Am. J. Pot Res (2009) 86:315–326 DOI 10.1007/s12230-009-9085-z

Metalaxyl-M-Resistant Species in Production Areas of the Pacific Northwest of the U.S.A.

Lyndon D. Porter & Philip B. Hamm & Nicholas L. David & Stacy L. Gieck & Jeffery S. Miller & Babette Gundersen & Debra A. Inglis

Published online: 3 April 2009 # Potato Association of America 2009

Abstract Several Pythium species causing leak on potato information is lacking on the distribution of MR isolates in are managed by the systemic fungicide metalaxyl-M. the Pacific Northwest. Soil samples from numerous fields Metalaxyl-M-resistant (MR) isolates of Pythium spp. have (312) cropped to potatoes in Idaho (140), Oregon (59), and been identified in potato production areas of the U.S.A., but Washington (113) were assayed using metalaxyl-M- amended agar for the presence of MR isolates of Pythium in 2004 to 2006. Altogether, 1.4%, 42.4% and 32.7% of the L. D. Porter (*) fields from these states, respectively, were positive for MR Vegetable and Forage Crops Research Unit, USDA-ARS, Pythium. Isolates of Pythium ultimum that were highly 24106 N. Bunn Road, Prosser, WA 99350, USA resistant to metalaxyl were recovered from 53 fields e-mail: [email protected] representing ID, OR, and WA. Greater than 50% of the : Pythium soil population consisted of MR isolates in ten of P. B. Hamm S. L. Gieck 64 fields from Oregon and Washington. Nine species of Department of Botany & , Hermiston Agricultural Research and Extension Center, Pythium were recovered from soil samples, of which MR P. Oregon State University, ultimum and P. spinosum were identified. Isolates of MR P. 2121 South First Street, ultimum recovered from soil were pathogenic on potato Hermiston, OR 97838, USA tubers and may pose a serious threat to the management of P. B. Hamm Pythium leak and seed rot of diverse crops rotated with e-mail: [email protected] potato. N. L. David Department of Plant Sciences, Resumen Varias especies de Pythium que causan la North Dakota State University, pudrición acuosa en papa son controladas por el fungicida NDSU, Dept. # 7670, sistémico metalaxilo-M. Cepas de metalaxilo-M-resistente P.O. Box 6050, Fargo, ND 58108, USA e-mail: [email protected] (MR) de Pythium spp. han sido identificadas en áreas de producción de papa de los Estados Unidos, pero no hay J. S. Miller información de la distribución de cepas MR en el Pacifico Miller Research LLC, Noroeste. Muestras de suelo de numerosos campos (312) 1175 E. 800 N, Rupert, ID 83350, USA cultivados con papa, en Idaho (140), Oregon (59), y e-mail: [email protected] Washington (113) fueron ensayadas utilizando agar : metalaxilo-M-enmendado para la presencia de Pythium B. Gundersen D. A. Inglis MR del 2004 al 2006. En total, 1.4%, 42.4% y 32.7% de NWREC, Washington State University, 16650 State Route 536, los campos de estos estados, respectivamente, dieron Mount Vernon, WA 98273, USA positivo a Pythium MR. Cepas de Pythium ultimum B. Gundersen altamente resistentes al metalaxilo fueron recuperadas de e-mail: [email protected] 53 campos representando a Idaho, Oregon y Washington. D. A. Inglis Más del 50% de la población de Pythium del suelo, e-mail: [email protected] consistió de cepas MR en 10 de los 64 campos de Oregon 316 Am. J. Pot Res (2009) 86:315–326 y Washington. Nueve especies de Pythium fueron recuper- Metalaxyl is a racemic fungicide that contains both R- and adas de muestras de suelo, de las cuales fueron identifica- S-enantiomers. Metalaxyl-M which contains 98% of the R- das P. ultimum y P. spinosum MR. Cepas de P. ultimum enantiomer (Nuninger et al. 1996) replaced metalaxyl as the resistente al metalaxilo-M recuperadas del suelo fueron active ingredient in Ridomil Gold EC (Syngenta Crop patogénicas en tubérculos de papa y pueden representar una Protection, Greensboro, NC) in 1997 and continues to be seria amenaza en el manejo de la pudrición acuosa por commonly used to manage Pythium and other Pythium y la pudrición de la semilla en diversos campos pathogens on potatoes. Metalaxyl-M is considered to be rotados con papa. more effective than the S-enantiomer in controlling oomy- cete plant pathogens (Hubele et al. 1983). The efficacy of Keywords Mefenoxam . Fungicide resistance . metalaxyl-M against Pythium leak has been called into Pythium paroecandrum . Pythium inflatum question under challenge inoculations because wounding appears to break the peripheral tuber barrier of metalaxyl and allows Pythium infection and leak symptoms to occur Introduction (Taylor et al. 2004). However, certain metalaxyl-M appli- cation methods have demonstrated some leak control, but it Several Pythium spp. are soilborne oomycete plant patho- is questionable whether the cost-benefit ratio of using gens that can cause major problems in potato production by metalaxyl strictly to manage Pythium leak is economically rotting potato seed pieces, and tubers in the field, at harvest favorable to potato growers (Taylor et al. 2004). or in storage facilities (Powelson et al. 1993; Salas and Resistance to metalaxyl in oomycete pathogens was first Secor 2001). Pythium ultimum is considered to be the reported in isolates of Pseudoperonospora cubensis recov- primary Pythium species causing Pythium leak on potato ered from greenhouse-grown cucumber plants in Israel in (Salas and Secor 2001). P. ultimum reportedly requires a 1980 (Reuveni et al. 1980) and under field conditions in wound to enter a potato tuber (Taylor et al. 2004), making isolates of P. infestans isolated from potatoes in 1981 in both tubers highly vulnerable to infection during harvest, Ireland (Dowley and O’Sullivan 1981) and the Netherlands transport and loading of potatoes into storage facilities. (Davidse 1981). Development of resistance to metalaxyl in In the Pacific Northwest (PNW; Idaho, Oregon and commercial agricultural fields or orchards has been con- Washington), potatoes are rotated with a diverse array of firmed in at least seven species of (Bruin and crops including: peas, , corn, beans, onions and Edgington 1981;FerrinandKabashima1991; Taylor et al. cereals that are susceptible to Pythium seed and seedling rot 2002; Timmer et al. 1998; Chauhan and Singh 1987;and (Higginbotham et al. 2004; Kraft and Burke 1971; Pscheidt Seemuller and Sun 1989); and in six other genera in the and Ocamb 2007; Paulitz and Adams 2003; Broders et al. order including Pythium (Taylor et al. 2002; 2007; Davis and Nunez 1999; Sumner et al. 1997; Hendrix Mazzola et al. 2002; Falloon et al. 2000; Wiglesworth et al. and Campbell 1973). The systemic fungicide metalaxyl-M 1988; Schettini et al. 1991; Herzog and Schuepp 1985; is commonly used as a foliar or in-furrow application to Molinero-ruiz 2003; Mazzola et al. 2002; White et al. 1988; manage Pythium leak and pink rot on potato, cavity spot on Cook et al. 1983;Hammetal.2004). Since the development , and as a seed treatment to manage Pythium pre- of metalaxyl resistance is common among oomycete plant emergence damping off on most vegetable seed and cereal pathogens, it is important to assess the Pythium population in crops grown in crop rotation with potato in the PNW potato production areas where metalaxyl is used, to (Pscheidt and Ocamb 2007). Therefore in the PNW, determine the current and future opportunity to effectively soilborne populations of Pythium spp. can be exposed to use this fungicide not only to help manage Pythium leak on metalaxyl in the soil on an annual basis. In some cases, potato but Pythium damping off on other crops in rotation where growers are planting two or more crops in the same with potato. Development of metalaxyl resistance in the field within the same growing season (i.e. peas and corn), Pythium population is particularly important in the PNW or where repeated foliar applications are used, isolates of where 550,500 acres of potatoes were grown in 2007 Pythium spp. may be exposed to metalaxyl-M multiple accounting for 56.6% of the commercial potato production times in a single growing season. in the USA (USDA-NASS 2008). Metalaxyl is a highly effective systemic fungicide with a Metalaxyl-resistant (MR) isolates of P. ultimum were single-site mode of action that inhibits ribosomal RNA previously recovered from 1 of 11 and 1 of 5 tubers with polymerases (Davidse et al. 1983) of several oomycete leak-like symptoms in Washington and Idaho in 1998 and pathogens. Metalaxyl has been used in the PNW since 1982 2000, respectively (Taylor et al. 2002). MR isolates of P. to manage oomycete pathogens on potatoes such as ultimum were also recovered from 5 of 57 infected tubers with Phytophthora infestans (cause of late blight), Phytophthora leak-like symptoms from Minnesota in 2000 (Taylor et al. erythroseptica (cause of pink rot), and Pythium ultimum. 2002), and from potato tubers located near Hermiston, OR in Am. J. Pot Res (2009) 86:315–326 317

2002 displaying abnormal development of severe symptoms all cropped to potatoes at the time samples were taken from prior to harvest (Hamm et al. 2004). Lack of additional the fields. A standard soil probe (2.9 cm diameter) was used effective fungicides to manage this tuber and seed-rotting to collect the subsamples from each sample point from the pathogen and unusually severe symptoms associated with MR surface to a soil depth of 30.5 cm. Pythium population isolates in Oregon prior to harvest without evidence of densities for each soil sample were assessed for both MR wounding, make development of MR isolates of P. ultimum Pythium isolates and total colony forming units of Pythium an important issue in the PNW. The purposes of the present per gram of soil (CFU/g) following a previously published research were to: i) determine how widespread MR isolates of protocol (Hansen et al. 1990). Briefly, ten grams of soil per Pythium spp. are in the PNW, ii) determine levels of sample was mixed with 90 ml of 0.1% water agar and further mefenoxam sensitivity among resistant isolates, iii) identify diluted stepwise in water agar up to 10−4g/ml. A 0.5-ml the Pythium species isolated during the survey, iv) quantify sample of each 10−4g/ml soil dilution per soil sample was the proportion of MR isolates compared to metalaxyl- thenaddedtoeachoffourPetriplates(100mmwide× sensitive (MS) isolates in selected fields cropped to potatoes, 15 mm deep) containing approximately 14 ml of ACV8 agar and v) verify the pathogenicity of MR Pythium isolates on either with or without 10µg/ml technical grade metalaxyl-M. potato tubers. The soil dilution sample was spread uniformly over the agar surface using a sterile glass rod in the shape of a hockey stick. The Petri plates were then incubated at 25ºC for 48 h in the Materials and Methods dark, washed with sterile distilled water to remove soil particles, and the number of Pythium CFUs growing on the Media Recipes amended and non-amended replicate plates per soil sample were recorded. Mean number of Pythium colonies per An Amended Clarified V8 agar medium (ACV8) was used to amended and non-amended plates was determined and then determine soil populations of Pythium. This medium con- expressed as the number of CFU/g dry soil. sisted of 30 g agar, 15 mg pimaricin, 15 mg rifampicin, Randomly selected representative samples (one to two 375 mg ampicillin, 30 mg rose bengal, and 180 mg PCNB samples per plate) of individual isolates of Pythium from the per liter of medium (Hansen et al. 1990). Metalaxyl-amended ACV8 metalaxyl-amended medium were transferred to a and non-amended medium of ACV8 at both 10µg/ml and CARP medium in 2004 to 2006 and from non-metalaxyl- 100µg/ml was used to assess the sensitivity of the Pythium amended medium in 2006. Pure cultures of these isolate were isolates to this fungicide. Metalaxyl was added to the obtained by hyphal-tipping and subculturing the isolates onto medium following autoclaving by creating a metalaxyl stock additional Petri plates with CARP medium until the cultures solution at 10,000µg/ml using sterile distilled water. The were free of contaminating bacteria. The resistance to stock solution was filtered through a 0.2µm filter and added metalaxyl-M of 82 Pythium isolates representing 64 individ- to the medium when the medium temperature was 50ºC. ual fields was confirmed by transferring approximately 3 mm3 A CARP medium was used to recover unidentified agar plugs from the leading edge of an expanding colony and Pythium isolates from the ACV8 agar soil isolation plates. placing it on the center of agar plates containing 0, 10µg/ml This medium contained 17 g Difco cornmeal agar, 250 mg and 100µg/ml metalaxyl-M. Each Petri plate was sealed with ampicillin, 10 mg pimarcin and 10 mg rifampicin per liter parafilm and randomly arranged on trays and allowed to grow of medium. Pimaricin and ampicillin were dissolved or for 48 h in the dark at 25°C. Three replicate plates were used suspended in 10 ml of sterile distilled water and rifampicin for each isolate and metalaxyl-M concentration. Colony in 1 ml DMSO. The antibiotics were added to the medium diameter of these isolates was measured at a single cross after it was autoclaved and cooled to 50ºC in a water bath. section after 2 days of incubation. Isolates with uninhibited growth compared to the non-amended control at 100µg/ml Collection of Samples and Population Counts were labeled as highly resistant (HR) to metalaxyl-M. Isolates with no growth at 100µg/ml but growth at 10µg/ml were Soil samples from 140, 59 and 113 individual commercial categorized as intermediately resistant (IR), and those with no agricultural fields representing 12, five, and seven counties growth at 10µg/ml and 100µg/ml but growth at 0µg/ml were within Idaho, Oregon and Washington, respectively, were labeled as susceptible (MS). surveyed for MR Pythium spp. from 2004 to 2006. The Infected potato tubers demonstrating Pythium leak-like selected fields had a history of potatoes grown in rotation with symptoms that were submitted to the Oregon State University diverse crops. Generally, ten soil sub-samples were evenly Disease Diagnostic Laboratory at Hermiston, OR by potato collected along a diagonal transect of each sampled field and growers were an additional source of Pythium isolates. A total homogenized into a single sample weighing between 76 g to of eight, one, and seven tubers were submitted in 2004, 86 g dry weight. Fields that were selected for sampling were 2005, and 2006, respectively, representing 3 (Umatilla, OR; 318 Am. J. Pot Res (2009) 86:315–326

Walla Walla, WA and Grant, WA), 1 (Umatilla, OR), and 2 and Ribeiro 1996, two agar plates per isolate) and allowed to (Umatilla, OR and Franklin, WA) counties and five, one, and grow at 25ºC for 2 days. Potato tuber tissue was then five individual fields from these years. Approximately removed from the stem and stolon end of a single tuber with 3mm3 of tuber tissue was excised with a sterile scalpel the aid of a cork borer (0.8 cm diameter) to a depth of 2 cm. from the interface of discolored and healthy looking tissue A mycelial plug measuring 5 mm in diameter and 3.5 mm in and dipped in a 10% solution of hypochlorite for five height was then removed from the leading edge of an seconds, rinsed in sterile distilled water for five seconds, expanding Pythium colony and inserted into the tuber. A blotted dry with paper towels and placed on CARP medium. single isolate was used to inoculate each tuber, with a plug Mycelium typical of Pythium spp. was then sub-cultured to from one culture plate being placed in the stem end and a fresh CARP medium in Petri plates after incubation at room plug from the second replicated plate being placed in the temperature (23–25°C) for 2–4 days. The isolates were stolon end. The tuber tissue cores were reinserted into the further subcultured by hyphal tipping onto CARP medium tubers, sealing in the mycelial plug, and a light surface and were maintained at 25ºC. application of pertroleum jelly was used to seal the cores in place. Tubers had been hand-washed in running tap water Identification of Pythium Isolates and then rinsed with running distilled water and allowed to dry for 24 h prior to use. Four additional Russet Burbank In 2004, MR Pythium isolates from metalaxyl-amended tubers were treated in the same manner as described above dilution plates were transferred to individual Petri plates except the agar plugs placed in the tubers were free of any containing CARP agar and placed into groups based on mycelia. These tubers were used as non-innoculated controls. growth rate, colony morphology and reproductive structures. Each tuber was labeled with a permanent marker, placed into Two groups were categorized based on this data. Represen- covered plastic containers lined with moist paper towels and tative samples from these groups were sent to North Carolina stored at 21ºC at 100% RH for 10 days, and then evaluated State University Plant Pathogen Identification Laboratory for leak-like symptoms. Tuber pathogenicity was scored as (Raleigh, NC) for species identification using morphological follows: 0=lack of disease symptoms; 1=disease symptoms and molecular identification techniques. In 2005, MR on one tuber end; 2=disease symptoms on both tuber ends. Pythium isolates recovered from metalaxyl-amended plates, To complete Koch’s Postulates, individual potato tubers were and in 2006 isolates from both metalaxyl-amended and non- inoculated with one of each of five randomly selected MR metalaxyl-amended plates, were tested with species-specific isolates of Pythium ultimum in 2005 and 2006 and reisolated primers for P. ultimum (Schroeder et al. 2006). The PCRs using CARP to verify the causal agent. The other Pythium were carried out for 30 cycles using the following isolates were tested for pathogenicity via development of conditions: 45 s denaturation at 92ºC (4 min denaturation Pythium leak symptoms on tubers, and pathogen recovery at 94ºC for the first cycle), annealing for 45 s at 63ºC, and was not attempted. primer extension for 60 s (7 min for final cycle) at 72ºC. The isolates testing negative for P. ultimum-specific primers were Statistical Analysis cultured (Wang and White 1997) and identified by extracting DNA (Wang and White 1997) from each isolate and Means and standard errors of the mean for number of IR/ sequencing the PCR amplicon produced using the universal HR isolates obtained per gram of soil from resistant fields fungi primers ITS1 and ITS4 (White et al. 1990), and were obtained using the Excel 2003 Statistical Package comparing the sequences with known isolates in GenBank. (Microsoft, Bellevue, WA). PCR amplicons were sent to Functional Biosciences Inc. (Madison, WI) for sequencing. Results Pathogenicity Test Collection of Samples and Population Counts Pathogenicity of P. ultimum isolates was tested by inoculating potato tubers (113–170 g) of the susceptible cultivar Russet Pythium species were isolated from 73, 97, and 94% of the Burbank. Pathogenicity was defined as the ability of the soil dilution plates from fields sampled from Idaho, pathogen to colonize the tuber tissue. A total of seven and 26 Oregon and Washington, respectively. Pythium isolates MR P. ultimum isolates recovered from individual fields in resistant to metalaxyl were isolated from 2, 25 and 37 of 2005 and 2006 (Table 1), respectively, and 93 MS Pythium the fields from Idaho, Oregon and Washington, represent- spp. isolates recovered in 2006 from 58, 3 and 32 individual ing one, two and six counties from within these states, fields from Idaho, Oregon and Washington (Table 2), respectively (Table 3,Fig.1). Of the 64 fields where MR respectively, were transferred to Clarified V8 agar (Erwin Pythium isolates were detected, 11, 44 and 9 of these Am. J. Pot Res (2009) 86:315–326 319

Table 1 Field identification a b c d number and year when isolate Field #/year County/state Metalaxyl sensitivity Pathogenicity score was recovered from soil, state and county where field was 2/2005 Benton, WA HR 2 located, metalaxyl sensitivity, 7/2005 Klickitat, WA HR 2 and pathogenicity of isolate on 8/2005 Benton, WA HR 2 potato, of selected metalaxyl- resistant Pythium ultimum 19/2005 Walla Walla, WA HR 2 isolates collected from potato 22/2005 Umatilla, OR HR 2 fields in Idaho, Oregon and 23/2005 Morrow, OR HR 2 Washington during 2005 26/2005 Morrow, OR HR 2 and 2006 1/2006 Morrow, OR HR 2 2/2006 Morrow, OR HR 2 4/2006 Morrow, OR IR 2 5/2006 Morrow, OR HR 2 28/2006 Power, ID HR 2 31/2006 Power, ID HR 2 153/2006 Grant, WA HR 2 162/2006 Grant, WA HR 2 163/2006 Grant, WA HR 2 a Field identification number and 168/2006 Grant, WA HR 0 year when the isolate was 173/2006 Grant, WA HR 2 recovered from soil, 178/2006 Adams, WA HR 2 b County and state of the field where the isolate was recovered, 181/2006 Adams, WA HR 2 c Metalaxyl sensitivity of 184/2006 Adams, WA HR 2 Pythium ultimum isolates: IR 187/2006 Adams, WA HR 2 intermediately resistant, HR 190/2006 Adams, WA HR 2 highly resistant. IR growth on agar plates amended with 192/2006 Adams, WA HR 2 10μg/ml metalaxyl but did not 199/2006 Adams, WA HR 2 grow on plates amended at 202/2006 Franklin, WA HR 2 100μg/ml; HR growth on 100μg/ml metalaxyl amended 203/2006 Adams, WA HR 2 agar with growth equal to 206/2006 Benton, WA IR 2 d nonamended plates, Pathoge- 208/2006 Benton, WA HR 2 nicity of isolates on potato 209/2006 Benton, WA HR 2 tubers was scored as follows: 0=lack of disease symptoms; 213/2006 Umatilla, OR HR 2 1=disease symptoms on one 214/2006 Umatilla, OR HR 2 tuber end; 2=disease symptoms 215/2006 Umatilla, OR HR 2 on both tuber ends isolates were categorized as IR, HR or undetermined, P. ultimum. The single tuber sample from 2005 contained a respectively (Table 3). The nine undetermined isolates sensitive isolate of P. ultimum. Potato tubers infected with were either IR or HR but the cultures were lost prior to MR isolates of P. ultimum originated from four fields in confirmation testing. The mean percentage of MR isolates Umatilla County in Oregon and 2, 2, and 1 field(s) from to the total number of Pythium CFU/g of soil from Walla Walla, Grant, and Franklin counties, respectively, in surveyed fields within individual counties ranged from a Washington. low of 2.2 to a high of 52.2% (Table 3). The highest percentage of the MR Pythium recovered from an Identification of Pythium Isolates individual field was 92.5%, and the low was 0.9% (Table 3). Ten fields had Pythium counts where the Representative isolates of unidentified MR Pythium species percentage of MR Pythium isolates was greater than 50% categorized into two groups and sent to the North Carolina of the total Pythium population, and four of these fields State University Plant Pathogen Identification Lab in 2004 were located in Umatilla County, OR (Table 4). were identified as P. ultimum and P. spinosum. MR isolates Isolations from tubers demonstrating Pythium leak-like of P. spinosum were isolated from a single field in Klickitat symptoms found in five of five fields (100%) in 2004 and County, WA. All but one MR Pythium isolate recovered four of five fields (80%) in 2006 contained MR isolates of from soil samples from 2005 to 2006 and rotted field tubers 320 Am. J. Pot Res (2009) 86:315–326

Table 2 Occurrence and pathogenicity on potato tubers of Pythium species recovered from soil dilution plates on non-metalaxyl-amended agar from fields cropped to potato in Idaho, Oregon and Washington in 2006

County, State P. deliense P. inflatum P. irregulare P. oligandrum P. paroecandrum P. sylvaticum P. ultimum

Bingham, ID 3 (3/3) Cassia, ID 2 (2/2) 7 (7/7) Elmore, ID 4 (4/4) Jefferson, ID 1a (1/1)b 1 (1/1) 2 (2/2) Jerome, ID 5 (5/5) Madison, ID 1 (1/1) Minidoka, ID 1 (1/1) 3 (3/3) Owyhee, ID 3 (3/3) Power, ID 1 (1/1) 15 (15/15) Twin Falls, ID 2 (2/2) 7 (7/7) Jefferson, OR 1 (1/1) 2 (2/2) Adams, WA 3 (3/3) 2 (2/2) 1 (1/1) 1 (1/1) Benton, WA 1 (1/1) 1 (1/1) Franklin, WA 1 (1/1) 2 (2/2) 1 (1/1)* Grant, WA 1 (1/1) 4 (1/4)*c 5 (5/5) 5 (5/5) 4 (4/4) Totals 3 (3/3) 4 (4/4) 9 (9/9) 4 (1/4)* 8 (8/8) 8 (8/8)* 57 (57/57) a Number of individual fields within a county where the Pythium species was recovered from soil, b Number of tubers that became infected at the stolon and bud end of a healthy tuber when the tuber was inoculated with the same isolate at both ends; one tuber/isolate, c “*” indicates that one of the tubers was only infected at either the bud or stolon end (pathogenicity rating of “1”) All other tubers were infected at both inoculated ends (pathogenicity rating of “2”) from 2004 and 2005 were confirmed to be P. ultimum using fields from Morrow and Umatilla counties in Oregon, species-specific primers. The single non-P. ultimum isolate respectively for a total of eighteen fields. could not be identified based on the PCR amplicon produced using the universal fungi primers ITS1 and ITS4 Pathogenicity Test (White et al. 1990), and comparing the sequences with known isolates in GenBank. Tests conducted on randomly selected MR P. ultimum isolates MS Pythium isolates from non-metalaxyl-amended plates determined that 7 of 7 and 25 of 26 MR isolates collected in 2006 consisted of seven Pythium species: P. deliense, P. from soil in 2005 and 2006, respectively were assigned a inflatum, P. irregulare, P. oligandrum, P. Paroecandrum, P. rating of two, indicating that potato tubers were highly sylvaticum and P. ultimum. P. deliense was recovered from susceptible to infection by these isolates (Table 1). Only one three fields, from three counties, representing two states (ID MR isolate in 2006 did not produce symptoms when placed and WA) (Table 2). P. inflatum was recovered from four into a potato tuber. All 93 MS Pythium isolates representing fields, from two counties in Washington. P. i rre gu la re was seven species recovered from soil dilution plates in 2006 recovered from nine fields, from six counties, representing received pathogenicity ratings of 1 or 2 indicating that they two states (ID and WA). P. oligandrum was recovered from were pathogenic on potato tubers, except for 3 of 4 isolates four fields, from one county in Washington. P. paroecan- of P. oligandrum (Table 2). Rot did not occur on any non- drum was recovered from eight fields, from three counties in inoculated control tubers during pathogenicity tests. P. Washington. P. sylvaticum was recovered from eight fields, ultimum was recovered from five tubers inoculated in 2005 from four counties, representing three states (ID, OR, WA). and 2006, verifying that P. ultimum was the causal agent for P. ultimum was recovered from 57 fields, from 13 counties, the expression of Pythium-leak-like symptoms on tubers. representing three states (ID, OR, WA). Resistant isolates of P. ultimum constituted 96.9% (62 of 64) of the MR isolates recovered from soil from individual Discussion fields within the three states from 2004 to 2006. Eighteen fields were found with mixed populations of IR and HR Pythium isolates resistant to metalaxyl were identified in isolates of P. ultimum. These fields included 1, 1, 2, 3, and Idaho, Oregon and Washington, and isolates of P. ultimum 3 fields from Grant, Walla Walla, Benton, Adams and were the predominant metalaxyl-resistant (MR) species Klickitat counties in Washington, respectively and 1 and 7 found in this potato production area of the Pacific Northwest. Am. J. Pot Res (2009) 86:315–326 321

Table 3 Year, state, county, number of fields, metalaxyl sensitivity and prevalence of metalaxyl resistant Pythium species isolated from soil from fields cropped to potatoes in the Pacific Northwest

Year State County No. of No. of No. of No. of Mean (%) no. of IR/HR No. of fields with percentage High Low fields MS IR HR/UD isolates per gram of soil of IR/HR isolates per gram resistancef resistanceg surveyed fieldsa Fieldsb Fieldsc from resistant fieldsd of soil > 50%e

2004 WA Adams 2 2 0 0/0 0 0 0 0 2004 WA Benton 22 14 0 7/1 7.9±6.25 0 18.9 1.0 2004 WA Grant 6 4 0 2/0 52.2±44.2 1 83.5 20.9 2004 WA Klickitat 2 0 0 2/0 2.2±1.85 0 3.5 0.9 2004 WA Skagit 8 8 0 0/0 0 0 0 0 2004 WA Walla Walla 3 1 0 2/0 47.5±63.6 1 92.5 2.5 2004 OR Morrow 13 12 0 1/0 84.7±0 1 84.7 84.7 2004 OR Umatilla 14 6 2 6/0 28.6±23.4 1 83.8 5.4 2004 ID Fremont 3 3 0 0/0 0 0 0 0 2004 ID Madison 1 1 0 0/0 0 0 0 0 2005 WA Benton 6 3 1 2/0 28.0±22.51 1 54.0 15.0 2005 WA Grant 1 1 0 0/0 0 0 0 0 2005 WA Klickitat 2 0 0 1/1 8.6±8.29 0 14.5 2.8 2005 WA Walla Walla 3 2 0 1/0 23.0±0 0 23.0 23.0 2005 OR Morrow 4 1 0 2/1 4.2±3.68 0 8.2 1.0 2005 OR Umatilla 4 2 1 0/1 10.3±12.86 0 19.4 1.2 2005 ID Fremont 3 3 0 0/0 0 0 0 0 2005 ID Madison 1 1 0 0/0 0 0 0 0 2005 ID Bingham 1 1 0 0/0 0 0 0 0 2005 ID Blaine 1 1 0 0/0 0 0 0 0 2006 WA Adams 14 7 3 4/0 29.4±15.95 1 51.6 14.0 2006 WA Benton 9 2 1 3/3 30.6±27.65 2 76.7 1.8 2006 WA Franklin 6 5 0 1/0 4.2±0 0 4.2 4.2 2006 WA Grant 31 27 0 4/0 12.6±6.71 0 20.7 4.9 2006 OR Crook 2 2 0 0/0 0 0 0 0 2006 OR Jefferson 3 3 0 0/0 0 0 0 0 2006 OR Klamath 7 7 0 0/0 0 0 0 0 2006 OR Morrow 5 1 1 1/2 13.8±10.76 0 29.3 4.5 2006 OR Umatilla 5 0 0 5/0 45.0±21.77 2 76.8 21.4 2006 ID Bingham 11 11 0 0/0 0 0 0 0 2006 ID Blaine 2 2 0 0/0 0 0 0 0 2006 ID Cassia 10 10 0 0/0 0 0 0 0 2006 ID Elmore 5 5 0 0/0 0 0 0 0 2006 ID Fremont 12 12 0 0/0 0 0 0 0 2006 ID Jefferson 5 5 0 0/0 0 0 0 0 2006 ID Jerome 12 12 0 0/0 0 0 0 0 2006 ID Madison 5 5 0 0/0 0 0 0 0 2006 ID Minidoka 11 11 0 0/0 0 0 0 0 2006 ID Owyhee 3 3 0 0/0 0 0 0 0 2006 ID Power 41 39 2 0/0 5.6±5.4 0 9.4 1.7 2006 ID Twin Falls 13 13 0 0/0 0 0 0 0 Total NAh NA 312 248 11 44/9 NA 10 NA NA a Number of soil samples from individual fields that contained only metalaxyl-sensitive (MS), b Number of fields that contained Pythium isolates that were intermediately resistant to metalaxyl (Isolates grew on metalaxyl amended agar plates at 10µg/ml but did not grow at 100µg/ml), c Number of fields that contained Pythium isolates that were either highly resistant (HR) to metalaxyl (Isolates grew on metalaxyl amended agar plates at 100µg/ml with growth comparable to plates with nonamended agar), or the number of Pythium isolates that were metalaxyl-resistant but the test to classify them as IR or HR was not conducted, therefore the metalaxyl sensitivity is undetermined (UD), d Mean percentage of Pythium isolates per gram of soil found with intermediate to high metalaxyl-resistance compared to the total Pythium population on amended agar plates from all fields surveyed within that specific county for a given year, e Number of fields where the mean percentage of Pythium isolates with intermediate to high metalaxyl resistance was greater than 50% of the total Pythium population recovered, f The highest mean percentage of Pythium isolates that were found with intermediate to high metalaxyl resistance per gram of soil compared to the total Pythium population within an individual field, g The lowest mean percentage of Pythium isolates that were found with intermediate to high metalaxyl resistance per gram of soil compared to the total Pythium population within an individual field, h NA Not applicable 322 Am. J. Pot Res (2009) 86:315–326

The occurrence of metalaxyl-resistant Pythium species other metalaxyl to manage potato late blight undoubtedly exposed than P. ultimum is considered to be very rare in fields the Pythium soil population in the Columbia Basin to cropped to potatoes in the PNW, since only two of the 64 metalaxyl which may account for the higher incidence of fields where MR Pythium isolates were detected contained MR Pythium found there, compared to soil populations in MR Pythium isolates that were not P. ultimum.MRP. Idaho. Although metalaxyl is not currently applied to ultimum isolates were most commonly isolated from soils potatoes in the Columbia Basin to manage P. infestans, cropped to potatoes in the Columbia Basin region of applications of metalaxyl are still used to manage pink rot Washington and Oregon and rarely isolated from fields in (Phytophthora erythroseptica) and Pythium leak in potatoes. Idaho (Fig. 1) at this time. Potential reasons for the In the two Idaho fields containing MR Pythium isolates, widespread occurrence of metalaxyl resistance in the resistance may have developed due to repeated use of this Columbia Basin of Washington and Oregon and not in material to manage severe pink rot problems caused by P. Idaho, may be due to long term and repeated exposure of P. erythroseptica. In contrast, although there is a history of ultimum populations to metalaxyl in the Columbia Basin. metalaxyl use in western Washington, no MR Pythium Late blight, caused by the oomycete Phytophthora infestans, isolates were detected in field soil from the present research is an annual problem in the Columbia Basin of WA and OR or from soil and potato tubers in a separate study (D. A. (Johnson et al. 1997) and foliar applications of metalaxyl Inglis, unpublished data) from that region. The MR resistant have been applied extensively to potatoes in this region, Pythium isolates appear to be confined to the sandy soil especially during the 1990s to manage this devastating regions of the Columbia Basin in northern Oregon and disease (Johnson et al. 1997, 2000). It was common in the central and southeastern Washington, and the Snake River Columbia Basin in the early 1990s for one, two or more Valley in Idaho (Fig. 1). foliar applications of metalaxyl to be applied in one season. Another possibility for the widespread development of MR Metalaxyl applications to manage P. infestans were reduced isolates in the Columbia Basin could be due to the distribution in 1995 in the Columbia Basin due to the development of of infected seed. Some countries recommend that metalaxyl MR strains of P. infestans in 1993 (Deahl et al. 1993;Hamm not be applied to fields used for potato seed production due to et al. 1994). In contrast to the high use of metalaxyl in the the risk of selecting for MR biotypes of P. infestans that could Columbia Basin (Johnson et al. 1997), metalaxyl applica- then be disseminated to potato production areas where the tions were not as common in the early 1990s in Idaho since seed is planted (Erwin and Ribeiro 1996). Exposure to widespread late blight outbreaks were infrequent (Henderson metalaxyl in potato seed production areas could also lead to et al. 2007). However, the widespread and common use of the development of MR Pythium spp. infecting potatoes that

Fig. 1 Incidence of metalaxyl- 1. Skagit, WA 13. Elmore, ID resistant (MR) Pythium ultimum 2. Grant, WA 14. Blaine, ID isolates (black marks) and 3. Adams, WA 15. Jerome, ID metalaxyl-resistant Pythium spinosum isolate (clear mark)in 4. Klickitat, WA 17. Twin Falls, ID Idaho, Oregon and Washington 1 5. Benton, WA 18. Cassia, ID of the Pacific Northwest. 6. Franklin, WA 19. Power, ID Shaded counties represent 7. Walla Walla, WA 20. Bingham, ID counties where soil samples 8. Morrow, OR 21. Jefferson, ID were assayed for MR Pythium 2 9. Umatilla, OR 22. Madison, ID 3 10. Jefferson, OR 23. Fremont, ID 6 11. Crook, OR 5 12. Klamath, OR 7 4 9 8

10 23 11 21 22 14 13 20

12 16 15 19

17 18 Am. J. Pot Res (2009) 86:315–326 323

Table 4 4 YearYear and and field field identification identification number, number, county county and and state state where wheremetalaxyl-resistant field was located, isolates, total Pythium and percentagepopulation of andthe population level of metalaxyl resistant fieldresistance, was located, level of total metalaxylPythium sensitivity,population species and identification level of metalaxyl of metalaxyl-resistantfrom fields cropped isolates, to and potato percentage from 2004 of tothe 2006 population in Idaho, resistant Oregonfrom and resistance,fields cropped level to potato of metalaxyl from 2004 sensitivity, to 2006 in species Idaho, identification Oregon and Washington of Washington

Year-Field County, State Total Pythiuma MR Pythiumb Metalaxyl sensitivityc Pythium speciesd Percent MR populatione

2004-122 Umatilla, OR 99 83 HR ultimum 83.8 2004-126 Walla Walla, WA 2812 2600 HR ultimum 92.5 2004-141 Morrow, OR 430 364 HR ultimum 84.7 2004-154 Grant, WA 230 192 HR ultimum 83.5 2005-42 Benton,WA 363 281 HR ultimum 54.0 2006-183 Adams, WA 366 189 IR ultimum 51.6 2006-215 Benton, WA 73 56 HR unknown 76.7 2006-219.3 Umatilla, OR 214 163 HR ultimum 76.8 2006-219.4 Umatilla, OR 196 152 HR ultimum 77.6 2006-219.5 Umatilla, OR 284 224 HR ultimum 55.0 a Mean number of total Pythium per gram of soil. Determined by plating soil on agar not amended with metalaxyl, b Mean number of metalaxyl- resistant Pythium colonies per gram of soil. Determined by plating soil on metalaxyl-amended agar plates (10µg/ml metalaxyl), c Metalaxyl sensitivity of Pythium isolates: IR intermediately resistant, HR highly resistant, d Pythium species of metalaxyl resistant isolate, e Percentage of the total Pythium population that was metalaxyl resistant could be disseminated to commercial potato fields. It is well The MR isolates represented 92.5%, 2.5% and 0% of the established that the movement of P. infestans from Mexico to total Pythium soil population in fields 1, 2 and 3 Europe was most likely by the shipment of infected tubers respectively. The determination to continue or discontinue (Goodwin and Drenth 1997). Information regarding seed the use of metalaxyl to manage pathogenic Pythium may source, soil type, fungicide application history, and crop need to be determined on a field by field basis since MR rotations need further investigation to determine if there are populations can vary among fields. In field 1 of the correlations among these factors and the incidence of MR example, the grower may seriously consider discontinuing Pythium isolates in the PNW. the use of metalaxyl due to the high incidence of resistant Isolates that were rated IR were isolated from eleven fields isolates. In field 2, the grower may choose to monitor the representing all three states in the current study. Presence or effectiveness of metalaxyl since 97.5% of the population absence of isolates with intermediate resistance may be still appears to be MS. In field 3, the grower should have no associated with the heterozygous state of the gene conferring reservations in using metalaxyl since there were no MR resistance to metalaxyl-M (Shattock 1988; Goodwin and isolates recovered. More work is needed to clarify how long McGrath 1995). Isolates with intermediate resistance were metalaxyl would still be efficacious in managing mixed commonly found in the PNW and differences in fitness and populations of MR and MS Pythium isolates. aggressiveness between IR and HR isolates of P. ultimum Research assessing the development of metalaxyl resis- need to be compared in future studies to determine potential tance by MS Pythium isolates under laboratory conditions has differences in fitness and aggressiveness. previously been assessed (Bruin and Edgington 1981). Single During the course of this research two question were isolates of P. aphanidermatum, P. arrhenomanes, P. grami- often raised by growers and crop consultants concerning the nicola, and P. vexans were exposed to varying concentrations presence of MR isolates of P. ultimum in commercial potato of metalaxyl from 0.1µg/ml to 300µg/ml and the isolates fields: 1) should they discontinue the use of metalaxyl as a were maintained on metalaxyl-amended agar and transferred control option to manage Pythium leak?, and 2) should they 12 times over an 8 month period. EC50 values assessed refrain from using metalaxyl-treated seed that prevents seed following this metalaxyl exposure period were determined rot and seedling damping off of crops grown in rotation for these isolates and P. aphanidermatum, P. arrhenomanes, with potatoes? Based on the present information, in many P. graminicola, and P. vexans went from pre-exposure EC50 fields where MR Pythium isolates were detected, a values of 0.2µg/ml, 0.4µg/ml, 10µg/ml and 0.1µg/ml to substantial population of MS isolates were also present post-exposure values of 25µg/ml, 45µg/ml, 140µg/ml, and and could presumably be managed by applications of 360µg/ml, respectively. However, after 12 consecutive trans- metalaxyl either as a foliar, in-furrow or seed treatment to fers on non-amended medium, all the isolates lost most of protect potato tubers or seeds of various crops found in this resistance returning to EC50 values of 0.8, not potato crop rotations. For example, in 2004 within Walla determined, 28, and 2, respectively. However, when three Walla County, WA (Table 3) three fields were surveyed. isolates of P. ca ps ic i were tested using the same methods as 324 Am. J. Pot Res (2009) 86:315–326 those for the Pythium isolates, one isolate of P. capsici isolates identified from commercial agricultural field soil. P. maintained a high resistance to metalaxyl (EC50=300µg/ml), spinosum is not considered to be a major pathogen of potato a second isolate maintained an intermediate resistance, and tubers in the PNW, but has been associated with root the third isolate returned to the original level of sensitivity, infections on bell peppers, corn, peanuts, and even after transfers on nonamended agar (Bruin and watermelon in the U.S. (Chellemi et al. 2000; Hollowell et al. Edgington 1981). In addition, in field tests of isolates of 1998; Njoroge et al. 2008; Zhang et al. 1998). All MR Phytophthora parasitica var nicotianeae exposed to metal- isolates of P. sp in os um were confined to a single field located axyl over a 3 year period, EC50 values steadily increased in Klickitat County, WA. The crop history of this field needs over the 3 year period as isolates of P. nicotianeae were to be evaluated to determine potential reasons for the continuously exposed to metalaxyl in the soil. EC50 values occurrence of metalaxyl resistance in this specie and confirm increased from 0.4µg/ml, 0.3µg/ml, 0.7µg/ml and 1.2µg/ml whether this specie is impacting the production of potato or during years 0, 1, 2, and 3, respectively, in soil that had other crops. previously not been exposed to metalaxyl (Shew 1985). P. deliense, P. in fl at um, P. ir re gu la re, P. p aro ec an dr um , P. Thus, continuous exposure of sensitive oomycete populations sylvaticum and P. ultimum isolated from potato fields in 2006 to sublethal levels of metalaxyl in soil can result in reduced all were consistently pathogenic on healthy wounded potato sensitivity by the pathogen to this fungicide. Sensitivity may tubers (Table 2). P. deliense (Levesque et al. 1998), P. be either temporary or permanent depending on the species. irregulare (Mendes et al. 1998), P. s ylv at ic um (Peters et al. Research studying the buildup of MR Pythium ultimum 2005)andP. ultimum (Salas and Secor 2001)have populations in soil over time where metalaxyl continues to be previously been reported to cause potato tuber rot. However, used as a management tool will be valuable in addressing the this is the first report of isolates of P. inflatum and P. rate of MR development and the permanence of metalaxyl paroecandrum causing potato tuber rot. This demonstrates resistance maintained by isolates in these soil populations. that other Pythium species besides those commonly associ- MS and MR isolates of P. ultimum did not appear to vary ated with Pythium leak are capable of rotting tubers and a in pathogenicity to potato since 57 of 57 MS isolates and 32 complete survey of Pythium species causing rot on potato of 33 MR isolates of P. ultimum were pathogenic on potato tubers in the PNW needs to be assessed to fully understand tubers, respectively, which may suggest an equal fitness for all the pathogens involved. survival. However, P. ultimum isolates that were HR to Studying the development of resistance by a pathogen to metalaxyl were found in higher numbers in some fields than a specific fungicide on a large regional scale is necessary to MS isolates which may indicate that MR isolates are more fit understand, develop and improve fungicide resistance (Table 4). Development of fungicide resistance by a pathogen management programs. In addition, to determine how is often associated with a reduction in fitness when resistant effective a fungicide resistance management program is, it isolates have been compared to sensitive isolates (Raposo is paramount that a resistance baseline be established. The et al. 2000;Dowleyetal.2002;Kadish and Cohen 1992). An present research established a baseline for the presence of example of this is when the survival of MR isolates of P. MR Pythium in the PNW that can be used in the future to infestans was compared to MS isolates. MS isolates were assess the continual spread and persistence of MR Pythium determined to be more fit due to a reduction in the frequency isolates in this region of the USA. of MR isolates when the use of metalaxyl was temporarily Documented occurrence of resistant isolates to metalaxyl suspended (Dowley et al. 2002). In contrast, in several cases in potato production areas may help explain the success or the fitness of MR isolates of oomycete plant pathogens has failure of this fungicide in managing tuber rot and damping been similar, or better than, MS isolates (Gent et al. 2008; off of metalaxyl-treated seed of crops found in rotation with Café-Filho and Ristaino 2008; Crute and Harrison 1988; Gisi potato. and Cohen 1996; Kadish and Cohen 1989; Porter et al. 2007). For example, MR isolates of Phytophthora infestans Acknowledgements The authors would like to thank the National (Kadish and Cohen 1989)andPhytophthora erythroseptica Potato Council for funding this research project, and Steve James Casey Royer, Brian Charlton and Mike Nielsen for collecting soil. The (Porter et al. 2007) have demonstrated signs of being more fit experiments associated with this research were in compliance with the and aggressive than MS isolates in sexual and asexual laws of the U.S.A. reproduction, increased growth rates, and abilities to infect and colonize host tissue. Future research will compare fitness and aggressiveness of MR and MS Pythium ulitmum isolates References recovered during this study. Among the Pythium spp. identified from field soils, in the Broders, K.D., P.E. Lipps, P.A. Paul, and A.E. Dorrance. 2007. Characterization of Pythium spp. associated with corn and present study, were MR isolates of Pythium spinosum.Toour seed and seedling disease in Ohio. Plant Disease 91: knowledge this is the first report of MR Pythium spinosum 727–735. Am. J. Pot Res (2009) 86:315–326 325

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