57PESTMANAGEMENT:DISEASESPythiumSpeciesAssociatedwithRiceStandEstablishmentProblemsinArkansasM.A.
Eberle, C.S. Rothrock, and R.D. CartwrightABSTRACTThe role of
seedling disease pathogens in stand-establishment problems of rice
was examined in feld 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 felds from these studies in Arkansas in 2006 and 2007
were identi-fed and characterized. Pythium isolates were evaluated
for pathogenicity (stand loss) andselected isolates wereidentifed
molecularlyusingthemitochondriallyencoded 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 in-cluded P. catenulatum, P. torulosum, and P. diclinum.
Virulence studies using isolates of different Pythium species in
artifcially infested, pasteurized soil confrmed 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 ar-rhenomanes was the most
frequently isolated and most virulent of the Pythium species in
Arkansas rice felds and also has been reported as an important
pathogen in other rice-production areas.INTRODUCTIONStand problems
consistently cause signifcant production losses and management
problems in Arkansas rice felds. Pythium species play an important
role in stand es-tablishment, especially under cool soil
temperatures (Rothrock et al., 2004). Previous research, funded by
the Arkansas Rice Research and Promotion Board, has identifed AAES
Research Series 56058cold-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 feld history, soil characteristics, and
environmental conditions shortly after planting
onstandestablishmentandtoidentifyandcharacterizeimportantPythiumspp.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.
PROCEDURESPythiumIsolatesPythium isolates were isolated from rice
seedlings from studies using 21 producers felds in 13 counties.
These studies included 9 feld experiments and controlled studies
using 12 soils and two environments. Fungicide seed treatment
trials in 2006 and 2007 were conducted in producers felds 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 felds 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
coleoptileswereassessedfordiseasefromallstudies.Rootsfromseednottreated
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
identifed to genus.IdentificationofPythiumSpeciesIdentifcation of
Pythium spp. was done using PCR-RFLP of the mitochondri-ally
encoded cytochrome oxidase II (cox II) gene. A total of 77 isolates
was chosen for identifcation. 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 amplifed
using Pythium-specifc 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 amplifed was digested with three
restric-tion enzymes: AluI , NlaIII, and RsaI. RFLP analyses were
conducted following the 59B.R. Wells Rice Research Studies
2007procedure of Martin and Tooley (2004). The Pythium isolates
were grouped according to banding patterns for identifcation
against known banding patterns (Fig. 1). In addi-tion, selected
isolates for a group were sent for sequencing of the ITS region to
assist in molecular identifcation.PathogenicityTestsPathogenicity
of Pythium isolates was evaluated initially in sterilized
vermiculite.
Pathogenicitywasdeterminedusingstandcountsfrominfestedpotscomparedtoa
noninfested control, after analysis of stands by the GLM procedure
using SAS.Identifed isolates of Pythium were evaluated for
virulence under two environ-ments 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 satura-tion. 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
har-vested 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.RESULTSANDDISCUSSIONPythium spp. were the most frequently
isolated organisms from rice seedlings. Isolation of Pythium spp.
from feld studies ranged from 44% of the seedlings in Faulkner Co.
to 100% of the seedlings in 4 of the 9 feld studies (Table 1).
Isolation was consis-tently high for both the cold- and
warm-environmental studies (Table 2). Results from
thesestudiesconductedin2006and2007wereingeneralagreementwithprevious
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
tothenoninfested control.Isolatesweretaken
fromeachgroupforidentifcation of species, emphasizing isolates in
the virulent
group.MoleculartechniqueswereusedtoidentifythedifferentPythiumspeciesiso-lated
from rice seedlings. Treatment of the DNA fragments of the mtDNA
cox II gene with the three enzymes allowed the differentiation and
identifcation of the different
isolatestocorrespondingspecies.Fromtheisolatesidentifed,fvedifferentspecies
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
speciesincludedPythiumtorulosum,Pythiumcatenulatum,Pythiumarrhenomanes,
and Pythium diclinum.The number of surviving plants was greater in
the warm environment compared
tothecoolenvironment,indicatingthatdiseasecausedbythesePythiumspeciesis
AAES Research Series 56060favored 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 environ-ment 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 environ-ments 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 inproducersfeldsin
Arkansas,hasbeenreportedtobeanimportantseedlingrice 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.SIGNIFICANCEOFFINDINGSField and controlled
environmental studies examined soils from 21 producers felds in
2006 and 2007. The importance of different seedling pathogens in
stand losses is being identifed by examining stand response to
specifc fungicides and isolation of
pathogensinthesestudies.FivedifferentPythiumspecieshavebeenidentifedand
their importance characterized: Pythium arrhenomanes, P.
irregulare, P. torulosum, P. catenulatum, and P.
diclinum.ACKNOWLEDGMENTS
Thisresearchwasconductedwiththesupportofthe ArkansasRiceResearch
and Promotion Board. LITERATURECITEDCorrell, J.C., D.D. Rhoads, and
J.C. Guerber. 1993. Examination of mitochondrial DNA restriction
fragment length polymorphism, DNA fngerprints, and randomly
amplifed 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. Identifcation of
Phytophthora isolates to species level using restriction fragment
length polymorphism analysis of a polymerase chain
reaction-amplifed region of mitochondrial DNA. Phytopathology
94:983-991.61B.R. Wells Rice Research Studies 2007Rothrock, C.S.,
R.L. Sealy, F.N Lee, M.M. Anders, and R.D. Cartwright. 2004.
Reac-tion 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 Experi-ment
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.Reac-tion 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 Sta-tion Research
Series 529:120-124. Fayetteville, Ark.Rothrock, C.S., R.L. Sealy,
F.N. Lee, J. Gibbons, and R.D. Cartwright. 2006. Rela-tionship 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 ofPythium species
from feld studies.YearCountyPythium isolation(%)2006Faulkner
442006Clark 702006Desha 632006Prairie
582006Poinsett1002006Mississippi1002007Poinsett612007Jackson1002007Crittenden100Table
2. Isolation frequency of Pythium species fromsoils used in
controlled environmental studies.Pythium
isolationYearCountyColdWarm -----------
(%)------------2006Clark95852006Prairie85942006Desha92902006Poinsett87902006Lonoke83832006Lafayette68982007Independence98972007Lonoke97992007Jackson89942007Poinsett84862007Cross81602007Jefferson8097AAES
Research Series 56062Fig. 1. RFLP banding pattern for Pythium
species using three enzymes: A = NlaIII,B = AluI, and C = RsaI.
Banding patterns for Pythium arrhenomanesare indicated.Fig. 2. Rice
plant stand in soil infested withselected Pythium species under two
environments.63B.R. Wells Rice Research Studies 2007Fig. 3. Root
weight of rice seedlings in soil infestedwith selected Pythium
species under two environments.Fig. 4. Root discoloration (%) of
rice seedlings in soilinfested with selected Pythium species under
two environments.