. oxysporum f. sp. ciceris 5 Chickpea I.A.S.18Mb F. oxysporum f. sp. melonis 1 Muskmelon I.N.I.A.1127b F. oxysporum f. sp. melonis 2 Muskmelon I.N.I.A.Fo-8e F. oxysporum f. sp. melonis 2 Muskmelon UCBA34e F. oxysporum f. sp. melonis 0 Muskmelon UCB275f F. oxysporum f. sp. melonis 1,2w Muskmelon C.I.D.A.G-60301 F. oxysporum f. sp. niveum 1 Watermelon I.P.O.

a I.N.I.A., Instituto Nacional de Investigacion Agraria, Madrid, Spain; ATCC, American Type Culture Collection, Rockville, Md.; I.A.S., Instituto de AgriculturaSostenible, Cordoba, Spain; UCB, University of California, Berkeley; C.I.D.A., Centro de Investigacion y Desarollo Agraria, Almeria, Spain; I.P.O., Research Institutefor Plant Protection, Wageningen, The Netherlands.

b Provided by J. Tello.c ND, not determined.d Provided by R. Jimenez Dıaz.e Provided by T. R. Gordon.f Provided by J. Gomez.

TABLE 2. Total PG activities in dialyzed culture filtrates ofF. oxysporum isolates grown in synthetic medium containing 1%citrus pectin for 24 h, as determined by a reducing sugar assay

Isolate PG activity(nkat ml21)a

42-87 .......................................................................................... 7.3 6 1.1281.............................................................................................. 2.1 6 0.577r .............................................................................................. 9.6 6 1.258110.......................................................................................... 3.5 6 0.715651.......................................................................................... 5.9 6 0.38503............................................................................................ 5.7 6 0.618M............................................................................................ 0.2 6 0.11127............................................................................................ 6.3 6 2.9Fo-8............................................................................................ 7.7 6 1.8A34............................................................................................. 0.9 6 0.6275.............................................................................................. 0.4 6 0.2G-60301 ..................................................................................... 6.2 6 2.0

a One nanokatal was defined as the amount of enzyme that produced 1 nmolof galacturonic acid equivalent per s at 37°C.

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internal PCR fragment (corresponding to nucleotides 114 to849) of the pg1 gene of F. oxysporum f. sp. lycopersici 42-87 (10)labelled with a nonisotopic digoxigenin kit (Boehringer Mann-heim). pg1 homologs were detected in most of the isolatesstudied (Fig. 2). A 2.3-kb HindIII fragment containing the pg1promoter and coding region (10) was remarkably conserved inthe majority of the isolates (Fig. 2B). One of the PG1-deficientisolates, 18M, produced band patterns consistent with the pres-ence of at least one additional EcoRV and HindIII site, indi-cating that there was a possible insertion in the pg1 gene.Isolates A34 and 275 did not exhibit detectable hybridizationwith pg1. The same result was obtained when hybridization wasperformed under low-stringency conditions (56°C, one wash in53 SSC [13 SSC is 0.15 M NaCl plus 0.015 M sodium citrate]),indicating that no other genes with homology to pg1 werepresent (results not shown). Stripping and reprobing of thefilters with the nit1 gene of F. oxysporum f. sp. lycopersici (12)resulted in the production of clear hybridizing bands by allisolates, demonstrating that the lack of hybridization with pg1observed with isolates A34 and 275 did not result from degra-dation of DNA (Fig. 2C). Nevertheless, when a Southern anal-ysis was performed with very large amounts of genomic DNA(.10 mg) and long exposure times, a high-molecular-weight

hybridizing band was observed in A34 that comigrated withundigested genomic DNA (data not shown). In this isolate,none of the restriction enzymes used (EcoRV, HindIII, MvnI,PvuII, SmaI) produced hybridizing bands with molecularweights lower than 20 kb, suggesting that the DNA in the pg1genomic region was not accessible to these enzymes. On theother hand, no hybridization signal was observed with isolate275 even when large amounts of DNA and long exposure timeswere used, suggesting that this isolate lacks pg1.

To confirm these results, 50 ng of genomic DNA from eachisolate was used for PCR amplification with the sense primerPG6 (59-CAC TAC TGC CGA TAA CGA CT-39) and theantisense primer PG7 (59-CAA GAA TGA GCC CTG AGATG-39), which spanned a 325-bp internal region (nucleotides326 to 650) of the pg1 gene of F. oxysporum f. sp. lycopersici(10). The PCR conditions used were as follows: denaturationat 94°C for 30 s, annealing at 55°C for 30 s, and extension at72°C for 1.5 min, with an initial denaturation step consisting of3 min at 94°C and a final elongation step consisting of 3 min at72°C. Aliquots (12 ml) of the PCR products were analyzed ona 2% agarose gel. The expected 325-bp band was amplifiedwith all of the isolates except 275, strongly suggesting that thisisolate lacked the pg1 gene (Fig. 3A).

An additional PCR analysis was performed with isolates42-87, 1127, 18M, A34, and 275 by using the following primercombinations: PG7 and sense primer PG8 (59-TCT TGT CTTTGT CTC ACT CG-39), which spanned 669 bp of the first partof the pg1 coding region (nucleotides 218 to 650); and PG6and antisense primer PG9 (59-AGT GAA CAG GGA GTGATG AT-39), which encompassed 1,027 bp from nucleotide

FIG. 2. Southern hybridization analysis of genomic DNAs of different F.oxysporum isolates. For lane contents see the legend to Fig. 1. DNA was digestedwith restriction enzymes EcoRV (A) and HindIII (B and C) and was probed witha digoxigenin-dUTP-labelled internal fragment of the pg1 gene (A and B) or withthe nit1 gene (C) of F. oxysporum f. sp. lycopersici. Lane 5 contained no DNA.The sizes of DNA marker bands are indicated on the left.

FIG. 3. PCR performed with genomic DNAs of different F. oxysporum iso-lates and primers specific for pg1. (A) Primers PG6 and PG7 (see Materials andMethods). For the contents of lanes 1 through 12 see the legend to Fig. 1.Aliquots of the PCR products were electrophoresed on a 2% agarose gel alongwith a 100-bp ladder marker (lane M). (B) PCR performed with primers PG7and PG8 (lanes 1 through 5) or primers PG6 and PG9 (lanes 6 through 11) andgenomic DNAs of isolates 42-87 (lanes 1 and 6), 1127 (lanes 2 and 7), 18M (lanes3, 8, and 11), A34 (lane 4 and 9), and 275 (lanes 5 and 10) by using an extensiontime of 2.5 min (lanes 1 through 10) or 4.5 min (lane 11). Aliquots wereelectrophoresed on a 1.4% agarose gel (or on a 0.7% agarose gel [lane 11]) alongwith lambda HindIII markers (lane M). The approximate sizes of amplifiedbands are indicated on the left.

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326 to 34 nucleotides downstream of the pg1 stop codon. ThePCR conditions were the same as those described above, ex-cept that the extension time was 2.5 min. Aliquots (12 ml) ofthe PCR products were analyzed on a 1.4% agarose gel. Withprimers PG7 and PG8 the expected fragment was amplifiedwith all of the isolates except 275, whereas with primers PG6and PG9, both the 18M preparation and 275 preparationlacked the expected amplification product (Fig. 3B). When aPCR was performed with primers PG6 and PG9 and a longerextension time (4.30 min), a 4.2-kb fragment instead of a 1-kbfragment was amplified from 18M, suggesting that this isolatecarries a 3.2-kb insertion in the second part of the pg1 gene. A0.8-kb EcoRV-HindIII fragment from this PCR amplificationproduct was subcloned into pBluescript/SK1, and both strandswere sequenced with a DyeDeoxy terminator cycle sequencingkit (Perkin-Elmer, Foster City, Calif.) and a model ABI Prism310 genetic analyzer (Applied Biosystems, Foster City, Calif.).The deduced amino acid sequence exhibited significant homol-ogy to the sequences of the hAT family transposable elements,particularly the transposon restless from the fungus Tolypocla-dium inflatum (13).

To study the possible correlation between PG1 productionand virulence, four F. oxysporum f. sp. melonis isolates, 18M,1127, A34, and 275, which differed in PG1 production, wereused in pathogenicity assays performed in a growth chamber.Ten-day-old seedlings of muskmelon (cultivar Amarillo Ca-nario; susceptible to all F. oxysporum f. sp. melonis races) in thefirst true leaf stage were inoculated by dipping the roots for 30min in a suspension containing 5 3 106 microconidia ml ofwater21. Control plants were immersed in sterile water. Tenseedlings per treatment were planted in minipots containingvermiculite and were incubated in a growth chamber at 25°Cwith a photoperiod consisting of 14 h of light and 10 h of dark.At different times after inoculation, the severity of diseasesymptoms was recorded by using a scale ranging from 1(healthy plant) to 5 (dead plant). The PG1 producer 1127 washighly virulent, killing the plants after 10 days (Fig. 4). PG1-

deficient isolate 18M exhibited the same degree of virulence as1127, whereas A34 and 275 exhibited lower levels of virulence(the disease symptoms were both delayed and not as severe asthe disease symptoms observed with the other strains). Theexperiment was repeated twice with similar results.

The data obtained from enzyme, Southern hybridization,and PCR analyses suggest that PG1 is widely distributed andhighly conserved in different formae speciales of F. oxysporum.This is consistent with the results of previous studies whichshowed that there is a close structural relationship between theendoPGs of F. oxysporum f. sp. lycopersici and Fusarium mo-niliforme (10). Three of the 12 isolates studied (18M, A34, and275) secreted little or no PG1. The absence of PG1 productionby isolate 275 presumably resulted from a lack of the pg1 gene.Conversely, in 18M the presence of additional restriction sitesin pg1, amplification of a 4.2-kb PCR fragment instead of a1.0-kb PCR fragment with pg1-specific primers, and homologybetween the sequence of this fragment and the sequence of afungal transposase indicate that the lack of PG1 in this isolatemay be due to disruption of the encoding gene by a transpos-able element. The presence of different types of mobile geneticelements in F. oxysporum has been described previously (5). Incontrast to 275 and 18M, A34 was not completely PG1 defi-cient, but the production of this enzyme by A34 was stronglyreduced. At the gene level, neither the highly conserved 2.3-kbHindIII fragment nor any other hybridizing restriction frag-ment smaller than 20 kb was detected. A possible explanationfor this is that hypermethylation of the surrounding DNAregion makes pg1 unaccessible to methylation-sensitive restric-tion enzymes. This status does not affect the total genomicDNA of the isolate, since no interference with restriction di-gests was detected in the nit1 region. It remains to be deter-mined whether the phenomenon observed in this study is dueto hypermethylation and whether this hypermethylation affectspg1 transcription, as reported for other eucaryotic genes (15),accounting for the low level of PG1 production in this isolate.

In a previous study, the PG1 activity band was detected instems of tomato plants infected with F. oxysporum f. sp. lyco-persici, suggesting that this enzyme may play a role in patho-genicity (7). The variability of PG1 production in isolates of F.oxysporum f. sp. melonis allowed us to study the correlationbetween in vitro secretion of the enzyme and virulence formuskmelon. The results indicate that at least in this formaspecialis, PG1 was not essential for pathogenicity since thestrong PG1 producer 1127 and the deficient isolate 18M ex-hibited the same degree of virulence for the cultivar tested.Thus, the reduced virulence of the remaining two isolatestested was probably due to factors other than PG1 production.Since F. oxysporum produces PGs other than PG1 in vitro, wecannot rule out the possibility that PG plays a role in patho-genicity, although PG1 does not appear to be involved.

We thank J. Gomez, T. R. Gordon, R. Jimenez Dıaz, and J. Tello forproviding F. oxysporum isolates and I. Huedo for photographic work.

This research was supported by grant BIO93-0923-CO2-01 from theComision Interministerial de Ciencia y Tecnologıa (CICYT) and bygrant HCM-CT93-0244 from the European Commission. A.D.P. wassupported by postdoctoral fellowship HCM-CT93-0244 from the Eu-ropean Commission. F.I.G.-M. was supported by a predoctoral fellow-ship from ICI. M.D.H.-G. and M.C.R.-R. were supported by predoc-toral fellowships from the Spanish Ministerio de Educacion y Ciencias,and A.S.B. was supported by an ERASMUS fellowship from the Eu-ropean Commission.

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FIG. 4. Incidence of Fusarium wilt caused by different isolates of F. oxyspo-rum f. sp. melonis on muskmelon plants (cultivar Amarillo Canario). The severityof disease symptoms was recorded at different times after inoculation by using ascale ranging from 1 (healthy plant) to 5 (dead plant). Symbols: �, isolate 1127;E, isolate 18M; F, isolate A34; ƒ, isolate 275; {, uninoculated control. Thevalues are the means of the values from two independent experiments, eachperformed with 10 plants per treatment. The error bars indicate standard devi-ations.

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