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European Journal of Plant PathologyPublished in cooperation with theEuropean Foundation for PlantPathology ISSN 0929-1873 Eur J Plant PatholDOI 10.1007/s10658-012-0148-5

Genetic variability of tolerance toVerticillium albo-atrum and Verticilliumdahliae in Medicago truncatula

A. Negahi, A. Sarrafi, A. Ebrahimi,P. Maury, J. M. Prospéri, C. Ben &M. Rickauer

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Genetic variability of tolerance to Verticillium albo-atrumand Verticillium dahliae in Medicago truncatula

A. Negahi & A. Sarrafi & A. Ebrahimi & P. Maury &

J. M. Prospéri & C. Ben & M. Rickauer

Accepted: 3 December 2012# KNPV 2013

Abstract Verticillium wilt caused by Verticilliumalbo-atrum and Verticillium dahliae is responsiblefor yield losses in many economically important crops.The capacity of pathogenic fungi to adapt to new hostsis a well-known threat to the durability of resistantcrop varieties. In the present work, 25 Medicago trun-catula genotypes from a core collection and six V.albo-atrum and V. dahliae strains were used to studythe potential of non-host Verticillium strains isolatedfrom different plant species to infect this legume plantand the plant’s susceptibility to the pathogens. Theexperiment was designed as factorial with randomisedcomplete blocks and with three repetitions. The wiltsymptoms caused by V. albo-atrum and V. dahliae

were scored on a disease index scale from 0 to 4,during 30 days after inoculation of 10-day-old plant-lets. Disease severity was quantified by maximumsymptom scores (MSS) and areas under the diseaseprogress curves (AUDPC), which integrate the timecourse of symptom development. Highly significantdifferences were observed among plant genotypes andfungal strains, and their interaction was also signifi-cant. The correlation between MSS and AUDPC was0.86. The most severe symptoms were caused by thealfalfa strain V. albo-atrum V31-2 and the least severeby V. dahliae JR2, as shown by mean values obtainedon the 25 genotypes. M. truncatula genotype TN8.3was the most susceptible genotype by mean values

Eur J Plant PatholDOI 10.1007/s10658-012-0148-5

A. Negahi :A. Sarrafi : C. Ben :M. RickauerUniversité de Toulouse; INP, UPS; EcoLab (LaboratoireD’Ecologie Fonctionnelle et Environnement); EcoleNationale Supérieure Agronomique de Toulouse,18 chemin de Borde Rouge, BP 32607Auzeville 31326Castanet-Tolosan, France

A. Negahi :A. Sarrafi : C. Ben :M. RickauerCNRS, UMR 5245EcoLab,31326 Castanet-Tolosan, France

P. MauryUniversité de Toulouse, INP, ENSAT, UMR AGIR,BP 52627, 31326 Castanet-Tolosan cedex, France

A. EbrahimiFaculty of Agriculture and Natural Resources,Science and Research Branch, Islamic Azad University,Ashrafi Esfehani Highway, BP 1477893855, Tehran, Iran

J. M. ProspériINRA, UMR AGAP 1334,Montpellier 34130 Mauguio, France

A. Sarrafi (*)Laboratoire Ecologie Fonctionnelle et Environnement(ECOLAB), UMR 5245 CNRS, Ecole NationaleSupérieure Agronomique de Toulouse,Pôle de Biotechnologie végétale 18 cheminde Borde Rouge, BP 32607Auzeville31326 Castanet-Tolosan, Francee-mail: Sarrafi@ensat.fr

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obtained with the six fungal strains, whereas F11013-3, F83005.9 and DZA45.6 were highly resistant to allstrains studied. The results can be used to chooseparents for studying the genetics of resistance inMedicago truncatula to Verticillium strains with dif-ferent levels of aggressiveness.

Keywords Disease . Resistance . Susceptibility .

Vascular wilt . Host-pathogen interaction . Non-host

Introduction

Verticillium wilt diseases are mainly caused by twospecies, Verticillium albo-atrum (Vaa) and Verticilliumdahliae (Vd) which differ in morphology, host range,and growth characteristics (Schnathorst 1981). Thesesoil-borne fungi are responsible for monocyclic vas-cular wilt diseases in over 200 dicotyledonous species,including economically important crops. The funguscolonises the vascular tissue of roots and shoots,which causes typical wilt symptoms and leads to thedeath of susceptible plants (Fradin and Thomma2006). Because of the localisation of the pathogen inthe central cylinder, vascular diseases are particularlydifficult to control and plants cannot be cured byfungicides once the symptoms are visible. In addition,survival structures may stay viable in the soil for along time. Vd and Vaa have different host rangeswhich are overlapping for some plant species such astomato (Solanum lycopersicum). Both fungal speciesare found to cause disease in temperate and subtropi-cal regions. Vd appears to be favoured by highertemperatures than Vaa, as can be deduced from itsgeographical distribution (Church and McCartney1995; Friebertshauser and De Vay 1982; Koike et al.1994). Both are pathogens of forage and grainlegumes (Aubé and Sackston 1964; Erwin 1973).Notably Vaa may cause significant damage to alfalfacultures, and in North America yield losses due toVerticillium wilt were reported to reach 50 %(Christen and Peaden 1981; Smith et al. 1995).Although many resistant cultivars are available today,they may not be best adapted to some specific con-ditions of soil or climate. Because of the high geneticvariability of pathogenicity in Verticillium, simulta-neous incorporation of several genes for resistancewould be needed to maintain effective cultivars whichcould be used over a large area (Klosterman et al.

2009). The foreseeable evolution of the pathogen andappearance of new races of V. albo-atrum will be aserious threat to alfalfa cultures, since resistance inmost cultivars was introduced from a small numberof parents (Acharya and Huang 2003). Breeding newresistant cultivars is necessary to control Verticilliumwilt in the present and future.

In several plant species, including alfalfa (Medicagosativa), cotton (Gossypium hirsutum), potato (Solanumtuberosum), strawberry (Fragaria vesca), sunflower(Helianthus annuus), and tomato, sources of geneticresistance to Verticillium have been described (reviewedin Fradin and Thomma 2006; Klosterman et al. 2009).Studies on the genetic variation for resistance toVerticillium wilt in alfalfa indicate that existing resis-tance levels are still insufficient and should be improvedthrough further selection (Huang et al. 1999; Vandemarket al. 2006). When twenty-five Medicago sativa popu-lations were investigated for their resistance to Vaa,three other fungal diseases and stem nematode, a greatvariability in susceptibility to the diseases was observed(Julier et al. 1996). Among 50 alfalfa varieties of aEuropean reference collection, less than 10 were resis-tant to Vaa (Molinéro-Demilly et al. 2007). Alfalfaresistance to Vaa can be quantitative or qualitative, anddistinct physiological features have been associated tothese two forms of resistance (reviewed by Pennypacker2000; Acharya and Huang 2003). In tomato, geneticvariability for resistance to Vaa was reported(Paternotte and Van Kesteren 1993) and it was foundthat one single gene confers protection towards Vd andVaa race 1 (Fradin et al. 2009).

Genetic studies of disease resistance in alfalfa aredifficult due to its outcrossing and tetraploid nature.The closely related speciesMedicago truncatula, whichis native in theMediterranean basin, has been developedas the first model legume plant. It is autogamous with ashort life cycle (6 months), and diploid (2n=2x=16)with a small genome (500 Mbp), which simplifies stud-ies on the genetic mechanisms of responses to abioticand biotic agents, notably with regard to the nitrogen-fixing symbiosis with rhizobia (Barker et al. 1990). M.truncatula shows high synteny with other legumeplants, especially with M. sativa (Choi et al. 2004).

Significant efforts have concentrated on M. trunca-tula as a model system to characterise legume-pathogen interactions (Tivoli et al. 2006), and variouspathosystems have been developed withM. truncatulaas a host plant, involving fungi, oomycetes, bacteria or

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insects. Genotype - strain interactions were observedwith Colletotrichum trifolii (Torregrosa et al. 2004),Erysiphe pisi (Foster-Hartnett et al. 2007), Fusariumoxysporum (Ramirez-Suero et al. 2010), Aphanomyceseuteiches (Djebali et al. 2009), Ralstonia solanacearum(Vailleau et al. 2007) or the pea aphid Acyrthosiphonpisum (Stewart et al. 2009).

M. truncatula has been shown to be a host plant forVaa in our laboratory and major QTLs for resistanceagainst the alfalfa strain VaaV31-2 of this pathogenwere identified (Ben et al. 2012). As far as we know,the interaction between M. truncatula and other, non-host Vaa and Vd strains is not reported in the literature.Pathogenic fungi are known for their capacity to rap-idly adapt to new environments or host plants. Hencewe aimed to study the potential of Verticillium strainsisolated from several crops which are cultivated inEurope or isolated from soil, to cause disease in thenon-host plant M. truncatula and to identify possiblesources of resistance in this species. In the presentresearch we studied the responses of 25 M. truncatulagenotypes from various geographical origins to sixstrains of V. albo-atrum and V. dahliae under con-trolled conditions.

Materials and methods

TheM. truncatula genotypes used in this study include acore collection of 16 lines (http://www1.montpellier.inra.fr/BRC-MTR/mauguio/mauguio.php) (Ronfort etal. 2006) and lines involved in crosses for genetic stud-ies (Table 1).Most of lines are sequenced as presented inTable 1. All the genotypes of M. truncatula are purelines and derived from one plant as single seed descend-ants. Line 2HA which is derived from Jemalong andconsidered a sister line of A17 was obtained from M.Chabaud, INRA Auzeville, France. Seeds were pro-duced at INRA Montpellier and Ecolab Toulouse inthe greenhouse. Plants for the experiments were grownin peat-substrate Jiffy pots (Jiffy France, Lyon, France)as described by Vailleau et al. (2007). They were used forinoculation after 10 days of growth in a phytotron with aphotoperiod of 16 h, a temperature of 25 °C/20 °Cand relative humidity of 75 %.

Six V. albo-atrum and V. dahliae strains isolated fromvarious plants as summarised in Table 2 were providedby B.P.H.J. Thomma (Wageningen, Netherlands) and A.von Tiedemann (Goettingen, Germany). Vaa and Vd

strains were grown on potato dextrose agar (PDA) inPetri dishes at 24 °C. Spores were harvested after21 days of growth by flooding the Petri dishes withsterile water. The concentration of spores was

Table 1 Medicago truncatula genotypes and their origin

Line Origin

A17b Unknown

F83005.5 a, b France

TN1.11 b Tunisia

TN1.21 b Tunisia

A10 b Morocco

A20 b Morocco

DZA45.6 a, b Algeria

DZA45.5 Algeria

2HA Unknown

TN6.18 b Tunisia

TN8.3 b Tunisia

F83005.9 b France

DZA315.16 a,b Algeria

SA09707 a,b Tunisia

ESP105-L a,b Spain

Salses71B a,b France

SA022322 a,b Syria

F11013-3 a,b France

SA024714 a,b Italy

DZA012-J a,b Algeria

DZA327-7 a,b Algeria

GRC020-B a,b Greece

DZA233-4 a,b Algeria

SA026063 a,b Morocco

SA028064 a,b Cyprus

a Lines from the core collection CC16b Sequenced lines

Table 2 Verticillium spp. strains used in this work

Strain Species Isolated from

Vd JR2 V. dahliae Tomato

Vd S26 V. dahliae Soil

Vaa VA1 V. albo-atrum Unknown

Vaa 5431 V. albo-atrum Tomato

Vaa V31-2 V. albo-atrum Alfalfa

Vaa LPP0323 V. albo-atrum Potato

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determined with a Malassez haemocytometer and ad-justed to 106 spores/ml with distilled water.

The experiment was a factorial design with random-ized complete blocks with eight plants per genotype ineach block, to test M. truncatula genotype effect,Verticillium strain effect and their interaction. Threeindependent repetitions were performed at three differ-ent dates. Plants were root-trimmed by cutting approx-imately 1 cm from the bottom of the Jiffy pot and wereinoculated by dipping the exposed roots for 30 min inthe fungal spore suspension. The inoculated plants werethen transferred to a growth chamber with a photoperiodof 16 h and a constant temperature of 20 °C. Thedevelopment of symptoms was checked every 3 daysduring 30 days after inoculation.

A disease index scale from 0 to 4 as established in ourlaboratory was used for the evaluation, based on thestandard test published by the North American AlfalfaImprovement Conference (http//www.naic.org/stdtests/vertical.htm) but adapted to the disease development onvery young plants. Score 0 corresponds to plants withoutsymptoms, score 1 when the first leaf is chlorotic, score 2when the first two leaves are wilted, score 3 when theentire plant is wilted, and 4 when the plant is dead.

The symptom score at the end of the experiment(maximum symptom score, MSS) and the area underdisease progress curve (AUDPC) which takes intoaccount the speed of symptom development and se-verity of symptoms, were determined for each plantper genotype per replication, and mean values werecalculated. The AUDPC was calculated according tothe equation of Campbell and Madden (1990). Theeffects of genotypes, strains and their interactions wereidentified by analysis of variance with the generallinear model (GLM) procedure in SAS software(SAS Institute, USA). For each analysis of variance,the normality of the residuals was tested by theShapiro - Wilk’s method. Comparison of means wasdone with the least significant difference (LSD) test.

Results

In the present work the interactions of 25 M. trunca-tula genotypes (Table 1) with six Verticillium isolates(Table 2) were studied. The interaction of M. trunca-tula with the French alfalfa strain Vaa V31-2, as wellas the reliability of the root inoculation protocol com-pared to the protocol used by alfalfa breeders on

trimmed aerial parts (Molinéro-Demilly et al. 2007),have been established previously (Ben et al. 2012).This strain was included in the present work forcomparison with five other strains which were isolatedfrom tomato, potato, soil or from unknown origin, andwhich have not been described to infect alfalfa orM. truncatula.

Symptoms started to develop between 7 and 10 daysafter inoculation of the plants, and reached score 4(dead plant) for highly susceptible lines at the end ofthe experiment. The effects of genotypes, strains andtheir interactions were tested for maximum symptomscore (MSS) which is the symptom score at the end ofthe experiment and for area under the disease progresscurve (AUDPC) which also considers the time courseof symptom development. Analysis of varianceshowed that the effects of genotype, and isolate aswell as of their interaction were highly significant forthese two parameters. We also detected a significanteffect of the repetition, which was not unexpectedsince experiments were repeated three times at differ-ent dates and environmental conditions may havechanged slightly (Table 3). The correlation betweenMSS and AUDPC was 0.86 and highly significant.Tables 4 and 5 summarise the effect of genotypes,strains and their interactions assessed by MSS andAUDPC respectively. A line was considered suscepti-ble when MSS values were ≥ 1.5 and AUDPC ≥ 22.Differential responses of the 25 genotypes towards thesix fungal strains were observed. When comparedwith Verticillium strains that were not isolated fromalfalfa, the disease was generally less severe. Only theinfection of TN8.3 with Vaa VA1 resulted in MSS andAUDPC values of 2.65 and 30.99 respectively, indic-ative of clear susceptibility, whereas all other combi-nations between lines and strains showed resistance orintermediate phenotypes. F83005.5 which is highlysusceptible to Vaa V31-2 was resistant to the otherfive Verticillium strains.

The effects of genotype and strain based on MSSand AUDPC mean values (Tables 4 and 5) showedthat genotype TN8.3 was the most susceptible line andF11013-3 the most resistant, respectively. Strain VaaV31-2 was the most aggressive and Vd JR2 the leastaggressive Verticillium strain. Among the 25 M. trun-catula genotypes we identified some with homoge-nous responses such as TN8.3 and F83005.9 thatwere respectively susceptible and resistant to allVerticillium strains, whereas others such as F83005.5,

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A17 and Salses 71B showed variable responses to thedifferent strains and were highly susceptible only to

strain Vaa 31–2, Vaa VA1 or three different strains,respectively (Table 5). These results show high genetic

Table 3 Analysis of variance for maximum symptom scores (MSS) and area under the disease progress curve (AUDPC) in Medicagotruncatula genotypes inoculated by Verticillium albo-atrum and Verticillium dahliae strains

MSS AUDPC

Source of variation d.f.a MSb Source of variation d.f. MS

Genotype 24 1.80*** Genotype 24 540.42***

Strain 5 14.06*** Strain 5 2114.64***

Genotype x strain 120 0.47*** Genotype x strain 120 64.17***

Replication 2 0.13** Replication 2 413.17***

Error 298 0.02 Error 298 17.40

a d.f. degree of freedombMS mean of squares** and *** significant at 0.01 and 0.001 levels, respectively

Table 4 Susceptibility of 25Medicago truncatula genotypesto six Verticillium strainsassessed by Maximum SymptomScores

a, b, …, n letters from a to n indi-cate comparison of mean for themain effect of genotypes andstrains. Genotypes or strainswith the same letter are in thesame group1 Genotype mean MSS value2 Strain mean MSS value3 Italic bold letters and numbersindicate the most resistant andsusceptible genotypes and themost aggressive and the least ag-gressive strains

Genotype Vaa V31-23 Vaa 5431 Vaa LPP0323 Vaa VA1 Vd JR23 Vd S26 Means1

A17 0.59 1.04 1.34 1.62 1.21 1.5 1.22efg

F83005.5 3.96 0.35 0.48 0.66 0.71 0.77 1.15def

TN1.11 1.63 1.41 1.25 1.18 0.98 1.04 1.25fgh

TN1.21 2.17 1.02 1.06 0.56 0.98 1.14 1.16def

A10 2.52 0.82 1.04 1.07 0.59 1.04 1.18def

A20 2.56 0.76 0.98 0.88 0.78 0.85 1.14de

DZA45.6 1.26 0.67 0.79 1.24 0.58 0.82 0.89b

DZA45.5 1.43 0.86 0.81 1.17 0.49 0.79 0.92b

2HA 0.54 0.4 1.14 1.32 1.04 1.27 0.95bc

TN6.18 1.66 1.02 1.31 1.47 1.38 1.47 1.39ij

TN8.33 3.44 1.64 1.83 2.65 1.53 1.61 2.12n

F83005.9 1.21 0.54 0.87 1.03 0.93 0.75 0.89b

DZA315.16 2.66 1.55 1.63 1.65 1.4 1.52 1.73m

SA09707 2.29 1.37 1.24 1.42 0.94 1.26 1.42j

ESP105-L 0.6 1.32 1.11 1.22 0.59 1.23 1.01bc

Salses71B 2.97 1.61 1.4 1.76 0.97 1.38 1.68lm

SA22322 3.04 1.44 1.28 1.56 1 1.34 1.61kl

F11013-33 1.37 0.4 0.61 0.53 0.4 0.78 0.68a

SA24714 2.79 1.12 0.87 1.28 1.04 0.87 1.33hij

DZA012-J 2.64 1.19 1 1.25 1.09 1.04 1.37hij

DZA327-7 2.99 1.38 1.42 1.12 1.21 1.12 1.54k

GRC020-B 2.12 1.35 1.29 1.2 0.92 0.84 1.29ghi

DZA233-4 2.07 1.31 1.23 1.43 1.03 1.06 1.35hij

SA26063 2.32 0.94 0.66 0.98 0.69 0.79 1.06cd

SA28064 2.07 0.91 0.76 0.82 0.87 0.87 1.05cd

Means 2 2.12d 1.06b 1.09b 1.24c 0.93a 1.09b

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variability of responses to Vaa and Vd among M.truncatula genotypes and significant statistical interac-tion between host plant and pathogen isolates. Throughsignificant interactions we can mention the case ofgenotypes 2HA and ESP105-L with isolates Vaa5431and VaaVA1, for MSS (LSD=0.30 at 0.01 level) andalso genotypes Salses71B and F11013-3 with isolatesVaaVA1 and VdJR2 for AUDPC (LSD=8.78 at 0.01level).

Discussion

The susceptibility of M. truncatula lines to differentVerticillium strains was estimated and analysed byMSS and AUDPC. There was a good correlationbetween these two parameters (R2=0.86). However,when values were near the threshold for susceptibility/

resistance, MSS and AUDPC gave sometimesopposite phenotypes. This was observed for examplewith genotype TN1.11 inoculated with Vaa Va1(susceptible by MSS but resistant by AUDPC values)and DZA315.16 inoculated with strains Vaa 5431(resistant by MSS but susceptible by AUDPC values)and also Vd JR2 (resistant by MSS but susceptible byAUDPC values). These differences between the twoparameters can be explained by the fact that the speedof symptom development is integrated in AUDPC.Lines which develop weak symptoms rapidly may havethe same AUDPC values as lines with slow symptomdevelopment but higher MSS.

Significant differences in aggressiveness amongstrains of V. albo-atrum and V. dahliae were alsorevealed in the present study. The most aggressiveand least aggressive strains were Vaa V31-2 and VdJR2, respectively. The highest aggressiveness of Vaa

Table 5 Susceptibility of 25Medicago truncatula genotypesto six Verticillium strainsassessed by AUDPC

a, b, …, g letters from a to g indi-cate mean comparison for geno-types and strains. Genotypes orstrains with the same letter are inthe same group.1 Genotype mean AUDPC value2 Strain mean AUDPC value3 Italic bold letters and numbersindicate the most resistant andsusceptible genotypes and themost aggressive and the least ag-gressive strains

Genotype Vaa V31-23 Vaa 5431 Vaa LPP0323 VaaVA1 Vd JR23 Vd S26 Means 1

A17 9.54 11.18 18.35 31.45 18.63 20.8 18.32e

F83005.5 39.96 2.81 4.56 16.64 4.79 6.62 12.56 abc

TN1.11 22.95 17.5 17.5 24.78 14.34 13.86 18.49 e

TN1.21 25.59 12.11 12.6 7.17 11.39 13.12 13.66bcd

A10 29.38 7.11 12.97 14.33 6.41 12.22 13.74bcd

A20 27.65 11.99 7.65 10.63 5.24 7.88 11.84 abc

DZA45.6 12.34 6.64 8.49 15.82 7.52 10.76 10.26ab

DZA45.5 15.19 9.76 11.22 18.87 9.17 9.78 11.83abc

2HA 9.04 5.3 13.88 16.26 12.98 16.69 12.36 abc

TN6.18 23.44 14.19 17.29 21.88 21.13 21.06 19.83 e

TN8.33 40.9 23.59 28.33 30.99 27.05 28.49 29.89g

F83005.9 16.01 5.29 10.9 13.73 9.99 8.98 10.82ab

DZA315.16 36.23 21.06 24.71 29.14 22.44 24.61 26.36f

SA09707 34.13 17.11 17.14 17.93 15.5 18.28 19.68 e

ESP105-L 9.49 10.34 13.26 16.46 8.61 16.62 12.46 abc

Salses71B 42.53 23.52 21.96 25.08 16.91 24.28 25.72f

SA22322 42.97 21.99 20.87 23.34 16.49 20.95 24.44f

F11013-33 21.49 4.3 6.92 7.47 5.07 9.03 9.05a

SA24714 35.87 14.41 11.98 15.15 11.83 13.17 17.07de

DZA012-J 30.07 13.57 13.43 17.26 13.51 13.83 16.94de

DZA327-7 35.41 19.42 19.5 15.1 15.71 14.77 19.98 e

GRC020-B 30.75 17.52 18.13 18.64 12.94 13.37 18.56 e

DZA233-4 28.65 18 19.51 18.79 14.51 15.95 19.24 e

SA26063 26.84 11.01 7.82 13.61 7.73 11.34 13.06 abc

SA28064 28.39 12.56 12.71 14.97 13.14 14.42 16.03cde

Means 2 26.99d 13.92a 14.87b 18.22c 12.72a 15.2b

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V31-2 is in agreement with the fact that it has beenisolated from alfalfa, which is closely related to M.truncatula. Alfalfa strains of V. albo-atrum are phylo-genetically different from non-alfalfa strains that havebeen described to cause little or no disease on alfalfa(Barbara and Clewes 2003). Among the five non-alfalfa strains studied in this work, Vaa strains wereonly slightly more aggressive than the Vd strains. Bhatand Subbarao (1999) reported that Vd strains fromvarious host plants were able to infect alfalfa, but thatdisease was less severe than with a Vaa strain fromalfalfa. It is interesting to note that Vd S26 which hasbeen isolated from soil and not from a diseased plant,was not the least aggressive strain and was able tocause disease in TN8.3. Similar results have beenreported for the interaction between M. truncatulaand Fusarium oxysporum, where “non-pathogenic”strains used for biological control of Fusarium wiltof tomato were found to cause disease symptoms inseveral lines (Ramirez-Suero et al. 2010).

Due to its large host range, Verticillium wilt causesproblems in many crops. Alfalfa is mainly affected byVaa, notably by alfalfa strains. However, fungal patho-gens are well known for their adaptability and capacityto overcome resistance genes, and to colonise newenvironments. The host range of several pathogenicfungi has been described to evolve due to horizontalgene flow between strains and species (Mehrabi et al.2011). In the case of Verticillium wilt of lettuce it hasbeen suggested that V. dahliae strains from spinach,which is not a close relative to lettuce phylogenetically,might be involved in the drastic outbreak of this diseasein California (Atallah et al. 2010). The fact thatM. truncatula plants could be infected by Verticilliumspecies considered as non-pathogens on Medicago isprobably transferable to alfalfa. Hence it should becarefully evaluated if legume plants like alfalfa couldbe used for crop rotation strategies in fields whereVerticillium wilt of a different crop has occurred. Asshown in this study, a M. truncatula line such as A17which is highly resistant to the alfalfa strain wasmoderately susceptible to a different Vaa strain.

Our results show that strains isolated from variousplant species were able to infect M. truncatula. The 25M. truncatula genotypes used in this study differedconsiderably in susceptibility and resistance to V. albo-atrum and V. dahliae strains. For example, TN8.3 wassusceptible to all strains of V. albo-atrum and V. dah-liae, whereas three genotypes, F11013-3, F83005.9

and DZA45.6, showed resistance to all Vaa and Vdstrains. The other genotypes showed variableresponses to Vaa and Vd strains. It should be notedthat lines 2HA and A17 which derive both from cul-tivar Jemalong and are often considered as sister lines,have a clearly distinct phenotype by AUDPC values,the difference being most pronounced with strain VaaVA1. Clearly the in-vitro culture involved in the creationof this highly embryogenic line (Rose et al. 1999) hasled to somaclonal variability, as shown by significantmodifications in protein expression patterns associatedwith defence against pathogens (Imin et al. 2005). Thisis also in agreement with observations that line 2HA isless susceptible than A17 to the wilt-inducing pathogensF. oxysporum and R. solanacearum (Ramirez-Suero andRickauer, unpublished results).

The statistical analysis showed interaction betweenM. truncatula genotypes and Vaa and Vd strains forboth MSS and AUDPC. Genotype A17 was resistantto Vaa V31-2 but susceptible to most other strains,whereas F83005.5 was susceptible to Vaa V31-2 andresistant to other strains. These two genotypes areoften contrasting in their response to pathogens, andare widely used to study differential defence responsesto pathogens (e.g., Ramirez-Suero et al. 2010;Jaulneau et al. 2010). A17 is reported to be resistantto Aphanomyces euteiches (Djebali et al. 2009), toColletotrichum trifolii and Erysiphe pisi (Ameline-Torregrosa et al. 2008), but susceptible to Fusariumoxysporum (Ramirez-Suero et al. 2010) and Ralstoniasolanacearum (Vailleau et al. 2007), whereasF83005.5 is susceptible to A. euteiches and C. trifoliiand resistant to F. oxysporum and R. solanacearum.Derived recombinant inbred lines from a cross be-tween these parents were used for identification ofQTLs involved in resistance to A. euteiches and R.solanacearum (Djebali et al. 2009; Vailleau et al.2007), as well as to Vaa V31-2 (Ben et al. 2012).

The M. truncatula genotypes used in this studyinclude a core collection of 16 lines representing thegenetic diversity of this species in the Mediterraneanbasin (Ronfort et al. 2006) and lines involved incrosses for genetic studies. Our results will be usedto choose parents with contrasted phenotypes forstudying the genetic mechanisms of resistance toVerticillium wilt caused by host and non-host strainsin M. truncatula. For example genotypes A17 andA20 respond differentially to strains Vaa V31-2, VaaVA1 and Vd S26, and populations of recombinant

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inbred lines from this cross are already in progress.Such knowledge could then be transferred to cultivat-ed alfalfa or other legume crops, thanks to macro-andmicro-synteny between the model plant and legumecrops. Resistant genes from M. truncatula could beintroduced into alfalfa by genetic engineering (Yang etal. 2008) or their sequences could be used for marker-assisted selection. In addition to the sequenced refer-ence line A17, twenty-two genotypes (annotated inTable 1) among the 25 used in our study were includedin a re-sequencing program and have been deep-sequenced recently by an international consortium aspublished by Branca et al. (2011); in total 384 linesfrom various germplasm collections will be sequencedeventually (www.medicagohapmap.org). The variabil-ity of responses to different strains observed in ourstudy highlights that M. truncatula has a wide spec-trum of susceptibility/resistance to Verticillium. Byanalysing the responses of a larger number of geno-types to host and non-host Verticillium strains it wouldbe possible to take advantage of the genomic toolsdeveloped for the model plant in order to identifygenomic regions conferring resistance to Verticilliumby genome-wide association studies. The knowledgeacquired by combination of genomics and geneticapproaches should then help to develop broad durableresistance in alfalfa against this pathogen.

Acknowledgments The authors thank the French Governmentfor support of A. Negahi with a PhD grant; B.P.H.J. Thomma(University ofWageningen, Netherlands) for providing strains VaaVA1, Vaa 5431, Vd JR2 and Vd S26; A. von Tiedemann (Georg-August University Göttingen, Germany) for providing strain VaaLPP0323; and L. Gentzbittel (ENSAT, Toulouse, France) forhelpful discussions.

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