Ali Chenari Bouket

Asadollah Babai-Ahari

Mahdi Arzanlou

Tojo Motoaki

Tojo, Motoaki

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Morphological and preliminary molecular characterizations of low temperature Pythium

National Institute of Polar Research Repository

  Morphological and preliminary molecular characterizations of low temperature Pythium  from grasslands in East-Azarbaijan province, Iran   Ali Chenari Bouket1,2, Motoaki Tojo1, Mahdi Arzanlou2 and Asadollah Babai-Ahari2 1Graduate School of Life and Environment Sciences, Osaka Prefecture University, Sakai, Japan 2Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Iran   The Genus Pythium contains more than 140 recognized species and is distributed worldwide in diverse environments including the cryosphere (Tojo and Newsham 2012, Kageyama 2014). Some species of low temperature Pythium cause severer snow rot on golf course turfgrass (Masumoto et al. 2010) and natural vegetation (Tojo et al. 2012). Snow rot has commonly been found in agricultural and natural grasslands in East-Azarbaijan province (Fig. 6), Iran (Chenari Bouket and Arzanlou, unpublished). We recently isolated several species of low temperature Pythium from grasses with snow rot symptoms (Fig. 1). Present study reports their morphology and primary molecular examination as the first step to clarify their taxonomical status.  Materials and Methods Isolation. – All isolates were recovered from snow covered grasslands in East-Azarbaijan province, Iran. Pieces of diseased grass leaves (Lolium perenne) were plated in NARM (Morita and Tojo 2007) and VP3 (Ali-Shtayeh et al. 1986), oomycete-specific media and incubated at 4C for 3 weeks. After purification, cultures were maintained in PCA slant vials at 4C. Morphological observations were made on structures produced on PCA as well as those on sterile grass blades floated in sterile pond water. DNA extraction, PCR and Electrophoresis. – DNA was extracted from mycelia grown on PDA (Moller et al. 1992). The ITS region of rDNA was amplified by polymerase chain reaction using primers ITS1 and ITS4 (White et al. 1990). Amplification was carried out in a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA) according to the following amplification program: 5 min at 96 °C for primary denaturation, followed by 40 cycles of 94 ºC for 30 s, 52 ºC for 30 s, 72 ºC for 60 s, with a final extension at 72 ºC for 7 min. PCR products were examined in 1.2% agarose Gel, at ~ 100V for 1h with 1Kb DNA Ladder (BioTools).   Results and Discussion Preliminary molecular evaluation and morphological observations showed that at least three species of Pythium were present in rotted grass leaves under the snow in East-Azarbaijan province (Fig. 2 and Fig. 3 to 5). They were tentatively identified as Pythium spp. 1, 2, and 3. Pythium sp. 1 was characterized by filamentous, spherical and intercalary sporangia. Subspherical sporangia produce apical papilla (Fig. 3). Pythium sp. 2 was characterized by spherical sporangia, oogonium with one and two antheridia and subspherical, filamentous structures that resembled appresoria. Sporangia germinated through papillae (Fig. 4). Pythium sp. 3 was characterized by subspherical and spherical sporangia, oogonium with one and two antheridia and plerotic oospores (Fig. 5).        These results demonstrated that low temperature Pythium were present in East-Azarbaijan province, Iran. This is the first report of low temperature Pythium in Iran. Their phylogenetic characterization is under way.                                                                                 References Ali – Shtayeh MS, Len LC, Dick MW (1986) An improved method and medium for quantitative estimates of populations of Pythium species from soil. Transactions of the British Mycological Society 86:39–47. Kageyama, K. 2014. Molecular taxonomy and its application to ecological studies of Pythium species. Journal of General Plant Pathology. Published online, DOI 10.1007/s10327-014-0526-2.  Masumoto S, Shigyo T, Tojo M (2009) Pythium snow blight of Kentucky bluegrass turf in a golf course in Hokkaido, Japan. Journal of Japanese Society of Turfgrass Science 38: 33-36. Morita Y, Tojo, M (2007) Modifications of PARP Medium Using Fluazinam, Miconazole, and Nystatin for Detection of Pythium spp. in Soil. Plant Disease 91(12): 1591-1599.  Moller EM, Bahnweg G, Geiger HH (1992) A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissues. Nuclear Acid Research 20, 6115–6116.  Tojo M, Newsham KK (2012) Snow mould in polar environments. Fungal Ecology 5: 395 -402. Tojo M., Van West P., Hoshino T., Kida K., Fujii H., Hakoda H., Kawaguchi Y., Mühlhauser H.A., Van den Berg A.H., Küpper F.C., Herrero M.L., Klemsdal S.S., Tronsmo A.M., and Kanda H. (2012) Pythium polare, a new heterothallic Oomycete causing brown discoloration of Sanionia uncinata in the Arctic and Antarctic. Fungal Biology 116: 756-768, 2012. White TJ, Bruns TD, Lee SB, Taylor JW (1990) Amplification and sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR-Protocols and Applications – A Laboratory Manual (eds. N Innis, D Gelfand, J Sninsky, TC White). Academic Press, New York 315–322.      

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Morphological and preliminary molecular characterizations of low temperature Pythium

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