Resistnce to Mefenoxm nd Metlxyl Among Field Isoltes of Phytophthor cpsici Cusing Phytophthor Blight of Bell Pepper Gregory Prr nd Jen Begle Ristino, Deprtment of Plnt Pthology, North Crolin Stte University, Rleigh 27695 ABSTRACT Prr, G., nd Ristino, J. B. 2001. Resistnce to mefenoxm nd metlxyl mong field isoltes of Phytophthor cpsici cusing Phytophthor blight of bell pepper. Plnt Dis. 85:1069-1075. Incidence of Phytophthor blight in bell pepper fields tht were spryed for the first time with Ridomil Gold (mefenoxm) ccording to lbeled recommendtions ws higher in North Crolin in 1997 thn in previous yers. Mefenoxm is the more ctive enntiomer contined in the rcemic fungicide metlxyl. A totl of 150 isoltes were obtined from 17 fields t eight grower loctions. Among isoltes from ll loctions, 30% were clssified s sensitive, 10% s intermedite, nd 59% were resistnt to mefenoxm. Mefenoxm-resistnt isoltes were found in 82% of the fields smpled (14 of 17 fields). The proportion of resistnt isoltes in individul fields rnged from 28 to 100%. The men effective concentrtion (EC 50 ) vlues for mefenoxmsensitive isoltes ws 0.568 µg ml 1 (rnging from 0.12 to 1.1 µg ml 1 ), wheres the men EC 50 vlue for mefenoxm-resistnt isoltes ws 366.5 µg ml 1 (rnging from 3 to 863 µg ml 1 ). The men EC 50 vlue for metlxyl-sensitive isoltes ws 0.27 µg ml 1 (rnging from 0.00002 to 1.3 µg ml 1 ) nd for metlxyl-resistnt isoltes ws 470.34 µg ml 1 (rnging from 10 to 966 µg ml 1 ). The gretest proportion of resistnt isoltes cme from fields where mefenoxm ws used lone rther thn in combintion with other fungicides. Both mting types were found mong resistnt isoltes, suggesting tht these isoltes my persist in soil in subsequent yers. Field isoltes of Phytophthor cpsici resistnt to mefenoxm on pepper hve not been reported previously nd now pose new chllenges for mngement of this importnt disese. Additionl keywords: epidemiology, fungicide resistnce, fungicide sensitivity, Phytophthor root nd crown rot Phytophthor blight, cused by the oomycete plnt pthogen Phytophthor cpsici, is n importnt disese of bell pepper nd cucurbit crops nd cuses significnt economic loss. The incidence of the disese hs incresed in recent yers in the United Sttes nd worldwide (19,35). P. cpsici cn infect ll prts of the plnt nd the pthogen cn be dispersed within the soil, with surfce wter, nd vi wter splshing from the soil to folige (36). The pthogen hs both sexul nd sexul life cycle, thus mking disese mngement difficult. Disese is often sptilly ggregted in fields nd initil disese foci cn quickly initite new foci vi pthogen spred in surfce wter (36). Mngement of P. cpsici currently relies on modifictions in culturl prctices, crop rottion, Corresponding uthor: J. B. Ristino E-mil: Jen_Ristino@ncsu.edu Funding for the reserch ws provided in prt by the North Crolin Vegetble Growers Assocition, the North Crolin Agriculturl Reserch Service, nd Syngent Chemicl Corportion. Accepted for publiction 14 June 2001. Publiction no. D-2001-0802-01R 2001 The Americn Phytopthologicl Society nd judicious use of selective fungicides (35). The phenyllmide fungicide metlxyl (trde nme: Ridomil; Syngent, Greensboro, NC) provides systemic protection ginst oomycete pthogens (7,8,40). Metlxyl ws introduced in 1977 nd provided excellent control of Phytophthor diseses (40). Its benefits included systemic ctivity, which enbled growers to extend spry intervls when used lone in disese mngement progrms (38). Metlxyl hs been used extensively for control of mny different oomycete pthogens, including P. infestns, Peronospor tbcin, nd Bremi lctuce (31). The intensive use of metlxyl led to the rpid selection for metlxyl-resistnt strins of Phytophthor infestns in Europe within 1 yer of its introduction (10,13,38). This ws primrily due to the use of metlxyl s curtive on lrge popultions of P. infestns (30). In the United Sttes, isoltes of P. infestns resistnt to metlxyl were first reported in Wshington Stte in 1991 (11,12). Isoltes of P. infestns resistnt to metlxyl lso hve been recovered from nontreted fields, demonstrting the importnce of migrtion of resistnt isoltes of P. infestns in disese spred (6,16,18,23). Metlxyl resistnce hs been reported mong mny oomycete pthogens, including Plsmopr viticol, Pseudoperonospor cubensis, Peronospor tbcin, B. lctuce, nd Pythium spp. (31). As result, metlxyl ws removed from some mrkets until new mngement strtegies were deployed, including its use in combintion with other fungicides (38). Metlxyl historiclly hs been used to control the soil nd crown (stem) rot phse of Phytophthor blight on bell pepper (20,21,28,32,36,39). In the erly 1990s, metlxyl received section 3 Federl registrtion for the control of Phytophthor blight of peppers in the United Sttes. Recently, metlxyl ws replced with mefenoxm (trde nme: Ridomil Gold; Syngent), the ctive enntiomer contined in the rcemic fungicide metlxyl. The fungicide is pplied t frequencies similr to those used with metlxyl, but t lower rtes. In 1997, mefenoxm ws widely used for the first time in field production of bell peppers in North Crolin. Resistnce of isoltes of Phytophthor cpsici to metlxyl hs been induced in lbortory studies by chemicl mutgenesis, ultrviolet irrdition, or exposure to sublethl concentrtions of metlxyl (1,3 5). Metlxyl-resistnt isoltes of P. cpsici tht were cross-resistnt in in vitro studies to other phenylmide fungicides lso hve been observed. However, crossresistnce hs not been observed in this pthogen to different clsses of fungicides (1,3). Adptive resistnce of isoltes of P. cpsici to metlxyl ws demonstrted in vitro fter successive trnsfer on metlxyl-mended medi, but some isoltes lost their insensitivity fter successive trnsfer on medi mended with sublethl concentrtions of metlxyl (4). It ws suggested tht sublethl exposure of propgules to the compound in soil might occur. Bsed on these studies nd the documented cses of field resistnce to metlxyl in other oomycetes, it ws predicted tht field resistnce to metlxyl by P. cpsici ws likely (5). Despite results from lbortory studies, development of fungicide resistnce in P. cpsici, which hs n importnt soilborne phse, hs been slower to develop thn in species of Phytophthor tht re primrily dispersed erilly nd infect folige. Production of peppers in the Southest cn require frequent fungicide pplictions to ensure qulity hrvest nd, therefore, gretly increse the risk of resistnce development. In the spring nd summer of Plnt Disese / October 2001 1069
1997, n lrming increse in Phytophthor blight occurred in North Crolin bell pepper fields tht were spryed with mefenoxm. Mny of the growers whose fields were ffected hd pplied mefenoxm for the first time in 1997 for the mngement of Phytophthor blight. High disese severity in fungicide-treted fields indicted the possibility of mefenoxm resistnce in field popultions of P. cpsici in North Crolin nd elsewhere (25,26,29). The objectives of this reserch were to collect isoltes of P. cpsici from fields where disese control filures hd occurred in North Crolin nd New Jersey nd to determine the in vitro sensitivity of these isoltes to mefenoxm. In ddition, the mting type nd pthogenicity of the isoltes were exmined. Evlution of the metlxyl sensitivity of isoltes of P. cpsici collected in the sme pepper-growing region of North Crolin prior to 1997 ws lso conducted. A preliminry report of this reserch hs been published (33). MATERIALS AND METHODS Culture collection nd mintennce. Plnts infected with P. cpsici were collected from bell pepper fields t 13 loctions mong seven frms in North Crolin (Tble 1). In ddition, Stephen Johnston (Rutgers University, Bridgeton, NJ) provided disesed plnts from four dditionl fields from one grower s frm in New Jersey. Initilly, isoltions from fields in North Crolin were from smll number of rndom smples submitted by county extension gents nd university extension fculty. Dt from our preliminry screen indicted tht severl isoltes of P. cpsici were highly resistnt to metlxyl (Ridomil 2E, 240 mg.i./ml). Subsequently, six of the North Crolin fields were smpled more extensively, nd 50 or more plnts were collected from ech site (Tble 1). Dt from the combined collections re shown in Tble 1. Sections of plnt stem tissue with visible blck lesions were surfce disinfested in 0.05% NOCl solution for 1 min, rinsed three times in sterile distilled wter, nd plted onto Knnwisher Mitchell gr mended with 50 µg ml 1 hymexzol (24). Petri dishes were incubted for 5 to 7 dys t 24 C in the drk nd colonies with growth chrcteristic of P. cpsici were trnsferred to clrified V8 juice gr (200 ml of V8 clrified juice, 800 ml of deionized wter, nd 17 g of gr). V8 juice ws clrified by filtrtion through Whtmn number 4 filter pper fter the ddition of 2 g of CCO 3, followed by centrifugtion t 4,340 g for 10 min. The pthogen ws identified bsed on colony morphology nd sporngil chrcteristics. Polymerse chin rection identifiction of isoltes. A subset of the isoltes ws tested by polymerse chin rection (PCR) to confirm identifiction s P. cpsici. DNA ws extrcted from mycelium of the isoltes by NOH method (42). DNA ws lso extrcted from 16 previously identified isoltes of P. cpsici in our collection for comprison (37). DNA ws mplified vi PCR utilizing the P. cpsici specific primer, PCAP. The PCAP primer, in combintion with the universl primer ITS1, mplifies n pproximtely 172-bp frgment of ribosoml DNA from isoltes of P. cpsici (37). PCR products were seprted by gel electrophoresis on 2% grose gels. Product size ws determined by comprison with moleculr size stndrd included in ech gel. Fungicide sensitivity nd EC 50 ssys. Thirty-five isoltes of P. cpsici collected prior to 1997 were ssyed for sensitivity to metlxyl. These isoltes were collected from pepper, tomto, pumpkin, eggplnt, cucumber, nd sqush (Tble 2). Stock cultures from the isoltes were grown on V8 juice gr for 10 dys in mbient light. Agr disks (6 mm in dimeter) were removed from ctively growing mrgins of the cultures nd trnsferred to clrified V8 juice gr medi previously mended with metlxyl (Ridomil 2E, 240 mg.i. ml 1 ) t 0, 0.1, 1.0, 5.0, 10.0, or 100 µg ml 1. The fungicide (emulsifible concentrte) ws diluted in sterile wter before dding it to the utoclved medi. Three replicte petri dishes per fungicide level were tested for ech isolte. Dishes were incubted t 24 C for 7 to 10 dys in constnt light. Colony dimeters of P. cpsici were mesured in two directions for ech individul dish, verged, nd compred to verge colony dimeters from non-mended medi. The percent growth of ech isolte reltive to the nonmended control ws plotted ginst the log 10 of the metlxyl concentrtion. The effective concentrtion (EC 50 ) for ech isolte ws clculted. Anlysis involved fitting regression lines for isolte rdil growth vlues expressed s percentge of the nonmended controls plotted ginst log 10 of the metlxyl concentrtion for ech isolte. The point on the regression line t which 50% of the isolte growth ws inhibited is the ED 50 vlue. Sttisticl nlysis ws conducted with the Sttisticl Anlysis System (SAS Institute, Inc., Cry NC) s described below. Tble 1. Sensitivity of isoltes of Phytophthor cpsici collected in 1997 from bell pepper fields in North Crolin nd New Jersey to the fungicide mefenoxm Number collected Number of isoltes Frm, county or stte Field Plnts Isoltes Sensitive Intermedite Resistnt Resistnt (%) Fungicide b 1, Wilson, NC 1 50 28 12 6 10 36 RG 2, Smpson, NC 1 62 13 4 2 7 53 R2E, M/Cu 2 54 8 3 2 3 38 RG 3 62 20 6 2 12 60 MBR, RG 4 7 4 2 0 2 50 RG 3, Smpson, NC 1 60 26 5 0 21 81 RG 2 57 15 1 0 14 93 RG 4, Smpson, NC 1 7 2 2 0 0 0 Chl, R2E 2 8 5 3 0 2 40 RG/Cu 3 1 1 1 0 0 0 RG 5, Smpson, NC 1 1 1 0 0 1 100 RG 6, Greene, NC 1 8 7 4 1 2 29 RG 3 7, Smpson, NC 1 1 1 1 0 0 0 No RG 8, New Jersey 1 3 3 0 0 3 100 RG 2 1 1 0 0 1 100 RG 3 5 5 0 1 4 80 RG 4 10 10 2 0 8 80 RG Totl 17 397 150 46 14 90 Isoltes were chrcterized s sensitive to mefenoxm if colony growth t 5 µg ml 1 ws less thn 40% of the isolte s growth on the nonmended medi. Intermedite isoltes exhibited growth greter thn 40% of the nonmended medi control t 5 µg ml 1, but less thn 40% of the nonmended medi control with mefenoxm t 100 µg ml 1. Resistnt isoltes exhibited growth greter thn 40% of the nonmended medi control with mefenoxm t 100 µg ml 1. b Use history during seson: RG = Ridomil Gold (mefenoxm), R2E = Ridomil 2E (metlxyl), M/Cu = Mneb/copper, MBR = methyl bromide/chloropicrin), Chl = Chloropicrin. 1070 Plnt Disese / Vol. 85 No. 10
In ll, 150 isoltes collected during the summer of 1997 were evluted for resistnce to mefenoxm (Tble 1). Isoltes were grown on clrified V8 juice gr for 10 dys t 24 C in mbient light prior to use. Clrified V8 juice gr ws mended with mefenoxm (Ridomil Gold EC, 480 mg.i. ml 1 ) t 0, 5, nd 100 µg ml 1 (11). Solutions of the fungicide were prepred in sterile wter prior to mendment of the gr medi. Agr disks from ech isolte were trnsferred onto two replicte petri dishes of medi mended with mefenoxm t ech of the three fungicide concentrtions. The dishes were incubted t 24 C in mbient light for 7 to 10 dys. Colony growth ws determined by mesuring two colony dimeters for ech replicte dish nd clculting the men dimeter for ech isolte. The experiment ws repeted two times for ech isolte. Isoltes were chrcterized s sensitive if colony growth on medi mended with 5 µg ml 1 of metlxyl or mefenoxm ws less thn 40% of the isoltes growth on nonmended medi. Intermedite isoltes exhibited growth on medi mended with 5 µg ml 1 greter thn 40% of tht on nonmended medi, but growth on medi mended with 100 µg ml 1 less thn 40% of tht on nonmended medi. Resistnt iso- Fig 1. Frequency of mefenoxm resistnce in isoltes of Phytophthor cpsici collected in 1997 in 13 loctions in North Crolin nd four loctions in New Jersey. Isoltes were grouped ccording to percentge of growth on mefenoxm-mended medi t 100 µg ml 1 reltive to nonmended control. Isoltes with intermedite resistnce hd growth with mefenoxm t 100 µg/ml less thn 40% of tht on nonmended medi. Resistnt isoltes hd growth with mefenoxm t 100 µg/ml greter thn 40% of tht on nonmended medi. Tble 2. Isolte designtion, county, stte, host, yer of isoltion, mting type nd effective concentrtion (EC 50 ) vlue on metlxyl-mended medi of isoltes of Phytophthor cpsici collected from pepper, cucumber, sqush, pumpkin, eggplnt, nd tomto between 1987 nd 1996 EC 50 (µg ml 1 ) Isolte number, nme County, stte Host Yer isolted Mting type Men Rnge 18 Fruit Smpson, NC Pepper 1989 A1 0.78 0.54 1.39 19 Wr 1 Smpson, NC Pepper 1989 A1 1.34 1.11 1.7 20 Wr 2 Smpson, NC Pepper 1989 A1 0.17 0.17 0.17 21 RT 403#1 Smpson, NC Pepper 1989 A1 0.60 0.53 0.7 22 RT 403 #2 Smpson, NC Pepper 1989 A1 1.20 0.86 1.96 23 B&W #1 Smpson, NC Pepper 1989 A1 0.97 0.89 1.06 25 King-1 Smpson, NC Pepper 1990 A1 0.67 0.53 0.94 26 King-2 Smpson, NC Pepper 1990 A1 9.15 7.62 11.45 27 King-3 Smpson, NC Pepper 1990 A1 0.88 NV 29 D. Wilson-1 Smpson, NC Pepper 1990 A1 1.32 1.15 1.53 30 Ruff 6-6 Polk, NC Pepper 1991 A1 2.09 1.36 4.48 31 Ruff 2-15 Polk, NC Pepper 1991 A1 0.74 0.58 1.04 32 Ruff 7-1 Polk, NC Pepper 1991 A1 1.19 0.066 5.62 33 Ruff 5-4 Polk, NC Pepper 1991 A1 0.73 0.65 0.83 34 1CA Cliforni Tomto 1986 A2 0.77 0.74 0.81 35 Tom2 Henderson, NC Tomto 1990 A2 0.39 0.32 0.51 36 Tom3 Henderson, NC Tomto 1990 A2 2.94 2.18 4.51 37 Tom4 Henderson, NC Tomto 1990 A1 0.19 NV 38 Tom5 Henderson, NC Tomto 1990 A2 1.95 1.71 2.27 39 Pump1 Cliforni Pumpkin 1986 A2 0.17 NV 40 Pump2 Wke, NC Pumpkin 1990 A2 0.76 0.55 1.22 41 Pump3 Wke, NC Pumpkin 1990 A2 0.67 0.55 0.87 42 Flim New Jersey Eggplnt 1988 A1 2.58 2.04 3.51 52 Acorn2 Smpson, NC Sqush 1988 A2 0.48 0.41 0.58 55 But2 Smpson, NC Sqush 1988 A1 2.66 2.17 3.45 57 Ptti4 Smpson, NC Sqush 1988 A1 0.89 0.78 1.04 59 Spg2 Smpson, NC Sqush 1988 A1 0.53 0.51 0.55 62 Ginseng Henderson, NC Ginseng 1991 A2 0.33 0.32 0.34 82 B92-1 Smpson, NC Pepper 1982 A1 2.51 2.06 3.23 83 B92-2 Smpson, NC Pepper 1992 A2 0.68 0.54 0.89 84 K92-1 Smpson, NC Pepper 1992 A1 7.81 6.96 8.88 85 K92-2 Smpson, NC Pepper 1992 A1 0.73 0.6 0.96 87 B1HB14 Smpson, NC Pepper 1993 A1 0.70 0.47 1.43 88 B2HH4 Smpson, NC Pepper 1993 A2 0.53 0.49 0.59 98 CL5-Phy2 Smpson, NC Cucumber 1994 A2 2.60 2.06 3.54 NV = no vrition, vlues were the sme between reps. Plnt Disese / October 2001 1071
ltes exhibited growth on medi mended with 100 µg ml 1 greter thn 40% of tht on nonmended medi (16). The EC 50 vlues for sensitivity to mefenoxm were estimted for six mefenoxm-resistnt (isoltes numbers 317, 363, 414, 428, 399, nd 391) nd six mefenoxm-sensitive (isolte numbers 358, 299, 376, 297, 378, nd 427) isoltes of P. cpsici. Clrified V8 juice gr ws mended with mefenoxm t levels of 0, 0.1, 1.0, 10, 100, nd 1,000 µg ml 1. Agr disks from ech of the 12 isoltes of P. cpsici were plced onto two replicte pltes of the mefenoxm-mended medi. The pltes were incubted in mbient light t 24 C for 7 to 10 dys. Colony growth ws determined by mesurement of colony dimeters in two directions for ech replicte petri dish. The experiment ws done three times for ech isolte. Percent growth ws determined reltive to the nonmended control for ech isolte. The percent growth of ech isolte compred to the nonmended control ws plotted ginst log 10 concentrtion of mefenoxm. EC 50 vlues were clculted for ech isolte. The experiment ws repeted substituting metlxyl (Ridomil 2E) for mefenoxm nd the EC 50 vlues for the sme 12 isoltes were determined. EC 50 vlues were clculted for ech isolte by nonliner regression using the Sttisticl Anlysis System Softwre (SAS Institute, Inc.). The log-logistic model (Pr (y > c) = 1/{1 + (λ*c)**p}, where EC 50 = 1/lmbd, provided the best fit of the dt for mefenoxm- nd metlxyl-sensitive isoltes. The Weibull distribution function (Pr (y > c) = exp { 1 (λ*c)**p}, where EC 50 = [(0.6931)**(1/p)]/λ), provided the best fit of the dt for mefenoxm- nd metlxyl-resistnt isoltes. Mting type. Mting type ws determined for subset of isoltes from ech field by piring ech isolte of P. cpsici with known A1 (isolte B1BH14) or A2 (isolte B2HH4) tester isolte. All crosses were conducted by Fig 2. Growth with mefenoxm t 100 µg/ml expressed s percentge of tht on nonmended medi versus log 10 mefenoxm concentrtion of A, six sensitive nd B, six resistnt isoltes of Phytophthor cpsici collected in 1997. Isoltes were grown on clrified V8 juice gr mended with mefenoxm t 0, 0.1, 1.0, 10, 100, or 1,000 µg/ml. Ech line represents different isolte Tble 3. Men effective concentrtion (EC 50 ) vlues of six isoltes of Phytophthor cpsici tht were sensitive or resistnt to mefenoxm nd metlxyl 1072 Plnt Disese / Vol. 85 No. 10 Mefenoxm (µg/ml) Metlxyl (µg/ml) Isolte number Isolte nme Mting type Men EC 50 Rnge EC 50 Men EC 50 Rnge EC b 50 Resistnt 414 SH A A1 512 170 840 723.07 551 1050 391 2950 A2 3 0.4 19 10.16 4.18 23.54 317 NJ SE A2 863 NV b 877.94 NV 399 SH(W)A A1 7 0.2 100 87.34 10.23 170.30 363 TriW2A A2 787 400 920 966 NV 428 JH(C)E ND c 27 0.5 >1,000 157.53 100.9 352.3 Sensitive 427 JH(C)G ND 0.12 0.02 0.29 0.0008 0 299 HAM A2 1.00 0.4 2.5 0.30 0.13 1.08 378 JH(C)D A2 0.12 0.07 0.19 0.003 0 376 SH(HS)J A2 0.94 0.6 1.5 0.0003 0 297 HAM A2 1.10 0.15 4.1 1.33 0.82 3.53 358 SH(HS)F A1 0.13 0.08 0.2 0.00002 0 Isoltes were previously chrcterized s sensitive or resistnt to mefenoxm on mefenoxm t 0, 5, nd 100 µg/ml. b NV = no vrition observed. c ND = not described.
plcing gr disks removed from 1-week-old cultures of ech isolte on clrified V8 juice gr nd incubting them for 21 to 28 dys in the drk t 24 C. In ech test, the bsence of oospores t the interfce between colonies indicted the sme mting type. The positive control ws cross between two tester isoltes of opposite mting type, wheres the negtive control ws cross between isoltes of the sme mting type. RESULTS PCR identifiction of isoltes. Isoltes originlly identified s P. cpsici through the use of selective medi, colony, nd sporngil morphology were confirmed s P. cpsici through the use of PCR nd the PCAP primer. All of the isoltes tested with the PCR technique yielded the expected 172-bp product. Fungicide sensitivity nd EC 50 vlues for isoltes collected between 1987 nd 1994. No isoltes of P. cpsici collected between 1987 nd 1994 were resistnt to metlxyl (Tble 2). In ll, 23 isoltes hd EC 50 vlues between 0.1 nd 1.0 µg ml 1, 10 isoltes hd EC 50 vlues between 1.0 nd 5.0 µg ml 1, nd 2 isoltes hd EC 50 vlues between 5 nd 10 µg ml 1 (Tble 2). These two isoltes were clssified s intermedite in sensitivity to metlxyl nd cme from the sme grower field in different yers with history of metlxyl use. Fungicide sensitivity nd EC 50 vlues for isoltes collected in 1997. Among 150 isoltes of P. cpsici collected from bell pepper fields in 1997, 30% were clssified s sensitive, 10% s intermedite, nd 59% s resistnt to mefenoxm (Tble 1). Mefenoxmresistnt isoltes were detected in 82% of the fields smpled (14 of 17 fields) nd the percentge of resistnt isoltes mong individul fields rnged from 29 to 100%. Isoltes tht were sensitive to mefenoxm were lso found in most fields smpled, nd the percentge of sensitive isoltes mong individul fields ws 7 to 100% (Tble 1). The lrgest frequency of isoltes exhibited growth greter thn 90% of the nonmended control on mefenoxm t 100 µg ml 1. (Fig. 1). Sixty-five isoltes were highly resistnt to mefenoxm nd exhibited enhnced growth of 90% or more on mefenoxm-mended medi when compred to growth on the nonmended control. Twenty percent of the isoltes exhibited enhnced growth of 100% or greter on mefenoxm t 100 µg ml 1 (Fig 1). More plnt smples were removed from three of the grower s frms where disese incidence ws highest (growers 1, 2, nd 3; Tble 1). Growers 1, 3, nd 8 pplied mefenoxm to their fields during the seson for mngement of Phytophthor spp., wheres growers 2 nd 4 pplied preplnt soil fumignt of methyl bromide/ chloropicrin or chloropicrin lone, followed by combintion of different fungicides, including metlxyl (Ridomil 2E), mneb/copper, metlxyl/copper (Ridomil 2E/copper), or mefenoxm (Ridomil Gold) during the seson (Tble 1). Highest percentges of mefenoxm-resistnt isoltes (>80%) cme from fields of growers 3 nd 8, where only mefenoxm ws pplied (Tble 1). However, in other fields where mefenoxm ws used lone (growers 1 nd 6), lower percentges of mefenoxmresistnt isoltes were found. A lower percentge of mefenoxm-resistnt isoltes (38 to 60%) ws found in fields of grower 2, where mixtures of fungicides were used during the seson (Tble 1). The men EC 50 vlue for subset of six sensitive isoltes collected in 1997 ws 0.568 µg ml 1 nd EC 50 vlues rnged from 0.12 to 1.1 µg ml 1 (Tble 3). The men EC 50 vlue for mefenoxm-resistnt isoltes ws 366.5 µg ml 1 nd EC 50 vlues rnged from 3 to 863 µg ml 1 (Tble 3). The reltive sensitivities to mefenoxm of six sensitive nd six resistnt isoltes re shown in Figure 2. Some of the mefenoxm-resistnt isoltes exhibited enhnced growth on medi mended with mefenoxm t 100 µg ml 1 (Fig. 2B), while sensitive isoltes hd reduced growth on medi with mefenoxm t 100 µg ml 1 ; Fig. 2A). The reltive sensitivities of six sensitive nd six resistnt isoltes of P. cpsici to metlxyl re shown in Figure 3. The men EC 50 vlue for the sensitive isoltes ws 0.27 µg ml 1 nd EC 50 vlues rnged from.00002 to 1.33 µg ml 1 (Tble 3). The men Fig 3. Growth with metlxyl t 100 µg/ml expressed s percentge of tht on nonmended medi versus metlxyl concentrtion of A, six sensitive nd B, six resistnt isoltes of Phytophthor cpsici collected in 1997. Isoltes were grown on clrified V8 juice gr mended with metlxyl t 0, 0.1, 1.0, 10, 100, or 1,000 µg ml 1. Ech line represents different isolte. Plnt Disese / October 2001 1073
EC 50 vlue for resistnt isoltes ws 470.34 µg ml 1 nd EC 50 vlues rnged from 10 to 966 µg ml 1 (Tble 3). Mting type determintion. A1 nd A2 mting types were recovered from most of the fields tht were smpled in North Crolin, s well s the fields in New Jersey (Tble 4). In nine fields locted on four grower frms, both A1 nd A2 mting types were found mong mefenoxm-resistnt isoltes. DISCUSSION Isoltes of P. cpsici collected between 1987 nd 1994 from rnge of vegetble crops in North Crolin fields were highly sensitive to metlxyl. Our dt show tht isoltes of P. cpsici collected in fields in 1997 in North Crolin nd New Jersey hve developed resistnce to the fungicide mefenoxm. Mefenoxm-resistnt isoltes were widespred in North Crolin fields nd occurred in 10 of 13 fields smpled. Metlxyl-resistnt isoltes were initilly discovered in 1997 vi routine screening for metlxyl sensitivity of isoltes obtined from the Plnt Disese nd Insect Clinic t North Crolin Stte University. Isoltes tht were resistnt to metlxyl were lso highly resistnt to mefenoxm. However, men EC 50 vlues were higher for metlxyl thn mefenoxm for ll isoltes tested. Metlxyl hs been used more frequently thn mefenoxm in the pst in both regions, which my explin the higher EC 50 vlues for metlxyl. Mefenoxm is pplied t lower rtes in the field thn metlxyl, but contins the more ctive enntiomer of metlxyl. The rte of resistnce development in popultions of the pthogen my vry for the two fungicides. The high percentge of mefenoxm-resistnt isoltes collected ws surprising considering tht neither metlxyl- nor mefenoxm-resistnt isoltes of P. cpsici hd been reported prior to 1997 in the field to our knowledge. After our initil report (33), isoltes of P. cpsici collected between 1992 to 1997 from greenhouse peppers grown in Itly were screened by others nd intermedite levels of resistnce to metlxyl were reported (34). These workers suggested tht cution should be exercised in the use of metlxyl in greenhouse-grown pepper crops in Itly. More recently, mefenoxm resistnce hs been reported in Michign on field-grown cucurbits nd on peppers nd cucurbits in Georgi (25,26,29). A shift in sensitivity to mefenoxm within popultions of P. cpsici hs occurred in bell pepper fields in North Crolin nd New Jersey in reltively short period of time. Due to the risk-verse nture of mny growers, it is possible tht more fields hve received pplictions of mefenoxm s disese incidence hs incresed in recent yers with high rinfll conditions in mny res of the northest nd southest. Frequently, bell pepper production in North Crolin is followed by cucurbit production in the sme field during the sme seson. Metlxyl nd mefenoxm re lso used for disese mngement in djcent tobcco fields. In Tble 4. Isoltes of Phytophthor cpsici clssified by mefenoxm sensitivity nd mting type within popultions from different loctions on eight grower frms in North Crolin nd New Jersey in 1997 Number of isoltes Sensitive Intermedite Resistnt Grower Field A1 b A2 b A1 A2 A1 A2 1 1 3 9 4 1 6 3 2 1 2 2 1 1 3 1 2 1 0 2 0 2 0 3 2 4 1 1 6 5 4 0 2 0 0 1 1 3 1 4 1 0 0 11 4 2 0 1 0 0 9 5 4 1 2 0 0 0 0 0 2 0 2 0 0 1 0 3 1 0 0 0 0 0 5 1 0 0 0 0 1 0 6 1 2 1 0 0 0 2 7 1 0 1 0 0 0 0 8 1 0 0 0 0 1 2 2 0 0 0 0 1 0 3 0 0 0 1 2 2 4 2 0 0 0 2 2 Totl 19 23 8 4 46 27 Isoltes were chrcterized s sensitive to mefenoxm if colony growth t 5 µg ml 1 ws less thn 40% of the isolte s growth on the nonmended control medi. Intermedite isoltes exhibited growth greter thn 40% of the nonmended medi control t 5 µg ml 1, but less thn 40% of the nonmended medi control with mefenoxm t 100 µg ml 1. Resistnt isoltes exhibited growth greter thn 40% of the nonmended medi control with mefenoxm t 100 µg ml 1. b A1 indictes A1 mting type nd A2 indictes A2 mting type determined by crosses with known tester isoltes. ddition, tobcco fields where phenylmide fungicides re used re rotted into vegetble production. Intensive use of phenylmide fungicides hs been shown to rpidly select for resistnce in other oomycetes (10). Other work on nonoomycete pthogens hs demonstrted tht pthogens cn become more resistnt to fungicide when it is used frequently over n extended period of time (43). Some mefenoxm-resistnt isoltes were not effectively controlled nd were pthogenic on plnts in greenhouse ssys when treted with lbeled rtes of the fungicide (J. B. Ristino, unpublished dt). Further reserch is needed to determine whether mefenoxm-resistnt isoltes re more fit thn sensitive isoltes nd to mesure their ggressiveness on plnts. Metlxyl cn promote growth of some resistnt isoltes of P. infestns nd cn cuse normlly heterothllic single mting type isoltes to form oospores in culture (41,44). Some studies hve demonstrted tht metlxylresistnt isoltes of P. infestns re more fit thn sensitive isoltes (6,8). However, other studies hve suggested tht no correltion exists between fitness nd metlxyl resistnce in P. infestns (17,27). Some of the mefenoxm-resistnt isoltes of P. cpsici in our study grew to greter extent thn the nonmended controls in the presence of mefenoxm (Fig. 1). Vrition in fitness could occur mong isoltes of P. cpsici nd fungicide resistnce my not present fitness cost in P. cpsici (17,25). The frequency of mefenoxm-resistnt isoltes of P. cpsici from cucurbits did not decrese over period of 2 yers in the bsence of mefenoxm use in Michign (26). Oospores re known to ply role in the survivl of P. cpsici in the field (35). Both mting types of P. cpsici were found mong resistnt isoltes within 9 of 17 grower fields smpled. Oospores survive in soil; therefore, mefenoxm-resistnt isoltes my persist for prolonged periods nd cuse disese in subsequent yers in the bsence of selection pressure from fungicide use. Further studies re needed to test individul fitness components of sensitive nd resistnt isoltes in quntittive ssys in vivo nd in soil. Mefenoxm-resistnt, intermedite, nd mefenoxm-sensitive isoltes of P. cpsici were found in single cucurbit fruit in Michign nd oospore progeny derived from isoltes from those fruit exhibited ll six mefenoxm sensitivity nd mting type combintions (25). Sexul reproduction of the pthogen enhnces genetic vrition nd my enhnce survivl of resistnt isoltes (25). It is believed tht the mode of ction of metlxyl is by the selective inhibition of ribosoml RNA synthesis (8,9,15). RNA polymerse is the trget site for metlxyl nd n ltertion of this trget site cn led to resistnce in some oomycete pthogens (9). Genetic studies of P. infestns nd P. soje hve indicted tht resistnce is 1074 Plnt Disese / Vol. 85 No. 10
linked to single dominnt gene, with vritions in resistnce ccounted for by the influence of minor genes (2,27). Recently, DNA mrkers tht re linked to loci ssocited with metlxyl resistnce in isoltes of P. infestns were reported (14,22). Loci found to confer metlxyl resistnce in isoltes of P. infestns from Mexico nd the Netherlnds were different thn in British isoltes of P. infestns, indicting tht metlxyl resistnce my hve developed independently in P. infestns isoltes in different res of the world (14). A single trget site for metlxyl resistnce in P. infestns is highly unlikely (22). Mefenoxm sensitivity in isoltes of P. cpsici from cucurbits is controlled by single, incompletely dominnt gene tht is not linked to mting type (25). Further work is needed to exmine the genetic mechnisms of mefenoxm resistnce in pepper isoltes of P. cpsici nd to determine whether the sme or different loci confer resistnce in popultions of P. cpsici found in different res or on different hosts (25). Mngement of Phytophthor blight in bell pepper will continue to rely on integrted pproches. Phenylmide fungicides should not be used for disese mngement in fields where mefenoxm-resistnt isoltes hve been found. Alterntive, low-risk fungicides with different modes of ction will be needed in problem fields. Phytophthor blight cn be most effectively mnged through the use of resistnt cultivrs nd culturl prctices, including wter mngement nd the use of cover crops (35). ACKNOWLEDGMENTS We thnk extension fculty members S. Johnston (Rutgers University), F. Louws nd G. Holmes (North Crolin Stte University), nd gents W. Jester nd A. Thornton (North Crolin Stte University) for providing some of the plnt smples for ssy from grower fields; nd L. Sujkowski nd R. French for screening popultions of P. cpsici collected prior to 1997. LITERATURE CITED 1. Abdelloui-Mne, S., Sing, J. M., Sndrenn, P., nd Bonpeix, G. 1988. Fosetyl-Al is effective ginst mutnts of P. cpsici resistnt to metlxyl. Cryptogm. 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