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Abstract. Several Pythium species causing leak on potato are managed by the systemic fungicide metalaxyl-M. Metalaxyl-M-resistant (MR) isolates of Pythium ...
Am. J. Pot Res (2009) 86:315–326 DOI 10.1007/s12230-009-9085-z

Metalaxyl-M-Resistant Pythium Species in Potato 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 are managed by the systemic fungicide metalaxyl-M. Metalaxyl-M-resistant (MR) isolates of Pythium spp. have been identified in potato production areas of the U.S.A., but

L. D. Porter (*) Vegetable and Forage Crops Research Unit, USDA-ARS, 24106 N. Bunn Road, Prosser, WA 99350, USA e-mail: [email protected] P. B. Hamm : S. L. Gieck Department of Botany & Plant Pathology, Hermiston Agricultural Research and Extension Center, Oregon State University, 2121 South First Street, Hermiston, OR 97838, USA P. B. Hamm e-mail: [email protected] N. L. David Department of Plant Sciences, North Dakota State University, NDSU, Dept. # 7670, P.O. Box 6050, Fargo, ND 58108, USA e-mail: [email protected] J. S. Miller Miller Research LLC, 1175 E. 800 N, Rupert, ID 83350, USA e-mail: [email protected] B. Gundersen : D. A. Inglis NWREC, Washington State University, 16650 State Route 536, Mount Vernon, WA 98273, USA B. Gundersen e-mail: [email protected] D. A. Inglis e-mail: [email protected]

information is lacking on the distribution of MR isolates in the Pacific Northwest. Soil samples from numerous fields (312) cropped to potatoes in Idaho (140), Oregon (59), and Washington (113) were assayed using metalaxyl-Mamended agar for the presence of MR isolates of Pythium in 2004 to 2006. Altogether, 1.4%, 42.4% and 32.7% of the fields from these states, respectively, were positive for MR Pythium. Isolates of Pythium ultimum that were highly resistant to metalaxyl were recovered from 53 fields representing ID, OR, and WA. Greater than 50% of the Pythium soil population consisted of MR isolates in ten of 64 fields from Oregon and Washington. Nine species of Pythium were recovered from soil samples, of which MR P. ultimum and P. spinosum were identified. Isolates of MR P. ultimum recovered from soil were pathogenic on potato tubers and may pose a serious threat to the management of Pythium leak and seed rot of diverse crops rotated with potato. Resumen Varias especies de Pythium que causan la pudrición acuosa en papa son controladas por el fungicida sistémico metalaxilo-M. Cepas de metalaxilo-M-resistente (MR) de Pythium spp. han sido identificadas en áreas de producción de papa de los Estados Unidos, pero no hay información de la distribución de cepas MR en el Pacifico Noroeste. Muestras de suelo de numerosos campos (312) cultivados con papa, en Idaho (140), Oregon (59), y Washington (113) fueron ensayadas utilizando agar metalaxilo-M-enmendado para la presencia de Pythium MR del 2004 al 2006. En total, 1.4%, 42.4% y 32.7% de los campos de estos estados, respectivamente, dieron positivo a Pythium MR. Cepas de Pythium ultimum altamente resistentes al metalaxilo fueron recuperadas de 53 campos representando a Idaho, Oregon y Washington. Más del 50% de la población de Pythium del suelo, consistió de cepas MR en 10 de los 64 campos de Oregon

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y Washington. Nueve especies de Pythium fueron recuperadas de muestras de suelo, de las cuales fueron identificadas P. ultimum y P. spinosum MR. Cepas de P. ultimum resistente al metalaxilo-M recuperadas del suelo fueron patogénicas en tubérculos de papa y pueden representar una seria amenaza en el manejo de la pudrición acuosa por Pythium y la pudrición de la semilla en diversos campos rotados con papa. Keywords Mefenoxam . Fungicide resistance . Pythium paroecandrum . Pythium inflatum

Introduction Several Pythium spp. are soilborne oomycete plant pathogens that can cause major problems in potato production by rotting potato seed pieces, and tubers in the field, at harvest or in storage facilities (Powelson et al. 1993; Salas and Secor 2001). Pythium ultimum is considered to be the primary Pythium species causing Pythium leak on potato (Salas and Secor 2001). P. ultimum reportedly requires a wound to enter a potato tuber (Taylor et al. 2004), making tubers highly vulnerable to infection during harvest, transport and loading of potatoes into storage facilities. In the Pacific Northwest (PNW; Idaho, Oregon and Washington), potatoes are rotated with a diverse array of crops including: peas, carrots, corn, beans, onions and cereals that are susceptible to Pythium seed and seedling rot (Higginbotham et al. 2004; Kraft and Burke 1971; Pscheidt and Ocamb 2007; Paulitz and Adams 2003; Broders et al. 2007; Davis and Nunez 1999; Sumner et al. 1997; Hendrix and Campbell 1973). The systemic fungicide metalaxyl-M is commonly used as a foliar or in-furrow application to manage Pythium leak and pink rot on potato, cavity spot on carrot, and as a seed treatment to manage Pythium preemergence damping off on most vegetable seed and cereal crops grown in crop rotation with potato in the PNW (Pscheidt and Ocamb 2007). Therefore in the PNW, soilborne populations of Pythium spp. can be exposed to metalaxyl in the soil on an annual basis. In some cases, where growers are planting two or more crops in the same field within the same growing season (i.e. peas and corn), or where repeated foliar applications are used, isolates of Pythium spp. may be exposed to metalaxyl-M multiple times in a single growing season. Metalaxyl is a highly effective systemic fungicide with a single-site mode of action that inhibits ribosomal RNA polymerases (Davidse et al. 1983) of several oomycete pathogens. Metalaxyl has been used in the PNW since 1982 to manage oomycete pathogens on potatoes such as Phytophthora infestans (cause of late blight), Phytophthora erythroseptica (cause of pink rot), and Pythium ultimum.

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Metalaxyl is a racemic fungicide that contains both R- and S-enantiomers. Metalaxyl-M which contains 98% of the Renantiomer (Nuninger et al. 1996) replaced metalaxyl as the active ingredient in Ridomil Gold EC (Syngenta Crop Protection, Greensboro, NC) in 1997 and continues to be commonly used to manage Pythium and other oomycete pathogens on potatoes. Metalaxyl-M is considered to be more effective than the S-enantiomer in controlling oomycete plant pathogens (Hubele et al. 1983). The efficacy of metalaxyl-M against Pythium leak has been called into question under challenge inoculations because wounding appears to break the peripheral tuber barrier of metalaxyl and allows Pythium infection and leak symptoms to occur (Taylor et al. 2004). However, certain metalaxyl-M application methods have demonstrated some leak control, but it is questionable whether the cost-benefit ratio of using metalaxyl strictly to manage Pythium leak is economically favorable to potato growers (Taylor et al. 2004). Resistance to metalaxyl in oomycete pathogens was first reported in isolates of Pseudoperonospora cubensis recovered from greenhouse-grown cucumber plants in Israel in 1980 (Reuveni et al. 1980) and under field conditions in isolates of P. infestans isolated from potatoes in 1981 in both Ireland (Dowley and O’Sullivan 1981) and the Netherlands (Davidse 1981). Development of resistance to metalaxyl in commercial agricultural fields or orchards has been confirmed in at least seven species of Phytophthora (Bruin and Edgington 1981; Ferrin and Kabashima 1991; Taylor et al. 2002; Timmer et al. 1998; Chauhan and Singh 1987; and Seemuller and Sun 1989); and in six other genera in the order Peronosporales including Pythium (Taylor et al. 2002; Mazzola et al. 2002; Falloon et al. 2000; Wiglesworth et al. 1988; Schettini et al. 1991; Herzog and Schuepp 1985; Molinero-ruiz 2003; Mazzola et al. 2002; White et al. 1988; Cook et al. 1983; Hamm et al. 2004). Since the development of metalaxyl resistance is common among oomycete plant pathogens, it is important to assess the Pythium population in potato production areas where metalaxyl is used, to determine the current and future opportunity to effectively use this fungicide not only to help manage Pythium leak on potato but Pythium damping off on other crops in rotation with potato. Development of metalaxyl resistance in the Pythium population is particularly important in the PNW where 550,500 acres of potatoes were grown in 2007 accounting for 56.6% of the commercial potato production in the USA (USDA-NASS 2008). Metalaxyl-resistant (MR) isolates of P. ultimum were previously recovered from 1 of 11 and 1 of 5 tubers with leak-like symptoms in Washington and Idaho in 1998 and 2000, respectively (Taylor et al. 2002). MR isolates of P. ultimum were also recovered from 5 of 57 infected tubers with leak-like symptoms from Minnesota in 2000 (Taylor et al. 2002), and from potato tubers located near Hermiston, OR in

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2002 displaying abnormal development of severe symptoms prior to harvest (Hamm et al. 2004). Lack of additional effective fungicides to manage this tuber and seed-rotting pathogen and unusually severe symptoms associated with MR isolates in Oregon prior to harvest without evidence of wounding, make development of MR isolates of P. ultimum an important issue in the PNW. The purposes of the present research were to: i) determine how widespread MR isolates of Pythium spp. are in the PNW, ii) determine levels of mefenoxam sensitivity among resistant isolates, iii) identify the Pythium species isolated during the survey, iv) quantify the proportion of MR isolates compared to metalaxylsensitive (MS) isolates in selected fields cropped to potatoes, and v) verify the pathogenicity of MR Pythium isolates on potato tubers.

Materials and Methods Media Recipes An Amended Clarified V8 agar medium (ACV8) was used to determine soil populations of Pythium. This medium consisted of 30 g agar, 15 mg pimaricin, 15 mg rifampicin, 375 mg ampicillin, 30 mg rose bengal, and 180 mg PCNB per liter of medium (Hansen et al. 1990). Metalaxyl-amended and non-amended medium of ACV8 at both 10µg/ml and 100µg/ml was used to assess the sensitivity of the Pythium isolates to this fungicide. Metalaxyl was added to the medium following autoclaving by creating a metalaxyl stock solution at 10,000µg/ml using sterile distilled water. The stock solution was filtered through a 0.2µm filter and added to the medium when the medium temperature was 50ºC. A CARP medium was used to recover unidentified Pythium isolates from the ACV8 agar soil isolation plates. This medium contained 17 g Difco cornmeal agar, 250 mg ampicillin, 10 mg pimarcin and 10 mg rifampicin per liter of medium. Pimaricin and ampicillin were dissolved or suspended in 10 ml of sterile distilled water and rifampicin in 1 ml DMSO. The antibiotics were added to the medium after it was autoclaved and cooled to 50ºC in a water bath. Collection of Samples and Population Counts Soil samples from 140, 59 and 113 individual commercial agricultural fields representing 12, five, and seven counties within Idaho, Oregon and Washington, respectively, were surveyed for MR Pythium spp. from 2004 to 2006. The selected fields had a history of potatoes grown in rotation with diverse crops. Generally, ten soil sub-samples were evenly collected along a diagonal transect of each sampled field and homogenized into a single sample weighing between 76 g to 86 g dry weight. Fields that were selected for sampling were

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all cropped to potatoes at the time samples were taken from the fields. A standard soil probe (2.9 cm diameter) was used to collect the subsamples from each sample point from the surface to a soil depth of 30.5 cm. Pythium population densities for each soil sample were assessed for both MR Pythium isolates and total colony forming units of Pythium per gram of soil (CFU/g) following a previously published protocol (Hansen et al. 1990). Briefly, ten grams of soil per sample was mixed with 90 ml of 0.1% water agar and further diluted stepwise in water agar up to 10−4 g/ml. A 0.5-ml sample of each 10−4 g/ml soil dilution per soil sample was then added to each of four Petri plates (100 mm wide × 15 mm deep) containing approximately 14 ml of ACV8 agar either with or without 10µg/ml technical grade metalaxyl-M. 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 dark, washed with sterile distilled water to remove soil particles, and the number of Pythium CFUs growing on the amended and non-amended replicate plates per soil sample were recorded. Mean number of Pythium colonies per amended and non-amended plates was determined and then expressed as the number of CFU/g dry soil. Randomly selected representative samples (one to two samples per plate) of individual isolates of Pythium from the ACV8 metalaxyl-amended medium were transferred to a CARP medium in 2004 to 2006 and from non-metalaxylamended medium in 2006. Pure cultures of these isolate were obtained by hyphal-tipping and subculturing the isolates onto additional Petri plates with CARP medium until the cultures were free of contaminating bacteria. The resistance to metalaxyl-M of 82 Pythium isolates representing 64 individual fields was confirmed by transferring approximately 3 mm3 agar plugs from the leading edge of an expanding colony and placing it on the center of agar plates containing 0, 10µg/ml and 100µg/ml metalaxyl-M. Each Petri plate was sealed with parafilm and randomly arranged on trays and allowed to grow for 48 h in the dark at 25°C. Three replicate plates were used for each isolate and metalaxyl-M concentration. Colony diameter of these isolates was measured at a single cross section after 2 days of incubation. Isolates with uninhibited growth compared to the non-amended control at 100µg/ml were labeled as highly resistant (HR) to metalaxyl-M. Isolates with no growth at 100µg/ml but growth at 10µg/ml were categorized as intermediately resistant (IR), and those with no growth at 10µg/ml and 100µg/ml but growth at 0µg/ml were labeled as susceptible (MS). Infected potato tubers demonstrating Pythium leak-like symptoms that were submitted to the Oregon State University Disease Diagnostic Laboratory at Hermiston, OR by potato growers were an additional source of Pythium isolates. A total of eight, one, and seven tubers were submitted in 2004, 2005, and 2006, respectively, representing 3 (Umatilla, OR;

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Walla Walla, WA and Grant, WA), 1 (Umatilla, OR), and 2 (Umatilla, OR and Franklin, WA) counties and five, one, and five individual fields from these years. Approximately 3 mm3 of tuber tissue was excised with a sterile scalpel from the interface of discolored and healthy looking tissue and dipped in a 10% solution of hypochlorite for five seconds, rinsed in sterile distilled water for five seconds, blotted dry with paper towels and placed on CARP medium. Mycelium typical of Pythium spp. was then sub-cultured to fresh CARP medium in Petri plates after incubation at room temperature (23–25°C) for 2–4 days. The isolates were further subcultured by hyphal tipping onto CARP medium and were maintained at 25ºC. Identification of Pythium Isolates In 2004, MR Pythium isolates from metalaxyl-amended dilution plates were transferred to individual Petri plates containing CARP agar and placed into groups based on growth rate, colony morphology and reproductive structures. Two groups were categorized based on this data. Representative samples from these groups were sent to North Carolina State University Plant Pathogen Identification Laboratory (Raleigh, NC) for species identification using morphological and molecular identification techniques. In 2005, MR Pythium isolates recovered from metalaxyl-amended plates, and in 2006 isolates from both metalaxyl-amended and nonmetalaxyl-amended plates, were tested with species-specific primers for P. ultimum (Schroeder et al. 2006). The PCRs were carried out for 30 cycles using the following conditions: 45 s denaturation at 92ºC (4 min denaturation at 94ºC for the first cycle), annealing for 45 s at 63ºC, and primer extension for 60 s (7 min for final cycle) at 72ºC. The isolates testing negative for P. ultimum-specific primers were cultured (Wang and White 1997) and identified by extracting DNA (Wang and White 1997) from each isolate and sequencing the PCR amplicon produced using the universal fungi primers ITS1 and ITS4 (White et al. 1990), and comparing the sequences with known isolates in GenBank. PCR amplicons were sent to Functional Biosciences Inc. (Madison, WI) for sequencing.

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and Ribeiro 1996, two agar plates per isolate) and allowed to grow at 25ºC for 2 days. Potato tuber tissue was then removed from the stem and stolon end of a single tuber with the aid of a cork borer (0.8 cm diameter) to a depth of 2 cm. A mycelial plug measuring 5 mm in diameter and 3.5 mm in height was then removed from the leading edge of an expanding Pythium colony and inserted into the tuber. A single isolate was used to inoculate each tuber, with a plug from one culture plate being placed in the stem end and a plug from the second replicated plate being placed in the stolon end. The tuber tissue cores were reinserted into the tubers, sealing in the mycelial plug, and a light surface application of pertroleum jelly was used to seal the cores in place. Tubers had been hand-washed in running tap water and then rinsed with running distilled water and allowed to dry for 24 h prior to use. Four additional Russet Burbank tubers were treated in the same manner as described above except the agar plugs placed in the tubers were free of any mycelia. These tubers were used as non-innoculated controls. Each tuber was labeled with a permanent marker, placed into covered plastic containers lined with moist paper towels and stored at 21ºC at 100% RH for 10 days, and then evaluated for leak-like symptoms. Tuber pathogenicity was scored as follows: 0=lack of disease symptoms; 1=disease symptoms on one tuber end; 2=disease symptoms on both tuber ends. To complete Koch’s Postulates, individual potato tubers were inoculated with one of each of five randomly selected MR isolates of Pythium ultimum in 2005 and 2006 and reisolated using CARP to verify the causal agent. The other Pythium isolates were tested for pathogenicity via development of Pythium leak symptoms on tubers, and pathogen recovery was not attempted. Statistical Analysis Means and standard errors of the mean for number of IR/ HR isolates obtained per gram of soil from resistant fields were obtained using the Excel 2003 Statistical Package (Microsoft, Bellevue, WA).

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 Burbank. Pathogenicity was defined as the ability of the pathogen to colonize the tuber tissue. A total of seven and 26 MR P. ultimum isolates recovered from individual fields in 2005 and 2006 (Table 1), respectively, and 93 MS Pythium spp. isolates recovered in 2006 from 58, 3 and 32 individual fields from Idaho, Oregon and Washington (Table 2), respectively, were transferred to Clarified V8 agar (Erwin

Pythium species were isolated from 73, 97, and 94% of the soil dilution plates from fields sampled from Idaho, Oregon and Washington, respectively. Pythium isolates resistant to metalaxyl were isolated from 2, 25 and 37 of the fields from Idaho, Oregon and Washington, representing one, two and six counties from within these states, respectively (Table 3, Fig. 1). Of the 64 fields where MR Pythium isolates were detected, 11, 44 and 9 of these

Am. J. Pot Res (2009) 86:315–326 Table 1 Field identification number and year when isolate was recovered from soil, state and county where field was located, metalaxyl sensitivity, and pathogenicity of isolate on potato, of selected metalaxylresistant Pythium ultimum isolates collected from potato fields in Idaho, Oregon and Washington during 2005 and 2006

a

Field identification number and year when the isolate was recovered from soil, b County and state of the field where the isolate was recovered, c Metalaxyl sensitivity of Pythium ultimum isolates: IR intermediately resistant, HR highly resistant. IR growth on agar plates amended with 10μg/ml metalaxyl but did not grow on plates amended at 100μg/ml; HR growth on 100μg/ml metalaxyl amended agar with growth equal to nonamended plates, d Pathogenicity of isolates on potato tubers was scored as follows: 0=lack of disease symptoms; 1=disease symptoms on one tuber end; 2=disease symptoms on both tuber ends

319 Field #/yeara

County/stateb

Metalaxyl sensitivityc

Pathogenicity scored

2/2005 7/2005 8/2005 19/2005 22/2005 23/2005 26/2005 1/2006 2/2006 4/2006 5/2006 28/2006 31/2006 153/2006 162/2006

Benton, WA Klickitat, WA Benton, WA Walla Walla, WA Umatilla, OR Morrow, OR Morrow, OR Morrow, OR Morrow, OR Morrow, OR Morrow, OR Power, ID Power, ID Grant, WA Grant, WA

HR HR HR HR HR HR HR HR HR IR HR HR HR HR HR

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

163/2006 168/2006 173/2006 178/2006 181/2006 184/2006 187/2006 190/2006 192/2006 199/2006 202/2006 203/2006 206/2006 208/2006 209/2006 213/2006 214/2006 215/2006

Grant, WA Grant, WA Grant, WA Adams, WA Adams, WA Adams, WA Adams, WA Adams, WA Adams, WA Adams, WA Franklin, WA Adams, WA Benton, WA Benton, WA Benton, WA Umatilla, OR Umatilla, OR Umatilla, OR

HR HR HR HR HR HR HR HR HR HR HR HR IR HR HR HR HR HR

2 0 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

isolates were categorized as IR, HR or undetermined, respectively (Table 3). The nine undetermined isolates were either IR or HR but the cultures were lost prior to confirmation testing. The mean percentage of MR isolates to the total number of Pythium CFU/g of soil from surveyed fields within individual counties ranged from a low of 2.2 to a high of 52.2% (Table 3). The highest percentage of the MR Pythium recovered from an individual field was 92.5%, and the low was 0.9% (Table 3). Ten fields had Pythium counts where the percentage of MR Pythium isolates was greater than 50% of the total Pythium population, and four of these fields were located in Umatilla County, OR (Table 4). Isolations from tubers demonstrating Pythium leak-like symptoms found in five of five fields (100%) in 2004 and four of five fields (80%) in 2006 contained MR isolates of

P. ultimum. The single tuber sample from 2005 contained a sensitive isolate of P. ultimum. Potato tubers infected with MR isolates of P. ultimum originated from four fields in Umatilla County in Oregon and 2, 2, and 1 field(s) from Walla Walla, Grant, and Franklin counties, respectively, in Washington. Identification of Pythium Isolates Representative isolates of unidentified MR Pythium species categorized into two groups and sent to the North Carolina State University Plant Pathogen Identification Lab in 2004 were identified as P. ultimum and P. spinosum. MR isolates of P. spinosum were isolated from a single field in Klickitat County, WA. All but one MR Pythium isolate recovered from soil samples from 2005 to 2006 and rotted field tubers

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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 Bingham, ID Cassia, ID Elmore, ID Jefferson, ID Jerome, ID Madison, ID Minidoka, ID Owyhee, ID Power, ID Twin Falls, ID Jefferson, OR Adams, WA Benton, WA Franklin, WA Grant, WA Totals

P. deliense

P. inflatum

P. irregulare

P. oligandrum

P. paroecandrum

P. sylvaticum

3 7 4 2 5

2 (2/2) 1a (1/1)b

P. ultimum

1 (1/1)

(3/3) (7/7) (4/4) (2/2) (5/5)

1 (1/1) 1 (1/1) 1 (1/1) 2 (2/2) 1 (1/1) 3 (3/3)

2 (2/2) 1 (1/1)

1 (1/1)

1 (1/1) 1 (1/1) 3 (3/3)

4 (4/4)

9 (9/9)

4 (1/4)* 4 (1/4)*

c

2 (2/2) 5 (5/5) 8 (8/8)

1 (1/1)* 5 (5/5) 8 (8/8)*

3 3 15 7 2 1 1

(3/3) (3/3) (15/15) (7/7) (2/2) (1/1) (1/1)

4 (4/4) 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 species-specific primers. The single non-P. ultimum isolate could not be identified based on the PCR amplicon produced using the universal fungi primers ITS1 and ITS4 (White et al. 1990), and comparing the sequences with known isolates in GenBank. MS Pythium isolates from non-metalaxyl-amended plates in 2006 consisted of seven Pythium species: P. deliense, P. inflatum, P. irregulare, P. oligandrum, P. Paroecandrum, P. sylvaticum and P. ultimum. P. deliense was recovered from three fields, from three counties, representing two states (ID and WA) (Table 2). P. inflatum was recovered from four fields, from two counties in Washington. P. irregulare was recovered from nine fields, from six counties, representing two states (ID and WA). P. oligandrum was recovered from four fields, from one county in Washington. P. paroecandrum was recovered from eight fields, from three counties in Washington. P. sylvaticum was recovered from eight fields, from four counties, representing three states (ID, OR, WA). P. ultimum was recovered from 57 fields, from 13 counties, 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 fields within the three states from 2004 to 2006. Eighteen fields were found with mixed populations of IR and HR isolates of P. ultimum. These fields included 1, 1, 2, 3, and 3 fields from Grant, Walla Walla, Benton, Adams and Klickitat counties in Washington, respectively and 1 and 7

fields from Morrow and Umatilla counties in Oregon, respectively for a total of eighteen fields. Pathogenicity Test Tests conducted on randomly selected MR P. ultimum isolates determined that 7 of 7 and 25 of 26 MR isolates collected from soil in 2005 and 2006, respectively were assigned a rating of two, indicating that potato tubers were highly susceptible to infection by these isolates (Table 1). Only one MR isolate in 2006 did not produce symptoms when placed into a potato tuber. All 93 MS Pythium isolates representing seven species recovered from soil dilution plates in 2006 received pathogenicity ratings of 1 or 2 indicating that they were pathogenic on potato tubers, except for 3 of 4 isolates of P. oligandrum (Table 2). Rot did not occur on any noninoculated control tubers during pathogenicity tests. P. ultimum was recovered from five tubers inoculated in 2005 and 2006, verifying that P. ultimum was the causal agent for the expression of Pythium-leak-like symptoms on tubers.

Discussion Pythium isolates resistant to metalaxyl were identified in Idaho, Oregon and Washington, and isolates of P. ultimum were the predominant metalaxyl-resistant (MR) species found in this potato production area of the Pacific Northwest.

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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

2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2005 2005 2005 2005 2005 2005 2005 2005 2005 2005 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 Total

WA WA WA WA WA WA OR OR ID ID WA WA WA WA OR OR ID ID ID ID WA WA WA WA OR OR OR OR OR ID ID ID ID ID ID ID ID ID ID ID ID NAh

Adams Benton Grant Klickitat Skagit Walla Walla Morrow Umatilla Fremont Madison Benton Grant Klickitat Walla Walla Morrow Umatilla Fremont Madison Bingham Blaine Adams Benton Franklin Grant Crook Jefferson Klamath Morrow Umatilla Bingham Blaine Cassia Elmore Fremont Jefferson Jerome Madison Minidoka Owyhee Power Twin Falls NA

a

No. of fields surveyed

No. of MS fieldsa

No. of IR Fieldsb

2 22 6 2 8 3 13 14 3 1 6 1 2 3 4 4 3 1 1 1 14 9 6 31 2 3 7 5 5 11 2 10 5 12 5 12 5 11 3 41 13 312

2 14 4 0 8 1 12 6 3 1 3 1 0 2 1 2 3 1 1 1 7 2 5 27 2 3 7 1 0 11 2 10 5 12 5 12 5 11 3 39 13 248

0 0 0 0 0 0 0 2 0 0 1 0 0 0 0 1 0 0 0 0 3 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 2 0 11

No. of HR/UD Fieldsc 0/0 7/1 2/0 2/0 0/0 2/0 1/0 6/0 0/0 0/0 2/0 0/0 1/1 1/0 2/1 0/1 0/0 0/0 0/0 0/0 4/0 3/3 1/0 4/0 0/0 0/0 0/0 1/2 5/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 44/9

Mean (%) no. of IR/HR isolates per gram of soil from resistant fieldsd

No. of fields with percentage of IR/HR isolates per gram of soil > 50%e

High resistancef

Low resistanceg

0 7.9±6.25 52.2±44.2 2.2±1.85 0 47.5±63.6 84.7±0 28.6±23.4 0 0 28.0±22.51 0 8.6±8.29 23.0±0 4.2±3.68 10.3±12.86 0 0 0 0 29.4±15.95 30.6±27.65 4.2±0 12.6±6.71 0 0 0 13.8±10.76 45.0±21.77 0 0 0 0 0 0 0 0 0 0 5.6±5.4 0 NA

0 0 1 0 0 1 1 1 0 0 1 0 0 0 0 0 0 0 0 0 1 2 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 10

0 18.9 83.5 3.5 0 92.5 84.7 83.8 0 0 54.0 0 14.5 23.0 8.2 19.4 0 0 0 0 51.6 76.7 4.2 20.7 0 0 0 29.3 76.8 0 0 0 0 0 0 0 0 0 0 9.4 0 NA

0 1.0 20.9 0.9 0 2.5 84.7 5.4 0 0 15.0 0 2.8 23.0 1.0 1.2 0 0 0 0 14.0 1.8 4.2 4.9 0 0 0 4.5 21.4 0 0 0 0 0 0 0 0 0 0 1.7 0 NA

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

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metalaxyl to manage potato late blight undoubtedly exposed the Pythium soil population in the Columbia Basin to metalaxyl which may account for the higher incidence of MR Pythium found there, compared to soil populations in Idaho. Although metalaxyl is not currently applied to potatoes in the Columbia Basin to manage P. infestans, applications of metalaxyl are still used to manage pink rot (Phytophthora erythroseptica) and Pythium leak in potatoes. In the two Idaho fields containing MR Pythium isolates, resistance may have developed due to repeated use of this material to manage severe pink rot problems caused by P. erythroseptica. In contrast, although there is a history of metalaxyl use in western Washington, no MR Pythium isolates were detected in field soil from the present research or from soil and potato tubers in a separate study (D. A. Inglis, unpublished data) from that region. The MR resistant Pythium isolates appear to be confined to the sandy soil regions of the Columbia Basin in northern Oregon and central and southeastern Washington, and the Snake River Valley in Idaho (Fig. 1). Another possibility for the widespread development of MR isolates in the Columbia Basin could be due to the distribution of infected seed. Some countries recommend that metalaxyl not be applied to fields used for potato seed production due to the risk of selecting for MR biotypes of P. infestans that could then be disseminated to potato production areas where the seed is planted (Erwin and Ribeiro 1996). Exposure to metalaxyl in potato seed production areas could also lead to the development of MR Pythium spp. infecting potatoes that

The occurrence of metalaxyl-resistant Pythium species other than P. ultimum is considered to be very rare in fields cropped to potatoes in the PNW, since only two of the 64 fields where MR Pythium isolates were detected contained MR Pythium isolates that were not P. ultimum. MR P. ultimum isolates were most commonly isolated from soils cropped to potatoes in the Columbia Basin region of Washington and Oregon and rarely isolated from fields in Idaho (Fig. 1) at this time. Potential reasons for the widespread occurrence of metalaxyl resistance in the Columbia Basin of Washington and Oregon and not in Idaho, may be due to long term and repeated exposure of P. ultimum populations to metalaxyl in the Columbia Basin. Late blight, caused by the oomycete Phytophthora infestans, is an annual problem in the Columbia Basin of WA and OR (Johnson et al. 1997) and foliar applications of metalaxyl have been applied extensively to potatoes in this region, especially during the 1990s to manage this devastating disease (Johnson et al. 1997, 2000). It was common in the Columbia Basin in the early 1990s for one, two or more foliar applications of metalaxyl to be applied in one season. Metalaxyl applications to manage P. infestans were reduced in 1995 in the Columbia Basin due to the development of MR strains of P. infestans in 1993 (Deahl et al. 1993; Hamm et al. 1994). In contrast to the high use of metalaxyl in the Columbia Basin (Johnson et al. 1997), metalaxyl applications were not as common in the early 1990s in Idaho since widespread late blight outbreaks were infrequent (Henderson et al. 2007). However, the widespread and common use of Fig. 1 Incidence of metalaxylresistant (MR) Pythium ultimum isolates (black marks) and metalaxyl-resistant Pythium spinosum isolate (clear mark) in Idaho, Oregon and Washington of the Pacific Northwest. Shaded counties represent counties where soil samples were assayed for MR Pythium

1. Skagit, WA

1

2 3

13. Elmore, ID

2. Grant, WA

14. Blaine, ID

3. Adams, WA

15. Jerome, ID

4. Klickitat, WA

17. Twin Falls, ID

5. Benton, WA

18. Cassia, ID

6. Franklin, WA

19. Power, ID

7. Walla Walla, WA

20. Bingham, ID

8. Morrow, OR

21. Jefferson, ID

9. Umatilla, OR

22. Madison, ID

10. Jefferson, OR

23. Fremont, ID

11. Crook, OR

6 5

12. Klamath, OR

7

4 9 8

10 23

11 21 14

13

20

12

15 17

16 18

19

22

Am. J. Pot Res (2009) 86:315–326

323

Table 44 Year Year and andfield fieldidentification identificationnumber, number,county county andand state state where where metalaxyl-resistant field was located, isolates, total Pythium and percentage population of and the population level of metalaxyl resistant field resistance, was located, level of total metalaxyl Pythium sensitivity, population species and identification level of metalaxyl of metalaxyl-resistant from fields cropped isolates,toand potato percentage from 2004 of to the2006 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

2004-122 2004-126 2004-141 2004-154 2005-42 2006-183 2006-215 2006-219.3 2006-219.4 2006-219.5

Umatilla, OR Walla Walla, WA Morrow, OR Grant, WA Benton,WA Adams, WA Benton, WA Umatilla, OR Umatilla, OR Umatilla, OR

Total Pythiuma

MR Pythiumb

99 2812 430 230 363 366 73 214 196 284

83 2600 364 192 281 189 56 163 152 224

Metalaxyl sensitivityc

Pythium speciesd

HR HR HR HR HR IR HR HR HR HR

ultimum ultimum ultimum ultimum ultimum ultimum unknown ultimum ultimum ultimum

Percent MR populatione 83.8 92.5 84.7 83.5 54.0 51.6 76.7 76.8 77.6 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 metalaxylresistant 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 established that the movement of P. infestans from Mexico to Europe was most likely by the shipment of infected tubers (Goodwin and Drenth 1997). Information regarding seed source, soil type, fungicide application history, and crop rotations need further investigation to determine if there are correlations among these factors and the incidence of MR Pythium isolates in the PNW. Isolates that were rated IR were isolated from eleven fields representing all three states in the current study. Presence or absence of isolates with intermediate resistance may be associated with the heterozygous state of the gene conferring resistance to metalaxyl-M (Shattock 1988; Goodwin and McGrath 1995). Isolates with intermediate resistance were commonly found in the PNW and differences in fitness and aggressiveness between IR and HR isolates of P. ultimum need to be compared in future studies to determine potential differences in fitness and aggressiveness. During the course of this research two question were often raised by growers and crop consultants concerning the presence of MR isolates of P. ultimum in commercial potato fields: 1) should they discontinue the use of metalaxyl as a control option to manage Pythium leak?, and 2) should they refrain from using metalaxyl-treated seed that prevents seed rot and seedling damping off of crops grown in rotation with potatoes? Based on the present information, in many fields where MR Pythium isolates were detected, a substantial population of MS isolates were also present and could presumably be managed by applications of metalaxyl either as a foliar, in-furrow or seed treatment to protect potato tubers or seeds of various crops found in potato crop rotations. For example, in 2004 within Walla Walla County, WA (Table 3) three fields were surveyed.

The MR isolates represented 92.5%, 2.5% and 0% of the total Pythium soil population in fields 1, 2 and 3 respectively. The determination to continue or discontinue the use of metalaxyl to manage pathogenic Pythium may need to be determined on a field by field basis since MR populations can vary among fields. In field 1 of the example, the grower may seriously consider discontinuing the use of metalaxyl due to the high incidence of resistant isolates. In field 2, the grower may choose to monitor the effectiveness of metalaxyl since 97.5% of the population still appears to be MS. In field 3, the grower should have no reservations in using metalaxyl since there were no MR isolates recovered. More work is needed to clarify how long metalaxyl would still be efficacious in managing mixed populations of MR and MS Pythium isolates. Research assessing the development of metalaxyl resistance by MS Pythium isolates under laboratory conditions has previously been assessed (Bruin and Edgington 1981). Single isolates of P. aphanidermatum, P. arrhenomanes, P. graminicola, and P. vexans were exposed to varying concentrations of metalaxyl from 0.1µg/ml to 300µg/ml and the isolates were maintained on metalaxyl-amended agar and transferred 12 times over an 8 month period. EC50 values assessed following this metalaxyl exposure period were determined for these isolates and P. aphanidermatum, P. arrhenomanes, P. graminicola, and P. vexans went from pre-exposure EC50 values of 0.2µg/ml, 0.4µg/ml, 10µg/ml and 0.1µg/ml to post-exposure values of 25µg/ml, 45µg/ml, 140µg/ml, and 360µg/ml, respectively. However, after 12 consecutive transfers on non-amended medium, all the isolates lost most of this resistance returning to EC50 values of 0.8, not determined, 28, and 2, respectively. However, when three isolates of P. capsici were tested using the same methods as

324

those for the Pythium isolates, one isolate of P. capsici maintained a high resistance to metalaxyl (EC50 =300µg/ml), a second isolate maintained an intermediate resistance, and the third isolate returned to the original level of sensitivity, even after transfers on nonamended agar (Bruin and Edgington 1981). In addition, in field tests of isolates of Phytophthora parasitica var nicotianeae exposed to metalaxyl over a 3 year period, EC50 values steadily increased over the 3 year period as isolates of P. nicotianeae were continuously exposed to metalaxyl in the soil. EC50 values increased from 0.4µg/ml, 0.3µg/ml, 0.7µg/ml and 1.2µg/ml during years 0, 1, 2, and 3, respectively, in soil that had previously not been exposed to metalaxyl (Shew 1985). Thus, continuous exposure of sensitive oomycete populations to sublethal levels of metalaxyl in soil can result in reduced sensitivity by the pathogen to this fungicide. Sensitivity may be either temporary or permanent depending on the species. Research studying the buildup of MR Pythium ultimum populations in soil over time where metalaxyl continues to be used as a management tool will be valuable in addressing the rate of MR development and the permanence of metalaxyl resistance maintained by isolates in these soil populations. MS and MR isolates of P. ultimum did not appear to vary in pathogenicity to potato since 57 of 57 MS isolates and 32 of 33 MR isolates of P. ultimum were pathogenic on potato tubers, respectively, which may suggest an equal fitness for survival. However, P. ultimum isolates that were HR to metalaxyl were found in higher numbers in some fields than MS isolates which may indicate that MR isolates are more fit (Table 4). Development of fungicide resistance by a pathogen is often associated with a reduction in fitness when resistant isolates have been compared to sensitive isolates (Raposo et al. 2000; Dowley et al. 2002;Kadish and Cohen 1992). An example of this is when the survival of MR isolates of P. infestans was compared to MS isolates. MS isolates were determined to be more fit due to a reduction in the frequency of MR isolates when the use of metalaxyl was temporarily suspended (Dowley et al. 2002). In contrast, in several cases the fitness of MR isolates of oomycete plant pathogens has been similar, or better than, MS isolates (Gent et al. 2008; Café-Filho and Ristaino 2008; Crute and Harrison 1988; Gisi and Cohen 1996; Kadish and Cohen 1989; Porter et al. 2007). For example, MR isolates of Phytophthora infestans (Kadish and Cohen 1989) and Phytophthora erythroseptica (Porter et al. 2007) have demonstrated signs of being more fit and aggressive than MS isolates in sexual and asexual 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 recovered during this study. Among the Pythium spp. identified from field soils, in the present study, were MR isolates of Pythium spinosum. To our knowledge this is the first report of MR Pythium spinosum

Am. J. Pot Res (2009) 86:315–326

isolates identified from commercial agricultural field soil. P. spinosum is not considered to be a major pathogen of potato tubers in the PNW, but has been associated with root infections on bell peppers, corn, peanuts, soybeans and watermelon in the U.S. (Chellemi et al. 2000; Hollowell et al. 1998; Njoroge et al. 2008; Zhang et al. 1998). All MR isolates of P. spinosum were confined to a single field located in Klickitat County, WA. The crop history of this field needs to be evaluated to determine potential reasons for the occurrence of metalaxyl resistance in this specie and confirm whether this specie is impacting the production of potato or other crops. P. deliense, P. inflatum, P. irregulare, P. paroecandrum, P. sylvaticum and P. ultimum isolated from potato fields in 2006 all were consistently pathogenic on healthy wounded potato tubers (Table 2). P. deliense (Levesque et al. 1998), P. irregulare (Mendes et al. 1998), P. sylvaticum (Peters et al. 2005) and P. ultimum (Salas and Secor 2001) have previously been reported to cause potato tuber rot. However, this is the first report of isolates of P. inflatum and P. paroecandrum causing potato tuber rot. This demonstrates that other Pythium species besides those commonly associated with Pythium leak are capable of rotting tubers and a complete survey of Pythium species causing rot on potato tubers in the PNW needs to be assessed to fully understand all the pathogens involved. Studying the development of resistance by a pathogen to a specific fungicide on a large regional scale is necessary to understand, develop and improve fungicide resistance management programs. In addition, to determine how effective a fungicide resistance management program is, it is paramount that a resistance baseline be established. The present research established a baseline for the presence of MR Pythium in the PNW that can be used in the future to assess the continual spread and persistence of MR Pythium isolates in this region of the USA. Documented occurrence of resistant isolates to metalaxyl in potato production areas may help explain the success or failure of this fungicide in managing tuber rot and damping off of metalaxyl-treated seed of crops found in rotation with potato. Acknowledgements The authors would like to thank the National Potato Council for funding this research project, and Steve James Casey Royer, Brian Charlton and Mike Nielsen for collecting soil. The experiments associated with this research were in compliance with the laws of the U.S.A.

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