PEST MANAGEMENT: DISEASES

Pythium Species Associated with Rice

Stand Establishment Problems in Arkansas
M.A. Eberle, C.S. Rothrock, and R.D. Cartwright
ABSTRACT
The role of seedling disease pathogens in stand-establishment problems of rice
was examined in field and controlled environmental studies using selective fungicides
and pathogens isolated from seedlings. Pythium spp. were the most common group of
seedling disease pathogens isolated in these studies. The Pythium spp. recovered from
over 20 producers fields from these studies in Arkansas in 2006 and 2007 were identified and characterized. Pythium isolates were evaluated for pathogenicity (stand loss)
and selected isolates were identified molecularly using the mitochondrially encoded
cytochrome oxidase II gene (mtDNA cox II). Pathogenic Pythium species were found
to be P. arrhenomanes and P. irregulare. Non- or less-pathogenic Pythium species included P. catenulatum, P. torulosum, and P. diclinum. Virulence studies using isolates
of different Pythium species in artificially infested, pasteurized soil confirmed that P.
arrhenomanes and P. irregulare caused greater stand losses than the other species, as
well as reduced plant weight and development of the surviving seedlings. Pythium arrhenomanes was the most frequently isolated and most virulent of the Pythium species
in Arkansas rice fields and also has been reported as an important pathogen in other
rice-production areas.
INTRODUCTION
Stand problems consistently cause significant production losses and management
problems in Arkansas rice fields. Pythium species play an important role in stand establishment, especially under cool soil temperatures (Rothrock et al., 2004). Previous
research, funded by the Arkansas Rice Research and Promotion Board, has identified
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cold-tolerant Pythium-resistant rice genotypes that hold the promise for more reliable
stand establishment for rice in Arkansas under marginal planting environments (Rothrock
et al., 2005; Rothrock et al., 2006).). These studies were designed to clarify the role of
field history, soil characteristics, and environmental conditions shortly after planting
on stand establishment and to identify and characterize important Pythium spp. and
other seedling pathogens. The objective of this portion of the study was to identify the
different Pythium spp. associated with seedling diseases of rice and characterize their
virulence on rice under different environmental conditions.
PROCEDURES
Pythium Isolates
Pythium isolates were isolated from rice seedlings from studies using 21 producers
fields in 13 counties. These studies included 9 field experiments and controlled studies
using 12 soils and two environments.
Fungicide seed treatment trials in 2006 and 2007 were conducted in producers
fields using the cultivar Wells. After 4 to 5 weeks, three 1-meter stand counts were
taken for each plot and approximately 25 seedlings were dug from the nontreated plots
for disease assessment and isolation of pathogens.
Soils were collected from six producers fields in 2006 and 2007 for the controlled
environmental studies. Two environments were used; cool/wet and warm/dry. Three
cultivars were used (Francis, Wells, and Cheniere) and four fungicide seed treatments
were used. Stand counts were taken and seedlings were removed from all containers
after 21 days in the warm environment and 28 days in the cool environment.
Rice seedlings were washed for 20 minutes in running tap water and roots and
coleoptiles were assessed for disease from all studies. Roots from seed not treated
with fungicides were disinfested in 0.5% NaOCl, blotted dry, and plated on water agar
(WArad). After 3 to 5 days, unique colony growth was transferred to potato dextrose
agar (PDArad) and identified to genus.
Identification of Pythium Species
Identification of Pythium spp. was done using PCR-RFLP of the mitochondrially encoded cytochrome oxidase II (cox II) gene. A total of 77 isolates was chosen
for identification. Each isolate was grown in V8 Juice-broth and the mycelium was
lyophilized and ground in liquid nitrogen. The mitochondrial DNA was extracted by a
procedure previously described by Correll et al. (1993).
The mitochondrially encoded cytochrome oxidase II gene (mtDNA cox II) was
amplified using Pythium-specific primers developed by Dr. Frank Martin (USDA-ARS,
Salinas, Calif.). The primers, PyRFLP-1 and PyRFLP-2, amplify the cox II gene and
the spacer of the mitochondrially encoded cox I and II gene clusters (Martin, 2000).
Each mtDNA cox II gene fragment that was amplified was digested with three restriction enzymes: AluI , NlaIII, and RsaI. RFLP analyses were conducted following the
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procedure of Martin and Tooley (2004). The Pythium isolates were grouped according
to banding patterns for identification against known banding patterns (Fig. 1). In addition, selected isolates for a group were sent for sequencing of the ITS region to assist
in molecular identification.
Pathogenicity Tests
Pathogenicity of Pythium isolates was evaluated initially in sterilized vermiculite.
Pathogenicity was determined using stand counts from infested pots compared to a
noninfested control, after analysis of stands by the GLM procedure using SAS.
Identified isolates of Pythium were evaluated for virulence under two environments by infesting a pasteurized soil from the Rice Research and Extension Center,
Stuttgart. The warm-environment experiments were in a greenhouse at 22.4C day and
17.1C night and soil water content was maintained between -30 Joules/kg and saturation. The cool-environment experiments were in growth chambers; two weeks at 15C
and three weeks at 20C. The soil moisture content was monitored gravimetrically and
maintained between -10 Joules/kg and saturation. Surviving rice seedlings were harvested and evaluated for root disease severity and plant development. Each experiment
was a randomized complete block design with four replications. Data were analyzed
using the GLM in SAS.
RESULTS AND DISCUSSION
Pythium spp. were the most frequently isolated organisms from rice seedlings.
Isolation of Pythium spp. from field studies ranged from 44% of the seedlings in Faulkner
Co. to 100% of the seedlings in 4 of the 9 field studies (Table 1). Isolation was consistently high for both the cold- and warm-environmental studies (Table 2). Results from
these studies conducted in 2006 and 2007 were in general agreement with previous
results that Pythium spp. play a large role in stand establishment.
Initial pathogenicity experiments divided isolates into three groups: virulent (little
or no stand), semi-virulent (moderate stand loss), and avirulent (no stand loss) relative
to the noninfested control. Isolates were taken from each group for identification of
species, emphasizing isolates in the virulent group.
Molecular techniques were used to identify the different Pythium species isolated from rice seedlings. Treatment of the DNA fragments of the mtDNA cox II gene
with the three enzymes allowed the differentiation and identification of the different
isolates to corresponding species. From the isolates identified, five different species
have been found. Pythium arrhenomanes and Pythium irregulare were found to be the
most virulent and common species. Semi-virulent to avirulent species or less common
species included Pythium torulosum, Pythium catenulatum, Pythium arrhenomanes,
and Pythium diclinum.
The number of surviving plants was greater in the warm environment compared
to the cool environment, indicating that disease caused by these Pythium species is

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favored by a cooler environment (Fig. 2). In addition to affecting stand, these isolates
also decreased root growth compared to the control (Fig. 3). Root weight decreased
drastically in the cool environment, including the control, showing that the environment also has an effect on rice development. Environment did not have much effect
on root discoloration, but species did (Fig. 4). Even species that did not have a large
impact on plant stand or seedling growth compared to the noninfested control caused
substantial root discoloration.
These studies demonstrated that Pythium spp. are a major part of the seedling
disease complex on rice. Rice planted and emerging under cooler and wetter environments is likely to suffer greater losses from Pythium spp. These studies also indicate
that these pathogens reduce root growth and plant development even at warmer soil
temperatures. P. arrhenomanes, one of the most important and common species found
in producers fields in Arkansas, has been reported to be an important seedling rice
pathogen in other states and countries. This research also indicated that a number of
other Pythium spp. may be important seedling disease pathogens in Arkansas.
SIGNIFICANCE OF FINDINGS
Field and controlled environmental studies examined soils from 21 producers
fields in 2006 and 2007. The importance of different seedling pathogens in stand losses
is being identified by examining stand response to specific fungicides and isolation of
pathogens in these studies. Five different Pythium species have been identified and
their importance characterized: Pythium arrhenomanes, P. irregulare, P. torulosum, P.
catenulatum, and P. diclinum.
ACKNOWLEDGMENTS
This research was conducted with the support of the Arkansas Rice Research
and Promotion Board.
LITERATURE CITED
Correll, J.C., D.D. Rhoads, and J.C. Guerber. 1993. Examination of mitochondrial
DNA restriction fragment length polymorphism, DNA fingerprints, and randomly
amplified polymorphic DNA of Colletotrichum orbiculare. Phytopathology
83:1199-1204.
Martin, F.N. 2000. Phylogenetic relationship among Pythium species inferred from
sequence analysis of the mitochondrially encoded cytochrome oxidase II gene.
Mycologia 92:711-727.
Martin, F.N. and P.W. Tooley. 2004. Identification of Phytophthora isolates to species
level using restriction fragment length polymorphism analysis of a polymerase
chain reaction-amplified region of mitochondrial DNA. Phytopathology 94:983991.
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B.R. Wells Rice Research Studies 2007

Rothrock, C.S., R.L. Sealy, F.N Lee, M.M. Anders, and R.D. Cartwright. 2004. Reaction of cold-tolerant adapted rice cultivars to seedling disease caused by Pythium
species. In: R.J. Norman, J.-F. Meullenet, and K.A.K. Moldenhauer (eds.). B.R.
Wells Rice Research Studies 2003. University of Arkansas Agricultural Experiment Station Research Series 517:207-210. Fayetteville, Ark.
Rothrock, C.S., R.L. Sealy, F.N. Lee, M.M. Anders, and R.D. Cartwright. 2005.Reaction of cold-tolerant rice genotypes to seedling disease caused by Pythium species.
In: R.J. Norman, J.-F. Meullenet, and K.A.K. Moldenhauer (eds.). B.R. Wells
Rice Research Studies 2004. University of Arkansas Agricultural Experiment Station Research Series 529:120-124. Fayetteville, Ark.
Rothrock, C.S., R.L. Sealy, F.N. Lee, J. Gibbons, and R.D. Cartwright. 2006. Relationship of cold-tolerance and Pythium resistance to rice stand establishment. In:
R.J. Norman, J.-F. Meullenet, and K.A.K. Moldenhauer (eds.). B.R. Wells Rice
Research Studies 2004. University of Arkansas Agricultural Experiment Station
Research Series 540:138-142. Fayetteville, Ark.
Table 1. Isolation frequency of
Pythium species from field studies.
Year

County

Pythium isolation

(%)

2006
2006
2006
2006
2006
2006
2007
2007
2007

Faulkner
Clark
Desha
Prairie
Poinsett
Mississippi
Poinsett
Jackson
Crittenden

44
70
63
58
100
100
61
100
100

Table 2. Isolation frequency of Pythium species from

soils used in controlled environmental studies.

Year

Pythium isolation
County

Cold

Warm

------------ (%)-----------2006
2006
2006
2006
2006
2006
2007
2007
2007
2007
2007
2007

Clark
Prairie
Desha
Poinsett
Lonoke
Lafayette
Independence
Lonoke
Jackson
Poinsett
Cross
Jefferson

95
85
92
87
83
68
98
97
89
84
81
80

85
94
90
90
83
98
97
99
94
86
60
97

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Fig. 1. RFLP banding pattern for Pythium species using three enzymes: A = NlaIII,
B = AluI, and C = RsaI. Banding patterns for Pythium arrhenomanes are indicated.

Fig. 2. Rice plant stand in soil infested with

selected Pythium species under two environments.

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Fig. 3. Root weight of rice seedlings in soil infested

with selected Pythium species under two environments.

Fig. 4. Root discoloration (%) of rice seedlings in soil

infested with selected Pythium species under two environments.

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