Population Genetic Structure of Phytophthora infestans in Ecuador

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1 Genetics Popultion Genetic Structure of Phytophthor infestns in Ecudor Gregory A. Fores, Ximen C. Escor, Ctlin C. Ayl, Jorge Revelo, Mri E. Ordoñez, Brr A. Fry, Kthrine Doucett, nd Willim E. Fry First nd fifth uthors: Interntionl Potto Center (CIP), P.O. Box , Quito, Ecudor; second, third, nd fourth uthors: Instituto Ncionl de Investigcion Agropecuri (INIAP), Box 340, Quito, Ecudor; nd sixth, seventh, nd eighth uthors: Deprtment of Plnt Pthology, 334 Plnt Science, Cornell University, Ithc, NY Accepted for puliction 19 Decemer ABSTRACT Fores, G. A., Escor, X. C., Ayl, C. C., Revelo, J., Ordoñez, M. E., Fry, B. A., Doucett, K., nd Fry, W. E Popultion genetic structure of Phytophthor infestns in Ecudor. Phytopthology 87: The popultion genetic structure of Phytophthor infestns in Ecudor ws ssessed from 101 isoltes collected from 1990 to 1992 nd 111 isoltes collected in All isoltes were nlyzed for mting type nd llozyme genotype. Both smples were dominted (>95%) y clonl linege (EC-1) defined from neutrl mrkers: 90/100 genotype for glucose-6-phosphte isomerse, 96/100 genotype for peptidse, A1 mting type, nd previously unreported nucler DNA fingerprint. The remining isoltes elonged to the US-1 clonl linege, which hs worldwide distriution. Isoltes in the 1993 smple were nlyzed for virulence nd metlxyl sensitivity. All representtives of EC-1 hd complex pthotypes, with three pthotypes representing >60% of the collection. There ws vrition for metlxyl sensitivity. There ws no evidence for geogrphicl sustructuring on the sis of neutrl mrkers, ut there ws evidence for limited sustructuring sed on metlxyl sensitivity nd specific virulence. We hypothesize tht EC-1 hs een recently introduced to Ecudor. Knowledge of the popultion genetic structure of Phytophthor infestns (Mont.) de Bry cn provide vlule insight into the overll disese epidemiology nd help to explin chnges in oserved disese severity. Recent displcement of endemic popultions of P. infestns y introduced genotypes hs een identified s potentil source of incresed losses due to greter ggressiveness of the pthogen, erlier outreks of disese, incresed resistnce to fungicides, nd the cpility of cusing severe disese on oth tomtoes nd pottoes with equl ggressiveness (5,6,17). Knowledge of the pthogen popultion structure is lso useful in screening for host plnt resistnce nd in the development of disese mngement strtegies (11). Both the clonl nture of this pthogen, in most prts of the world, nd the existence of roust genetic mrkers hve permitted workers to mp the glol migrtions of P. infestns (5). Anlysis of limited glol collection of isoltes demonstrted tht single clonl linege (US-1) is present in most countries in which potto is grown (7). This linege is defined y prticulr restriction frgment length polymorphism (RFLP) nding pttern with the proe RG57, the glucose-6-phosphte isomerse (Gpi) genotype 86/ 100, nd the peptidse (Pep) genotype 92/100 (7). All Europen isoltes collected prior to 1980 were US-1. This, together with its worldwide distriution, hs led to hypothesis tht US-1 is n old linege tht my dte ck to the introduction of the fungus into Europe in the mid 1840s (7). In Mexico, the presumed center of origin of P. infestns, the fungus reproduces sexully, nd the concept of clonl linege hs very limited utility. Sexul reproduction is now thought to occur in severl prts of Europe (3,19). In this glol picture, Ecudor ws n interesting cse. A smple collected in the 1980s of the popultion of P. infestns in Peru ws exclusively US-1 (7,22). In contrst, three isoltes collected from the province of Antioqui in northern Colomi in 1990 were not US-1 (G. A. Fores, unpulished dt). The Colomin Corresponding uthor: G. A. Fores; E-mil ddress: foresecip.org.ec Puliction no. P R 1997 The Americn Phytopthologicl Society isoltes hd the A1 mting type, ut did not hve either the 86 llele for Gpi or the 92 llele for Pep, which re chrcteristic of US-1. These oservtions led us to hypothesize tht Ecudor my e n re in which US-1 from Peru nd the other popultion from Colomi re converging nd tht genetic sustructuring my exist long north-south xis in the country. The following study ws conducted to test this hypothesis nd to chrcterize the popultion in Ecudor using existing mrkers. Becuse recent review of the literture on popultion genetic studies of plnt pthogens concluded tht they re chrcteristiclly wek in smple design (11), we pplied rigorous smpling pproch. MATERIALS AND METHODS Smpling. Two smpling strtegies were used. The first ws designed to provide generl overview of the popultion structure. For this purpose, 101 isoltes were tken from the P. infestns collection of the Interntionl Potto Center (CIP), which is mintined in the CIP s highlnd reserch sttion in Quito, Ecudor. These isoltes hd een collected on numer of different excursions etween 1990 nd 1992 in the provinces of Crchi in northern Ecudor nd Pichinch in northern-centrl Ecudor. Isoltion nd isolte mintennce procedures re descried elow. The second smple set ws collected etween April nd July of 1993 to test the hypothesis of popultion sustructuring long north-south xis in Ecudor. Smples were collected in three geogrphiclly seprte production zones tht primrily correspond to the provinces of Crchi in the north, Chimorzo in the centrl prt, nd Loj in the south (Fig. 1). Since our ojective ws to test for popultion sustructuring long north-south xis, we chose to strtify our smpling to hve equl proilities of finding rre genotypes in the northern, centrl, nd southern prts of the country. Smple sizes for ech of the three production zones were determined sed on the premise of finding rre genotype which occurs t frequency of 0.1 with known proility of filure (e.g., = 0.01 or = 0.05) using the following formul: N = log( ) / log( 1 p) Vol. 87, No. 4,

2 in which N = smple size, = the ccepted proility of filure, nd p = the frequency of the rre genotypes (13). Assuming rre genotypes occur rndomly, smple sizes etween 28 nd 43 in ech production zone would give us levels of proility of filure etween 0.05 nd 0.01, respectively, which we considered pproprite. In Crchi nd Chimorzo, 40 smples were collected; in Loj, only 31 could e found, ecuse of dry conditions. For the first level of strtifiction, we used mps t scle of 1:200,000, ech of which siclly covered one production zone in the northern, centrl, nd southern prts of the country (Fig. 1). Smple size t this strtum rnged from 31 to 40 s descried ove. Ech mp of 1:200,000 ws then divided into 16 smller lnd-use mps (1:50,000), which indicted the res dedicted to mjor crops including potto. The 1:50,000 mps primrily delimited one politicl unit known s cnton nd will e referred to herefter s cnton mps. The numer of cnton mps tht contined pottogrowing res in ech production zone ws four for Crchi, six for Chimorzo, nd one for Loj, where potto production is of minor importnce (Tle 1). The 31 to 40 smples from ech production zone were ssigned to the individul cnton mps sed on their reltive levels of production (Tle 1). Smple sizes for cnton mps rnged from 1 to 31. Cnton mps were divided into 486 qudrts, ech representing 4 km 2. Qudrts were then chosen t rndom until sufficient numer of them (out 50) were identified tht overlid potto production s indicted on the lnd-use mps. For the purpose of nlysis, the experimentl unit consisted of one isolte from ech qudrt. The smpling scheme is represented grphiclly (Fig. 1). When collections were mde in the field, qudrt res were visited in their rndomized order until the sufficient numer of smples were collected for ech cnton (Tle 1). One potto field ws picked t rndom within ech qudrt to e smpled. Five to 10 leflets with single lesions were collected from ech field long trnsect. These were mintined t pproximtely 5 C for 1 to 2 dys until isoltion. In the lortory, isoltion ttempts were mde on five of the lesions, ut only one rndomly chosen isolte ws used in ll the nlyses. Single-lesion isoltes hve een pproprite for llozyme nd DNA nlyses nd pper to generlly e single genotypes (8). Isoltion nd mintennce of the pthogen. Isoltes of P. infestns were trpped y plcing 1-cm 2 pieces of infected tissue under potto slices in petri dishes. This step ids in seprting P. infestns from sprophytes even when the infected tissue is old. Pure cultures were mde y picking off smll pieces of mycelium tht were generlly growing undntly on the upper side of the potto slice fter 7 dys of incution t 15 C. Mycelil pieces were plted on rye B gr (2). After 1 to 2 weeks, growing colonies were trnsferred to rye A gr (2) nd mintined t 5 or 15 C until used. Mting type. Mting type ws ssessed y plcing gr plugs of four isoltes t equl distnce round n gr plug of the known A1 isolte on 10% clrified V8 gr (9). After 14 dys of incution t 18 C, petri dishes were ssessed regulrly during 4 weeks for the formtion of oospores. Virulence. Virulence ws determined only for the 1993 smple. A differentil set of potto cultivrs representing the 11 known mjor genes (R genes) for resistnce (14,15) ws provided y L. Turkensteen (Reserch Institute for Plnt Protection [IPO-DLO], Wgeningen, the Netherlnds). Inoculum for virulence tests ws isoltes t this level Scle = 1:200, isoltes t this level Scle = 1:50,000 1 isolte t this level 2 km 2 km Fig. 1. Qudrts tht pproximtely represent the re covered y mps t scle of 1:200,000 re superimposed on the mp of Ecudor to indicte the loction of the three production zones tht were smpled: Crchi, Chimorzo, nd Loj. Smple size per production zone ws 31 to 40. The ox in the upper right corner shows how ech 1:200,000 mp ws divided into 16 smller cnton mps of scle 1:50,000. The selection of cnton mps nd the determintion of smple size per cnton mp is descried in Mterils nd Methods. The ox t the center right shows how the cnton mps were divided into 486 qudrts. These were chosen t rndom nd hve smple size of one nd, thus, represent the experimentl unit of the mrker nlyses. Shding on the mp of Ecudor indictes res of intense potto production. 376 PHYTOPATHOLOGY

3 grown on potto tuer slices for 6 to 7 dys t 15 C in seled plstic trys. After incution, tuer slices were wshed gently with wter to lierte sporngi. These were rinsed severl times with sterile wter over 12-µm mesh filter. The sporngil suspension ws plced in refrigertor for pproximtely 2 h to induce zoospore formtion. Zoospores were seprted from sporngi y filtrtion through 12-µm mesh nd diluted to concentrtion of /ml. Leflets were selected from plnts tht were 6 to 10 weeks old nd hd not yet egun to flower. The differentils were grown in screen house in the CIP instlltions ner Quito, Ecudor, t 3,050 m ove se level. Fvorle growing conditions nd dy length (out 12 h) re constnt yer round in Quito. Dy length ws extended to 16 h with hlogen lights for the differentils, some of which do not grow well under short dys. Two 10-µl drops of inoculum were plced on the dxil side of ech leflet to e tested. These were incuted with 12 h of fluorescent light (out 3,000 lux) in inverted petri dishes contining 1.5% wter gr in the se. Approximtely 10 isoltes were tested for virulence t one time, ut ll virulence ssys were done with two control isoltes. One control ws virulent on ll of the known 11 R genes. The second ws one of two isoltes, oth of which re virulent on other differentil cultivrs including R2, R4, R10, nd R11. These re the R genes tht we consider the most difficult to score (G. A. Fores, unpulished dt). Virulence ws scored 5 to 6 dys fter inocultion. A specific isolte ws considered virulent on differentil cultivr when sporulting lesion of more thn 1 cm developed within 6 dys. Nonsporulting lesions of less thn 1 cm were considered virulent. Amivlent interctions were repeted. If ny control interctions were mivlent, tht dy s ssy ws repeted. Pthotype frequencies were compred mong potto production zones using chi-squre sttistic of ssocition etween rows nd columns (pthotypes nd zones). The significnce of the sttistic ws determined in two wys. First, we exmined the level of proility from stndrd chi-squre test (SAS PROC FREQ, relese 6.11; SAS Institute, Inc., Cry, NC). However, this test my not e pproprite when frequencies re low for ny comintion of fctors, nd severl pthotypes in our study were rre or did not occur in ech zone. Therefore, we lso compred the oserved chi-squre sttistic with distriution of chi-squre sttistics generted from 10,000 rndom permuttions of the dt set (18). Pthotype frequencies were lso compred mong zones with Hill s diversity numers, N1 nd N2 (12). N1 ws clculted from the Shnnon index, Hʹ, y the formul N1 = e Hʹ. The Shnnon index ws estimted from the dt s S n i ni Hʹ = ln i = 1 n n in which S = pthotype, n i = the frequency of isoltes with pthotype i, nd n = the totl numer of isoltes in the zone (12). N2 ws clculted from the Simpson index, λ, y the formul N2 = 1/λ. The Simpson index ws estimted from the dt s λ = S ni( ni 1) i 1 n( n 1) in which S = pthotype, n i = the frequency of isoltes with pthotype i, nd n = the totl numer of isoltes in the zone (12). Allozyme mrkers. Isoltes were cultivted in still culture of 10% V8 roth or pe roth (9) for 7 dys t 18 C. Protein ws extrcted from fresh mycelium s descried y Goodwin et l. (6). Buffer systems, gel concentrtions, current specifictions, nd stining procedures for electrophoretic nlyses of Gpi nd Pep were s previously descried (6). Allozyme lleles re reported in migrtion distnces reltive to commonly used control tht hs ritrrily een given vlue of 100. In smll numer of isoltes (seven in totl), the 96/100 genotype for Pep ws not lwys clerly distinguishle from 100/100. However, the DNA fingerprint of every such isolte nlyzed ws identicl to tht of isoltes with 96/100, so these isoltes were ssigned Pep genotype of 96/100. Metlxyl resistnce. Metlxyl resistnce ws ssessed only for the 1993 smple. Assys were done t 18 C in the drk on 10% V8 gr mended with 5 or 100 ppm of metlxyl. Isoltes were clssified s susceptile, intermeditely resistnt, or resistnt sed on rdil colony growth reltive to metlxyl-free controls. To estlish the clssifiction criteri, we first plotted the rdil growth t 5 ppm ginst tht of 100 ppm for ech isolte. We then superimposed oundry lines on the grph tht were sed on the criteri used y Therrien et l. (21): susceptile = rdil growth less thn 40% of control with oth concentrtions; intermedite = greter thn 40% with 5 ppm, ut less thn 40%, with 100 ppm; nd resistnt = greter thn 40% with oth concentrtions. These were then modified slightly to void cses flling on clssifiction orders (Fig. 2). TABLE 1. Loction nd numer of isoltes collected in Ecudor from 1990 to 1993 Production zone 1:50,000 mp No. of isoltes 1990 to 1992 North Sn Griel 58 Centrl-north Pichinch 43 Totl ( ) North (Crchi) Tufino 10 Huc 8 Sn Griel 12 Tulcon4 10 Centrl (Chimorzo) Guero 17 Guno 9 Riom 3 Gunto 2 Gurnd 1 Sicolp 8 South (Loj) Chuquirim 31 Totl (1993) 111 Nme of cnton most closely ssocited with ech mp. The numer of isoltes ssessed from ech cnton ws determined y reltive potto production. Fig. 2. Rdil growth of isoltes with 100 ppm of metlxyl plotted ginst rdil growth with 5 ppm of metlxyl. Vlues re given s proportion rdil growth of metlxyl-free control. Smples from northern, centrl, nd southern Ecudor re represented s oxes, open circles, nd solid circles, respectively. Intermeditely resistnt isoltes re delimited y the lrge ox, resistnt ones re to the right nd ove the ox, nd susceptile ones re to the left nd elow. Vol. 87, No. 4,

4 DNA fingerprinting. Cultures were grown in pe roth t 18 C in drk incutor. After 10 dys, the fungl tissue ws hrvested, frozen t 80 C for severl hours, nd then lyophilized. Lyophilized tissue ws then frozen with liquid nitrogen in mortr nd ground with pestle. DNA ws extrcted from the powdered mycelium s previously descried (9). Gel electrophoresis, hyridiztion with 32 P rndom-primed proe RG57 (8), nd utordiogrphy were ll performed ccording to stndrd techniques (16). RESULTS 1990 to 1992 smple. The erly smple ws chrcterized y predominnce of one dilocus llozyme genotype representing 96 of the 101 isoltes. Forty-nine isoltes with the predominnt llozyme genotype were nlyzed for RFLP fingerprint, nd ll ut one were identicl, the vrint differing t only one locus (Tle 2). We hve nmed the predominnt genotype EC-1, nd it is defined y the Gpi nd Pep vlues of 90/100 nd 96/100, respectively, nd n RFLP nding pttern differing from US-1 y three nds (Tle 2). The one isolte differing y one RFLP nd is ssumed to hve risen within the EC-1 linege y muttion or mitotic recomintion. Five isoltes hd the llozyme genotype of US-1. Three of these were nlyzed for RFLP fingerprint, nd ll were identicl to the pulished US-1 fingerprint (7) smple. The EC-1 dilocus llozyme genotype ws even more predominnt in the second smple, representing 110 of the 111 isoltes tested. A suset of 17 of these ws nlyzed for RFLP fingerprint, nd ll ut one were EC-1. The one vrint gin differed t only one locus, ut different one thn tht found in the TABLE 2. Dilocus llozyme genotypes of glucose-6-phosphte isomerse (Gpi) nd peptidse (Pep) lleles, nd restriction frgment length polymorphism (RFLP) fingerprints of isoltes of Phytophthor infestns collected in Ecudor Dilocus llozyme genotype RFLP fingerprint Smple Numer Gpi Pep Numer Bnds 1 to 25 Linege 1990 to /100 92/ US /100 96/ EC /100 96/ EC /100 92/100 NT c US /100 96/ EC /100 96/ EC-1 Clonl lineges were determined y DNA fingerprint nd llozyme genotype. Differs from predominnt EC-1 genotype y one locus (underlined). c Not tested. TABLE 3. Pthotypes of Phytophthor infestns collected in three production zones in Ecudor in PHYTOPATHOLOGY Crchi Chimorzo Loj Totl Pthotype No. % No. % No. % No. % R1,2,3,4,7,8,10, R1,3,4,7,10, R1,3,4,7,8,10, R3,4,7,8,10, R1,2,3,4,7,10, R3,4,7,10, R1,2,3,4,6,7,8,10, R1,3,7,10, R2,3,4,7,10, R2,3,4,7,8,10, R2,4,7,10, R3,4,7, R3,7, R1,2,3,4,5,6,7,8,9,10, R1,2,3,4,7, R1,3,4,10, R1,3,4,7,8, R1,3,4,8,10, R1,3, R3,4,5,7,8,10, R12,3,4,6,7,8,9,10, R1,3,4, R1,3,4,7, r (no virulence) Totl c Numer of pthotypes Hill s N1 d Hill s N2 e The chi-squre sttistic for dependence of pthotypes nd zones ws 64.3 (46 degrees of freedom), which ws significnt t P = (SAS PROC FREQ, relese 6.11; SAS Institute, Inc., Cry, NC), nd t P < when compred with distriution of chi-squre sttistics generted from 10,000 rndom permuttions of the dt. Numers represent known R genes overcome y tht pthogen phenotype. c Dt were inconclusive for three of the 111 isoltes. d Hill s N1 is n indiction of the numer of undnt pthotypes. It is clculted s N1 = e Hʹ, in which Hʹ is the Shnnon index of diversity (12). e Hill s N2 is n indiction of the numer of very undnt pthotypes (12). It is clculted s N2 = 1/λ, in which λ is the Simpson index of diversity (12).

5 erly smple (Tle 2). Only one US-1 dilocus llozyme genotype ws found in It ws not nlyzed for RFLP fingerprint. Most isoltes in the 1993 smple were virulent on six or more R genes. The specific pthotypic structure differed significntly mong the three production zones sed on chi-squre nlysis (Tle 3). In spite of this, the three most common pthotypes, hving eight, seven, nd eight virulence fctors, respectively, represented 62.9% of the smple nd were found in reltively high frequency in ll three production zones. Numerous other pthotypes were unique to one of the zones, ut these were only found once or in low frequencies. One isolte ws virulent on ll R genes, nd this ws the only US-1 isolte found in the 1993 smple. Pthotype diversity ws gretest in the northern zone (Crchi), intermedite in the centrl zone, nd lowest in the southern zone (Tle 3). Hill s diversity numer N1, which is n estimte of the numer of undnt pthotypes (12), ws twice s lrge in the north (N1 = 10.25) s in the south (N1 = 4.71). The diversity numer N2, which is mesure of very undnt pthotypes (12), indicted pttern of generlly liner decrese in diversity from north to south. Approximtely one-third (34%) of ll isoltes from 1993 were highly resistnt to metlxyl (Tle 4 nd Fig. 2). The frequency of resistnce ws similr in the north (Crchi) nd south (Loj) of Ecudor, ut lower in the centrl prt (Chimorzo), which ws the only zone in which intermeditely resistnt isoltes were not rre (20%). This disprity resulted in highly significnt (P = 0.002) chi-squre test for homogeneity of metlxyl resistnce mong production zones (Tle 4). All isoltes from oth smples were A1 mting type. DISCUSSION The A2 mting type hs not een found mong isoltes of P. infestns tken from cultivted potto in Ecudor, so clonl popultions re to e expected. Our survey provides strong evidence tht this popultion is predominntly mde up of one linege, which we hve nmed EC-1 in ccordnce with the nomenclture estlished y Goodwin et l. (7). Therefore, we reject the hypothesis tht frontier exists within Ecudor etween the Peruvin US-1 popultion smpled in the 1980s (7,22) nd distinct popultion tht we hd susequently detected in Colomi. Rther, the potto production zones in ll prts of Ecudor re chrcterized y lmost complete predominnce of EC-1. In the strtified smple of 1993, ll ut one isolte were EC-1. Within EC-1, there ws some vrition for neutrl mrkers. For exmple, one isolte in ech smple vried from the predominnt EC-1 genotype y one RFLP nd (Tle 2). Goodwin et l. (7) found single locus devition of US-1 in the Philippines, which they designted s US-1.1. The popultion in the Philippines is lso sexul, nd the single-locus chnge ws ssumed to e the result of muttion or mitotic recomintion. Similrly, in our study, the vrints were identicl to EC-1 for mting type nd dilocus llozyme genotype, so we ssume they rose within the EC-1 linege either y muttion or mitotic recomintion. The frequency of the vrints ws low (one in 17 in the strtified smple), nd we do not elieve it is pproprite or necessry, t this point, to nme specific genotypes within the EC-1 linege. Although our study did not indicte geogrphicl sustructuring in the popultion of P. infestns in Ecudor for neutrl mrkers, there ws difference in metlxyl resistnce mong the production zones nd less noticele, ut sttisticlly significnt, difference mong zones for specific virulence. Differences in fungicide-use ptterns my explin the regionlity of the fungicide resistnce dt, ut we hve no informtion on fungicide usge in different prts of Ecudor. Fry et l. (4) found tht levels of metlxyl resistnce were higher in popultions of P. infestns coming from production fields in the Netherlnds, where metlxyl ws frequently used, thn in popultions coming from home grdens were metlxyl ws seldom used. The diversity of virulence pthotype mong isoltes nd mong regions is not immeditely ttriutle to ny specific fctor(s). A sttisticl test indicted tht pthotype structure ws ssocited with production zones, nd diversity mesures indicted strong north-south trend. It is not cler why this pttern should exist, ut it could e n indiction tht EC-1 hs migrted within Ecudor from north to south. The southern popultion of EC-1 could represent suset of wht is in the north. Overll, the level of diversity mong pthotypes within EC-1 is similr to the diversity within clonl lineges recently reported from the United Sttes (10) nd from Polnd (20). Selection y R gene-contining pottoes (10) in Ecudor is proly not the reson for the presence of complex pthotypes in EC-1. Studies done recently t the CIP experiment sttion in Quito indicte tht most Ecudorin cultivrs re either free of R genes or contin R genes 1 nd 3 (P. J. Oyrzun nd G. A. Fores, unpulished dt). Furthermore, cultivrs grown in the south of Ecudor re free of known R genes, nd isoltes we collected there in 1993 were lso highly virulent. Excess virulence in EC-1 ws proly imported with the linege nd hs remined unused. Thus, the sitution in Ecudor prllels tht in the Netherlnds in tht the level of specific virulence incresed drmticlly fter the detection of immigrtion (4). Other workers hve lso found high levels of virulence in popultions of P. infestns tht cn not e explined y the R gene constitution of the potto cultivrs eing grown (14). These oservtions lend support to the rgument tht stilizing selection, i.e., selection ginst the ccumultion of unnecessry virulence (24), is not importnt in the P. infestnspotto pthosystem (23). Dt from this study led us to suggest new hypothesis concerning the genetic structure of P. infestns in Ecudor: EC-1 is recently introduced clonl linege tht hs displced US-1 on pottoes. This hypothesis is consistent with the conclusion of Goodwin et l. (7) tht most continents were dominted y clonl popultions of US-1 until recently. Erly dt from Peru re consistent with tht conclusion; collections from the erly 1980s identified only US-1 from Peru (22). In ddition to the dt from nlysis of neutrl mrkers, the pthotypic dt re lso consistent with our new hypothesis. In studies with isoltes collected from pottoes in Ecudor in the 1970s (1) nd from pottoes in Peru in the 1980s (22), highly complex pthotypes were not found, nd most isoltes were virulent on pottoes with R genes. In contrst, in our study with isoltes collected in the erly 1990s, we found most isoltes to e highly complex. Thus, the current popultion of P. infestns in Ecudor seems very different from the popultion tht existed in the 1970s. Also consistent with our hypothesis is our finding tht isoltes of EC-1 were highly complex, ut tht the only completely virulent isolte detected in our study ws n isolte of US-1. ACKNOWLEDGMENTS This reserch ws supported, in prt, y grnt HRN-5600-G in the progrm in Science nd Technology Coopertion, Office of the TABLE 4. Numers of isoltes of Phytophthor infestns collected in three production zones of Ecudor tht were either resistnt, modertely resistnt, or sensitive to metlxyl Level of resistnce Production zones Resistnt Intermedite Sensitive Totl Crchi Chimorzo Loj Totl Chi-squre nlysis of ssocition etween zones nd resistnce clsses ws highly significnt: χ 2 = , P = 0.002, nd degrees of freedom = 4. One isolte could not e ssessed. Vol. 87, No. 4,

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