J Gen Plant Pathol (2005) 71:174–182 © The Phytopathological Society of Japan DOI 10.1007/s10327-005-0184-5 and Springer-Verlag Tokyo 2005 FUNGAL DISEASES Koji Kageyama · Ayako Nakashima · Yuki Kajihara Haruhisa Suga · Eric B. Nelson Phylogenetic and morphological analyses of Pythium graminicola and related species Received: May 19, 2004 / Accepted: September 19, 2004 Abstract Isolates of Pythium graminicola and related spe- matum with large oogonia and aplerotic oospores was cies were differentiated using restriction fragment length not related to the morphologically similar species P. polymorphism (RFLP) analyses of the internal transcribed myriotylum. Results suggest that P. graminicola and related spacer (ITS) regions of rDNA and the cytochrome c oxi- species are phylogenetically distinct, and molecular analy- dase subunit II (COX II) gene. These sequences were used ses, in addition to morphological analyses, are necessary in subsequent phylogenetic analyses. Finally, the phylo- for the accurate taxonomic placement of species in this genetic placement of species was compared to that deter- complex. mined from morphological characteristics. The 62 isolates tested were divided into seven groups, A–G, based on Key words Pythium graminicola · rDNA-ITS regions · RFLP analysis of the rDNA-ITS region. In the RFLP analy- Cytochrome oxidase gene · Phylogenetic relationship · sis of the COX II gene, isolates were divided into groups Morphology · Identification similar to those based on ITS-RFLP. Groups A and B were each separated into two additional subgroups. Grouping of isolates based on RFLP analyses agreed with the morphological differentiation. Groups A, B, D, E, F, and Introduction G were identified as P. graminicola, P. arrhenomanes, P. aphanidermatum, P. myriotylum, P. torulosum, and The genus Pythium is complex, containing more than 100 P. vanterpoolii, respectively. Group C was closely related species and consisting of plant and animal pathogens, to group B based on phylogenetic analysis of the rDNA- mycoparasites, and saprophytes. The identification of ITS region and the COX II gene and is similar to P. Pythium species has been based traditionally on mor- arrhenomanes. Each of the other species occupied their own phological characteristics (Dick 1990; Matthews 1931; individual clades. Although P. arrhenomanes is morpho- Middleton 1943; Van der Plaats-Niterink 1981; Waterhouse logically similar to P. graminicola, our phylogenetic analy- 1967). The major morphological criteria for species ses revealed that it was evolutionarily distant from P. identification are based on qualitative characteristics that graminicola and more closely related to P. vanterpoolii. Our may vary depending on the culture conditions and the iso- analysis also revealed that P. torulosum with smaller oogo- late tested (Dick 1990; Matthews 1931; Middleton 1943; nia is more closely related to P. myriotylum with large oogo- Van der Plaats-Niterink 1981; Waterhouse 1967). The event nia than to P. vanterpoolii, which forms smaller oogonia and makes accurate species placement extremely confusing and is morphologically similar to P. torulosum. P. aphanider- difficult, although species identification is one of the most important for diagnosing diseases and developing control strategies. K. Kageyama (*) · A. Nakashima · Y. Kajihara Pythium graminicola Subramaniam, one of the more River Basin Research Center, Gifu University, 1-1 Yanagido, Gifu important plant pathogenic species, is distributed world- 501-1193, Japan wide. The pathogen causes seed and root rots and seedling Tel. ϩ81-58-293-2063; Fax ϩ81-58-293-2062 e-mail: [email protected] damping-off especially in gramineous crops such as rice, corn, sugarcane, and turf grasses. P. graminicola belongs to H. Suga Life Science Research Center, Gifu University, Gifu, Japan a large group of species with smooth-walled oogonia and inflated filamentous sporangia (Van der Plaats-Niterink E.B. Nelson 1981). This group contains other important plant patho- Department of Plant Pathology, New York State College of Agriculture and Life Science, Cornell University, Ithaca, New York, genic species, such as P. arrhenomanes Drechsler, P. USA aphanidermatum (Edson) Fitzp, P. myriotylum Drechsler, 175 and P. vanterpoolii V. Kouyeas and H. Kouyeas. These were transferred to potato dextrose broth (Difco, Detroit, species are distinguished on the basis of various quantitative MI, USA). After 7–10 days of incubation in the dark characteristics, such as the number of antheridia per oogo- at 25°C, mycelial mats were collected on filter paper placed nium and the size of the oogonia, each of which often in a Buchner funnel and rinsed several times with distilled displays considerable variation among isolates, culture water. After removing excess water, mycelial mats were conditions, and growth media, making it difficult to assign frozen at Ϫ80°C for at least 1 day and ground to powder species to this group accurately. P. graminicola and P. with a sterilized, prefrozen mortar and pestle. The mycelial arrhenomanes have often been confused with one another powder was suspended in 250µl of extraction buffer because of the variability in the number of antheridia per (100mM Tris·HCl (pH 9.0), 40mM EDTA), 50µl of 10% oogonium. It is necessary in plant pathology and in Pythium sodium dodecyl sulfate (SDS), 10µl of 20% skim milk solu- taxonomy to know whether these species are the same or tion (Difco), and 5µl of RNase A (10mg/ml) (Nippongene, they should be separated. Toyama, Japan) and then vigorously vortexed for 1min. Restriction fragment length polymorphic (RFLP) analy- Benzyl chloride (150µl) was added to the tube, which was sis of the internal transcribed spacer (ITS) regions between vigorously vortexed again for 2min and then incubated nuclear ribosomal RNA genes (rDNA) has become a useful at 50°C for 1h. Following this incubation, 150µl of 3M tool for identifying closely related Pythium species (Chen NaOAc was added to the suspension, lightly vortexed, and Hoy 1993; Matsumoto et al. 2000) that lack sexual and incubated on ice for 15min. This suspension was organs (Kageyama et al. 1998, 2003). Moreover, the analysis cleared by centrifugation at 18000g for 10min, and the of ITS sequences has provided a means of clarifying supernatant was transferred to a new tube. This step was phylogenic relationships in the genus Pythium (Matsumoto repeated twice. DNA was subsequently precipitated with et al. 1999). These data suggest that the sporangial form is two volumes of cold ethanol and collected by centrifugation the most important characteristic in Pythium taxonomy. at 18000g for 20min. The resulting pellet was rinsed with The cytochrome oxidase subunit II (COX II) gene is a 70% ethanol and dried under vacuum. The DNA pellet was housekeeping gene and is thought to accumulate mutations dissolved in 200µl of TE buffer/10mM Tris·HCl (pH 7.5), through evolution, indicating that this gene might be useful 1mM EDTA. for determining phylogenic relationships. Moreover, be- cause the gene is encoded in mitochondria, it may clarify phylogenic relationships among the species in relation to RFLP analyses of rDNA-ITS regions and COX II gene another genetic background. In species of both Pythium and Phytophthora, sequence analyses of the COX II gene The ITS region of nuclear rDNA was amplified with primer corroborates findings from sequence analysis of the ITS pair ITS1–ITS4 described by White et al. (1990) and the region (Martin 2000; Martin and Tooley 2003). COX II gene with FM58 and FM66 described by Martin The objectives of the present study were to distinguish (2000). The 50-µl reaction mixture contained 1µM of each isolates of P. graminicola from closely related species using primer, 1.25 units of rTaq DNA polymerase (Takara Shuzo, RFLP analyses of the ITS regions of rDNA and of the Shiga, Japan), 0.2mM dNTP mixture, 1ϫ polymerase chain COX II gene, determine phylogenic relationships based reaction (PCR) buffer (10mM Tris·HCl, pH 8.3, 50mM on these sequences, and compare sequence analyses with KCl, and 1.5mM MgCl2), and 200ng of DNA template. morphological characteristics as a means of identifying P. Reactions were carried out with a DNA Thermal Cycler graminicola and related species. (Applied Biosystems, Norwalk, CT, USA). The tempera- ture cycling parameters for the ITS regions were pro- grammed for one cycle of 3min at 94°C followed by 30 Materials and methods cycles of 1min at 94°C, 1min at 55°C, 2min at 72°C, and one cycle of 10min at 72°C. The program for the COX II Isolates was the same as the one for the ITS regions except for the annealing temperature at 52°C. PCR products were Thirty isolates of Pythium graminicola, seven of P. electrophoresed in 1.2% agarose LO3 (Takara Shuzo) gel aphanidermatum, five each of P. aristosporum and P. in TAE buffer (40mM Tris·HCl, pH 7.5, 19mM glacial arrhenomanes, ten of P. myriotylum, two of P. torulosum, acetic acid, and 2mM EDTA) and then stained with and three of P. vanterpoolii were used in this study (Table ethidium bromide. The amplified DNA was used for restric- 1). A P. dissotocum isolate was used as outgroup for phylo- tion enzyme analysis. Digestions with four restriction genetic analysis. Many isolates were collected from various enzymes – EcoRI, TaqI, HaeIII, HinfI (Toyobo, Osaka, hosts and geographic origins and were maintained on corn- Japan) – were carried out for the ITS regions according meal agar (CMA) at 25°C. to the manufacturer’s specifications and three additional enzymes – HhaI, MboI, RsaI – were used for the COX II gene. The restriction fragments were electrophoresed DNA extraction in 3.5% NuSieve (3:1) agarose gel (FMC BioProducts, Rockland, MN, USA) in TAE buffer followed by staining Three agar plugs were removed from the growing margin of with ethidium bromide and visualizing under ultraviolet 2-day-old cultures on CMA using a 1-cm cork borer and light and photography. 176 Table 1. Pythium isolates belonging to each DNA group (see Table 2) DNA Species Isolate Host/habitat Locale Sourcea Accession no.