Ultrastructure and phylogeny of Ustilago coicis

Ultrastructure and phylogeny of Ustilago coicis 1 first-author †E-mail:jzzhang@zju.edu.cn †E-mail:jzzhang@zju.edu.cn jzzhang@zju.edu.cn 1 2 3 1 4 Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China Guizhou Institute of Plant Protection, Guiyang 550006, China Guizhou Institute of Plant Protection, Guiyang 550006, China Key Laboratory of Systematic Mycology and Lichenology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China Key Laboratory of Systematic Mycology and Lichenology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China Institutue of Excellence in Fungal Research, School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand Institutue of Excellence in Fungal Research, School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand Ustilago coicis causes serious smut on Coix lacryma-jobi in Dayang Town, Jinyun County, Zhejiang Province of China. In this paper, ultrastructural assessments on fungus-host interactions and teliospore development are presented, and molecular phylogenetic analyses have been done to elucidate the phylogenetic placement of the taxon. Hyphal growth within infected tissues was both intracellular and intercellular and on the surface of fungus-host interaction, and the fungal cell wall and the invaginated host plasma membrane were separated by a sheath comprising two distinct layers between the fungal cell wall and the invaginated host plasma membrane. Ornamentation development of teliospore walls was unique as they appeared to be originated from the exosporium. In addition, internal transcribed spacer (ITS) and large subunit (LSU) sequence data showed that U. coicis is closely related to Ustilago trichophora which infects grass species of the genus Echinochloa (Poaceae). Fungus-host interaction Molecular phylogenetics Smut Teliospore wall development Introduction 1 Job’s tears ( Coix lacryma-jobi ), also called as Chinese pearl barley or adley, is a grass (Poaceae) cultivated as a nourishing food eaten in the same way as rice and is believed to have medicinal value (Chang, et al., 2003 2003 ). Job’s tears smut is one of the most important diseases affecting this plant and occurs in many countries (Titatarn et al., 1983 1983 ). The causal agent of Job’s tears smut was named as Ustilago coicis Bref. by Small ( 1927 1927 ). Although subsequently Mundkur ( 1940 1940 ) named the smut fungus infectious on Job’s tears as C. lacryma-jobi Mundkur, Chowdhury ( 1946 1946 ) reported it as a synonym with U. coici s. Smut genera Ustilago and Sporisorium (Ustilaginales) show a great diversity on grasses (Poaceae) (Stoll et al., 2005 2005 ). Generic delimitation can be characterized by single teliospores/teliospore balls, sori with or without columella and peridium, and sterile cells between the teliospores (Vánky, 1987 1987 ; Stoll et al., 2003 2003 ). However, intermediate character combinations have made it difficult for consistent delimitation of these two closely related genera (Vánky, 1985 1985 ; 1998 1998 ; Stoll et al., 2003 2003 ; 2005 2005 ). Supplementary ultrastructural characters of teliospore wall development of smut fungi have been reported and an attempt is made to research their taxonomic relationships (Piepenbring et al., 1998 1998 ; Stoll et al., 2003 2003 ), but these studies have not included U. coicis . Molecular data have been used to delimit genera and species in Ustilaginaceae (Bauer et al., 1997 1997 ; Begerow et al., 1997 1997 ; Stoll et al., 2003 2003 ; 2005 2005 ). Such studies have led to the revisions of the taxonomic position of several species but did not involve U. coicis . In this study, we systemically described interactions between U. coici s and C. lacryma-jobi , and the teliospore development at the ultrastructural level. In addition, we investigated the phylogenetic relationships of U. coicis with closely related taxa based on the internal transcribed spacer (ITS) and large subunit (LSU) region sequence analysis. Materials and methods 1 Plants 2 Samples were collected from naturally infected leaves and inflorescences of Job’s tears at different development stages in a field in Dayang Town (Jinyun County, Zhejiang Province, China) in August 2010. Electron microscopy 2 Tissue samples (1 mm×2 mm) with mycelia of U. coicis and also with teliospore sori were fixed in glutaraldehyde (2.5%) in 0.1 mol/L sodium phosphate buffer (pH 7.0), overnight at 4 °C, including 5 min vacuum-infiltration. Samples were washed in the same buffer, and postfixed in osmium tetroxide (0.01 g/ml) in buffer for 2 h at room temperature. Tissues were dehydrated in a graded ethanol series and were then embedded in Spurr’s epoxy resin, and polymerized at 70 °C for 9 h. Infection hyphae within or between cells at precise stages of development were identified by light microscopy and ultrathin sections were cut on a Reichert-Jung Ultracut-E ultramicrotome, collected onto Formvar-coated slot copper grids, and stained with uranyl acetate and lead citrate. Observations were made using a transmission electron microscope at 80 kV and a Gatan 832 CCD camera (Hitachi H-7650, Tokyo, Japan). DNA extraction, amplification, and sequencing 2 The teliospores were collected from samples germinated at 28 °C on potato sucrose agar (PSA) with chloramphenicol and the single sporidia were separated as abundant sporidia were produced on PSA. The single-spore cultures were inoculated in 100 ml of liquid growth media (potato sucrose both (PSB)) in 250-ml flasks, and shaken at 150 r/min at 25 °C for 48 h. Fungal bodies were harvested by filtration, freeze-dried, ground to a final powder in liquid nitrogen, and then stored at −70 °C. About 50 mg of fungal powder was removed into a sterile 1.5 ml microcentrifuge tube, rehydrated in 600 μl of 2× CTAB buffer (100 mmol/L Tris (pH 8.0), 1.4 mol/L NaCl, 30 mmol/L ethylenediaminetetraacetic acid (EDTA), 2% hexadecyltrimethylammonium bromide) and incubated in a water bath at 65 °C for 30–60 min. Following a phenol/chloroform extraction, the genomic DNA was precipitated by isopropanol in the presence of sodium acetate. Genomic DNA was visualized in 1% agrose gels (0.01 g/ml) after ethidium bromide staining (Xie et al., 2010 2010 ). The ITS and LSU regions were amplified using ITS1 and ITS4 as well as NL1 and NL4, respectively (O′Donnell, 1993 1993 ). The purified polymerase chain reaction (PCR) products were inserted into pMDTM19-T vector (TaKaRa Co., Japan) and transformed into Escherichia coli DH5α competent cells. The positive clone was propagated and the recombinant plasmids were extracted using AxyPrep plasmid miniprep kit according to the manufacturer’s instructions (Axygen, USA). In the meantime, they were identified by PCR and restriction endonuclease enzyme digestion. The sequence determination of recombinant plasmid was carried out by the Hangzhou Genomics Institute in both directions. The accession numbers of all sequences used for comparison are listed in Table 1 1 . ITS/LSU: ITS sequence and LSU sequence or single accession numbers with contiguous sequences (ITS and LSU) GD: Günter Deml; RB: Robert Bauer; Ust. Exs.: Ustilaginales Exsiccata; H.U.V.: Herbarium Ustilaginales Vánky; M: München, Germany; TUB: Tübingen, Germany; MP: Meike Piepenbring; HB: Hansjörg Prillinger; F: Franz Oberwinkler; MS: Matthias Stoll Species Host Origin GenBank acc. No. (ITS/LSU) 1 1 Source 2 2 Sporisorium aegypticum (A.A. Fisch. Waldh.) Vánky Schismus arabicus Iran AY344970/AY740129 Ust. Exs. 756 (M) S. andropogonis (Opiz) Vánky Bothriochloa saccharoides (as Andropogon saccharoides ) Ecuador AY740042/AY740095 56588 (M) S. andropogonis (Opiz) Vánky Bothriochloa saccharoides Bolivia AY740043/AY740096 Stoll et al., 2005 2005 S. andropogonis-micranthi (L. Ling) L. Guo (as S. capillipedii ) Capillipedium spicigerum Australia AY740047/AY740100 56595 (M) S. anthracoideisporum Vánky & R.G. Shivas Pseudoraphis spinescens Papua; New Guinea AY740044/AY740097 Stoll et al., 2005 2005 S. apludae-aristatae (B.V. Patil & Thirum.) Vánky Apluda mutica India AY740045/AY740098 56590 (M) S. arthraxonis (Pat.) L. Guo Arthraxon lanceolatus China AY740046/AY740099 56592 (M) S. bursum (Berk.) Vánky Themeda quadrivalvis India AY740154 Ust. Exs. 844 (M) S. cenchri (Lagerh.) Vánky Cenchrus pilosus Nicaragua AY344972/AF453943 MP 1974 (TUB) S. chrysopogonis Vánky Chrysopogon fulvus Sri Lanka AY344973/AY740131 Ust. Exs. 407 (M) S. consanguineum (Ellis & Everhart) Vánky Aristida uruguayensis Argentina AY740048/AY740101 H.U.V. 19145 (TUB) S. cordobense (Spegazzini) Vánky Digitaria insularis Bolivia AY740155 Stoll et al., 2005 2005 S. cruentum (J.G. Kühn) Vánky Sorghum halepense USA AY344974/AF453939 Ust. Exs. 687 (M) S. cruentum (J.G. Kühn) Vánky Sorghum bicolor Nicaragua AY740156 Stoll et al., 2005 2005 S. culmiperdum (J. Schröt.) Vánky Andropogon gerardii Honduras AY344975/AF133580 MP 2060 (TUB) S. destruens (Schltdl.) Vánky Panicum miliaceum Romania AY344976/AY747077 Ust. Exs. 472 (M) S. dimeriae-ornithopodae Vánky & C. Menge Dimeria ornithopoda India AY344977/AY740132 Ust. Exs. 848 (M) S. elionuri (Henn. & A. Evans) Vánky Elionurus muticus Bolivia AY740157 Stoll et al., 2005 2005 S. enteromorphum (McAlpine) Vánky Themeda triandra South Africa AY740158 56602 (M) S. erythraeense (Syd. & P. Syd.) Vánky Hackelochloa granularis India AY740049/AY740102 Ust. Exs. 849 (M) S. everhartii (Ellis & Galloway) M. Piepenbr Andropogon virginicus Cuba AY740159 Stoll et al., 2005 2005 S. fastigiatum Vánky Andropogon angustatus Nicaragua AY344978/AY740133 Stoll et al., 2005 2005 S. formosanum (Sawada) Vánky Panicum repens Taiwan, China AY344979/AY740134 Ust. Exs. 688 (M) S. foveolati (Maire) Vánky Eremopogon foveolatus Canary Islands AY740050/AY740103 MP 2365 (TUB) S. holwayi (G.P. Clinton & Zundel) Vánky Andropogon bicornis Panama AY344980/AF453941 Stoll et al., 2005 2005 S. hwangense Vánky & C. Vánky Sporobolus panicoides Zimbabwe AY740051/AY740104 56607 (M) S. lacrymae-jobi (Mundkur) Vánky Coix lacryma-jobi India AY740052/AY740105 56611 (M) S. lepturi (Thüm.) Vánky Hemarthria uncinata Australia AY344981/AY740135 Ust. Exs. 966 (M) S. lepturi (Thüm.) Vánky Hemarthria uncinata Australia AY740160 56613 (M) S. loudetiae-pedicellatae Vánky & C. Vánky Loudetia pedicellata South Africa AY740053/AY740106 56615 (M) S. manilense (Syd. & P. Syd.) Vánky (as S. sacciolepidis ) Sacciolepis indica India AY740059/AY740112 Ust. Exs. 854 (M) S. mishrae Vánky Apluda mutica India AY344983/AY740136 Ust. Exs. 967 (M) S. modestum (Syd.) H. Scholz Enneapogon avenaceus Australia AY740054/AY740107 56617 (M) S. monakai (Mishra) Vánky Isachne globosa India AY740161 56618 (M) S. moniliferum (Ellis & Everh.) L. Guo Heteropogon contortus Indonesia AY344984/AF453940 Ust. Exs. 851 (M) S. nealii (Ellis & F.W. Anderson) Vánky Heteropogon melanocarpus India AY740055/AY740108 56621 (M) S. neglectum (Niessl) Vánky Setaria pumila Germany AY740056/AY740109 RB 2056 (TUB) S. nervosum Vánky, C. Vánky & R.G. Shivas Sehima nervosum Australia AY740057/AY740110 56622 (M) S. occidentale (Seym.) Vánky & Snets Andropogon gerardii USA AY344985/AY740137 Ust. Exs. 758 (M) S. ophiuri (Henn.) Vánky Rottboellia cochinchinensis Unknown AY740019/AJ236136 HB 20 S. ovarium (Griffiths) Vánky Urochloa fasciculata (Sw.) R. Webster Mexico AY740020/AJ236137 Stoll et al., 2005 2005 S. paspali-notati (Henn.) M. Piepenbr Paspalum notatum Cuba AY344982/AF453944 Stoll et al., 2005 2005 S. penniseti (Maire) Vánky (as S. catharticum ) Pennisetum setaceum Canary Islands AY344971/AY740130 MP 2367 (TUB) S. polliniae (Magnus) Vánky Andropogon distachyos Greece AY344987/AY740138 Ust. Exs. 690 (M) S. provinciale (Ellis & Galloway) Vánky & Snets A. gerardii USA AY344988/AY747076 Ust. Exs. 759 (M) S. pseudechinolaenae Vánky & C. Menge Pseudechinolaena polystachya Indonesia AY344989/AY740139 Ust. Exs. 853 (M) S. puellare (Syd. & P. Syd.) G. Deml Hyparrhenia hirta Canary Islands AY740058/AY740111 MP 2372 (TUB) S. pulverulentum (Cooke & Massee) Vánky Saccharum strictum Yugoslavia AY740162 56627 (M) S. reilianum (J.G. Kühn) Langdon & Full. Sorghum halepense Greece AY740163 Ust. Exs. 527 (M) S. scitamineum (Syd.) M. Piepenbr., M. Stoll & Oberw (as Ustilago scitaminea ) Saccharum sp. Cuba AY345007/AY740147 Stoll et al., 2005 2005 S. scitamineum (Syd.) M. Piepenbr., M. Stoll & Oberw (as U. scitaminea ) Saccharum sp. Costa Rica AY740070/AJ236138 Stoll et al., 2005 2005 S. sorghi Ehrenb. ex Link Sorghum bicolor Nicaragua AY740021/AF009872 Stoll et al., 2005 2005 S. themedae-arguentis Vánky Themeda arguens Indonesia AY344991/AY740140 Ust. Exs. 855 (M) S. trachypogonicola Vnky & C. Vnky Trachypogon plumosus Cuba AY344992/AY740141 Stoll et al., 2005 2005 S. trachypogonis-plumosi Vánky T. plumosus Venezuela AY740060/AY740113 56635 (M) S. tumefaciens (McAlpine) Vánky (as Sorosporium tumefaciens ) Chrysopogon aciculatus Sri Lanka AY344969/AY740128 Ust. Exs. 231 (M) S. veracruzianum (Zundel & Dunlap) M. Piepenbr Panicum viscidellum Costa Rica Y344993/AY740114 MP 960 S. veracruzianum (Zundel & Dunlap) M. Piepenbr P. viscidellum Costa Rica AY747075/AY740142 MP 735 (USJ) Ustilago affinis Ellis & Everh Stenotaphrum secundatum Costa Rica AY344995/AF133581 Stoll et al., 2005 2005 U. alcornii Vánky Tripogon loliiformis Australia AY740165 56514 (M) U. altilis Syd. Triodia pungens Australia AY740166 Ust. Exs. 418 (M) U. austro-africana Vánky & C. Vánky Enneapogon cenchroides Zimbabwe AY740061/AY740115 56516 (M) U. avenae (Pers.) Rostr. Arrhenaterum elatius Germany AY740063/AY740117 DB 559 (TUB) U. avenae (Pers.) Rostr. A. elatius Germany AY740062/AY740116 RB 3092 (TUB) U. avenae (Pers.) Rostr. Avena barbata Canary Islands AY344997/AF453933 MP 2362 (TUB) U. avenae (Pers.) Rostr. A. barbata Italy AY344996/AJ236140 F 946/GD 1292 (TUB) U. bouriquetii Maubl. & Roger Stenotaphrum dimidiatum Réunion AY740167 56517 (M) U. bromivora (Tul. & C. Tul.) A.A. Fisch. Waldh Bromus catharticus Argentina AY740064/AY740118 H.U.V. 19322 U. bullata Berk B. diandrus Canary Islands AY344998/AF453935 MP 2363 (TUB) U. calamagrostidis (Fuckel) G.P. Clinton Calamagrostis epigeios Bulgaria AY740065/AY740119 56518 (M) U. coicis Bref Coix lacryma-jobi China JX219371/JX219374 In this study U. crameri Körnicke Setaria italica India AY344999/AY740143 Ust. Exs. 995 (M) U. cynodontis (Pass.) P. Henn Cynodon dactylon Mexico AY345000/AF009881 Stoll et al., 2005 2005 U. cynodontis (Pass.) P. Henn C. dactylon Taiwan, China AY740168 MS 1 (TUB) U. davisii Liro Glyceria multiflora Argentina AY740169 H.U.V. 19252 U. drakensbergiana Vánky Digitaria tricholaenoides South Africa AY740170 56523 (M) U. echinata J. Schröt. Phalaris arundinacea Germany AY345001/AY740144 Ust. Exs. 540 (M) U. esculenta P. Henn Zizania latifolia Taiwan, China AY345002/AF453937 Ust. Exs. 590 (M) U. esculenta P. Henn Zizania latifolia China JX219372/JX219375 In this study U. esculenta P. Henn Zizania latifolia China JX219373/JX219376 In this study U. filiformis (Schrank) Rostr Glyceria fluitans Germany AY740066/AY740120 RB 3011 (TUB) U. hordei (Pers.) Lagerh (as U. kolleri ) Avena sativa Spain AY740068/AY740122 F 947/GD 1300 U. hordei (Pers.) Lagerh Hordeum vulgare Iran AY345003/AF453943 Ust. Exs. 784 (M) U. ixophori Durán Ixophorus unisetus Costa Rica AY740067/AY740121 Stoll et al., 2005 2005 U. maydis (Link) Unger Zea mays L. Germany AY345004/AF453938 RB 3093 (TUB) U. nuda (Jens.) Rostr Hordeum leporinum Unknown AY740069/AJ236139 H.U.V. 17782 U. pamirica Golovin Bromus gracillimus Iran AY345005/AY740145 Ust. Exs. 789 (M) U. schroeteriana Henn Paspalum paniculatum Costa Rica AY345006/AY740146 Ust. Exs. 887 (M) U. spermophora Berk. & M.A. Curtis Eragrostis ferruginea Namibia AY740171 F 565/H.U.V. 13634 U. striiformis (Westend.) Niessl Alopecurus pratensis Germany AY740172 H.U.V. 18286 U. syntherismae (Schwein.) Peck Digitaria ternata India AY740071/AY740123 Ust. Exs. 998 (M) U. tragana Zundel Tragus berteronianus Zimbabwe AY740072/AY740124 56562 (M) U. trichophora (H.F. Link) F. Körnicke Echinochloa colona Cuba AY345009/AY740148 Stoll et al., 2005 2005 U. trichophora (H.F. Link) F. Körnicke E. colona Mexico AY740023/AJ236141 Stoll et al., 2005 2005 U. trichophora (H.F. Link) F. Körnicke E. colona India AY740073/AY740125 56564 (M) U. triodiae Vánky Triodia microstachya Australia AY740074/AY740126 H.U.V. 17662 U. triodiae Vánky T. microstachya Australia AY740075/AY740127 56566 (M) U. turcomanica Tranzschel Eremopyrum distans Iran AY345011/AF453936 F 585/H.U.V. 23 U. vetiveriae Padwick Vetiveria zizanioides Unknown AY345011/AY740149 H.U.V. 17954 U. xerochloae Vánky & R.G. Shivas Xerochloa imberbis Australia AY345012/AY740150 Ust. Exs. 1000 (M) Phylogenetic analyses 2 Phylogenetic analyses were performed using PAUP* 4.0b10 (Swofford, 2002 2002 ). Phylogenetic trees were built using the maximum parsimony (MP) method. In the MP analyses, trees were inferred using the heuristic search option with tree bisection and reconnection (TBR) branch swapping and 1 000 random sequence additions. Gaps were treated as missing data and characters were equally weighted. Maxtrees were unlimited, branches of zero length were collapsed, and all parsimonious trees were saved. Bootstrap analyses were based on 1 000 replications, each with 10 replicates of random stepwise addition of taxa. Kishino-Hasegawa (KH) tests were performed in order to determine whether trees were significantly different (Kishino and Hasegawa, 1989 1989 ). Trees were figured using TreeView (Page, 1996 1996 ). Results 1 Interaction between Ustilago coicis and Coix lacryma-jobi 2 Before teliospore formation, transmission electron micrographs (TEMs) showed that hyphae of U. coicis were present between and within plant cells in parenchyma tissue of an ovary. The intracellular hypha growing in a cell was surrounded by a sheath comprising two distinct layers, which separated the fungal cell wall from the invaginated host plasma membrane (Fig. 1a 1a ). The plant plasma membrane was continuous but occasionally it was convoluted at some sites. The outer layer of a sheath was an electron-opaque layer surrounded by the plant plasma membrane and an inner electron-dense layer covering the hyphal wall. However, the sheaths were variable between and within parenchyma tissue. The intercellular hypha development caused intercellular space enlargement and elongation of plant cell walls but intercellular hyphal walls were occasionally only coated by an inconspicuous electron-dense layer (Fig. 1b 1b ). (a) Transverse section of an intracellular hypha. A hypha is surrounded by a sheath comprising electron-opaque layer (arrowhead) and electron-dense layer (arrow), which is located between the plant plasma membrane (PPM) and the fungal cell wall (FCW). (b) Transverse section of an intercellular hypha. Hyphae induce intercellular space enlargement (*) and FCW is covered by an inconspicuous electron-dense layer in some areas (arrowheads). (c) Sporogenous hyphae. (d) A magnified sporogenous hyphae. (e) Abundant hyphae in a host cell. (f) Disintegrated host tissues with fungal hyphae In some cells with intracellular fungal development, the hyphae produced short or lateral branches (Fig. 1c 1c ). In this case the host protoplast appeared to be disintegrated and these hyphae were not surrounded by a sheath (Figs. 1c and 1d 1c and 1d ). They grew and branched extensively until individual host cells became filled with contorted and coiled hyphae, which constituted the hyphal aggregations (Fig. 1e 1e ). These hyphae were sporogenous hyphae and their hyphal aggregations were the sites of teliosporogenesis. Subsequently, the aggregations caused host cells to disintegrate, resulting in large intercellular spaces in the parenchyma tissue of an affected ovary. Teliospore development 2 In a large intercellular space, abundant hyphal fragments were seen under the light microscope (Fig. 2a 2a ) and they had originated from the sporogenous hyphae. Under TEM, the hyphal fragments began to plasmolyze (Figs. 2b and 2c 2b and 2c ) and the initial exosporia of the young teliospores were produced on the plasma membranes of the plasmolyzed hyphal fragments (Fig. 2d 2d ). In this case, a young teliospore was in the completely plasmolyzed cell wall, but the fungal cell wall was still intact. With teliospore development, the electron-opaque warty ornamentation produced from the exosporium of a young teliospore (Fig. 2e 2e ) but it appears to be homogenous with the exosporium. Fungal cell walls began to disintegrate but some remnants of fungal cell wall were retained on top of warts (Fig. 2f 2f ) and young teliospores were irregular. With further development, the warty ornamentation became electron-dense and young teliospores were regularly spherical or subspherical with a few lipid globules (Figs. 2g and 2h 2g and 2h ). At this time, the exosporium also became electron-dense and was covered by spiny ornamentation, and young teliospores exhibited more lipid globules (Fig. 2i 2i ). Subsequently, an electron-opaque endosporium in a young teliospore developed beneath the exosporium (Fig. 2j 2j ). The mature teliospore wall was comprised of an endosporium of mostly constant thickness and an exosporium covered by warts (Fig. 2k 2k ). (a) Light microscopy. Abundant hyphal segments produced from sporogenous hyphae in a large intercellular space. (b–k) TEM. (b, c) Plasmolyzed process of hyphal segments: (b) a hyphal segment being plasmolyzed; (c) a completely plasmolyzed hyphal segment. (d) Exosporium (arrowhead) of a young teliospore produced on plasma membrane in plasmolyzed process. (e) Warty ornamentation formation and fungal cell walls being degraded. (f) A magnified view of warty ornamentation (*) with the remnant (arrowheads) of fungal cell wall in (e). (g) A regularly young teliospore with electron-dense warty ornamentation. (h) Magnified view of a conical wart in (g). (i) A young teliospore with electron-dense exosporium and spines. (j) A well-developed wall of teliospore with endosporium, exosporium, and warts. (k) Magnified view of a teliospore wall in (j) As teliospores of U. coicis matured, sori became darker and eventually black. SEM of sori revealed masses of matured teliospores (Fig. 3a 3a ). Individual teliospores were fine spines or warts (Figs. 3b and 3c 3b and 3c ) and usually between 9 and 11 μm in diameter. (a) Sorus. (b, c) Mature teliospores clearly showing the surface spines or warts Phylogenetic analysis 2 Sequence analysis showed that ITS/5.8S and LSU regions from three isolates of U. coicis were identical and one of them, isolate UCDY01, had been submitted to the GenBank database (Table 1 1 ). The final ITS/5.8S and LSU dataset included 95 sequences with 1 316 characters after alignment. Parsimony analysis resulted in 52 equally parsimonious trees. The KH test showed that these trees were not significantly different. One of these trees is shown in Fig. 4 4 . The taxa name and GenBank number of each sequence are given on the tree. One isolate of U. coicis formed a monophyletic lineage and clustered together with U. trichophora with 84% bootstrap support when Moesziomyces bullatus (AB369259) was used as the outgroup taxon. Teliospores of U. trichophora are globose and ornamented with spines, and are similar to those of U. coicis (7–14 μm wide compared with 6–14 μm wide in U. trichophora ) (Fullerton and Langdon, 1968 1968 ; Titatarn et al., 1983 1983 ). U. trichophora is a pathogen infecting the ovaries and vegetable parts of Echinochloa grass species and exhibits intermediate morphlogical characters between Ustilago and Sporisorium . Bootstrap values ≥50% are shown above or below branches Discussion 1 U. coicis can infect the ovaries and leaves of C. lacryma-jobi and cause Job’s tears smut. In infected tissues, hyphal distribution is both intercellular and intracellular. At the ultrastructural level, it can be seen that intracellular hypha at the interface of fungus-host interaction is encased by a sheath with an outer electron-opaque layer and an inner electron-dense layer, as shown in other species of Ustilago , such as U. esculenta (Zhang et al., 2012 2012 ). The teliosporogenesis process in U. coicis is similar to those of many species of Ustilago -infecting members of Poaceae. Teliospores are covered by fine to coarse warts or by warts of different sizes on the same teliospore. However, ornamentation development in U. coicis is unique. During teliosporogenesis in almost all smut fungi, the outer layers are usually deposited first. At the beginning of ornamentation formation, the plasma membrane may be smooth or undulated, carrying the developing ornaments on its tips or in its depressions (Piepenbring et al., 1998 1998 ). By contrast, the ornamentation of teliospores in U. coicis appeared to be produced on the exosporium of a young teliospore (Fig. 5 5 ), never from the plasma membrane, being different from developmental patterns of ornamentation of Ustilago (Piepenbring et al., 1998 1998 ; Zhang et al., 2012 2012 ). This shows that teliospore development in Ustilago is diverse. Parts of walls are shown in section, and ornamentation also in surface view. The process of a teliospore wall development is plasmolyzation of hyphal segment, exosporium deposition, formation of the ornamentation, and endosporium deposition The phylogenetic relationship between U. coicis and its closely related species in Ustilago was analyzed based on combined ITS and LSU sequence data. The phylogenetic analysis clearly indicates that U. coicis is closely related to U. trichophora . Although peridia, columella, spore balls, and sterile cells have all been used as morphological characters to distinguish Ustilago from Sporisorium , sori of U. trichophora contain a columella and are packed by a peridium (Fullerton and Langdon, 1968 1968 ), which are typical Sporisorium characters (Piepenbring, 2003 2003 ), so U. trichophora was considered to demonstrate intermediate morphological characters (Piepenbring et al., 1998 1998 ) between these genera. In Ustilago spp., U. trichophora and U. coicis have the same ornamentation with spines and could be not well differentiated by overlapping teliospore size (Fullerton and Langdon, 1968 1968 ; Titatarn et al., 1983 1983 ). Piepenbring ( 2004 2004 ) and Stoll et al. ( 2005 2005 ) found that it was because morphological characters were non-homologous and had been misused as a taxonomic standard for defining the genera, resulting in the genus complex, as shown in Fig. 4 4 . Therefore, additional molecular loci are needed to resolve the Ustilago - Sporisorium complex, and should include morphological characters. Conclusions 1 In this article, we showed, for the first time, that there is a unique development of teliospore walls of U. coicis in Ustilaginaceae. In addition, phylogeny and fungus-host interactions of U. coicis were ultra-structurally assessed. Therefore, this research can provide more valuable information for taxonomy and biology in Ustilaginaceae. 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1200239 Project supported by the National Natural Science Foundation of China (No. 31070123) and the Special Fund for Agro-scientific Research in the Public Interest of China (No. 201003004) 2012-09-06 2013-03-15 2012-11-09 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2013 2013 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 04 14