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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
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-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
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
Am. J. Pot Res (2009) 86:315–326
DOI 10.1007/s12230-009-9085-z
L. D. Porter (*)
Vegetable and Forage Crops Research Unit, USDA-ARS,
24106 N. Bunn Road,
Prosser, WA 99350, USA
e-mail: lyndon.porter@ars.usda.gov
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: philip.b.hamm@oregonstate.edu
N. L. David
Department of Plant Sciences,
North Dakota State University,
NDSU, Dept. # 7670,
P.O. Box 6050, Fargo, ND 58108, USA
e-mail: nicholas.david@ndsu.edu
J. S. Miller
Miller Research LLC,
1175 E. 800 N,
Rupert, ID 83350, USA
e-mail: jeff@millerresearch.com
B. Gundersen :D. A. Inglis
NWREC, Washington State University,
16650 State Route 536,
Mount Vernon, WA 98273, USA
B. Gundersen
e-mail: gunde@cahnrs.wsu.edu
D. A. Inglis
e-mail: dainglis@wsu.edu
y Washington. Nueve especies de Pythium fueron recuper-
adas de muestras de suelo, de las cuales fueron identifica-
das P. ultimum yP. 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 patho-
gens 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 pre-
emergence 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.
Metalaxyl is a racemic fungicide that contains both R- and
S-enantiomers. Metalaxyl-M which contains 98% of the R-
enantiomer (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 oomy-
cete 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 appli-
cation 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 recov-
ered 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 con-
firmed in at least seven species of Phytophthora (Bruin and
Edgington 1981;FerrinandKabashima1991; 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;Hammetal.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
316 Am. J. Pot Res (2009) 86:315–326
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 metalaxyl-
sensitive (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 con-
sisted 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
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
thenaddedtoeachoffourPetriplates(100mmwide×
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-metalaxyl-
amended 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 individ-
ual fields was confirmed by transferring approximately 3 mm
3
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 0 µg/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;
Am. J. Pot Res (2009) 86:315–326 317
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
3mm
3
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. Represen-
tative 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 non-
metalaxyl-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.
Pathogenicity Test
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
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
Collection of Samples and Population Counts
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, represent-
ing 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
318 Am. J. Pot Res (2009) 86:315–326
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
Field #/year
a
County/state
b
Metalaxyl sensitivity
c
Pathogenicity score
d
2/2005 Benton, WA HR 2
7/2005 Klickitat, WA HR 2
8/2005 Benton, WA HR 2
19/2005 Walla Walla, WA HR 2
22/2005 Umatilla, OR HR 2
23/2005 Morrow, OR HR 2
26/2005 Morrow, OR HR 2
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
168/2006 Grant, WA HR 0
173/2006 Grant, WA HR 2
178/2006 Adams, WA HR 2
181/2006 Adams, WA HR 2
184/2006 Adams, WA HR 2
187/2006 Adams, WA HR 2
190/2006 Adams, WA HR 2
192/2006 Adams, WA HR 2
199/2006 Adams, WA HR 2
202/2006 Franklin, WA HR 2
203/2006 Adams, WA HR 2
206/2006 Benton, WA IR 2
208/2006 Benton, WA HR 2
209/2006 Benton, WA HR 2
213/2006 Umatilla, OR HR 2
214/2006 Umatilla, OR HR 2
215/2006 Umatilla, OR HR 2
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 metalaxyl-
resistant 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
Pathoge-
nicity 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
Am. J. Pot Res (2009) 86:315–326 319
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. i r r e gu l a r e 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. paroecan-
drum 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 non-
inoculated 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.
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 1
a
(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”)
320 Am. J. Pot Res (2009) 86:315–326
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
fields
surveyed
No. of
MS
fields
a
No. of
IR
Fields
b
No. of
HR/UD
Fields
c
Mean (%) no. of IR/HR
isolates per gram of soil
from resistant fields
d
No. of fields with percentage
of IR/HR isolates per gram
of soil > 50%
e
High
resistance
f
Low
resistance
g
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 NA
h
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 10 µg/ml but did not grow at 100 µg/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
Am. J. Pot Res (2009) 86:315–326 321
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.MRP.
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 applica-
tions 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
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
1
4
3
2
5
6
7
8
9
10
11
12
13 14
15
1. Skagit, WA 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
12. Klamath, OR
17 18
16 19
20
21 22
23
Fig. 1 Incidence of metalaxyl-
resistant (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
322 Am. J. Pot Res (2009) 86:315–326
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 resis-
tance by MS Pythium isolates under laboratory conditions has
previously been assessed (Bruin and Edgington 1981). Single
isolates of P. aphanidermatum, P. arrhenomanes, P. grami-
nicola, 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. EC
50
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 EC
50
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 trans-
fers on non-amended medium, all the isolates lost most of
this resistance returning to EC
50
values of 0.8, not
determined, 28, and 2, respectively. However, when three
isolates of P. c a p s ic i were tested using the same methods as
Table 4 Year and field identification number, county and state where field was located, total Pythium population and level of metalaxyl
resistance, level of metalaxyl sensitivity, species identification of metalaxyl-resistant isolates, and percentage of the population resistant from
fields cropped to potato from 2004 to 2006 in Idaho, Oregon and Washington
Year-Field County, State Total Pythium
a
MR Pythium
b
Metalaxyl sensitivity
c
Pythium species
d
Percent MR population
e
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 (10 µg/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
Table 4 Year and field identification number, county and state where
field was located, total Pythium population and level of metalaxyl
resistance, level of metalaxyl sensitivity, species identification of
metalaxyl-resistant isolates, and percentage of the population resistant
from fields cropped to potato from 2004 to 2006 in Idaho, Oregon and
Washington
Am. J. Pot Res (2009) 86:315–326 323
those for the Pythium isolates, one isolate of P. capsici
maintained a high resistance to metalaxyl (EC
50
=300 µg/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 metal-
axyl over a 3 year period, EC
50
values steadily increased
over the 3 year period as isolates of P. nicotianeae were
continuously exposed to metalaxyl in the soil. EC
50
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;Dowleyetal.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)andPhytophthora 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.Toour
knowledge this is the first report of MR Pythium spinosum
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. s p i n o s u m 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. i n f l a t um ,P. i r re g u l a r e ,P. p a r o ec a n d r u m ,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. s y l v a t i c u m (Peters et al.
2005)andP. 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 associ-
ated 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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