Genetic diversity of <em>Fusarium oxysporum</em> and related species pathogenic on tomato in Algeria and other Mediterranean countries

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species pathogenic on tomato in Algeria and other Mediterranean countries

Veronique Edel-Hermann, Nadine Gautheron, Christian Steinberg

To cite this version:

Veronique Edel-Hermann, Nadine Gautheron, Christian Steinberg. Genetic diversity of Fusarium oxysporum and related species pathogenic on tomato in Algeria and other Mediterranean countries.

Plant Pathology, Wiley, 2012, 61 (4), pp.787-800. �10.1111/j.1365-3059.2011.02551.x�. �hal-02642550�

Genetic diversity of Fusarium oxysporum and related species pathogenic on tomato in Algeria and other Mediterranean countries

V. Edel-Hermann*, N. Gautheron and C. Steinberg

INRA, Universite´ de Bourgogne, UMR 1229 Microbiologie du Sol et de l’Environnement, 21065 Dijon Cedex, France

In order to characterize the pathogen(s) responsible for the outbreak of fusarium diseases in Algeria, 48 Fusarium spp. iso- lates were collected from diseased tomato in Algeria and compared with 58 isolates of Fusarium oxysporum originating from seven other Mediterranean countries and 24 reference strains. Partial sequences of the translation elongation factor EF- 1a gene enabled identification of 27 isolates as F. oxysporum, 18 as F. commune and three as F. redolens among the Alge- rian isolates. Pathogenicity tests confirmed that all isolates were pathogenic on tomato, with disease incidence greater at 28C than at 24C. All isolates were characterized using intergenic spacer (IGS) DNA typing, vegetative compatibility group (VCG) and PCR detection of the SIX1 (secreted in xylem 1) gene specific to F. oxysporum f. sp. lycopersici (FOL). No DNA polymorphisms were detected in the isolates of F. redolens or F. commune. In contrast, the 27 Algerian isolates of F. oxyspo- rum were shown to comprise nine IGS types and 13 VCGs, including several potentially new VCGs. As none of the isolates was scored as SIX1+, the 27 isolates could be assigned to F. oxysporum f. sp. radicis-lycopersici (FORL). Isolates from Tuni- sia were also highly diverse but genetically distinct from the Algerian isolates. Several Tunisian isolates were identified as FOL by a PCR that detected the presence of SIX1. The results show that isolates from European countries were less diverse than those from Tunisia. Given the difference between Algerian populations and populations in other Mediterranean coun- tries, newly emergent pathogenic forms could have evolved from local non-pathogenic populations in Algeria.

Keywords: Fusarium commune, Fusarium oxysporum, Fusarium redolens, molecular characterization, pathogenicity, tomato

Introduction

Fusarium diseases of tomato plants (Solanum esculen- tum) are known to be mainly caused by two formae speci- ales of the morphospecies Fusarium oxysporum, F. oxysporum f. sp. radicis-lycopersici (FORL), responsi- ble for crown and root rot, and F. oxysporum f. sp. lyco- persici (FOL), responsible for a vascular wilt disease.

Members of the species complex F. solani may cause some crown and root rots of solanaceous plants such as potato and tomato under tropical conditions (Romberg

& Davis, 2007; Blancard et al., 2009) and some other spe- cies may be potentially responsible for pre-emergence damping off of various crops including tomato (Palmero et al., 2009). In contrast to these species, formae speciales FORL and FOL have strict host specificity. Within FOL, three races have been identified, differentiated by their specific pathogenicity to different cultivars carrying spe- cific resistance genes (Grattidge & O’Brien, 1982). How-

ever, no races are known within FORL. Both formae speciales display a considerable genetic diversity. Like many formae speciales in F. oxysporum, FORL and FOL are composed of several vegetative compatibility groups (VCG) and are known to be polyphyletic (Kistler, 1997;

O’Donnell et al., 2009). To date, nine VCGs have been identified among FORL isolates (VCG 0090 to 0094 and 0096 to 0099) and five VCGs among FOL isolates (0030 to 0033 and 0035) (Katan, 1999; Cai et al., 2003). The two formae speciales can be identified and distinguished by time-consuming pathogenicity tests on tomato and by the disease symptoms. VCG can also help identify formae speciales but this method requires obtaining mutants and pairing them with reference strains until a heterokaryon is detected, which is also very time-consuming (Correll et al., 1987). However, VCG grouping remains the refer- ence method to characterize pathogenic strains of F. oxy- sporum because it makes it possible to compare new isolates with reference strains and previously collected isolates. Recently, a rapid molecular method has been proposed to identify FOL, based on the PCR amplifica- tion of specific markers corresponding to the SIX genes (secreted in xylem) (van der Does et al., 2008; Lievens et al., 2009). Genetic diversity within formae speciales can be effectively characterized by targeting different

*E-mail: veronique.edel@dijon.inra.fr

Published online 31 October 2011

ª2011 INRA

DNA regions, the ribosomal intergenic spacer (IGS) being one of the most polymorphic (Lori et al., 2004; Abo et al., 2005; O’Donnell et al., 2009).

Fusarium diseases of tomato have caused severe dam- age in tomato growing areas all over the world. During the past decade, diseases caused by FOL have decreased in European countries due to the availability of resistant cultivars. However, resistance genes can be overcome, resulting in the emergence of new races. Only tolerant cultivars are available for FORL but the disease can be effectively prevented using grafted material (Blancard et al., 2009). However, this strategy is not always used and fusarium disease due to FORL is still present in many Mediterranean countries including Turkey (Can et al., 2004) and Malta (Porta-Puglia & Mifsud, 2005), where it was recently reported for the first time.

Tomato is an important and common crop in North Africa. Contrary to the decline in several other countries, fusarium diseases are still frequent in tomato-growing areas in Tunisia (Hibar et al., 2007). Similarly, an out- break of fusarium diseases of tomato was recently discov- ered in Algeria (N. Hamini-Kadar, University of Oran, Algeria, personal communication). However, nothing is known about the pathogen(s) responsible for this out- break of disease. This study was therefore initiated to determine: (i) whether the pathogens are diverse or are composed of a limited number of genotypes; and (ii) whether they are clonally related to other known strains of FOL and FORL found in neighbouring countries or are newly emergent pathogenic forms. These questions were addressed by characterizing the genetic diversity of the fungal isolates responsible for fusarium diseases in Alge- ria, and by comparing them with isolates originating from other Mediterranean countries.

Material and methods

Fungal isolates

From 2006 to 2008, surveys were conducted in tomato- growing areas of Algeria, mainly around Alger and Oran.

Diseased plants showed discoloration of stem vascular tissue, wilting of the lower leaves, and crown and root rot. Isolates with morphological characteristics similar to those of F. oxysporum (Nelson et al., 1983) were col- lected from diseased plants or soil and preserved in the MIAE collection (Microorganisms of Interest for Agricul- ture and Environment, INRA Dijon, France).

The 48 isolates collected in Algeria were compared with 58 isolates of F. oxysporum originating from seven other Mediterranean countries: seven isolates previously identified as FOL, 34 isolates previously identified as FORL and 17 isolates of F. oxysporum originating from diseased tomato for which the forma specialis was unknown (Table 1). Single-spore isolations were conducted for the 106 isolates. In addition, 24 strains were used as reference strains in VCG tests and ⁄ or as controls in the molecular characterization and pathoge- nicity tests (Table 1). To characterize VCGs, reference

strains of all known VCGs within the formae speciales ly- copersici and radicis-lycopersici were used, with the exception of VCG 0035 and VCG 0097, which were not available from any collection.

Molecular characterization

Fungal DNA was extracted from cultures on potato dex- trose agar (PDA) using a rapid minipreparation proce- dure (Edel et al., 2001). Some isolates were identified at the species level using a molecular strategy based on sequences of a portion of the translation elongation factor EF-1a gene (Geiser et al., 2004) and sequences of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA).

Part of the EF-1a gene was amplified by PCR using the primers ef1 (5¢-ATG GGT AAG GA(A ⁄ G) GAC AAG AC-3¢) and ef2 (5¢-GGA (G ⁄ A)GT ACC AGT (G ⁄ C)AT CAT GTT-3¢) (O’Donnell et al., 1998b) in a final volume of 25 lL containing 1 lL of DNA, 0Æ1 l

of each primer, 150 l

dNTP, 3 U Taq DNA polymerase and PCR reac- tion buffer. Amplifications were conducted in a Master- cycler (Eppendorf) with an initial denaturation of 7 min at 95C followed by 38 cycles of 60 s denaturation at 95C, 75 s annealing at 57C, 60 s extension at 72C and a final extension of 10 min at 72C. Presence of PCR products was confirmed by gel electrophoresis. EF-1a amplicons were sequenced by Beckman Coulters Genom- ics using the two PCR primers as sequencing primers. For each PCR product, sequences from both strands were assembled using S

M

6Æ0 (DNASTAR Lasergene, GATC Biotech). Sequence identities were determined using

analysis from the National Center for Bio- technology Information (NCBI) available online.

Sequences were aligned and compared with the Kimura’s two parameters distance model and the neighbour-join- ing (NJ) method using the program S

V

(Gouy et al., 2010). The topology of the resulting tree was tested by NJ bootstrapping with 1000 re-samplings of the data.

The ITS region was amplified by PCR using primers ITS1F (Gardes & Bruns, 1993) and ITS4 (White et al., 1990) in a final volume of 25 lL containing 1 lL of DNA, 0Æ5 l

of each primer, 150 l

dNTP, 3 U Taq DNA polymerase (Q-Biogen) and PCR reaction buffer.

Amplifications were conducted in a Mastercycler with an initial denaturation of 3 min at 94C followed by 35 cycles of 1 min denaturation at 94C, 1 min annealing at 50C, 1 min extension at 72C and a final extension of 10 min at 72C. Presence of PCR products was confirmed by gel electrophoresis. For each PCR product, sequences from both strands were assembled and sequence identi- ties were determined as above.

All isolates were characterized by restriction fragment

length polymorphism (RFLP) analysis of the IGS region

as previously described (Edel et al., 1995, 1997). A 1Æ7 kb

fragment of the IGS was amplified by PCR with oligonu-

cleotide primers PNFo (5¢-CCC GCC TGG CTG CGT

CCG ACT C-3¢) and PN22 (5¢-CAA GCA TAT GAC

TAC TGG C-3¢) and digested with seven restriction

Table1IsolatesofFusariumanalysedinthisstudy SpeciesIsolate MIAE accession number Geographicorigin Originalcode (source)aHost Yearof isolation IGS typebVCGcSIX1PCRd

Percentageof diseasee CountryRegionTownAt24CAt28C FusariumcommuneNH100101AlgeriaNorthwestOran(NHK)Tomato200678A)8040 NH200102AlgeriaNorthwestOran(NHK)Tomato200678A)100Nd NH300103AlgeriaNorthwestOran(NHK)Soil200678A)80Nd NH400104AlgeriaNorthwestOran(NHK)Tomato200678A)88Nd NH500105AlgeriaNorthwestMascara(NHK)Tomato200578A)60Nd NH600106AlgeriaNorthwestMascara(NHK)Soil200578A)0100 NH700107AlgeriaNorthwestMascara(NHK)Tomato200578Nd)090 NH800108AlgeriaNorthwestSidiBelAbbes(NHK)Tomato200778A)90Nd NH900109AlgeriaNorthwestSidiBelAbbes(NHK)Tomato200778HSI)090 NH1000110AlgeriaNorthwestSidiBelAbbes(NHK)Tomato200778Nd)0100 NH1200112AlgeriaNorthwestOran(NHK)Tomato200678Nd)0100 NH1300113AlgeriaNorthwestOran(NHK)Soil200678A)3090 NH1400114AlgeriaNorthwestSidiBelAbbes(NHK)Tomato200778A)1040 NH1500115AlgeriaNorthwestSidiBelAbbes(NHK)Tomato200778A)070 NH1600116AlgeriaNorthwestOran(NHK)Soil200678A)50Nd NH1700117AlgeriaNorthwestSidiBelAbbes(NHK)Tomato200778A)3020 NH1800118AlgeriaNorthwestSidiBelAbbes(NHK)Tomato200778A)100Nd NH2000120AlgeriaNorthwestMostaganem(NHK)Tomato200878A)1020 F.redolensNH2900129AlgeriaNorthAlger(NHK)Tomato200880HSI)40100 NH3000130AlgeriaNorthAlger(NHK)Tomato200880sm)2080 NH3100131AlgeriaNorthAlger(NHK)Tomato200880HSI)50100 F.oxysporumNH1100111AlgeriaNorthwestSidiBelAbbes(NHK)Soil200720B)1050 NH1900119AlgeriaCentreAdrar(NHK)Tomato200720B)090 NH2100121AlgeriaNorthwestSidiMaarouf(NHK)Tomato200774sm)3050 NH2200122AlgeriaNorthwestSidiMaarouf(NHK)Tomato20075HSI)2030 NH2300123AlgeriaNorthwestAinTemouchent(NHK)Tomato200879C)5020 NH2400124AlgeriaNorthwestAinTemouchent(NHK)Tomato200879C)1060 NH2500125AlgeriaNorthwestAinTemouchent(NHK)Tomato200879C)4080 NH2600126AlgeriaNorthwestAinTemouchent(NHK)Tomato200879C)3080 NH2700127AlgeriaNorthwestSidiMaarouf(NHK)Tomato20085HSI)3030 NH2800128AlgeriaNorthwestSidiMaarouf(NHK)Tomato20085HSI)060 NH3200132AlgeriaNorthAlger(NHK)Tomato20083sm)1080 NH3300133AlgeriaNorthAlger(NHK)Tomato20085D)80Nd NH3400134AlgeriaNorthwestTlemcen(NHK)Tomato200867sm)2060 NH3500135AlgeriaNortheastAnnaba(NHK)Tomato20083E)100Nd NH3600136AlgeriaNorthwestTlemcen(NHK)Tomato20085sm)7080

Table1Continued SpeciesIsolate MIAE accession number Geographicorigin Originalcode (source)aHost Yearof isolation IGS typebVCGcSIX1PCRd

Percentageof diseasee CountryRegionTownAt24CAt28C NH3700137AlgeriaNorthAlger(NHK)Tomato20085F)6090 NH3800138AlgeriaNortheastAnnaba(NHK)Tomato200812G)4020 NH3900139AlgeriaNortheastAnnaba(NHK)Tomato20085F)5080 NH4000140AlgeriaNortheastAnnaba(NHK)Tomato20085F)5070 NH4100141AlgeriaNortheastAnnaba(NHK)Tomato200812G)2020 NH4200142AlgeriaNortheastAnnaba(NHK)Tomato200816sm)3070 NH4300143AlgeriaNorthwestTlemcen(NHK)Tomato20085D)10100 NH4400144AlgeriaNorthAlger(NHK)Tomato20085H)2080 NH4500145AlgeriaNortheastAnnaba(NHK)Tomato20083E)80Nd NH4600146AlgeriaNortheastAnnaba(NHK)Tomato200812G)2040 NH4700147AlgeriaNorthAlger(NHK)Tomato20085H)5070 NH4800148AlgeriaNorthAlger(NHK)Tomato200851sm)5050 Fol1500504TunisiaTomato1968250030+90Nd KH6600506TunisiaCoˆteFOM-05(KH)Tomato2005250030+2080 KH6700514TunisiaCoˆteChottMeriamFo4-ch-06(KH)Tomato200667Nd)040 KH6800505TunisiaCentreFol-04(KH)Tomato2004250030+70Nd KH6900507TunisiaEastMonastirFoB1Æ05(KH)Tomato2005250030+100Nd KH7000508TunisiaEastMonastirFoB2-05(KH)Tomato2005250030+80Nd KH7100515TunisiaCentreFo1B308(KH)Tomato200825HSI+90Nd KH7200516TunisiaCentreFoB3Æ08(KH)Tomato2008250030+90Nd KH7300509TunisiaSouthFo2-05(KH)Tomato2005250090)100Nd KH7400510TunisiaSouthFo3-05(KH)Tomato2005250090)3040 KH7500511TunisiaSouthFo4-05(KH)Tomato20052I)3040 KH7600512TunisiaCentreFo1SB-05(KH)Tomato200581J)4020

Table1Continued SpeciesIsolate MIAE accession number Geographicorigin Originalcode (source)aHost Yearof isolation IGS typebVCGcSIX1PCRd

Percentageof diseasee CountryRegionTownAt24CAt28C KH7700513TunisiaCentreFo2SB-05(KH)Tomato200581J)1010 KH7800539TunisiaSouthFo4-07(KH)Tomato20072I)70Nd Forl7100459CreteLerapetraLasithiForl78(DV)Tomato198523Nd)80Nd Fox700480GreeceF623(KE)Tomato200120Nd)80Nd Forl9000481TurkeyEastMediterraneanMersin-1(SY)Tomato2006320094)80Nd Forl9100517TurkeyEastMediterraneanCeyhan-1(SY)Tomato2007250090)90Nd Forl9300518IsraelSouthAravaValleyForl(MR)Soil2008250090)100Nd Fox400502SpainAguilasAFA241-2(MJ)Tomato2007250092)3040 Fox500503SpainAguilasFORLaguilas(MJ)Tomato2000250091)8090 Forl7200537SpainFORL(ILN)Tomato<199025Nd)80Nd Fol2000538SpainSouthFOL(ILN)Tomato1995250030+80Nd Forl7300501ItalySardiniaS6(QM)1998250090)90Nd Forl7400487ItalySardiniaS13(QM)1998250090)70100 Forl7500488ItalySardiniaS7(QM)1998250090)100Nd Forl7600489ItalySardiniaDP95(QM)1998250090)100Nd Forl7700490ItalySardiniaDP83(QM)1998250091)100Nd Forl7800491ItalySardiniaDP93(QM)1998250092)90Nd Forl7900492ItalySicilyDP282(QM)1997250096)100Nd Forl8000485ItalySardiniaS16(QM)1998250090)70Nd Forl8100493ItalySardiniaS3(QM)1995250090)020 Forl8200484ItalySardiniaS9(QM)1998250090)5030 Forl8300494ItalySardiniaS10(QM)1998250090)4020 Forl8400495ItalySardiniaS11(QM)1998250090)100Nd Forl8500496ItalySardiniaS12(QM)1997250090)70Nd Forl8600486ItalyCalabriaDP61(QM)1998250091)100Nd Forl8700497ItalyCalabriaDP63(QM)1998250091)5020 Forl8800498ItalySicilyDP238(QM)1996⁄97250091)60Nd Forl8900499ItalySicilyDP31(QM)1996⁄97250091)0100

Table1Continued SpeciesIsolate MIAE accession number Geographicorigin Originalcode (source)aHost Yearof isolation IGS typebVCGcSIX1PCRd

Percentageof diseasee CountryRegionTownAt24CAt28C Fol2700462FranceSouthwestPerpignanSoil1980250030+100Nd Fol2800463FranceSouthwestPerpignanSoil1980250030+90Nd Fol2900464FranceSouthwestTomato1981250030+100Nd Fol3000467FrancePerpignanTomato1985230031+100Nd Fol3200468FranceNorthwestBrestTomato1985230031+80Nd Forl100477FranceNorthwestBrest(AM)Tomato1991320094)70Nd Forl200469FranceSouth(AM)Tomato1991250091)90Nd Forl500470France(AM)Tomato1993250091)50Nd Forl700471FranceSouthEgragues(AM)Tomato1994250091)90Nd Forl1000472France(AM)Tomato1995320094)30Nd Forl1500474FranceSouth(AM)Tomato19943Nd)50Nd Forl1700473FranceSouthwestAlenyaTomato1984250090)100Nd Forl1800465FranceSouthAlenyaTomato1984250090)100Nd Forl2100466FranceSouthAvignon-BerreTomato1984250091)40Nd Forl3000479FranceSouthwestTomato1997320094)100Nd Forl4500475FranceNorthwestBrestTomato1998320094)100Nd Forl9200476FranceSouthForlref41(AB)Tomato1995⁄9825Nd)70Nd Fox00500FranceSouthFotomate(AB)Tomato2006250091)100100 Referencestrains F.oxysporumFo4700047FranceSouthChaˆteaurenardSoil197822HSI)00 F.oxysporumf.sp. lycopersici

Fol800046FranceCentreVillefranchesSao

ˆne

Tomato197123HSI+100100 Fol2400460FranceNorthwestNantesTomato1977230031+100Nd Fol2600461FranceNantesTomato1977230031+100Nd Fol100*00521USAOhioOSU451B(LRG)230031+NdNd Fol101*00525FolR(TK)250030+NdNd Fol102*00526Folm(TK)250030+NdNd Fol103*00522USAArkansasFolMM59⁄5(TK)250032+NdNd Fol104*00523USAArkansasFolMM66⁄6(TK)250032+NdNd Fol105*00524USANorthCarolinaFolRG1⁄5(TK)200033+NdNd Fol106*00527FolMN27⁄9(TK)200033+NdNd

Table1Continued SpeciesIsolate MIAE accession number Geographicorigin Originalcode (source)aHost Yearof isolation IGS typebVCGcSIX1PCRd

Percentageof diseasee CountryRegionTownAt24CAt28C F.oxysporumf.sp. radicis-lycopersici

Forl1200045MoroccoTomato1995250091)100100 Forl100*00519USAFloridaCL7(LRG)1995320098)100Nd Forl101*00520USAFloridaPB114(LRG)1996200099)90Nd Forl102*005280-1090(TK)250090)NdNd Forl103*00529ForlIID(TK)250090)NdNd Forl106*00530C544(TK)250091)NdNd Forl107*00531C758(TK)250091)NdNd Forl120*00532CRNK676(TK)250092)NdNd Forl121*00533CRNK678(TK)250092)NdNd Forl122*0053401150-6(TK)320094)NdNd Forl123*0053501152-31(TK)320094)NdNd Forl129*00483IsraelC204⁄3(TK)600093)NdNd Forl132*00536C623⁄35250096)NdNd *VCGtesters. MIAE,MicroorganismsofInterestforAgricultureandenvironment,INRADijon,France. aAB,A.Buffie`re,InstitutNationaldelaRechercheAgronomique(INRA),Montfavet,France;AM,A.Moretti,INRA,Montfavet,France;DV,D.Vakalounakis,NationalAgricultureResearchFoundation (NAGREF),PlantProtectionInstitute,Heraklio,Crete,Greece;ILN,I.LarenaNistal,DepartmentofPlantProtection,Madrid,Spain;KE,K.Elena,DepartmentofPhytopathology,Athens,Greece;KH,K. Hibar,EcoleSupe´rieured’HorticulturedeChott-Mariem,Sousse,Tunisia;LRG,L.RosewichGale,USDA-ARS,UniversityofMinnesota,StPaul,USA;MJ,M.Jacquet,Vilmorin,CentredeRecherchedela Costie`re,Ledenon,France;NHK,N.Hamini-Kadar,Universite´d’Oranes-Se´nia,Oran,Algeria;QM,Q.Migheli,DipartimentodiProtezionedellePiante,UniversitadegliStudidiSassari,Sassari,Italy;SY, S.Yu¨cel,MinistryofAgriculture,PlantProtectionInstitute,Adana,Turkey;TK,T.Katan,HebrewUniversityofJerusalem,Jerusalem,Israel. bIntergenicspacer(IGS)typesaredefinedinTable3. cIsolateswiththesamenumberorthesameletterbelongtothesamevegetativecompatibilitygroup(VCG);HSI,heterokaryonself-incompatibility;sm,single-memberVCG;Nd,notdeterminedbecause nitmutantscouldnotbeobtained. d+indicatesthata990bpampliconwasobtainedwithprimersdesignedtoamplifypartofthesecretedinxylem(SIX1)gene;)indicatesthatnoampliconwasdetected. ePercentageofdiseasedanddeadplants.Nd,notdetermined.

enzymes: AluI, HaeIII, HinfI, MspI, RsaI, ScrFI and XhoI.

Each isolate was assigned to an IGS type defined by the combined restriction patterns obtained with the seven enzymes (Edel et al., 1997, 2001; Lori et al., 2004; Abo et al., 2005).

All isolates were tested for the presence of the SIX1 gene specific to forma specialis lycopersici by PCR using primers P12-F2B (5¢-TAT CCC TCC GGA TTT TGA GC-3¢) and P12-R1 (5¢-AAT AGA GCC TGC AAA GCA TG-3¢) (van der Does et al., 2008). Amplifications were performed in a final volume of 25 lL containing 1 lL of DNA, 0Æ2 l

of each primer, 200 l

dNTP, 3 U Taq DNA polymerase and PCR reaction buffer. Amplifica- tions were conducted in a Mastercycler with an initial denaturation of 2 min at 94C followed by 30 cycles of 45 s denaturation at 94C, 45 s annealing at 64C, 45 s extension at 72C and a final extension of 10 min at 72C. Strain Fol8 of F. oxysporum f. sp. lycopersici was used as positive control in each PCR experiment. The PCR products were checked by electrophoresis. Strains were scored as SIX1+ if amplification of their DNA yielded a 990 bp fragment or as SIX1) if no amplification product was obtained.

Vegetative compatibility testing

Vegetative compatibility was determined by pairing com- plementary nitrate non-utilizing (nit) mutants derived from each strain, as previously described by Correll et al.

(1987). Briefly, mutants were first generated for each strain on PDA-potassium chlorate medium (PDA 30 g L

, KClO

15 g L

) and then classified as nit1, nit3 or nitM based on their phenotype on minimal med- ium (MM) containing one of three different nitrogen sources (nitrate, nitrite and hypoxanthine). The composi- tion of MM was (per litre distilled water): sucrose 30 g, KH

PO

1 g, MgSO

.7H

O 0Æ5 g, KCl 0Æ5 g, FeS- O

.7H

O 10 mg, NaNO

2 g, agar 20 g and trace ele- ments solution 0Æ2 mL. The trace element solution contained (per 95 mL distilled water): citric acid 5 g, ZnSO

.7H

O 5 g, Fe(NH

)

.6H

O 1 g, CuSO

.5H

O 0Æ25 g, MnSO

.H

O 50 mg, H

BO

50 mg and Na- MoO

.2H

O 50 mg.

For some isolates for which the different types of mutants could not be obtained, the concentration of potassium chlorate in the medium was increased from 15 to 30 g L

. Mutants of different types were paired on MM in 5 cm Petri dishes and scored as compatible if dense aerial growth was formed at the point of contact between the two paired mutants, corresponding to the formation of a heterokaryon. Vegetative compatibility was determined by pairing complementary nit mutants derived from all 106 isolates in all pairwise combinations.

For each pairwise combination, at least two pairings were performed: (i) a nit1 mutant of isolate A was paired with a nit3 or nitM mutant of isolate B, and (ii) a nit3 or nitM mutant of isolate A was paired with a nit1 mutant of iso- late B. When two mutants formed a visible and robust heterokaryon, indicated by the presence of dense aerial

mycelium, the corresponding strains were placed in the same VCG. Once the VCGs were identified, representa- tive isolates of each group were checked for compatibility with the tester strains of FOL and FORL (Table 1). Con- trol pairings between complementary mutants derived from the same parental strain were made to detect any strain that might be heterokaryon self-incompatible (Correll et al., 1987).

Pathogenicity tests

The 106 isolates of Fusarium together with six reference strains were tested for their pathogenicity on tomato cv.

63Æ5 Montfavet (Table 1). For each isolate, 10 12-day-old seedlings of tomato were inoculated by cutting the roots 7 mm below the crown and dipping the plants for 5 min in 5 mL of a suspension of 10

conidia mL

in liquid minimal medium (LMM, i.e. MM without agar). Plants were transplanted into perlite and the 5 mL of conidial suspension were poured onto the perlite around the crown. For each of the isolates tested, the 10 seedlings were inoculated with 10 independent conidial suspensions. Controls consisted of plants inoculated with reference strains of F. oxysporum Fo47 (non-patho- genic strain), Fol8 (f. sp. lycopersici) and Forl12 (f. sp. ra- dicis-lycopersici), and with LMM instead of a conidial suspension as the negative control. Plants were incubated in a growth chamber at 24C during the day (16 h) and 20C at night. Three weeks after inoculation, the percent- age of affected plants (dead plants together with plants showing dark brown lesions on crown and roots) was determined. For all of the isolates that induced a low per- centage of disease, pathogenicity tests were performed a second time, but at a daytime temperature of 28C instead of 24C.

Results

Species identification

Microscopic observations showed that numerous isolates

originating from Algeria originally identified as F. oxy-

sporum were atypical. Macroconidia appeared longer

and thinner than those generally observed for F. oxyspo-

rum. Thus the 48 Algerian isolates were identified by their

EF-1a sequences (GenBank accession nos HM584898 to

HM584901 and JN000865 to JN222908).

analysis

using 99–100% sequence identity as the threshold for

species limits indicated that 27 isolates were F. oxyspo-

rum, 18 were F. commune and three were F. redolens

(Table 1). Sequence alignments revealed that the 18

EF-1a sequences of F. commune were identical, as were

the three EF-1a sequences of F. redolens. Representative

isolates of F. commune (MIAE00101 and MIAE00112)

and of F. redolens (MIAE00129 and MIAE00131) were

identified by their ITS sequences (GenBank accession

Nos HM584894 to HM584897). Their identification at

the species level was confirmed by

analysis with

100% sequence identity with corresponding sequences.

Evolutionary relationships of the 48 EF-1a sequences were inferred in a NJ tree, which revealed eight different sequence types among the 27 Algerian isolates of F. oxy- sporum (Fig. 1).

Molecular characterization

Oligonucleotide primers PNFo and PN22 successfully amplified a single DNA fragment of about 1Æ7 kb for each of the 130 isolates (106 tomato or soil isolates and 24 reference strains). The PCR products were digested individually with each of the seven restriction enzymes. Depending on the enzyme, two to 11 differ- ent restriction patterns were obtained among the 130 isolates of Fusarium (Table 2). In all, 18 combinations of patterns representing 18 IGS types were identified among the 130 isolates (Table 3). Among the Algerian isolates, the 17 isolates of F. commune and the three isolates of F. redolens were assigned to separate unique IGS types, 78 and 80 respectively. The 27 Algerian isolates of F. oxysporum were distributed in nine out of the 16 IGS types found within F. oxyspo- rum in this study. Among these nine, six (5, 12, 16, 51, 74 and 79) were not found among tomato isolates originating from other countries. IGS type 25 was the

most abundant type, which was represented by 55 of the F. oxysporum isolates analysed. It was not found in Algeria; however it was present and dominant in its neighbouring country Tunisia.

Of the Algerian isolates characterized by their EF-1a sequence and their IGS type, a high correspondence was found between the two grouping methods: all isolates in the same IGS type possessed the same EF-1a sequence type, with the exception of isolates belonging to IGS type 3 (MIAE00132, MIAE00135 and MIAE145) that were differentiated into two EF-1a sequence types (Fig. 1).

Conversely, three isolates (MIAE00132, MIAE00134 and MIAE00142) grouped in the same EF-1a sequence type could be differentiated by their IGS type.

PCR amplification with primers P12-F2B and P12-R1 yielded a 990 bp fragment of the SIX1 gene for the 10 ref- erence strains of FOL (SIX1+) but no PCR product for the 13 reference strains of FORL (SIX1 ) ). Among the 48 Algerian isolates and the 58 isolates originating from seven other Mediterranean countries, 13 isolates were scored as SIX1+. These included seven isolates previously identified as FOL, and six isolates recently collected in Tunisia (Table 1). The 93 remaining isolates classified as FORL or as unknown forma specialis were found to be SIX1 ) .

MIAE00129

MIAE00120 MIAE00118 MIAE00117 MIAE00116 MIAE00115 MIAE00114 MIAE00113 MIAE00112 MIAE00110 MIAE00109 MIAE00108 MIAE00107 MIAE00106 MIAE00105 MIAE00104 MIAE00103 MIAE00102

MIAE00101 99MIAE00145MIAE00135MIAE00119MIAE00111MIAE00147MIAE00144MIAE00143MIAE00140MIAE00139MIAE00137MIAE00136MIAE00133MIAE00128MIAE00127MIAE00122 MIAE00142

MIAE00134 MIAE00132

76

MIAE00126 MIAE00125 MIAE00124 MIAE00123 99

MIAE00138 MIAE00141 MIAE00146 MIAE00121 89

MIAE00148

100 100

MIAE00131 MIAE00130

100

0·01

Fusarium redolens

F. oxysporum

F. commune

Figure 1Bootstrapped neighbour-joining tree (Kimura two-parameter distance) inferred from 48 partial translation elongation factorEF-1a gene sequences ofFusariumspp. isolated from tomato or soil in Algeria. The origin of the isolates is given in Table 1. Bootstrap values (>75 %) are indicated on the internodes.

Vegetative compatibility grouping

One isolate of F. commune, two isolates of F. redolens and three isolates of F. oxysporum were found to be hetero-

karyon self-incompatible (HSI) (Table 1). Among the 17 remaining isolates of F. commune originating from Alge- ria, 14 isolates were assigned to the same VCG and three isolates could not be assigned to any VCG because of the lack of some types of mutants, despite repeated attempts to obtain them. The 24 Algerian isolates of F. oxysporum that were not HSI sorted into 13 VCGs. None of these VCGs corresponded to previously described groups; these new VCGs were designated by letters. VCG C included four isolates, VCG F and G each included three isolates, VCG B, D, E and H each included two isolates. The six remaining VCGs were each represented by a single isolate.

The 12 Tunisian isolates of F. oxysporum that were not HSI sorted into four VCGs. VCG 0030 of FOL included six isolates also scored as SIX1+, VCG 0090 of FORL included two isolates, and two new VCGs each grouped two isolates. These new VCGs (I and J) were different from the new VCGs identified among the Algerian isolates.

Among the European isolates, seven French isolates collected before 1990 and one Spanish isolate collected in 1995 were assigned to VCG 0030 or 0031 of FOL; these eight isolates were also scored as SIX1+. All of the other European isolates, previously identified as FORL or from unknown forma specialis, were assigned to one of the fol- lowing VCGs of FORL: 0090, 0091, 0092, 0094 and 0096, with the exception of five isolates for which the VCG could not be determined. Isolates in the same VCG also possess the same IGS type. All of the isolates in VCG 0090, 0091, 0092, 0096 of FORL and VCG 0030, 0031, 0032 of FOL were identified as IGS type 25. Isolates in VCG 0094 and 0098 of FORL were grouped in IGS type 32. Isolates in VCG 0099 of FORL and 0033 of FOL were grouped in IGS type 20. Finally, the isolate of VCG 0093 was placed in IGS type 60.

Pathogenicity tests

The references strains of FOL and FORL induced 90 to 100% of disease (including death) in the bioassay conducted at 24C (Table 1). No symptoms were observed on uninoculated plants and plants inoculated with strain Fo47 known to be non-pathogenic. Among

Table 2Restriction patterns of PCR-amplified intergenic spacer (IGS) fragments ofFusariumisolates

Enzymes^a Fragments (bp)

AluI

1 850, 500, 270, 85

2 540, 500, 320, 270, 85

7 500, 370, 300, 235, 145, 135

21 690, 500, 270, 145, 85

23 760, 500, 375, 85

HaeIII

1 460, 145, 130, 115*, 100, 95*, 85, 80, 60 6 460, 145, 115*, 100*, 95*, 85, 80, 60 7 350, 150, 145, 115, 110, 100, 95*, 85, 75, 60 8 460, 145, 130, 115, 100, 95*, 85, 80, 70, 60 11 480, 145, 130, 115*, 100, 95*, 85, 80, 60 13 470, 175, 145, 130, 115, 100, 95*, 85, 80, 60 19 460, 175, 145, 130, 115, 100, 95*, 85, 80, 60 21 460, 195, 145, 130, 115, 100, 95*, 85, 60 22 460, 175, 145, 130, 115, 100, 95*, 85, 60 28 460, 145, 130, 115*, 100, 95, 85, 60 29 600, 145, 130, 115, 100, 95*, 85, 80, 60 HinfI

2 700, 550, 210, 120, 80

3 670, 530, 330, 160

5 700, 580, 210, 120, 80

20 700, 550, 210, 130, 80

22 700, 550, 220, 195

MspI

1 560, 275, 200, 105, 95, 85, 75, 60*

4 420, 275, 200, 135, 105, 95, 85, 75, 60*

6 560, 275, 200, 105, 95, 75, 60*

11 560, 275, 200, 115, 105, 85, 75, 60*

18 640, 275, 200, 140, 105, 75, 60*

19 590, 275, 200, 105, 95, 85, 75, 60*

27 625, 275, 200, 120, 105, 75, 60*

28 500, 275, 200, 175, 105, 95, 75, 60*

RsaI

1 610, 560, 400, 90

3 1200, 400, 90

4 650, 560, 400, 90

5 520, 400, 320, 275, 90

6 610, 540, 400, 90

9 650, 540, 400, 90

22 560, 400, 375, 275, 90

ScrFI

1 460, 215, 180, 170, 135, 110, 90, 85, 60 2 460, 305, 180, 170, 135, 110, 90, 60 9 420, 305, 180, 170, 135, 110*, 60 11 460, 215, 180, 170, 135, 110*, 85, 60 12 460, 215, 180, 170, 135, 90, 85*, 60 13 470, 215, 180, 170, 135, 120, 90, 85, 60 21 460, 215, 180, 170, 135, 120, 85*, 60 24 490, 240, 145, 135, 125, 110*, 60 XhoI

1 1300, 370

2 1670

Table 2Continued

aFor each restriction enzyme, the various patterns are indicated by numbers in the first column and the sizes in base pairs (bp) of the corresponding restriction fragments in the second column.

Estimates of fragment sizes were determined by electrophoresis in 4–6% Nusieve 3:1 agarose (FMC) and by comparison with the molecular weight marker VIII (Roche Diagnostic) with measurements rounded to the nearest 5 bp. This was done for purposes of comparison among isolates; the values do not reflect absolute base pair fragment sizes. Restriction fragments less than 60 bp were not taken into consideration because they were not clearly resolved by electrophoresis. Numbers corresponding to the different restriction patterns follow those previously described (Aboet al., 2005; Edel et al., 1997; Edelet al., 2001; Loriet al., 2004).

*Two restriction fragments of the same size (doublet).

the 106 isolates tested, 11 isolates induced no disease symptoms at all, 24 isolates induced 10–30% of disease, 20 isolates induced 40–60% of disease and 50 isolates induced 70–100% of disease. All of the isolates that induced a low percentage of disease were tested again in a bioassay conducted at 28C during the day instead of 24C. Among the 53 isolates tested at 28C, 41 isolates induced a higher percentage of disease than that induced at 24C. Particularly among the Algerian isolates, the eight isolates that produced no disease at all at 24C induced 60% (one isolate), 70% (one isolate), 90% (three isolates) and 100% (three isolates) of disease at 28C.

The disease symptoms observed on plants inoculated with F. commune and F. redolens were similar to those observed on plants inoculated with FORL: plants showed dark brown lesions on crown and roots and dead plants.

The pathogenicity of the latter two species was recently reported on tomato in Algeria (Hamini-Kadar et al., 2010).

Discussion

A surprisingly high level of genetic diversity was found among the isolates responsible for fusarium diseases in Algeria collected between 2005 and 2008. Besides several genotypes within F. oxysporum, it was also found that F. redolens and F. commune were patho- genic to tomato. Fusarium redolens is closely related to

F. oxysporum. The latter two species were thought to be conspecific by Snyder & Hansen (1940) but they were recognized as two distinct species by Gerlach (1981). Later, the two species were clearly differenti- ated by molecular phylogenetic studies (Baayen et al., 1997; O’Donnell et al., 1998a); however, the two spe- cies remain difficult to distinguish using only morpho- logical characters. Apart from the present study, only one other report of F. redolens causing disease on tomato plants was found, which was in the UK 40 years ago (Wilcox & Jackson, 1970). In the study here, the three isolates identified as F. redolens all originated from Algeria. They shared the same IGS type, and all three were more pathogenic in the bioassay conducted at 28C than at 24C. As two of them were HSI in the VCG test, there is no more information concerning the intraspecific diversity, which might be further resolved by amplified fragment length polymorphism (AFLP) analysis (Baayen et al., 2000). Whether the concept of forma specialis could be applied to these isolates of F. redolens is not known because their host specificity has not yet been demonstrated. This concept was previ- ously applied to Fusarium isolates responsible for car- nation diseases, including F. oxysporum f. sp. dianthi and F. redolens f. sp. dianthi (Baayen et al., 1997).

Eighteen Algerian isolates were identified as F. com- mune. This recently described species is closely related to, but phylogenetically distinct from, F. oxysporum

Table 3Intergenic spacer (IGS) types and restriction patterns ofFusariumisolates revealed by restriction fragment length polymorphism analysis of PCR- amplified IGS sequences

Species IGS type^a Representative isolate^b

Restriction patterns of amplified IGS fragments digested with enzymes^c

Geographic distribution AluI HaeIII HinfI MspI RsaI ScrFI XhoI

Fusarium oxysporum 2 MIAE00511 1 1 2 1 1 1 1 Tunisia

3 MIAE00132 1 1 2 1 1 1 2 Algeria, France, USA

5 MIAE00122 1 1 2 1 1 2 2 Algeria

12 MIAE00138 1 6 2 4 1 1 2 Algeria

16 MIAE00142 2 1 2 1 6 1 2 Algeria

20 MIAE00524 2 1 2 1 3 1 2 Algeria, Spain, Italy, USA

22 MIAE00047 1 11 2 11 4 11 2 France

23 MIAE00046 1 1 2 1 3 1 2 Crete, USA

25 MIAE00045 1 13 2 1 1 13 2 Tunisia, Morocco, Turkey, Israel,

Spain, France, USA

32 MIAE00534 2 1 2 1 3 12 2 Turkey, France, USA

51 MIAE00148 1 21 5 19 1 1 2 Algeria

60 MIAE00483 1 29 2 1 3 13 2 Israel, USA

67 MIAE00514 21 22 20 27 9 21 2 Algeria, Tunisia

74 MIAE00121 1 8 2 6 1 1 2 Algeria

79 MIAE00123 1 1 2 1 3 13 2 Algeria

81 MIAE00512 2 19 2 1 1 1 2 Tunisia

F. commune 78 MIAE00101 23 28 22 18 22 9 1 Algeria

F. redolens 80 MIAE00129 7 7 3 28 5 24 2 Algeria

aIGS types represent the combination of patterns obtained with seven restriction enzymes. The numbers assigned to IGS types 2–60 follow those previously described (Aboet al., 2005; Edelet al., 1997; Edelet al., 2001; Loriet al., 2004). The restriction patterns are described in Table 2.

bIsolates are described in Table 1.

cNumbers designate the various patterns obtained for each restriction enzyme and follow those previously described (Aboet al., 2005; Edel et al., 1997; Edelet al., 2001; Loriet al., 2004; Schoutenet al., 2004).

(Skovgaard et al., 2003). This is the first report of F. commune isolated from and pathogenic on tomato.

Because F. commune and F. oxysporum are morpholog- ically similar (Skovgaard et al., 2003), previous cases of fusarium diseases on tomato may have been due to F. commune. The species has been isolated from soil and from different host plants including carrot, barley, corn, carnation, pea, pine and Douglas-fir (Skovgaard et al., 2003; Stewart et al., 2006). In this study, unlike F. redolens which was only found in the north near Alger, F. commune was collected only in the northwest region in several towns around Oran. The 18 isolates shared the same EF-1a sequence and comparison with other sequences available in GenBank showed that they were identical with several other isolates of F. commune originating from different host plants. However, at the present time, it is not known if there is any host speci- ficity in F. commune. In addition to the shared EF-1a sequence, the 18 isolates of F. commune also shared the same IGS type. Also 14 of the 18 isolates grouped in the same VCG, corresponding to what could be called a clo- nal population (Klein & Correll, 2001). However, vari- ability in the percentages of diseased plants suggests intraspecific diversity that could be assessed by AFLP.

As with isolates of F. redolens, disease induced by the F. commune isolates was generally greater at 28C than at 24C, which might reflect adaptation to the warmer climatic conditions in Mediterranean countries.

The 27 remaining isolates originating from Algeria were identified as F. oxysporum, which showed a high level of intraspecific diversity, including nine IGS types and 13 VCGs. All 27 isolates were pathogenic on tomato and exhibited diverse levels of disease incidence ranging from 20–100%. As none of the isolates was scored as SIX1+, the 27 isolates can be assigned to FORL (van der Does et al., 2008). However, none of the Algerian isolates could be assigned to a known VCG of FORL. They were distributed in seven multiple-member VCGs and six sin- gle-member VCGs, but are all closely related genetically to previously characterized strains of F. oxysporum with which they share the same IGS type or at least common RFLP patterns. IGS 79 was the only novel type, but it matched known restriction patterns (Edel et al., 1997, 2001; Lori et al., 2004; Abo et al., 2005).

High diversity was also found among isolates of F. oxy- sporum originating from the neighbouring country Tuni- sia, with 14 isolates distributed in four IGS types.

Although they were collected at the same period in both countries, the F. oxysporum isolates pathogenic on tomato were completely different. Several Tunisian iso- lates were identified as FOL both by their SIX1+ PCR and their VCG (0030). Among the other isolates identified as FORL, the typical VCG 0090 was found in Tunisia. IGS type 25, that includes VCG 0030 of FOL and VCG 0090 of FORL, was the dominant type among the Tunisian iso- lates; the dominance of IGS type 25 in the survey corrobo- rates similar findings by Hibar et al. (2007). In the present study, IGS type 25 was also dominant among isolates collected in other countries including France, Spain and

Italy. In these countries, the forma specialis FOL was mostly detected among the oldest isolates collected before 1985. The more recent isolates were all identified as FORL with a known VCG. In contrast to what was observed in both North African countries, the diversity in IGS types was much lower in European countries, with two IGS types found within FOL (IGS types 20 and 25) and four IGS types within FORL (IGS types 20, 25, 32, 60). Some isolates of FOL and FORL shared the same IGS type, whereas isolates of the same forma specialis were distributed in different IGS types, which may reflect their polyphyletic origins (Cai et al., 2003; O’Donnell et al., 2009). In addition to the different climatic conditions, the intensive cropping of tomato in European countries could have changed the balance among populations of F. oxy- sporum linked to tomato crops.

The combination of the two loci EF-1a and IGS made it possible to identify the Fusarium isolates to species and to characterize them at the intraspecific level. O’Donnell et al. (2009) have recently proposed the same strategy to type F. oxysporum isolates by sequencing both loci. Thus the number of types in the survey might increase by sequencing the IGS region. Concerning the identification of the formae speciales of F. oxysporum pathogenic on tomato, the results confirm that it is now possible to iden- tify more rapidly FOL and FORL by combining the spe- cific SIX PCR described by van der Does et al. (2008) with the rapid bioassay described in this study. Within formae speciales, although laborious, VCG testing remains a helpful method to characterize the genetic diversity of F. oxysporum populations. It also has several limitations, the first being the fact that it depends on refer- ence strains that could be lost from collections. This study was unable to find representative strains of one VCG of FOL (0035) and one VCG of FORL (0097). Thus, it is possible that one of the new VCG identified here is VCG 0097.

Considerable diversity was found among the isolates of

Fusarium pathogenic on tomato in Algeria, including spe-

cies never (F. commune) and rarely (F. redolens) isolated

from tomato, and several possibly new VCGs. Also, this

diversity was different from the one observed in Tunisia,

and both were different from that observed in European

countries. The fact that numerous isolates collected in

Algeria correspond to new genotypes never detected

before and not detected in other countries may be due to

the tomato cultivars used in Algeria. These might be dif-

ferent from the ones used in other countries and are

diverse because farmers have long produced their own

seeds using old cultivars; it is thus difficult to identify the

different cultivars used. Given the difference between

pathogenic isolates in Algeria and in other Mediterranean

countries, it is hypothesized that newly emergent patho-

genic forms may have evolved from local non-pathogenic

populations or were previously misidentified. This ques-

tion could be addressed by investigating the genetic diver-

sity of soilborne populations in Algerian tomato growing

areas. Genetic similarity between pathogenic and

co-occurring non-pathogenic isolates may indicate that

pathogenic isolates could be recent derivatives from local non-pathogenic populations.

Acknowledgements

The authors thank N. Hamini-Kadar and M. Begin for technical assistance, A. Buffie`re, K. Elena, N. Hamini-Ka- dar, K. Hibar, M. Jacquet, I. Larena Nistal, A. Moretti, Q. Migheli, M. Raviv, D. Vakalounakis and S. Yucel for providing fungal strains and C. Alabouvette for helpful comments on the manuscript.

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