Distribution of Pathotypes with Regard to Host Cultivars in French Wheat Leaf Rust Populations

Transcription

1 Genetics nd Resistnce Distribution of Pthotypes with Regrd to Host Cultivrs in French Whet Lef Rust Popultions Henriette Goyeu, Robert Prk, Brigitte Scheffer, nd Christin Lnnou First nd fourth uthors: INRA, Lbortoire de Pthologie Végétle, BP 01, Thivervl-Grignon, Frnce; second uthor: University of Sydney, Plnt Breeding Institute, Cmden NSW, Austrli; nd third uthor: Unité de recherche Mthémtiques et Informtique Appliquées INRA Domine de Vilvert Jouy-en-Joss Cedex, Frnce. Accepted for publiction 15 October ABSTRACT Goyeu, H., Prk, R., Scheffer, B., nd Lnnou, C Distribution of pthotypes with regrd to host cultivrs in French whet lef rust popultions. Phytopthology 96: Isoltes of whet lef rust collected from durum nd bred whet cultivrs in Frnce during were nlyzed for virulence on 18 Thtcher lines with single genes for lef rust resistnce (Lr genes). Smpling focused on the five most widely grown bred whet cultivrs (two susceptible nd three resistnt) to llow sttisticl comprison of diversity indexes between the cultivrs. Lef rust popultions from durum nd bred whets were different. The diversity of the bred whet lef rust pthotypes, s mesured by the Shnnon index, rnged from 2.43 to 2.76 over the 4 yers. Diversity for whet lef rust resistnce ws limited in the host since we postulted only seven seedling resistnce genes in the 35 cultivrs most widely grown during Lef rust popultions were strongly differentited for virulence within bred whet cultivrs, nd diversity ws higher on those tht were resistnt, minly due to more even distribution of virulence phenotypes thn on susceptible cultivrs. The pthogen popultion on the susceptible cv. Soissons ws lrgely dominted by single pthotype (073100), wheres ll other pthotypes virulent on cv. Soissons either decresed in frequency or remined t low frequency during the period studied. Severl pthotypes including the most complex one were found only on resistnt cultivrs, even though most of them were virulent on the susceptible cv. Soissons. Specific interctions were necessry, but not lwys sufficient, to ccount for pthotype distribution nd frequencies on the cultivrs, suggesting tht selection for virulence to host resistnce genes is blnced by other selective forces including selection for ggressiveness. Corresponding uthor: H. Goyeu; E-mil ddress: goyeu@grignon.inr.fr DOI: /PHYTO This rticle is in the public domin nd not copyrightble. It my be freely reprinted with customry crediting of the source. The Americn Phytopthologicl Society, Whet lef rust, cused by Puccini triticin Eriks, ws mong the most dmging folir diseses of whet in Europe t the end of the 20th century. Dmging disese levels hve been noted regulrly in western Europe (except the UK), Itly, nd estern nd southestern Europe (nmely Yugoslvi nd Romni) (25). During the lst 30 yers, prticulrly in western Europen countries, fungicides hve been used to control diseses including lef rust in most yers (25). Although selection for resistnce to lef rust hs remined n issue for whet breeders, it hs not been top priority. This probbly explins the limited studies on this pthosystem in Europe s compred with other regions like North Americ (12,14,20) nd Austrli (27). Pthogen popultions hve been monitored nd host resistnce genes postulted from 1966 to 1993 in the Czech Republic (4). A more recent study by Mesterhzy et l. (23) tht ttempted to coordinte 4-yer survey of P. triticin in western, southern, nd estern Europe showed gret vrition in the distribution of virulence phenotypes between yers nd countries. However, no interprettion of the observed diversity could be provided becuse of lck of knowledge of the host resistnce genes used in these countries. Prk et l. (29) postulted lef rust resistnce genes in western Europen whet cultivrs grown in 1995, nd relted this to survey of P. triticin popultions in the sme yer. In this study, it ws noted tht western Europe comprised different epidemiologicl units, with different climtes nd different host popultions, nd tht the role of host selection on the pthogen ws difficult to estimte becuse virulence on the genes with the highest frequencies in the host popultion, Lr13 nd Lr14, hd not been monitored. Moreover, this study ws limited to 1 yer. The necessity to plce the results of popultion studies in perspective of severl yers ws stressed by Brown (5). In this pper, we describe 4-yer study of host nd pthogen popultions in which we plced specil emphsis on selection by the host. To chrcterize host selection s ccurtely s possible, the smpling scheme ws designed to collect lef rust isoltes from identified cultivrs, providing more informtion thn wht would be gined from pooling results from mny different cultivrs (36). The pthogen popultion ws chrcterized through two complementry pproches: (i) clssicl survey of virulences on durum whet cultivrs nd minor bred whet cultivrs to provide brod view of virulences in P. triticin popultions in Frnce; nd (ii) detiled smpling on the five min bred whet cultivrs. We focused our smpling on the most widely grown cultivrs, whether resistnt or susceptible, to obtin smple sizes lrge enough to be considered representtive of the popultion present on ech of these cultivrs. The study ws limited t the sptil scle to one country, Frnce, ssuming tht it could be regrded s single epidemiogicl unit to overcome the bove-mentioned difficulties of heterogeneity t the continentl scle. MATERIALS AND METHODS Smpling. Becuse 98% of whet fields in Frnce re spryed with fungicides (3), smples were collected from network of unspryed nurseries plnted nnully in pproximtely 50 different loctions throughout the whet-growing re by breeders (the French Whet Breeders group Club des Cinq ) or extension services (Arvlis-Institut du Végétl). For bred whet, the smpling effort focused on the most widely grown cultivrs (Tble 1) 264 PHYTOPATHOLOGY

2 plnted s checks in ll nurseries, i.e., Soissons, Isengrin, nd Trémie from 1999 to 2002, nd Apche nd Orvntis from 2001 to On ech site nd for ech cultivr, few infected leves were collected during My or June ( single collection dte per loction) from smll plots (10 to 20 m 2 ) from which one singlepustule isolte ws selected nd incresed, giving one isolte per cultivr nd loction. In ddition, in the sme nurseries, we collected lef rust smples from minor bred nd durum whet cultivrs. Since the minor cultivrs were not present in ech loction, the smple size for ech minor cultivr ws less thn 10. The different popultions considered were (i) the popultion P d growing on durum whet cultivrs; (ii) the popultion P b growing on ll bred whet cultivrs, including five mjor nd severl minor cultivrs; nd (iii) the five popultions (P 1 to P 5 ) growing on the five mjor bred whet cultivrs, P 1 to P 5 being subpopultions of P b. Selection by host could thus be evluted for the influence of host species by compring P d nd P b nd the influence of resistnce genes within the bred whet species by compring no mtter wht given popultion (from P 1 to P 5 ), to nother. Moreover, P b llowed comprison of our results with the published dt on popultions of P. triticin in other countries, where the cultivr is not tken into ccount in the smpling scheme. The smple from P d ws collected in 1999, 2000, nd 2001 from 8, 7, nd 16 cultivrs, respectively, yielding 19, 16, nd 47 isoltes, respectively, with totl of 82 isoltes for the 3 yers. A minimum smple size of 50 ws chieved on cvs. Soissons (P 1 ) nd Isengrin (P 2 ), wheres collections from resistnt cultivrs (Trémie, Apche, nd Orvntis, P 3, P 4, nd P 5, respectively) were more limited nd yielded smple size from 18 to 55 depending on the yer (Tble 2). The smple from P b ws obtined from 18 different bred whet cultivrs in 1999 nd 2000, 13 cultivrs in 2001, nd 21 cultivrs in 2002, yielding smple size from 168 to 234 depending on the yer (Tble 2) with totl of 821 isoltes for the 4 yers. Pthotype determintion. Prior to inocultion, ll helthy mteril ws grown in ir-filtered cbinets in glsshouse t tempertures between 15 nd 25 C nd with 14-h photoperiod (dylight supplemented by 400 W N-lmps). TABLE 1. Percentge of whet re plnted to five cultivrs in Frnce during Cultivr Registrtion yer Pedigree Soissons (Lr14) 1988 Hybride nturel dns 35/Ien Isengrin (Lr14) 1997 Apollo/Soissons Trémie (Lr10, Lr13) 1992 SE32/Moulin Apche (Lr13, Lr37) 1998 Axil/NRPB Orvntis (Lr10, Lr13, Lr37) 1999 Thésée/Disponent/Monopol/ 3 Torfrid 4 The percentge of the most commonly grown cultivr for ech yer is given in bold. TABLE 2. Clcultion of indices of richness, diversity, nd evenness for popultions of Puccini triticin collected from bred whet cvs. Soissons (P 1 ), Isengrin (P 2 ), Trémie (P 3 ), Apche (P 4 ), nd Orvntis (P 5 ) during Sttistic P 1 P 2 P 3 P b b P 1 P 2 P 3 P b P 1 P 2 P 3 P 4 P 5 P b P 1 P 2 P 3 P 4 P 5 P b Infected field-collected leves were wiped gently on 7-dy-old seedlings of the whet cv. Michign Amber tht hd been treted with 20 ml of mleic hydrzide solution (0.25 g of mleic hydrzide per liter of H 2 O) to prevent emergence of secondry leves nd increse the size of uredini. Inoculted seedlings were plced for 24 h in dew chmber t 15 C, then in the glsshouse. One week fter inocultion, the seedlings were trimmed so tht only one plnt with one uredinium remined in ech pot. Cellophne bgs were then plced on the pots to prevent contmintion between isoltes. Spores from single uredinium were collected with cyclone collector into 00 geltin cpsule. Spores were incresed by dding 0.5 ml of light minerl oil to ech cpsule nd sprying the resulting spore suspension onto 7-dy-old Michign Amber seedlings. The spores collected 10 dys fter inocultion were divided into two btches, one to inoculte differentil set nd one for storge t 80 C. The differentil sets were sown in pressed-pet pots (3 3 cm 2 ) contining commercil compost (pet substrte; Gebr. Brill Substrte, Georgsdorf, Germny) with four seedlings per pot nd two pots per line (eight seedlings for ech differentil line). The differentil set comprised 17 Thtcher differentils, Lr1, Lr2, Lr2b, Lr2c, Lr3, Lr3bg, Lr3k, Lr10, Lr13, Lr14, Lr15, Lr16, Lr17, Lr20, Lr23, Lr26, Lr37, the Austrlin cv. Hrrier crrying Lr17b (32) nd the susceptible check cv. Morocco. After inocultion with spore suspension, the sets were plced in dew chmber for 24 h t 15 C nd then for 9 dys in growth chmber mintined t 22 C with 16-h photoperiod given by metl hlide lmps (350 µe). Infection types on the differentils were red 10 dys fter inocultion ccording to Stkmn et l. (35). Ech isolte ws ssigned six-digit code dpted from the system described by Gilmour (7): the 18 differentil isolines were rrnged in six sets of three to ssign to ech isolte n octl pthotype code. Postultion of resistnce genes. All cultivrs belonging to the list of the 10 most widely grown cultivrs in Frnce, in ny of the yers , were nlyzed to determine which lef rust resistnce genes were present. Nine of these cultivrs were tested t the Plnt Breeding Institute, The University of Sydney, Austr- Smple size Richness H c Confidence intervl d N G Evenness c (E5) Confidence intervl d P 4 nd P 5 could not be smpled in 1999 nd 2000 becuse cvs. Apche nd Orvntis, recently registered, were not present in the nurseries. b P b indices were clculted from smple including isoltes from ll bred whet cultivrs (minor cultivrs nd five mjor cultivrs). c The underlined vlues, clculted from the observed smple, were not included in the confidence intervl clculted with the BC method. d Confidence intervls were clculted by the bootstrpping pproch (BC method) by Grünwld et l. (8) Vol. 96, No. 3,

3 li, following the procedure described by Prk et l. (29). For the remining cultivrs, multipthotype tests were conducted in growth room under the conditions described bove for pthotype determintion. A selection of 15 pthotypes of P. triticin ws used (Tble 3). Spore suspension (3 mg of spores per ml of minerl oil) ws spryed on the first leves of 12 seedlings of ech cultivr using 0.7 ml per try of 240 seedlings. A set of differentil cultivrs ws included in ech test to check the identity nd purity of the isolte s well s the infection type. Infection types were scored ccording to Stkmn et l. (35). Dt nlysis. Diversity for virulence phenotypes in the popultions ws estimted by the Shnnon index H = p i ln(p i ) in which p i is the frequency of the ith phenotype. Since differences in n index of diversity cn reflect vrition in richness (i.e., the number of genotypes observed), evenness (i.e., the distribution of the genotypes), or both, we hve clculted these indices nd computed mens nd confidence intervls whenever possible. Richness ws evluted by the number of phenotypes, nd evenness evluted by the index E 5 proposed by Grünwld et l. (9): E 5 = (G 1)/(N 1 1) in which G is the Stoddrt nd Tylor index, G = 1/ p 2 i, nd N 1 = e H. We used the bootstrpping pproch recommended by Grünwld et l. (9) to obtin confidence intervls on the diversity index H (Shnnon index) nd evenness index E 5, using the BC (ccelerted bootstrp procedure) with the R softwre (R Development Core Tem (2004). R is lnguge nd environment for sttisticl computing. (R Foundtion for Sttisticl Computing, Vienn, Austri; ISBN , However, in our clcultions, this method twice yielded confidence intervl tht did not include the popultion men. In these two cses (underlined in Tble 2), the rtio of richness to smple size ws very high. Becuse the theory is bsed on symptotic pproximtion, our smple size might hve been too smll in these two cses. Index N 1 = e H represents the number of eqully common genotypes tht would produce the sme diversity s H. Becuse G weighs the number of bundnt genotypes more strongly nd N 1 weighs rrer genotypes more strongly (9), different rnkings of the popultions were obtined in some cses depending whether H (nd N 1 ), or G were considered. Clcultion of diversity index ws performed for the bred whet popultions P 1 to P 5 nd P b, but not for the durum whet popultion P d becuse of the smll smple size. RESULTS Comprison of P d nd P b. In the durum whet popultion P d, totl of 16 pthotypes ws identified (Tble 4), of which seven were found very rrely (only once yer in the smples) or not t ll on bred whet. The most frequent pthotype ws , virulent on three genes only (Lr2c, Lr10, nd Lr20), wheres this pthotype ws identified only three times in P b (Tble 5). In the bred whet popultion P b, totl 104 pthotypes ws identified (Tble 5), with 34 to 45 different pthotypes identified ech yer, of which 16 to 25 were found only once in the smple. The proportion of these pthotypes found only once in the yer ws roughly constnt, from 47 to 60% cross the 4 yers. Virulence frequencies on Lr13, Lr14, Lr16, nd Lr20 (Tble 6) were significntly different between durum whet nd bred whet. Pthotype distribution on the five mjor whet cultivrs. On the two susceptible cvs. Soissons nd Isengrin, disese level ws high (80 on the modified Cobb scle; 30) in the 4 yers the dy smples were collected. In contrst, there ws t most, one pustule per flg lef on cvs. Trémie (Lr10 nd Lr13) nd Orvntis (Lr10, Lr13, nd Lr37) in some yers, nd the mximum disese level on these cultivrs ws between 1 nd 5. Cv. Apche ws more vrible in its lef rust response with disese level usully between 1 nd 10, but reching 50 t one loction in The distribution of the pthotypes in the popultions P 1 to P 5 is plotted in Figure 1. Pthotypes found only once t most in ech yer were pooled in group nmed rre. As soon s pthotype ws found t lest twice in 1 yer, it ws plotted individully. Becuse the distributions on cvs. Soissons nd Isengrin were rther similr, the first nine pthotypes on the x xis re the sme for these two cultivrs. There were two dominnt pthotypes in 1999: (i) pthotype (virulent on Lr2c, Lr3, Lr3bg, Lr3k, Lr10, nd Lr14; frequency 28 nd 33%); nd (ii) pthotype (virulent on Lr2c, Lr13, Lr14, Lr26, nd Lr17b; frequency 19 nd 25%) for cvs. Soissons nd Isengrin, respectively. The frequency of pthotype decresed over the following yers, dropping to 0 in 2002 tht resulted in popultion dominted by pthotype tht represented 54 to 72% of the isoltes. The popultion on these two cultivrs ws lso composed of five to nine pthotypes found t low frequencies, between 2 nd 13%, nd rre pthotypes found only once, representing 2 to 11 pthotypes ech yer, i.e., between 5 nd 26% of the popultion. All pthotypes found on cvs. Soissons (except pthotype ; Fig. 1) nd Isengrin (except for four belonging to the group of rre pthotypes) possessed virulence for the gene Lr14 postulted in these two cultivrs. TABLE 3. Stndrd isoltes of Puccini triticin used in multipthotype tests for postultion of resistnce genes Isolte Pthotype Virulence t the seedling stge b B9405-2B Lr2c, Lr3, Lr3bg, Lr3k, Lr13, Lr14, Lr15, Lr17, Lr17b, Lr20, Lr26, Lr37 B9407-1CA Lr2c, Lr3bg, Lr14, Lr20, Lr37 B9201-2C Lr2c, Lr13, Lr14, Lr17b, Lr26 B9387-1A Lr2c, Lr3, Lr3bg, Lr3k, Lr10, Lr14 B9506-2B Lr2c, Lr13, Lr14, Lr17b, Lr26 B Lr2c, Lr3, Lr3bg, Lr3k, Lr10, Lr14 B9384-1C Lr2c, Lr3, Lr3bg, Lr3k, Lr13, Lr14, Lr15, Lr17, Lr17b, Lr20, Lr26, Lr37 B A Lr2c, Lr10, Lr17b, Lr20, Lr37 B9414-1CA Lr1, Lr2c, Lr3, Lr3bg, Lr14, Lr17b, Lr37 B8907-3B Lr2c, Lr3, Lr3bg, Lr14, Lr20, Lr37 B950506A Lr1, Lr10, Lr13, Lr15, Lr17, Lr17b, Lr20 B77SB Lr1, Lr2, Lr2b, Lr2c, Lr3, Lr3bg, Lr3k, Lr13, Lr15, Lr17, Lr17b, Lr20, Lr26, Lr37 B Lr1, Lr2, Lr2b, Lr2c, Lr3, Lr3bg, Lr3k, Lr10, Lr13, Lr15, Lr17, Lr20, Lr37 B9834-E Lr2c, Lr10, Lr13, Lr14, Lr16, Lr37 B00M057-A Lr1, Lr3, Lr3bg, Lr10, Lr13, Lr14, Lr15, Lr17, Lr17b, Lr20, Lr37, Lr27 + Lr31 Six-digit code bsed on 18-Lr genes differentil set (Thtcher lines Lr1, Lr2, Lr2b, Lr2c, Lr3, Lr3bg, Lr3k, Lr10, Lr13, Lr14, Lr15, Lr16, Lr17, Lr20, Lr23, Lr26, the Austrlin cv. Hrrier (Lr17b), nd Lr37. b Tested for pthogenicity on the 18 lines listed bove in ddition to the Thtcher lines with the resistnce genes Lr9, Lr19, Lr24, line CS2A/2M (Lr28), nd the Austrlin cv. Gtcher (Lr10, Lr27 + Lr31). 266 PHYTOPATHOLOGY

4 The popultion P 3 from cv. Trémie ws composed of three to four pthotypes with intermedite frequencies of 9 to 31%, then three to seven pthotypes with low frequencies (2 to 7%), nd some rre pthotypes representing 7 to 25% of the popultion (Fig. 1). Pthotypes , , nd , combining virulences for Lr10 nd Lr13 present in Trémie, were the min components of P 3 in 1999 nd The popultion then chnged with the rpid emergence of new group of pthotypes. This group included three closely relted pthotypes lso virulent on Lr10 nd Lr13, pthotype tht ws found only twice in 2000 nd incresed to 13% of P 3 in 2001 nd pthotypes (differing from by virulence on Lr26) nd (differing from by virulence on Lr23) representing 16% nd 9%, respectively, of P 3 in This group of three pthotypes ccounted for 38% of P 3 in Pthotypes nd were not detected in 2002, but pthotype remined t frequency of 25%. Pthotype , lthough virulent on Lr13 (one of the resistnce genes of cv. Trémie), ws found ech yer in P 3 t frequencies between 3 nd 14%. Of the 32 pthotypes found during the 4 yers on cv. Trémie, 13 were virulent either on Lr10 (four pthotypes) or Lr13 (eight pthotypes) or on both genes (one pthotype). The distribution of pthotypes ws more blnced on cv. Apche (Lr13 nd Lr37), with the popultion comprising eight pthotypes t frequencies between 3 nd 16% nd group of rre pthotypes representing 19 to 26% of the popultion. The popultion on cv. Orvntis (Lr10, Lr13, nd Lr37) comprised the lowest number of pthotypes, with only four pthotypes t frequencies between 6 nd 33% nd rre pthotypes representing 33% of the popultion. All four pthotypes found on cv. Orvntis were present on Apche. In ddition, one found on Apche (pthotype ) ws lso found on Trémie nd second (pthotype virulent on Lr2c, Lr3, Lr3bg, Lr3k, Lr13, Lr14, Lr15, Lr17, Lr26, Lr17b, nd Lr37 t the seedling stge) ws not found on ny other cultivr. Agin, s for Trémie, lthough pthotype ws virulent on Lr13, it ws present on Apche nd Orvntis t frequency s much s 17%. Diversity indices. The highest richness index, clculted s the number of phenotypes in the smple, ws lwys obtined on the popultion P b (Tble 2). In 1999, the lowest richness ws observed on cv. Trémie (P 3 ), but in 2000 it ws observed on cv. Soissons (P 1 ). In 2001 nd 2002, richness ws lmost constnt cross the cultivrs, rnging between 10 nd 14. Diversity nd evenness for ech yer cross cultivrs. The diversity index H ws higher in popultion P b thn in popultions P 1 to P 5 for the 4 yers nd this difference ws significnt in most cses (12 of 16). For the 4 yers, diversity on cv. Soissons ws the sme s on cv. Isengrin. In 1999, diversity on cv. Trémie ws significntly lower thn on cvs. Soissons nd Isengrin, wheres in 2000, it ws higher thn on cv. Soissons nd the sme s on cv. Isengrin. In 2001 nd 2002, diversity on the two susceptible cultivrs ws significntly lower thn on the three resistnt cvs. Trémie, Apche, nd Orvntis. In 1999, evenness ws the sme cross the cultivrs. In 2000, evenness on Trémie ws not only higher thn on other cultivrs, but lso on the globl popultion. In 2001, evenness on Soissons, Isengrin, nd the globl popultion ws significntly lower thn on the resistnt cvs. Trémie, Apche, nd Orvntis. In 2002, the sitution ws not so cler, differences being significnt in only one-hlf of the cses. Evenness on Isengrin ws lower thn on ny other cultivr except Soissons. Diversity nd evenness for ech cultivr cross yers. In 1999, diversity nd evenness on cv. Soissons were higher thn in the three other yers. Diversity on cv. Isengrin ws rther constnt cross the yers, but ws lowest in Some vritions in evenness were observed, which ws highest in 1999 nd lowest in For cv. Trémie, diversity ws lowest in 1999, reched mximum in 2000, nd then decresed in 2001 nd 2002, wheres evenness ws constnt cross the 4 yers. Diversity nd evenness were stble for cvs. Apche nd Orvntis between 2001 nd For the popultion P b, diversity ws very stble cross the yers, vrying between 2.43 nd 2.76, wheres evenness vried, with mximum (0.595) in 1999, minimum (0.350) in 2000, nd then incresed to rech in 2002 the sme level (0.507) s in Reltive importnce of the different resistnce genes in the host popultion. Resistnce gene Lr13 ws the most commonly detected gene, found in 25 of 35 cultivrs nlyzed (Tble 7) nd ws lso the most widely deployed gene present in verge on 40.1% of the whet re during Lr14, lthough found in only seven of the cultivrs, ws similr to Lr13 by the importnce of cultivtion (35% of the whet re) becuse of the dominnce of cv. Soissons (Lr14). Fourteen of 35 cultivrs exmined hd either Lr13 or Lr14 lone. In ddition, resistnce genes Lr3, Lr10, Lr20, Lr26, nd Lr37 were postulted in the cultivrs (Tble 7). Three cultivrs hd no detectble resistnce TABLE 4. Number of isoltes of Puccini triticin collected from durum whet (popultion P d ) in Frnce in , identified for virulence phenotype on 18 differentil hosts Virulence phenotype Virulences (Lr genes) Totl c, 10, c, 3, 3bg, 3k, 10, c, 10, 20, 23, 17b c, 3, 3bg, 3k, 13, 14, 15, 17, 20, 26, 17b, c, 3, 3bg, 3k, 10, 13, 14, 15, 17, 26, 17b, c, 10, 14, , 2, 2b, 2c, 3, 3bg, 3k, 15, 17, 20, 26, 17b, c, 14, c, 10, c, 13, 14, 26, 17b , 2, 2b, 2c, 3, 3bg, 3k, 13, 15, 17, 20, 26, 17b, c, 3, 3bg, 3k, 15, 16, 17, 26, c, 10, 13, 16, c, 3, 3bg, 3k, 13, 14, 15, 17, 26, 17b, c, 10, 14, 20, 17b, c, 10, 14, 20, 17b 1 1 Totl number of isoltes Totl number of pthotypes Six-digit code bsed on 18 Lr genes differentil set (Thtcher lines Lr1, Lr2, Lr2b, Lr2c, Lr3, Lr3bg, Lr3k, Lr10, Lr13, Lr14, Lr15, Lr16, Lr17, Lr20, Lr23, Lr26, the Austrlin cv. Hrrier (Lr17b), nd Lr37. Yer Vol. 96, No. 3,

5 gene. Combintions of two resistnce genes were postulted for 13 cultivrs, nd combintions of three resistnce genes were postulted for only three cultivrs, of which two were registered recently (Orvntis in 2000 nd Cphorn in 2001). DISCUSSION Since virtully ll commercil whet fields in Frnce re spryed with fungicide we hd to collect isoltes from unspryed blocks in experimentl nurseries. As evluted with the Shnnon index, nursery collections were more diverse thn field collections in the pririe re in Cnd in 1990 (15), however, bsed on the Rogers index nd contingency tble nlysis, they were regrded s similr. Although it ws stted tht nurseries could not ccurtely reflect the level of diversity occurring in commercil fields (18), it ws concluded in lter study (19) tht in most res there were no consistent differences in pthotype diversity between field nd nursery collections. TABLE 5. Number of isoltes of Puccini triticin collected from bred whet (popultion P b ) in Frnce during nd identified by virulence phenotype on 18 differentil hosts Virulence phenotype Virulences (Lr genes) Totl c, 3, 3bg, 3k, 10, c, 10, 13, 16, c, 10, 13, 15, 17b, c, 10, 13, 14, 16, c, 13, 14, 26, 17b c, 3, 3bg, 3k, 10, 13, 14, 15, 17, 26, 17b, c, 3, 3bg, 3k, 13, 14, 15, 17, 26, 17b, , 3, 3bg, 10, 13, 14, 15, 17, 20, 17b, , 3, 3bg, 10, 13, 14, 15, 17, 20, 26,17b, c, 10, 13, 14, 16, 26, c, 14, 26, 17b c, 3, 3bg, 3k, 10, 14, c, 10, 13, 14, 16, c, 3, 3bg, 3k, 13, 14, 15, 16, 17, 26, 17b, c, 3, 3bg, 3k, , 3, 3bg, 10, 13, 14, 15, 17, 20, 23, 17b, c, 13, 14, 16, 26, 17b c, 10, 13, 14, c, 10, 13, c, 13, , 2c, 10, 13, 15, 17b, c, 3, 3bg, 3k, 10, 13, 14, 15, 17, 20, 26, 17b, c, 3, 3bg, 3k, 13, 14, 15, 17, 20, 26, 17b, c, 10, 17b c, 10, 13, 14, 16, 26, 17b c, 10, c, 10, 13, 14, , 2, 2b, 2c, 3, 3bg, 3k, 10, 13, 15, 16, 17, 20, c, 3, 3bg, 3k, 10, 13, 14, c, 13, 15, 17b, c, 10, c, 3, 3bg, 3k, 14, 16, c, 3, 3bg, 3k, 14, 15, 26, 17b c, 10, 17b, c, 10, 15, 17b, c, 10, 13, 16, 26, c, 10, 13, 14, 15, 17b, , 13, 14, 16, 17b, c, 10, 13, 15, 17b c, 10, 16, c, 14, 16, 17b c, 10, 13, 15, 17, 17b, c, 3, 3bg, 3k, 10, 13, 14, 15, c, 3, 3bg, 13, 14, 17, c, 3, 3bg, 3k, 10, 14, 16, c, 3, 3bg, 3k, 10, 14, 15, c, 3, 3bg, 3k, 10, 14, c, 3, 3bg, 3k, 14, 15, 17, 20, 26, 17b, c, 10, 13, 16, 26, 17b, c, 10, 15, 20, 17b, c, 13, 14, 17b Number of isoltes Number of pthotypes Number of pthotypes found once in four yers b Unique pthotypes c Six-digit code bsed on n 18-Lr genes differentil set (Thtcher lines Lr1, Lr2, Lr2b, Lr2c, Lr3, Lr3bg, Lr3k, Lr10, Lr13, Lr14, Lr15, Lr16, Lr17, Lr20, Lr23, Lr26, the Austrlin cv. Hrrier (Lr17b), nd Lr37. b Pthotypes found only once in 4 yers hve been pooled in group nd their virulence formul is not given here. c Number of pthotypes found only once for ech yer. Yer 268 PHYTOPATHOLOGY

6 Diversity nd evenness in P b were stble cross the 4 yers. The diversity in P b, s mesured by the Shnnon index, rnged between 2.43 nd These vlues were very similr to those observed in some epidemiologicl res of the United Sttes (18,19). In the estern nd pririe res of Cnd in the period , the Shnnon index ws mostly between 1 nd 2 nd it dropped below 1 in the priries fter the introduction of resistnt cultivrs (14), but the use of only four differentil lines resulted in lower diversity s compred with other studies using more differentil lines. In Isrel, with isoltes derived from durum whet nd bred whet nd in n re where high diversity could be expected becuse of the presence of indigenous wild Triticum nd Aegilops spp. reltives, the Shnnon index vlues were very close to those clculted in our study, rnging between nd in (21). The Shnnon index clculted for Frnce in previous study in 1995 (29) ws lower thn the vlues obtined here, rnging between 0.75 nd In the 1995 study, smple sizes were smller thn in our study. The Shnnon index underestimtes diversity in smll smples s compred with big smples (19). Although Prk et l. (29) used 25 differentils while we used only 18, they did not include Lr13 nd Lr14, the most frequent resistnce genes (Tble 7) in the French whet host popultion. Using these resistnce genes in our differentil set my hve permitted discrimintion of more pthotypes. Although the differentil set we used differed from tht used by Prk nd Felsenstein (28) for few lines, it is likely tht the two predominnt pthotypes 17 nd 39 (28) found in Europe in 1995 were the sme s pthotypes nd in our study, respectively. On the other hnd, pthotypes 3 nd 6, common in Frnce in the 1995 study, were not found in our study. Aeril spore trpping s used in the 1995 study hs been shown to reflect minly pthogen popultions from fields bordering the smpling route (10). The spores trpped from pthotypes 3 nd 6 might therefore, hve come minly from Siderl (Lr13 nd Lr14), the second most widely grown cultivr in these res in The shrp decline in the cultivtion of Siderl from 1998 nd lck of smple from it in the current study my explin why pthotypes 3 nd 6 were not detected during the period. Comprison between durum whet (P d ) nd bred whet (P b ). The first level of differentition mde in French whet lef rust popultions ws between isoltes originting from durum nd bred whet. Although virulence frequencies (Tble 5) could be bised becuse of the smller size of the smple derived from P d, the frequency of virulence on Lr20 ws much higher on P d thn on P b, nd the frequency of virulence on Lr13, Lr14, nd Lr16 ws lower on P d thn on P b. Resistnce gene Lr20 is infrequent in bred whet cultivrs (Tble 7). Informtion bout Lr resistnce genes in the durum whet cultivrs is missing so we do not know if the high virulence frequency for Lr20 in P d results from selection by host. The most frequent pthotype on durum whet, , represented 47% of ll pthotypes collected in 3 yers for durum whet, wheres it ws only 0.3% of pthotypes collected in 4 yers from bred whet, which is not surprising given tht did not hve the virulences mtching the resistnce genes crried by the min bred whet cultivrs. Almost one-hlf of the pthotypes found on durum whet were rre or not found t ll on bred whet. Thus, we concluded tht lef rust popultions from durum nd bred whet were different s ws shown in Morocco (6), Mexico (33), nd interntionl collections (11,26). Pthotype , found in 2001 in Frnce (Tble 5), ppers to be the sme s the new rce BBG/BN detected in 2001 on durum whet in Mexico (34). With the differentil set used, we were ble to detect differences between P d nd P b, thus, to conclude to differentil selection exerted by host species. A study of selection by host within P d would require the chrcteriztion of the resistnce genes in the durum whet cultivrs, nd the definition of corresponding differentil set. Moreover, it is possible tht P. triticin nd P. recondit occur on durum whet in Frnce since both species occur in the Mediterrnen re, (Itly, Morocco, Spin, nd Portugl) (2). Diversity nd distribution of pthotypes in the bred whet lef rust popultions (P 1 to P 5 ). Differences between the yers could be found in diversity or evenness for some cultivrs, but no yer ws chrcterized by generl increse or decrese in these two indexes. Diversity ws similr in the two popultions from susceptible cvs. Soissons (P 1 ) nd Isengrin (P 2 ) in the 4 yers, but it ws lower thn in the three popultions from resistnt cvs. Trémie (P 3 ), Apche (P 4 ), nd Orvntis (P 5 ) in ll yers except 1999 where it ws lower in P 3 thn in P 1 nd P 2. Since evenness ws the sme cross the cultivrs in 1999, the difference between P 3 on one hnd nd P 1 nd P 2 on the other hnd in tht yer ws due to lower richness in P 3. In the two following yers, 2000 nd 2001, the significnt differences in diversity between susceptible nd resistnt cultivrs were ttributed to differences in TABLE 6. Virulence frequencies on Lr genes in whet lef rust popultions collected from durum whet (P d ) nd bred whet (P b ) cultivrs in Frnce in Gene Durum Bred Durum Bred Durum Bred Bred Lr Lr Lr2b Lr2c Lr Lr3bg Lr3k Lr Lr13 b Lr14 b Lr Lr16 b Lr Lr17b Lr20 b Lr Lr Lr Durum lef rust popultion ws not studied in b The virulence frequencies were significntly different t P < 0.05 between durum whet nd bred whet (one-wy nlysis of vrince with the yers considered s replictes). Vol. 96, No. 3,

7 evenness becuse evenness on the resistnt cultivrs ws lwys higher thn on the susceptible cultivrs. The sitution ws similr in 2002, lthough differences in evenness between cultivrs were not lwys significnt. However, it is difficult to ttribute the significnt differences in diversity to the significnt differences in richness becuse richness vlues for the different cultivrs were very close, being between 11 nd 14. The smll smple sizes of isoltes collected from resistnt cultivrs could hve been responsible for the lck of significnce in some cses. Despite this, the generl trend from these results ws tht diversity ws higher on resistnt cultivrs thn on susceptible cultivrs, nd this difference ws due to evenness s it is shown clerly on Figure 1. Evenness on the susceptible cultivrs ws low becuse of the dominnce of two pthotypes, nd in 1999, nd single pthotype, in , the frequency of which vried between 54 nd 72%. In contrst, in the Cndin pririe re in 1990, the Shnnon index for resistnt cultivrs (pooled popultion smpled from 13 cultivrs) ws significntly lower thn for susceptible cultivrs (pooled popultion smpled from five cultivrs), lthough the Rogers index nd contingency tble nlysis showed tht there ws no significnt difference between popultions from resistnt nd susceptible cultivrs (15). The distribution of pthotypes ws very similr on the two susceptible cvs. Soissons nd Isengrin tht crried the lrgely ineffective resistnce gene Lr14, both of which lso hve very similr genetic bckground. The popultions P 1 (Soissons) nd P 2 (Isengrin) were dominted by two pthotypes in 1999, fter which one (014103) decresed in frequency nd disppered in 2002, while the other (073100) represented more thn 70% of the popultion. The pthotype distribution ws more blnced on the resistnt cultivrs s reflected by the evenness indexes being higher thn on susceptible cultivrs. A strong nd rpid chnge in P 3 ws detected when two of the most frequent pthotypes, nd , dropped in frequency in 2001 nd were replced by group of three very similr pthotypes designted s group 166. This group, bsent in 1999, represented 6% of P 3 in 2000 nd then 38% in Group 166 comprised the most virulent pthotypes (virulent on 12 of 18 differentil lines) nd differed gretly from the other pthotypes common in Frnce by virulence on Lr2c, virulence on Gtcher (results not presented here), combined virulence on Lr1, Lr10, nd Lr13, combined virulence on Lr3 nd Lr3bg, nd virulence on Lr3k. The most similr pthotypes described in the literture were pthotypes MBD/SM nd MCD/SM, dominnt in CIMMYT nurseries in 270 PHYTOPATHOLOGY (continued on next pge) Fig. 1. Frequencies of Puccini triticin pthotypes on five mjor bred whet cultivrs (popultions P 1 to P 5 ) in Frnce during Blck brs = pthotype crrying the virulence gene(s) mtching ll the resistnce gene(s) of the cultivr. Shded brs nd corresponding pthotype nme in bold = pthotype lcking t lest one of the virulence gene(s) mtching resistnce gene(s) of the cultivr. The pthotypes found only once t most for ech yer hve been pooled in the ctegory rre. Susceptible cultivrs re Soissons (P 1 ) nd Isengrin (P 2 ). Resistnt cultivrs re Trémie (P 3 ), Apche (P 4 ), nd Orvntis (P 5 ).

8 1990 (33), but with the difference tht these ltter pthotypes were virulent on Lr28, wheres group 166 pthotypes were virulent on Lr28 (results not presented here). Virulence for Lr28 is chrcteristic of North Americn popultions of P. triticin becuse the frequency of virulence for this gene is very high in these popultions (33). Rces with the sme virulence s group 166 were found in North Americ strting in the mid-1990s. These rces were found to hve very distinct mplified frgment length polymorphism (AFLP) ptterns compred with other rces (17). The isoltion of pthotype , virulent on Lr13, from three cultivrs crrying this gene (Trémie, Apche, nd Orvntis) ws not unexpected becuse it ws known tht this resistnce gene does not completely prevent sporultion by virulent pthotypes. Although 98% of the whet fields in Frnce re spryed with fungicide, inoculum is present every yer s shown by epidemics occurring on ny untreted plot. On cultivr s susceptible s Soissons, infections cn still be observed fter tretments when environmentl conditions re conducive. Infections cn lso be esily observed in newly plnted commercil fields in the fll nd it is likely tht some inoculum survive on volunteers during the summer. The stbility of the pthotype distribution on cv. Soissons over 4 yers lso suggested survivl of locl inoculum. The differences between pthotype distributions observed in popultions from cvs. Soissons nd Trémie suggested tht strong selection ws exerted on the inoculum by the cultivr. Distribution of pthotypes nd resistnce fctors in the cultivrs. In severl cses, the distribution of some pthotypes on the different cultivrs could not be explined by the selection exerted by specific host resistnce genes only. Ten to eighteen pthotypes, lthough virulent on Lr14, nd therefore, virulent on cvs. Soissons nd Isengrin crrying only Lr14 nd no dult plnt resistnce gene, occurred in P 1 nd P 2 but remined t frequencies much lower thn the dominnt pthotype Severl virulence gene combintions, including the most complex one, were found only on resistnt cultivrs even though these multivirulent pthotypes were primrily virulent on the susceptible cultivrs nd despite the fct tht our smples were collected from smll plot nurseries, where exchnge of inoculum between plots is expected to be frequent. The two cultivrs from which popultions were similr (Soissons nd Isengrin) hd the sme resistnce gene nd similr genetic bckground. It is prticulrly surprising tht pthotype remined t high nd stble frequency during the 4 yers, wheres ll other virulent pthoytpes either decresed in frequency (014103) or never incresed in frequency in the popultion. Given tht Soissons ws lrgely dominnt cultivr in the period nd considering tht Fig. 1. (continued from preceding pge) Vol. 96, No. 3,

9 pthotype ws not frequent on the other dominnt cultivrs (except on Isengrin, which hs the sme resistnce gene nd genetic bckground close to tht of Soissons), it is unlikely tht the dominnce of in the globl popultion could be ccounted for by nother reson thn strong ggressiveness on cvs. Soissons nd Isengrin. This hypothesis is reinforced by considering the trnsition between 1999, where coexisted with , nd yer 2000, where remined the only dominnt pthotype. Although this hs not been tested, this high frequency of , virulent on Lr13 in 1999, might result from the development of epidemics on cv Thésée (Lr13) tht ws lrgely cultivted from 1988 to 1997 nd on which is virulent. It ppers then tht lef rust epidemics on the susceptible cultivrs were minly cused by single pthotype (073100) despite the presence of firly lrge diversity of other virulent pthotypes in the globl popultion, nd this for severl yers. In this sitution, the knowledge of the frequency nd distribution of resistnce genes in the host popultion nd of the virulence combintions in the pthogen popultion is not sufficient to predict or explin the distribution nd frequency of the pthotypes. When isoltes of P. grminis f. sp. tritici, virulent on commonly grown cultivrs, were found t very low frequencies (31), it ws suggested by Alexnder et l. (1) tht negtive ssocitions between virulence nd trits ffecting spore survivl nd reproduction my be responsible. In our cse, converse hypothesis my be proposed, in which higher ggressiveness of pthotype could led to strong dominnce of this pthotype over other pthotypes virulent on Lr14. A few popultion studies show tht the dominnce of subpopultion (pthotype, clonl linege) in globl popultion cn be ccounted for by differences in ggressiveness (24). This cn sometimes be explined by better dpttion to specific climtic conditions (13). This ltter explntion is unlikely here, given the high frequency of over severl consecutive yers. Other studies hve concluded tht rust popultion structure is likely to be influenced by chrcters other thn the interction of host resistnce genes nd corresponding genes in the pthogen (22,29). High levels of diversity mintined in North Americn whet lef rust popultions indicte tht selection for virulence to TABLE 7. Men percentge of whet re plnted during with the different Lr genes postulted in the 35 most commonly grown cultivrs Lr gene combintion Men percentge of whet re No. of cultivrs No gene Lr10, Lr Lr10, Lr13, Lr Lr10, Lr13, Lr Lr Lr13, Lr13, Lr Lr13, Lr Lr13, Lr Lr Lr14, Lr Lr Lr Lr3, Lr Totl Cumultive percentge of whet re for ech Lr gene Lr gene Lr Lr Lr No gene 4.3 Lr Lr Lr3 1.4 Lr The totl is not 100% becuse the minor cultivrs re not included in this tble. host resistnce genes is effectively blnced by other selective forces (16). Group 166, virulent on Lr10 nd Lr13, occurred on cvs. Trémie (Lr10 nd Lr13) nd Apche (Lr13 nd Lr37) but not on cv. Orvntis (Lr10, Lr13, nd Lr37). Thus gin, interctions with specific resistnce gene(s) cnnot explin the bsence of this group on cv. Orvntis, unless cvs. Apche nd Orvntis hve unknown nd different dult-plnt resistnce gene(s). Pthotype , lthough virulent on Lr10 nd Lr13, ws bsent or rre on cv. Trémie. For these popultions, derived from resistnt cultivrs nd where the structure observed could not be fully explined by interction of known genes for resistnce nd virulence, possible differences in ggressiveness my contribute to pthogen popultion composition, s for the dominnce of on cvs. Soissons nd Isengrin. However, unlike for the susceptible cultivrs, it is not known here whether specific dultplnt resistnce is present in the resistnt cultivrs. For exmple, Brtos et l. (4) concluded tht rce frequency in the former Czechoslovki could not be explined on the bsis of host selection, but the study of Prk et l. (29) showed tht mny cultivrs registered there crried Lr13, gene tht hd not been considered in their study. In Frnce, unexplined differences between lef rust popultions from cvs. Soissons nd Thésée in (8) my well hve been due to the presence of the resistnce genes Lr14 in Soissons nd Lr13 in Thésée, s virulence for these genes ws not monitored in these yers. In summry, P. triticin popultions in Frnce were diverse for virulence despite the presence of only few specific resistnce genes in host bred whet cultivrs. The potentil occurrence of recombintion, which could explin the observed diversity, still hs to be tested. Popultions were strongly differentited between durum whet nd bred whet cultivrs nd within bred whet cultivrs. Diversity ws high on those tht were resistnt, minly due to n evenness found to be higher for resistnce s compred with susceptible cultivrs. The popultion structure described in our study stresses the importnce for smpling procedures to tke into ccount the host cultivr for mesuring the influence of selective fctors on whet lef rust popultions, s completely different virulence or pthotype frequencies could be obtined, depending on the host cultivrs represented in the smple. Lst, we showed with lrge-scle study tht specific interctions in the lef rust whet pthosystem were necessry but not sufficient to ccount for pthotype distribution nd frequency on the cultivrs. Other fctors like ggressiveness, could ccount for the dominnce of one or few pthotypes on some cultivrs over severl yers. ACKNOWLEDGMENTS We thnk A.-C. Zippert for excellent technicl ssistnce in collecting, isolting, mintining, nd testing cultures of Puccini triticin. We grtefully cknowledge the French Whet Breeders group Club des Cinq for permitting collection of isoltes on their field tril network nd Arvlis Institut du Végétl for their help in collecting smples. LITERATURE CITED 1. Alexnder, H. M., Groth, J. V., nd Roelfs, A. P Virulence chnges in Uromyces ppendicultus fter five sexul genertions on prtilly resistnt cultivr of Phseolus vulgris. Phytopthology 75: Anikster, Y., Bushnell, W. R., Eilm, T., Mnisterski, J., nd Roelfs, A. P Puccini recondit cusing lef rust on cultivted whets, wild whets, nd rye. Cn. J. Bot. 75: Anonymous Enquête sur les prtiques culturles en Agreste Chiffres et Données Agriculture 159:38. Ministère de l Agriculture, de l Alimenttion, de l Pêche et des Affires Rurles. 4. Brtos, P., Stuchlikov, E., nd Hnusov, R Adpttion of whet rusts to the whet cultivrs in former Czechoslovki. Euphytic 92: Brown, J. K. M Surveys of vrition in pthogen popultions nd their ppliction to disese control. Pges in: The Epidemiology 272 PHYTOPATHOLOGY

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