Pathogenicity and vegetative compatibility grouping among Indianpopulations of Fusarium oxysporum f. sp. ciceris causing chickpeawilt

Parasappa R. Saabale & Sunil C. Dubey

Received: 16 April 2013 /Accepted: 8 January 2014# Springer Science+Business Media Dordrecht 2014

Abstract Thirty-nine isolates of Fusarium oxysporumf. sp. ciceri – the causal agent of chickpea (Cicerarietinum) wilt – collected from different parts of Indiaand representing eight races of the pathogen, were ana-lyzed for virulence and classified on the basis of vege-tative compatibility grouping (VCG). Thewilt incidenceranged from 24% to 100% on a highly susceptiblecultivar JG 62. Six isolates, from Delhi, Gujarat, Karna-taka, Punjab and Rajasthan and belonging to six differ-ent races of the pathogen, caused 100% wilt incidence.Five isolates belonging to four different races, namely,Foc 143 from Andhra Pradesh, Foc 161 from Chhattis-garh, Foc 146 from Karnataka, Foc 158 from MadhyaPradesh and Foc 50 from Rajasthan, caused low wiltincidence. For VCG analysis, nitrate non-utilizing mu-tants (nit) were obtained by culturing wild-type isolateson 2.5% potassium chlorate and selecting resistant sec-tors. Complementary nit mutants were paired in allpossible combinations to determine varying degrees ofheterokaryon formation within the isolates, whichshowed that most of the isolates were self-compatible.Pairing of all the mutants showed that the isolates in-cluded in the present study belonged to a single VCG

(0280). Thus, in spite of variability in the virulence, theIndian populations of the pathogen have only one VCG.

Keywords Chickpea .Fusariumwilt .Nitmutants .

Self-compatible . VCG . Virulence

Introduction

Fusarium wilt caused by Fusarium oxysporum f. sp.ciceris (Padwick)Matauo and K. Sato is one of the mostimportant biotic stresses of chickpea (Cicer arietinumL.), with the potential to cause 100% yield loss. It is themajor factor limiting chickpea production worldwideand is widely distributed in chickpea-growing areas(Haware & Nene 1982a). Pathogenic and genetic vari-ability in the pathogen were characterized using differ-ential cultivars of chickpea and DNAmarkers (Dubey&Singh 2008; Haware & Nene 1982b). Cultivar special-ization of F. oxysporum f. sp. ciceris (Foc) was firstreported in India (Haware & Nene 1982a). Based ondisease reactions on ten chickpea differential cultivars,eight races of the pathogen have been reported. Of these,six races (1A, 2, 3, 4, 5 and 6) cause wilting, while tworaces (0 and1B/C) cause yellowing syndrome (Jimenez-Diaz et al. 1993). Races 1A, 2, 3 and 4 have beenreported from India (Haware & Nene 1982b). Virulenceanalysis of 64 isolates of Foc on international differen-tials suggested the presence of more than one race inevery state of India. It was also suggested to modify theinternational differentials in order to obtain clear cutdifferential responses (Dubey & Singh 2008; Dubey

PhytoparasiticaDOI 10.1007/s12600-014-0383-8

P. R. Saabale (*)Division of Crop Protection, Indian Institute of PulsesResearch,Kanpur 208024, Indiae-mail: sparashu@gmail.com

S. C. DubeyDivision of Plant Pathology, Indian Agricultural ResearchInstitute,New Delhi 110012, India

et al. 2010). Recently, Dubey et al. (2012) standardizeda new set of chickpea differential cultivars for identifi-cation of changed Indian populations of Foc and char-acterized them into eight races. Knowledge of geneticdiversity within a fungus is needed for deployment ofresistant cultivars in breeding programs.

A vegetative compatibility group (VCG) approachwas developed to assess the genetic diversity inF. oxysporum. Vegetative compatibility phenotypes arenaturally occurring genetic markers, which have beenused to differentiate isolates of F. oxysporum (Baayenet al. 1998; Puhalla 1985). Genetic variability is con-trolled by heterokaryon incompatibility (het) or vegeta-tive incompatibility (vic) specific loci (Anagnostakis1982). Strains with matching alleles at each locus arevegetatively compatible (Correll et al. 1986). VCGswithin some formae speciales have significant correla-tion with the virulence level (Katan & Katan 1988;Zamani et al. 2004). Owing to the lack of sexual stage,they can be utilized for differentiating pathogenic andnon-pathogenic strains of F. oxysporum (Larkin et al.1990) and understanding the origin and relatedness, andgenetic diversity of the fungus (Ploetz & Shepard 1989).

Only one report is available on VCG grouping ofFocisolates collected from different parts of the world, anddespite variations in geographical distribution, race andsymptom types, all the pathogenic isolates were placedin a single VCG (Nogales-Moncada et al. 2009). India isa major chickpea-growing country with a maximumnumber of typical wilt-producing races of the pathogenwith a high level of variability, but information is notavailable on VCGs of the pathogen. With this back-ground the objective of the present study was to deter-mine the regional diversity among Indian isolates of Focrepresenting eight races based on the VCGs and rela-tionship between virulence and VCGs.

Materials and methods

Fusarium oxysporum f. sp. ciceris isolates Single sporecultures of 39 isolates of Foc isolated from 12 differentchickpea-growing states of India belonging to eightraces (Dubey et al. 2012) and maintained at the PulseLaboratory of the Division of Plant Pathology, IndianAgricultural Research Institute, New Delhi, India, wereselected for the present study (Fig. 1). The isolates andtheir origins are listed in Table 1. The cultures weremaintained on PDA slants at 4°C.

Virulence analysis A pot experiment was conducted todetermine the virulence of the isolates included in thepresent study. Twelve seeds of the chickpea variety JG62, highly susceptible to wilt, were sown in 15-cm-diamsurface-sterilized plastic pots (0.1% mercuric chloride)filled with 4 kg sterilized soil (1.0% formalin for 15 days)and inoculated with 15-day-old cultures of Foc isolatesmultiplied on sorghumgrains (10 g kg-1 soil; 2x109 cfu g-1)7 days before sowing. The grains were soaked in tap waterfor 12 h, strained and filled into 500 ml conical flasks(250 g per flask). The flasks containing grains wereautoclaved for two subsequent days at 1.1 kg cm-2 for 30minutes, inoculated with a 5-day-old culture of Foc andincubated for 15 days at 25+1ºC. The required inoculummultiplied on sorghum grains was thoroughlymixed in potsoil; at 7 days after inoculation, the pots were sown withseeds of chickpea (Dubey & Singh 2008). Pots with un-inoculated soils were maintained as control for compari-son. Thewilt incidencewas recorded at 15-day intervals upto maturity of the crop plants. The experiment was con-ducted in two replications. The data were analyzed as perthe procedure of completely randomized design (Gomez&Gomez 1984). The transformed angular values of the datarecorded in percentage were used for the analysis. The datawere subjected to analysis of variance (ANOVA) by usingSAS Software (SAS Institute, version 9.1, Cary, NC,USA). Statistical significance was assessed at P<0.05,and Fisher’s least significance difference test was used toseparate the means.

Vegetative compatibility grouping A system proposedby Puhalla (1985) for the analysis, classification andgenetic diversity based on vegetative compatibilitygrouping technique was used in the present study.

Induction and selection of nitrate non-utilizingmutants Chlorate-resistant sectors for each isolate ofthe pathogen were recovered from 2.5% KClO3 withminimal medium (MM) and maintenance, and comple-mentation tests were carried out on MM (per liter: 30 gsucrose, 2 g NaNO3, 1 g KH2PO4, 0.5 g KCl, 0.5 gMgSO4·7H2O, 0.01 g FeSO4·7H2O, 200 μl trace ele-ment solution (5 g ZnSO4·7H2O, 5 g citric acid, 1 gFe(NH4)2(SO4)2·6H2O, 0.25 g CuSO4·5H2O, 0.05 gNaMoO4·2H2O, 0.05 g MnSO4·H2O, 0.05 g H3BO4,and 95 ml distilled water and 20 g agar). Nitrate non-utilizing (nit) mutants were selected by Puhalla’s meth-od with slight modification. For this, 39 isolates of Focrepresenting eight races were grown on quarter-strength

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potato dextrose agar instead of MM for 3–4 days. Twomm blocks of fungal mycelium were transferred to9 mm petri plates containing MM with 2.5% potassiumchlorate (KClO3) (Correll et al. 1987; Pasquali et al.2004; Puhalla 1985) and incubated at 28±1°C for 2–3weeks. The colony growth in the medium was restrictedfor the first week, and after that fast growing sectorsappeared. These fast growing sectors from initially re-stricted colonies were then transferred to a MM thatcontained sodium nitrate as a nitrogen source. Wild typecells used the chlorate ion, an analog of nitrite, andconverted it to chlorite. Mutant cells that could notutilize nitrate emerged as fast growing sectors on chlo-rate, and sectors having thin and expanding areal myce-lium on MM were considered to be nit mutants.

Characterization and storage of nit mutants All the nitmutants of each isolatewere further characterized into nit 1,

nit 3 and nitMmutants based on the utilization of differentnitrogen sources (Correll 1991; Nogales-Moncada et al.2009), namely, sodium nitrate (2 g l-1), sodium nitrite (0.5 gl-1), ammonium tartrate (1 g l-1), hypoxanthin (0.2 g l-1) anduric acid (0.2 g l-1). The mutants that showed thin andsparse growth on MM containing sodium nitrate weredesignated as nit1. NitM mutants showed sparse growthon the medium containing hypoxanthin and sodium ni-trate, whereas nit3 showed similar growth on the mediumcontaining sodium nitrate and sodium nitrite (Correll et al.1986) (Table 2). These nit 1, nit 3 and nit M mutants ofeach isolate were selected and stored at 4°C in MM slantsand later used for complementation tests.

Determina t ion o f VCG by complemen tarypairing Vegetative compatibility was determined byobserving complementation reaction between two nitmutants leading to heterokaryon formation and

Fig. 1 Map of India showingareas of collection of isolates ofFusarium oxysporum f. sp ciceris

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production of wild-type growth, while the un-complementation reaction showed thin expansive

growth. Several complementary nitM and nit1 or nit3mutants from each isolate were paired to determine

Table 1 Fusarium oxysporum f. sp. ciceris isolates, place of origin, races and wilt incidence caused by them on chickpea variety JG 62

Sl.No. States Places Isolates Race Wilt incidence (%)z

1 Andhra Pradesh Hyderabad Foc 117 1 45 (42.1)f

2 Andhra Pradesh Guntur Foc 143 1 25 (30.0)i

3 Bihar Doli Foc 125 2 62.5 (52.2)d

4 Chhattisgarh Bilaspur Foc 127 6 36 (36.9)gh

5 Chhattisgarh Raipur Foc 161 6 25 (30.0)i

6 Delhi New Delhi Foc 43 4 60 (50.8)de

7 Delhi MD4C New Delhi Foc 53 4 100 (90.0)a

8 Gujarat Junagarh Foc 172 7 60 (50.8)de

9 Gujarat Anand Foc 173 7 100 (90.0)a

10 Gujarat Anand Foc 174 7 80 (63.4)b

11 Haryana Sikophpur Foc 9 4 55 (47.9)e

12 Haryana Hissar Foc 64 4 80 (63.4)b

13 Haryana Nilhati Foc 92 4 65 (53.7)cd

14 Jharkhand Dumka Foc 26 6 55.5 (48.1)e

15 Jharkhand Ranchi Foc 44 6 80 (63.4)b

16 Jharkhand Darisai Foc 100 6 45 (42.1)f

17 Karnataka Dharwad Foc 121 1 100 (90.0)a

18 Karnataka Raichur Foc 146 1 25 (30.0)i

19 Maharashtra Badanapur Foc 124 7 55.5 (48.1)e

20 Maharashtra Dhula Foc 166 4 43.5 (41.3)f

21 Maharashtra Badanapur Foc 176 7 60.5 (51.1) de

22 Madhya Pradesh Indore Foc 156 6 44.5 (41.8)f

23 Madhya Pradesh Sehore Foc 158 3 24 (29.3)i

24 Madhya Pradesh Jabalpur Foc 170 6 100 (90.0)a

25 Punjab Faridkot Foc 13 8 55 (47.9)e

26 Punjab Firojpur Foc 34 3 55 (47.9)e

27 Punjab Ludiana Foc 63 3 100 (90.0)a

28 Punjab Gurdaspur Foc 94 3 77.5 (61.7)b

29 Rajasthan Sikar Foc 02 5 55 (47.9)e

30 Rajasthan Jaipur Foc 07 5 59.5 (50.5) de

31 Rajasthan Udaipur Foc 50 5 30.5 (33.5)hi

32 Rajasthan Sardargarh Foc 69 5 100 (90.0)a

33 Rajasthan Churu Foc 84 5 81 (64.1)b

34 Uttar Pradesh Kanpur Foc 119 2 81.5 (64.5)b

35 Uttar Pradesh Jalun Foc 132 2 70.5 (57.1)c

36 Uttar Pradesh Mahoba Foc 134 2 60 (50.8)de

37 Uttar Pradesh Gorakpur Foc 138 2 70 (56.8)c

38 Uttar Pradesh Gazipur Foc 139 2 60 (50.8) de

39 Uttar Pradesh Fazabad Foc 145 4 39.5 (38.9)fg

z The values with a common letter do not differ significantly at 5% level using Fisher’s least significance difference test

The numbers in parentheses in the right column are arc-sine transformed values

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whether the isolate was self-compatible or self-incompatible. Two-mm blocks of complementary nitmutants were transferred fromMM to a fresh 9 cm petridish in daisy configuration with a nit M mutant at thecenter and nit1 and nit3 mutants from different isolatesat the outer circle 4 cm apart, and incubated at 28±1°Cfor 10–15 days. Three types of scorings, namely, strongreaction (++), moderate or weak reaction (+) and noreaction (-), were made (Correll et al. 1986). If strongand moderate reactions occurred at the intersection ofthe colonies, then the isolates were of the same VCG; ifit remained thin, then the isolates were of differentVCGs. Pairing of nit1, nit3 and nitM mutants of differ-ent isolates was done twice in all possible combinations.

Results

Thirty-nine isolates of Foc obtained from different geo-graphical locations of India and belonging to eight raceswere used for the vegetative compatibility study.

Virulence analysis The results (Table 1) showed that theisolates were highly variable in causing wilt incidenceon the chickpea variety JG 62. The wilt incidenceranged from 24% to 100%. Significantly high wilt inci-dence was caused by six isolates, namely, Foc 53 fromDelhi, Foc 173 from Gujarat, Foc 121 from Karnataka,Foc 63 and 170 from Punjab and Foc 69 from Rajasthanbelonging to six different races of the pathogen. Statis-tically similar and low wilt incidence was caused by fiveisolates, namely, Foc 143 from Andhra Pradesh, Foc161 from Chhattisgarh, Foc 146 from Karnataka, Foc158 from Madhya Pradesh and Foc 50 from Rajasthanbelonging to four different races of the pathogen.

Induction and phenotypic characterization of nitmutants A total of 499 chlorate-resistant sectors wereobtained from 39 single spore isolates of Foc with amean of 12.8 sectors per isolate (Table 3). The isolatesof Foc varied in their frequency of chlorate-resistantsectors produced on chlorate medium. Most of the sec-tors appeared in the second and third weeks after incu-bation from the initially restricted colonies. Of the 499chlorate-resistant sectors, 379 (75.9 sectors that grewthin expansive colonies on MM were designated as nitmutants. The nit mutants could be classified into nit 1,nit 3 and nit M by their colony morphology on MMcontaining different nitrogen sources. The predominantphenotype was nit1 (73.3%), followed by nit 3 (15.6 andnitM (9.5%). The nit 1 phenotypes were obtained fromall the 39 isolates. Nit 3 was produced from 21 isolates,and nit M was produced from 19 isolates.

De t e rm i na t i o n o f VCG by compa t i b i l i t ytests Complementations of nitmutants occurred in com-patible isolates by forming a wild-type growth at thepoint of contact. Initially, different nit phenotypes ofeach isolate were paired to check the self-incompatibility. Of the 39 isolates, 36 isolates wereself-compatible and the remaining three isolates wereself-incompatible (Foc 09, Foc 173 and Foc 176) asthey did not form a heterokaryon with mutants obtainedfrom the same parental strain. All self-compatible iso-lates of nit 1, nit 3 and nit M mutants of different Focisolates showed varying degrees of heterokaryon forma-tion in all possible combinations. In the present study,several nitmutants of an isolate were able to anastomizewith the mutants of other isolates and some isolatesacted as bridging strains between two vegetative incom-patible strains. VCGs were identified through the com-plementation of nit mutants in interstrainal pairings on

Table 2 Classification of nit mutants based on utilization of nitrogen sources

Growth of different nitrogen sources Phenotypic class

Sodium nitrate Sodium nitrite Ammonium tartarate Hypoxanthine Uric acid

- + + - + Nit M

- - + + + Nit 3

- + + + + Nit 1

- - + - - Unknown

+ + + + + Wild type

-thin growth, +wild type growth

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Table 3 Frequency of nit phenotypes generated from the isolates of Fusarium oxysporum f. sp. ciceris

Isolates Chlorate-resistant sectors nit mutants Nit mutant phenotypes VCG

nit 1 nit3 nitM Unknown

Foc 02 13 11 7 2 2 - 0280

Foc 07 14 11 6 5 - - 0280

Foc 09 16 12 6 5 1 - 0280

Foc 13 11 8 8 - - - 0280

Foc 26 13 10 10 - - - 0280

Foc 34 6 5 3 - 2 - 0280

Foc 43 11 7 5 1 1 - 0280

Foc 44 16 11 8 1 2 - 0280

Foc 50 15 11 7 3 1 - 0280

Foc 53 9 8 4 4 - - 0280

Foc 63 10 7 5 2 - - 0280

Foc 64 10 9 6 - 2 1 0280

Foc 69 8 6 2 2 2 - 0280

Foc 84 16 13 13 - - 1 0280

Foc 92 16 14 11 - 3 - 0280

Foc 94 17 12 10 - - 2 0280

Foc 100 19 13 8 2 1 2 0280

Foc 117 9 7 5 2 - - 0280

Foc 119 10 9 6 - 2 1 0280

Foc 121 14 11 11 - - - 0280

Foc 124 19 14 14 - - - 0280

Foc 125 12 8 8 - - - 0280

Foc 127 14 10 4 2 4 - 0280

Foc 132 13 8 7 - 1 - 0280

Foc 134 12 12 8 4 - - 0280

Foc 138 19 15 8 3 4 - 0280

Foc 139 13 11 8 - 3 - 0280

Foc 178 9 6 6 - - - 0280

Foc 145 13 7 4 3 - - 0280

Foc 146 15 13 10 3 - - 0280

Foc 156 8 7 3 4 - - 0280

Foc 158 18 12 10 - 2 - 0280

Foc 161 17 13 13 - - - 0280

Foc 166 11 7 6 1 - - 0280

Foc 170 11 9 6 3 - - 0280

Foc 172 6 5 3 2 - - 0280

Foc 173 11 8 7 - 1 - 0280

Foc 174 14 10 10 - - - 0280

Foc 176 11 9 3 5 1 - 0280

Total 499 379 278 59 36 6

Mean 12.79 9.71 7.12 1.51 0.92 0.15

Share (%) 73.35 15.56 9.49 1.58

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MM. When the nit mutants from distinct isolates wereobserved to be complementary, these isolates wereplaced in the same VCG. The pattern of heterokaryonformation between the mutants of different isolatesshowed the existence of a single VCG even though thecompatibility between the isolates was variable.

Discussion

Vegetative compatibility and virulence were analyzedfor 39 Indian isolates of Foc representing eight racesprevalent in the country. The range of wilt incidence(24–100%) caused by the isolates showed variability inthe virulence of the pathogen even on a single suscep-tible variety of chickpea. The isolates that caused highwilt incidence belonged to six races of the pathogenoriginating from six different states of India and indicat-ed the prevalence of highly pathogenic populations ofthe pathogen across the country. Even the isolates orig-inating from a single state showed variable wilt inci-dence. The present finding is supported by the observa-tions made by earlier workers that Indian populations ofthe pathogen are highly variable (Dubey & Singh 2008;Dubey et al. 2012; Jimenez-Diaz et al. 1993).

The growth of wild-type strains was restricted inchlorate-containing medium (Bosland & Williams1987) owing to the toxic effect of chlorite. In the presentstudy, 75.9% chlorate-resistant sectors yielded nit mu-tants. The present study also indicated that the isolatesdiffered considerably in yielding nit mutants. The nitmutants were classified into three phenotypic classes,namely, nit 1, nit 3 and nit M. The ratio of tester nit Mmutants was relatively low as compared with others.Previous studies also reported the occurrence of a lower

proportion of nitM (Bayraktar et al. 2010; Mohammadiet al. 2012). In the present study, only three isolates wereself-incompatible. This finding is in accordance with theearlier observations of rare occurrence of self-incompatibility in various formae speciales ofF. oxysporum (Bayraktar et al. 2010; Gilardi et al.2008). The isolates upon daisy pairing produced weakand strong heterokaryon formation based on the numberof allelic differences in each locus which is controllingthe vegetative compatibility (Fig. 2). In the presentstudy, the paring of nit1, nit3 and nitM mutants of allIndian isolates representing eight new races groupedthem into a single VCG, arbitrarily designated as0280. Some of the isolates behaved as bridging isolatesas they were compatible with two vegetative incompat-ible isolates and thus grouped into the same VCG. Suchtypes of bridging strains were also reported by earlierresearchers (Katan et al. 1991; Katan & Katan 1999).The isolates were moderate to highly virulent and theVCG naming for them was given according to thesystemic numbering proposed by Katan (1999). Ourresults are in accordance with the findings of Baayenet al. (1998), who reported that all the isolates ofF. oxysporum included in a VCGwere highly pathogenicto their host plants. Based on the study of 47 isolates ofFoc in Spain, one VCG was identified (Nogales-Moncada et al. 2009). Genealogy studies also supportthe hypothesis of monophyletic origin of Foc. It wasobserved that six race pathotypes showed identical se-quences for introns of genes EF1α, β-tubulin, histone 3,actin and calmodulin (Jiménez-Gasco et al. 2002).There was no correlation between VCGs and races, asreported in other formae speciales (Bosland &Williams1987; Roebroeck & Mess 1992). Many of the formaespeciales had more than one VCG and a few were

1 2

Strong heterokaryon formation

Weak heterokaryon formation

No heterokaryon formation

Fig. 2 Compatibility test of nitmutants (forming aerial myceliumat the border between two isolates). Plate 1: Pairing between Foc44 nit M and Foc 92 nit 1 strong heterokaryon formation andpairing between Foc 44 nitM and Foc 26 nit 1weak heterokaryon

formation, Plate 2: Pairing between Foc 92 nitMandFoc 100 nit 1strong heterokaryon formation Pairing between Foc 92 nit M andFoc 161 nit 1&170 nit 1 no heterokaryon formation

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included in a single VCG. Zamani et al. (2004) assignedthree VCGs for 15 Iranian isolates of F. oxysporum, thecausal agent of yellow disease of chickpea. Anotherimportant pigeonpea wilt pathogen (F. udum) belongedto a single VCG (VCG 1) with two subgroups, VCG 1 Iand VCG 1 II (Kiprop et al. 2002). A high level of VCGdiversity was also observed in a few formae speciales ofF. oxysporum (Alves-Santos et al. 1999; Bayraktar et al.2010; Elmer & Stephens 1989). The study helps in ourunderstanding of the relationship between pathogenicgroups within formae speciales and the phylogeneticorigin of Foc. The presence of multiple pathogenicgroups within a single VCG can be explained based onthe fact that there is a high degree of genetic homologyamong the isolates. Jacobson & Gordon (1988) reportedthat pathogenic races within a VCG may differ at arelatively small number of loci. Vegetative compatibilityin F. oxysporum is thought to be controlled by multiplevegetative incompatibility (vic) loci (Correll 1991); al-leles at each of the corresponding vic loci must beidentical for the isolates to be vegetatively compatible.A mutation at a single locus would result in closelyrelated isolates being vegetatively incompatible. It isalso possible that distantly related isolates have the sameincompatibility loci and are vegetatively compatibleeven though they are not genetically similar (Correll1991).

In conclusion, the pathogenicity test and the phylo-genetic analysis using the VCG tool revealed that allFoc populations, which are moderate to highly patho-genic, originated from a common clone.

Acknowledgments The study was supported by ICAR and theIndian Agricultural Research Institute, New Delhi, India.

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