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.