J Gen Plant Pathol (2014) 80:66–72 DOI 10.1007/s10327-013-0477-z FUNGAL DISEASES Various species of Pyricularia constitute a robust clade distinct from Magnaporthe salvinii and its relatives in Magnaporthaceae Nobuaki Murata • Takayuki Aoki • Motoaki Kusaba Yukio Tosa • Izumi Chuma • Received: 19 March 2013 / Accepted: 28 June 2013 / Published online: 4 September 2013 Ó The Phytopathological Society of Japan and Springer Japan 2013 Abstract In a phylogenetic analysis of species of Magnaporthaceae based on nucleotide sequences of rDNA-ITS and the RPB1 gene, isolates of the tested species were divided into two clusters with high bootstrap support. One group was composed of Pyricularia spp.; the other was composed of Magnaporthe salvinii, M. rhizophila, M. poae, Gaeumannomyces graminis, and G. incrustans. On the basis of this result, we concluded that Pyricularia spp. constitute a large but distinct phylogenetic species group that is not congeneric with Magnaporthe salvinii, the type species of Magnaporthe. Keywords Magnaporthe
Pyricularia
Gaeumannomyces The generic name, Pyricularia, has been assigned to the anamorphs of filamentous fungi that cause blast disease on monocot species. The best known species is P. oryzae Cavara, pathogenic on staple crops including rice, wheat, Electronic supplementary material The online version of this article (doi:10.1007/s10327-013-0477-z) contains supplementary material, which is available to authorized users. N. Murata
Y. Tosa
I. Chuma (&) Laboratory of Plant Pathology, Graduate School of Agricultural Sciences, Kobe University, Kobe 657-8501, Japan e-mail: chuizm@kobe-u.ac.jp T. Aoki Genetic Resources Center, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan M. Kusaba Laboratory of Plant Pathology, Faculty of Agriculture, Saga University, Saga 840-8502, Japan 123 and millet (Kato et al. 2000). Among its close relatives are P. grisea Saccardo, pathogenic on crabgrass, Pyricularia sp. (LS) isolated from Leersia/Setaria, and Pyricularia sp. (CE) isolated from Cenchrus/Echinochloa (Hirata et al. 2007). These four species are members of the P. oryzae/ grisea species complex and indistinguishable in conidial morphology. Pyricularia also includes several species defined by morphological features such as P. zizaniaecola Hashioka, pathogenic on Zizania, P. zingiberi Y. Nisikado on Zingiber, P. higginsii Luttr. on Cyperus, Pyricularia sp. (SsPb) on Sasa/Phyllostachys (Hirata et al. 2007). In the 1970s, teleomorphs of P. grisea and P. oryzae were discovered in several laboratories (Hebert 1971; Kato et al. 1976; Ueyama and Tsuda 1975; Yaegashi and Nishihara 1976). These species produced nonstromatic black perithecia with long necks and four-celled, spindleshaped ascospores. The teleomorphs of P. grisea and P. oryzae were, at first, collectively designated as Magnaporthe grisea (T.T. Hebert) M.E. Barr (Barr 1977; Yaegashi and Udagawa 1978), but they are now called M. grisea and M. oryzae B.C. Couch, respectively (Couch and Kohn 2002). The blast fungi are primarily airborne and colonize leaves and panicles of host plants. However, the genus Magnaporthe also includes soilborne, root-infecting species such as M. rhizophila D.B. Scott & Deacon (Scott and Deacon 1983), and M. poae Landschoot & N. Jackson (Landschoot and Jackson 1989). In addition, the family Magnaporthaceae (Cannon 1994), typified by the genus Magnaporthe, includes Gaeumannomyces spp., widely distributed soilborne pathogens. These two groups (airborne and soilborne species) also differ in their anamorphs; the blast fungi produce pyriform conidia, whereas M. rhizophila, M. poae, and Gaeumannomyces spp. produce Phialophora-like conidia (Zhang et al. 2011). Furthermore, Species Isolatea Host Locality Isolation year Allele rDNA-ITS Pyricularia oryzae RPB1 Guy11 Oryza sativa Guiana (Combi) 1978 IT1 RP1 Ken54-20 (MAFF235005) O. sativa Japan (Yamaguchi) 1954 IT1 (AB274418) RP1 (AB818003) Ina72 (MAFF235003) O. sativa Japan (Nagano) 1957 IT2 (AB274420) RP1 0903-4 O. sativa Japan (Tochigi) 1976 IT3 (AB818014) RP1 CHNOS 59-6-1 O. sativa China (Yunnan) 1989 IT1 RP1 CHNOS 60-8-1 Br10 O. sativa O. sativa China (Yunnan) Brazil (Parana) 1989 1990 IT1 IT3 RP1 RP1 PO-04-7501 O. sativa Indonesia (Jawa Timur) 1975 IT1 RP1 GFSI1-7-2 Setaria italica Japan (Gifu) 1977 IT4 (AB274422) RP1 NRSI3-1-1 S. italica Japan (Nara) 1977 IT4 RP1 NNSI3-2-1 S. italica Japan (Nagano) 1984 IT4 RP1 IN77-20-1-1 S. italica India (Mysore) 1977 IT4 RP1 Z2-1 (MAFF244064) Eleusine coracana Japan (Kagawa) 1977 IT5 RP2 Br58 Avena sativa Brazil (Parana) 1990 IT5 (AB274424) RP2 (AB818004) Br7 Triticum aestivum Brazil (Parana) 1990 IT5 RP2 Br48 (IMI368172) T. aestivum Brazil (Mato Grosso do Sul) 1990 IT5 RP2 Br115.7 T. aestivum Brazil (Parana) 1992 IT5 RP2 Br118.2D T. aestivum Brazil (Parana) 1992 IT5 RP2 WK3-1 (MAFF244065) Lolium perenne Japan (Yamaguchi) 1996 IT5 RP2 Br35 Brachiaria plantaginea Brazil (Parana) 1990 IT4 RP2 NI986 (MAFF244066) Eragrostis lehmanniana Japan (Kumamoto) 1975 IT6 (AB818015) RP2 Pyricularia sp. (CE)b Br38 Echinochloa colonum Brazil (Parana) 1990 IT7 (AB818016) RP3 (AB818005) P. grisea Dig41 Digitaria sanguinalis Japan (Hyogo) 1990 IT8 (AB274428) RP4 (AB818006) NI907 D. sanguinalis Japan (Tochigi) 1974 IT9 (AB274429) RP4 IBDS4-1-1 (MAFF244068) D. sanguinalis Japan (Ibaraki) 1985 IT8 RP4 NI980 Digitaria smutsii Japan (Kumamoto) 1975 IT9 RP4 Br29 (IMI368175) Digitaria horizontalis Brazil (Sao Paulo) 1990 IT9 RP4 Br33 Digitaria horizontalis Brazil (Parana) 1990 IT10 (AB274430) RP4 P. zizaniaecola IBZL3-1-1 Zizania latifolia Japan (Ibaraki) 1985 IT11 RP5 KYZL201-1-1 Z. latifolia Japan (Kyoto) 2003 IT11 (AB274432) RP5 (AB818007) P. zingiberi HYZiM101-1-1-1 Zingiber mioga Japan (Hyogo) 1990 IT12 (AB274433) RP6 (AB818008) Z. mioga Japan (Hyogo) 2002 IT13 (AB274434) RP7 (AB818009) Z. mioga Japan (Hyogo) 2003 IT12 RP6 HYZiM201-1-1 Z. mioga Japan (Hyogo) 2003 IT13 RP6 67 123 HYZiM201-0-1 HYZiM202-1-2 J Gen Plant Pathol (2014) 80:66–72 Table 1 Isolates used in this study 123 Refer to Hirata et al. (2007) b Accession numbers in public culture collections are in parentheses. (MAFF, Microorganisms Section of the NIAS Genebank, National Institute of Agrobiological Sciences Tsukuba, Japan; IMI, CABI Bioscience, UK Centre, Egham, UK.) RP11 (AB818013) IT17 (AB274438) 2002 Japan (Hyogo) Cyperus iria HYCI201-1-1 P. higginsii a RP10 RP10 (AB818012) IT16 (AB818017) IT16 2003 2003 Japan (Fukuoka) Japan (Fukuoka) Kyllinga brevifolia K. brevifolia FKKB201-1-5 FKKB201-3-2 (MAFF244067) Pyricularia sp. (Kb)b RP8 (AB818010) IT14 (AB274435) IT15 (AB274436) 1993 1992 Japan (Aichi) Sasa sp. Phyllostachys bambusoides INA-B-92-45 INA-B-93-19 Pyricularia sp. (SsPb)b Japan (Aichi) rDNA-ITS Allele Isolation year Locality Host Isolatea Species Table 1 continued RP9 (AB818011) J Gen Plant Pathol (2014) 80:66–72 RPB1 68 M. salvinii (Catt.) R.A. Krause & R.K. Webster, the type species of the genus (Krause and Webster 1972), is different from both of the two groups; its infection cycle is primarily dependent on sclerotia that colonize the leaf sheath although it forms bicolored conidia. This circumstantial evidence led Zhang et al. (2011) to question whether Magnaporthe and Gaeumannomyces were monophyletic taxa. On the basis of the results from multilocus phylogenetic analyses, they considered that both Magnaporthe and Gaeumannomyces were polyphyletic and suggested that anamorphic and ecological features were more informative than the teleomorphic characters in defining monophyletic natural groups. However, they used only four M. oryzae/P. oryzae isolates as representatives of Pyricularia for their analysis. As mentioned already, several phylogenetically distinct, morphological species (Hirata et al. 2007) have been described in Pyricularia. Their teleomorphs have not yet been discovered, and their phylogenetic positions in Magnaporthaceae have been still unclear. At the nomenclatural sessions of the International Botanical Congress in Melbourne, 2011, it was decided that, after 1 January 2013, one fungus can only have one correct name (Hawksworth 2011) and that other names will be considered synonyms. Based on the new nomenclatural code for algae, fungi, and plants (McNeill et al. 2012), all legitimate fungal names are treated equally for the purposes of establishing priority (Wingfield et al. 2012). Therefore, P. oryzae and P. grisea as former anamorphic names may compete with former teleomorphic names M. oryzae and M. grisea, respectively, for priority. In the present study, we performed phylogenetic analyses using diverse blast fungi including various species. As a result of the obtained phylogenetic structure of Magnaporthaceae, we discuss which generic name should be adopted for the blast fungi, Magnaporthe or Pyricularia. The Pyricularia isolates tested are listed in Table 1. Genomic DNA was extracted as described by Nakayashiki et al. (1999). According to the number of informative sites reported by Zhang et al. (2011), two loci were selected for analysis: a portion of the nu-rRNA gene repeat (rDNA-ITS: ITS1, 5.8S and ITS2) and a portion of the largest subunit of RNA polymerase II gene (RPB1). The rDNA-ITS region was amplified with primers ITS5 (50 -GGAAGTAAAAG TCGTAACAAGG-30 ) and ITS4 (50 -TCCTCCGCTTATT GATATGC-30 ) (White et al. 1990) as described by Hirata et al. (2007). The RPB1 gene was amplified in a 20 lL reaction containing 1 U rTaq DNA polymerase (TOYOBO, Osaka, Japan), 1 9 PCR buffer provided by the manufacturer, 200 lM each dNTP, 0.2 lM primers RPB1-Ac (50 -G ARTGYCCDGGDCAYTTYGG-30 ) and RPB1-Cr (50 -CC NGCDATNTCRTTRTCCATRTA-30 ) (Zhang et al. 2011), 1.5 mM MgCl2, and 1 ng of template DNA using a Species Isolatea Host Locality GenBank accession rDNA-ITS Cryphonectria parasitica Gaeumannomyces graminis var. graminis EP155 Reference RPB1 Cryphonectria parasitica EP155 v2.0 Castanea dentata b M33 Stenotaphrum secundatum USA (Florida) JF710374 JF710442 Zhang et al. (2011) M53 unknown USA (Florida) JF414847 JF710443 Zhang et al. (2011) M54 unknown USA (Florida) JF414848 JF710444 Zhang et al. (2011) M57 Stenotaphrum secundatum USA (Florida) JF414849 JF710446 Zhang et al. (2011) JF710445 Zhang et al. (2011) G. graminis var. tritici M55 Triticum sp. USA (Montana) JF414850 G. incrustans R3-111a-1 M51 Triticum aestivum Zoysia matrella USA (Kansas) Magnaporthe comparative Database c JF414846 JF710440 Zhang et al. (2011) M26 (ATCC64417) Cynodon sp. USA (Kansas) JF414842 Magnaporthe oryzae M. poae M. rhizophila M. salvinii JF710435 Zhang et al. (2011) M35 unknown unknown JF414843 JF710437 Zhang et al. (2011) M45 Poa pratensis USA (New Jersey) JF414844 JF710438 Zhang et al. (2011) 70-15 – – Magnaporthe grisea Database d M25 Oryza sativa unknown JF414839 JF710449 Zhang et al. (2011) M60 Festuca arundinacea USA (New Jersey) JF414840 JF710447 Zhang et al. (2011) M61 Festuca arundinacea USA (New Jersey) JF414841 JF710448 Zhang et al. (2011) 73-15 (ATCC64411) Triticum aestivum Magnaporthe comparative Database c M1 unknown USA (New Jersey) JF414827 JF710425 Zhang et al. (2011) M12 Poa annua USA (Pennsylvania) JF414828 JF710426 Zhang et al. (2011) M14 unknown unknown JF414829 JF710427 Zhang et al. (2011) M15 Poa annua USA (Pennsylvania) JF414830 JF710428 Zhang et al. (2011) M16 unknown USA (Pennsylvania) JF414831 JF710429 Zhang et al. (2011) M17 M47 Poa annua Poa pratensis USA (New Jersey) USA (New Jersey) JF414832 JF414836 JF710430 JF710433 Zhang et al. (2011) Zhang et al. (2011) M48 Poa pratensis USA (New Jersey) JF414837 JF710434 Zhang et al. (2011) M22 unknown unknown JF414833 JF710431 Zhang et al. (2011) M23 Poa pratensis unknown JF414834 JF710432 Zhang et al. (2011) M46 Poa pratensis unknown JF414845 JF710439 Zhang et al. (2011) M21 (ATCC44754) Oryza sativa Japan JF414838 JF710441 Zhang et al. (2011) ATCC American type culture collection, Manassas, Virginia, USA b http://genomeportal.jgi-psf.org/Crypa2/Crypa2.home.html c http://www.broadinstitute.org/annotation/genome/magnaporthe_comparative/MultiHome.html d http://www.broadinstitute.org/annotation/genome/magnaporthe_grisea/MultiHome.html 69 123 a J Gen Plant Pathol (2014) 80:66–72 Table 2 GenBank accessions used in this study 70 J Gen Plant Pathol (2014) 80:66–72 Fig. 1 Maximum likelihood (ML) phylogenetic tree based on rDNAITS (ITS1, 5.8S and ITS2) and RPB1 nucleotide sequences of Magnaporthaceae isolates. The tree was rooted using Cryphonectria parasitica EP155 as an outgroup. Numbers at nodes represent bootstrap support [75 % from 500 replicates Mastercycler (Eppendorf, Hamburg, Germany). PCR cycling conditions for RPB1 were 1 min at 95 °C; 30 cycles of 1 min at 94 °C, 1 min at 60 °C, 1 min at 72 °C; and 5 min at 72 °C. PCR products were purified with USB ExoSAP-IT (Affymetrix, Santa Clara, CA, USA), and sequenced directly with the same primers as in the 123 J Gen Plant Pathol (2014) 80:66–72 71 amplification using BigDye Terminator v3.1 Cycle Sequencing Kit and ABI PRISM 3130xl Genetic Analyzer (Life Technologies, Carlsbad, CA, USA). The sequences were assembled with SeqMan II (DNASTAR, Madison, WI, USA) and deposited in GenBank (see Table 1). The nucleotide sequences of rDNA-ITS and RPB1 reported by Zhang et al. (2011) and those of model organisms (Cryphonectria parasitica EP155, Gaeumannomyces graminis var. tritici R3-111a-1, Magnaporthe oryzae 70-15 and M. poae ATCC 64411) were downloaded from GenBank and genome databases (Table 2). Nucleotide sequences of each locus were aligned with the program CLUSTAL W (Thompson et al. 1994), and manually optimized using the program MEGA 5.10 (Tamura et al. 2011). Combined alignments were analyzed using maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI) methods. Cryphonectria parasitica EP155 was used as an outgroup. The MP analysis was performed with MEGA 5.10 using the CloseNeighbor-Interchange (CNI) algorithm at a search level of 1. The initial tree for the CNI search was created by random addition for 10 replications. Nodal supports were assessed using 500 bootstrap replicates. The best-fit model for the ML and BI analyses was selected using the corrected Akaike information criteria in the program jModelTest 2.1.1 (Darriba et al. 2012; Guindon and Gascuel 2003). The ML analysis with the GTR ? G model was carried out using MEGA 5.10. Nodal supports were assessed using 500 bootstrap replicates. Bayesian analysis was conducted with the program MrBayes 3.2.1 (Ronquist et al. 2012) using the GTR ? G model and consisted of two runs of four chains each. The two runs were performed for 500,000 generations, sampling every 100 generations. Average standard deviations of split frequency values lower than 0.01 were taken as an indication that convergence had been achieved. After the first 1,250 trees were discarded, a 50 % majority rule consensus tree was constructed based on the remaining samples. The tree was visualized using the program FigTree v1.4.0 (available at http://tree.bio.ed.ac.uk/software). The ML tree is shown in Fig. 1. The isolates of Magnaporthaceae species tested were divided into two clusters with high bootstrap support. One cluster was composed of the blast fungi and harbored all of the morphologically distinct Pyricularia species (Fig. 2). The other was composed of soilborne pathogens, M. rhizophila, M. poae, G. graminis, and G. incrustans. Magnaporthe salvinii was included in the latter cluster. Gaeumannomyces was split into two subclusters; one was composed of all isolates of G. graminis var. graminis and G. graminis var. tritici, while the other was G. incrustans, grouped together with soilborne Magnaporthe species. Similar results were obtained from the MP and BI analyses (Online resources 1 and 2). As mentioned already, the species analyzed in the present study is divided into three groups based on the ecological and anamorphic features; (1) Pyricularia spp. mainly colonizing leaves and panicles, (2) Gaeumannomyces spp. and soilborne Magnaporthe spp. colonizing roots, and (3) M. salvinii colonizing leaf sheath. The present study showed that M. salvinii, the type species of Magnaporthe, is clustered with group (2) and distinct from group (1) (the blast fungi). The high bootstrap support at the nodes of the two clusters suggests that each of them is a monophyletic clade derived from a common ancestor. Although species classified in the same genus Magnaporthe are found in both clades, their differences in anamorphs and infection behaviors are not so trivial as those found among species of the same genus. We suggest that the two clades should logically be separated as distinct genera and have different generic names. Tsuda and Ueyama (1982), one of the three teams that discovered the teleomorph of P. oryzae (Ueyama and Tsuda 1975), found that the teleomorph of the blast fungus was different from typical Magnaporthe in morphology of ascospores, especially structure at their tips, and mode of their germination. Based on these observations, they concluded that the teleomorph of the blast fungus should not Fig. 2 Pyriform conidia produced by Pyricularia spp. a P. oryzae (Triticum isolate, Br48). b P. grisea (Digitaria isolate, Dig41). c P. zizaniaecola (Zizania isolate, KYZL201-1-1). d P. zingiberi (Zingiber isolate, TKZiM202-1). e P. higginsii (Cyperus isolate, HYCI201-1-1). Bars = 10 lm 123 72 be designated as Magnaporthe and argued that a new teleomorph genus should be established for it. Our phylogenetic analyses also suggest that Magnaporthe, typified by M. salvinii, should not be connected with the blast fungi, that have long been called Pyricularia species. Luo and Zhang (2013) suggested synonymizing M. salvinii to Nakataea oryzae (Cattaneo) J. Luo & Zhang, recombined from Sclerotium oryzae Cattaneo, an anamorphic synonym of M. salvinii. According to this treatment, the generic name Magnaporthe will correspondently be synonymized to Nakataea Hara. It should be noted that Nakataea is apparently different from Pyricularia, especially in conidial shape and pigmentation (Luo and Zhang 2013). Based on their morphological and phylogenetic considerations, they suggested that the blast fungus should not be congeneric with the type species of Magnaporthe. Taken together, we conclude that the blast fungi composed of various species constitute a large, but distinct phylogenetic group of fungi that is not congeneric with M. salvinii, the type species of Magnaporthe. If Magnaporthe is adopted as the generic name of the blast fungi, therefore, the type species of Magnaporthe must be replaced with some species in the clade of blast fungi. On the basis of these considerations, we propose that the clade of the blast fungi should be designated Pyricularia simply based on its priority, as suggested by Luo and Zhang (2013), and that the name Magnaporthe should be used for its type or closely related species in the M. salvinii clade. Acknowledgments We thank Dr. H. Kato, former professor of Kobe University, for providing the isolates and valuable suggestions. We also thank Dr. B. Valent, Kansas State University, for critical reading the manuscript and valuable suggestions and Dr. N. Zhang, Rutgers University for helpful comments and information that improved the manuscript. References Barr ME (1977) Magnaporthe, Telimenella, and Hyponectria (Physosporellaceae). Mycologia 69:952–966 Cannon PF (1994) The newly recognized family Magnaporthaceae and its interrelationships. Syst Ascomycet 13:25–42 Couch BC, Kohn LM (2002) A multilocus gene genealogy concordant with host preference indicates segregation of a new species, Magnaporthe oryzae, from M. grisea. Mycologia 94:683–693 Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9:772 Guindon S, Gascuel O (2003) A simple, fast and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704 Hawksworth DL (2011) A new dawn for the naming of fungi: impacts of decisions made in Melbourne in July 2011 on the future publication and regulation of fungal names. IMA Fungus 2:155–162 Hebert TT (1971) The perfect stage of Pyricularia grisea. Phytopathology 61:83–87 123 J Gen Plant Pathol (2014) 80:66–72 Hirata K, Kusaba M, Chuma I, Osue J, Nakayashiki H, Mayama S, Tosa Y (2007) Speciation in Pyricularia inferred from multilocus phylogenetic analysis. Mycol Res 111:799–808 Kato H, Yamaguchi T, Nishihara N (1976) The perfect state of Pyricularia oryzae Cav. in culture. Ann Phytopath Soc Jpn 42:507–510 Kato H, Yamamoto M, Yamaguchi-Ozaki T, Kadouchi H, Iwamoto Y, Nakayashiki H, Tosa Y, Mayama S, Mori N (2000) Pathogenicity, mating ability and DNA restriction fragment length polymorphisms of Pyricularia populations isolated from Gramineae, Bambusideae and Zingiberaceae plants. J Gen Plant Pathol 66:30–47 Krause RA, Webster RK (1972) The morphology, taxonomy, and sexuality of the rice stem rot fungus, Magnaporthe salvinii (Leptosphaeria salvinii). Mycologia 64:103–114 Landschoot PJ, Jackson N (1989) Magnaporthe poae sp. nov., a hyphopodiate fungus with a Phialophora anamorph from grass roots in the United States. Mycol Res 93:59–62 Luo J, Zhang N (2013) Magnaporthiopsis, a new genus in Magnaporthaceae (Ascomycota). Mycologia 105:1019–1029 McNeill J, Barrie FR, Buck WR, Demoulin V, Greuter W, Hawksworth DL, Herendeen PS, Knapp S, Marhold K, Prado J, Prud’homme van Reine WF, Smith GF, Wiersema JH, Turland NJ (eds) (2012) International Code of Nomenclature for algae, fungi, and plants (Melbourne Code) adopted by the Eighteenth International Botanical Congress Melbourne, Australia, July 2011. Koeltz Scientific Books, Koenigstein Nakayashiki H, Kiyotomi K, Tosa Y, Mayama S (1999) Transposition of the retrotransposon MAGGY in heterologous species of filamentous fungi. Genetics 153:693–703 Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61:539–542 Scott DB, Deacon JW (1983) Magnaporthe rhizophila sp.nov., a dark mycelial fungus with a Phialophora conidial state, from cereal roots in South Africa. Trans Br Mycol Soc 81:77–81 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680 Tsuda M, Ueyama A (1982) A comment from a taxonomical viewpoint on the perfect state of the blast fungus. Ann Phytopathol Soc Jpn (abstract in Japanese) 48:340 Ueyama A, Tsuda M (1975) Formation of the perfect state in culture of Pyricularia sp. from some gramineous plants (preliminary report). Trans Mycol Soc Jpn 16:420–422 White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, San Diego, pp 315–322 Wingfield MJ, De Beer ZW, Slippers B, Wingfield BD, Groenewald JZ, Lombard L, Crous PW (2012) One fungus, one name promotes progressive plant pathology. Mol Plant Pathol 13:604–613 Yaegashi H, Nishihara N (1976) Production of the perfect stage in Pyricularia from cereals and grasses. Ann Phytopathol Soc Jpn 42:511–515 Yaegashi H, Udagawa S (1978) The taxonomical identity of the perfect state of Pyricularia grisea and its allies. Can J Bot 56:180–183 Zhang N, Zhao S, Shen Q (2011) A six-gene phylogeny reveals the evolution of mode of infection in the rice blast fungus and allied species. Mycologia 103:1267–1276