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Phylogenetic analysis of Tilletia and allied genera in order Tilletiales (Ustilaginomycetes; Exobasidiomycetidae) based on large subunit nuclear rDNA sequences

     USDA ARS Systematic Botany and Mycology Laboratory, 10300 Baltimore Avenue, Beltsville, Maryland 20705-2350

Lori M. Carris

     Department of Plant Pathology, Washington State University, Pullman, Washington 99164–6430

Kálmán Vánky

     Herbarium Ustilaginales Vánky, Gabriel-Biel-Str. 5, D-72076 Tübingen, Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

The order Tilletiales (Ustilaginomycetes, Basidiomycota) includes six genera (Conidiosporomyces, Erratomyces, Ingoldiomyces, Neovossia, Oberwinkleria and Tilletia) and approximately 150 species. All members of Tilletiales infect hosts in the grass family Poaceae with the exception of Erratomyces spp., which occur on hosts in the Fabaceae. Morphological features including teliospore ornamentation, number and nuclear condition of primary basidiospores and ability of primary basidiospores to conjugate and form an infective dikaryon were studied in conjunction with sequence analysis of the large subunit nuclear rDNA gene (nLSU). Analysis based on nLSU data shows that taxa infecting hosts in the grass subfamily Pooideae form one well supported lineage. This lineage comprises most of the reticulate-spored species that germinate to form a small number of rapidly conjugating basidiospores and includes the type species Tilletia tritici. Two tuberculate-spored species with a large number of nonconjugating basidiospores, T. indica and T. walkeri, and Ingoldiomyces hyalosporus are also included in this lineage. Most of the species included in the analysis with echinulate, verrucose or tuberculate teliospores that germinate to form a large number (>30) of nonconjugating basidiospores infect hosts in the subfamilies Panicoideae, Chloridoideae, Arundinoideae and Ehrhartoideae. This group of species is more diverse than the pooid-infecting taxa and in general do not form well supported clades corresponding to host subfamily. The results of this work suggest that morphological characters used to segregate Neovossia, Conidiosporomyces and Ingoldiomyces from Tilletia are not useful generic level characters and that all included species can be accommodated in the genus Tilletia.

Key words: Conidiosporomyces, Erratomyces, germination, Ingoldiomyces, molecular systematics, Neovossia, smut and bunt fungi

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The genus Tilletia Tul. & C. Tul. comprises ca. 140 species restricted to hosts in the grass family (Poaceae) and is the largest genus in order Tilletiales (Basidiomycota, Ustilaginomycetes, Exobasidiomycetidae) (Vánky 2002). Tilletia is characterized by the formation of pigmented teliospores intermingled with hyaline sterile cells, and in most species the teliospores are formed in host ovaries. Teliospore ornamentation ranges from reticulate, echinulate, verrucose, tuberculate to smooth. In many species teliospore masses have a fetid, herring brine odor due to the production of trimethylamine. Teliospores germinate to form an aseptate basidium, frequently with multiple retraction septa, and a terminal whorl of aerial primary basidiospores (FIG. 1). The type species, T. tritici, produces 8–12 filiform to narrowly falcate monokaryotic basidiospores (Goates 1996). Most of the basidiospores conjugate while attached to the basidium to form an "H-body," giving rise to dikaryotic mycelium that infects host plants at seedling stage, resulting in a systemic infection (Vánky 1994). A second type of germination pattern, consisting of the production of large numbers of nonconjugating primary basidiospores, is found in species of Neovossia Körn., Conidiosporomyces Vánky (Vánky and Bauer 1992) and some species of Tilletia, such as T. indica, T. horrida and T. walkeri (Castlebury and Carris 1999, Durán 1987). Oberwinkleria Vánky & R. Bauer produces nonconjugating primary basidiospores (Vánky and Bauer 1995) but their nuclear condition was not reported.

View larger version (27K): [in this window] [in a new window]   FIG. 1. Tilletia tritici germinated teliospore with conjugating primary basidiospores. Bar = 20 µm.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Spore types from various species in the Tilletiales. A. Blastospores from T. ixophori. B. Denticulate sporogenous cells from T. kimberleyensis. C. Formation of ballistospores and proliferation of ballistospore. D. Formation of blastospores. E. Proliferating blastospores. F. Uninucleate and multinucleate primary basidiospores. G. Y-shaped conidia formed in culture of C. verruculosus.

Members of Tilletiales have been poorly represented in previous phylogenetic analyses of the smut fungi (Begerow et al 1997, Begerow et al 2000). The analysis of Begerow et al (1997) included only four type species, Ingoldiomyces hyalosporus, Conidiosporomyces ayresii, Tilletia tritici and Erratomyces patelii. In that analysis T. tritici and I. hyalosporus were related most closely and formed a sister group of C. ayresii, with E. patelii basal to these species. The present study, using nLSU sequence data, was initiated to determine phylogenetic relationships among species of Tilletia and segregate genera. Data on teliospore morphology, teliospore germination, primary and secondary basidiospore morphology, and nuclear condition, when available, are presented.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Isolation, maintenance and deposition of cultures and voucher specimens.— – Species used in this study are listed (TABLE I). All available taxa for which teliospores could be germinated were included. Teliospores were germinated after soaking in water for 2 d and surface sterilization in 0.26% NaClO (5% v/v commercial bleach) on 2% water agar at room temperature (20–25 C), 15 C or 5 C depending on the species. Teliospores of T. controversa were germinated at 5 C under a 8/16 h daylight/dark regimin. Primary basidiospores were fixed and stained with Giemsa-HCl following Durán (1980) to determine nuclear condition or were transferred to potato-sucrose agar (PSA) or M-19 agar (Trione 1964) to establish colonies for nucleic acid extraction.

The nLSU genes were amplified in 50 µL reactions on a GeneAmp 9700 thermal cycler (Applied Biosystems, Foster City, California) under these reaction conditions: 10–15 ng of genomic DNA, 200 µM each dNTP, 2.5 units Amplitaq Gold (Applied Biosystems, Foster City, California), 25 pmoles each of primers LR0R and LR7 (Vilgalys and Hester 1990, Rehner and Samuels 1994) and the supplied 10 x PCR buffer with 15 mM MgCl₂. The thermal cycler program was: 10 min at 95 C followed by 35 cycles of 30 s at 94 C, 30 s at 55 C, 1 min at 72 C, with a final extension for 10 min at 72 C. After amplification, the PCR products were purified with QIAquick columns (QIAGEN Inc., Chatsworth, California) according to the manufacturer’s instructions. Amplified products were sequenced with the BigDye terminator kit (Applied Biosystems, Foster City, California) on an automated DNA sequencer with these primers: LR0R, LR3R, LR5R, LR7, LR5, LR3 (Vilgalys and Hester 1990, Rehner and Samuels 1994, 1995).

Sequence analysis.— – Raw sequences were edited with Sequencher version 4.1.4 for Windows (Gene Codes Corp., Ann Arbor, Michigan). Alignments were adjusted manually with GeneDoc 2.6.001 (http://www.psc.edu/biomed/gene-doc/). The alignment included sequences from 57 isolates, with three species of Entyloma de Bary and one species of Graphiola Poit. (WSP 71169) as outgroup taxa and consisted of 1345 positions. Entyloma and Graphiola also are contained within the Exobasidiomycetidae in different orders and have been placed close to the Tilletiales in previous analyses (Begerow et al 1997). The sequence alignment was deposited in TreeBase.

Trees were inferred by the neighbor joining (NJ) method (Kimura 2-parameter distance calculation) and by maximum parsimony (MP) using the heuristic search option with the random addition sequence (1000 replications, maximum of 100 trees saved per replicate) and the branch swapping (tree bisection-reconnection) option of PAUP* 4.0b10 (Swofford 2002). All aligned positions were included in the analyses. All characters were unordered and given equal weight. Gaps were treated as missing data in the parsimony analysis and the neighbor joining analysis; missing or ambiguous sites were ignored for affected pairwise comparisons. Relative support for branches was estimated with 1000 bootstrap replications (Felsenstein 1985) with multrees and TBR off and 10 random sequence additions for the MP bootstraps.

Phylogenetic trees also were inferred with Bayesian inference as implemented in MrBayes (http://morphbank.ebc.uu.se/mrbayes/) with these commands: number of generations = 500 000, sample frequency = 100, number of chains = 4, temperature = 0.2, save branch lengths = yes, starting tree = random. Likelihood model assumptions were as determined with Modeltest version 3.06 (Posada and Crandall 1998) with the Akaike Information Criterion (AIC) under the GTR+I+G model: base frequencies A = 0.2664, C = 0.1971, G = 0.2907, T = 0.2458; number substitution types = 6; proportion of invariable sites = 0.6672; gamma shape parameter = 0.5947; rate matrix = 0.6253, 2.4765, 0.8936, 0.2228, 5.4558, 1.000. The first 100 000 generations were discarded as the chains were converging (burn-in). Three independent analyses, each starting from a random tree, were run under the same conditions.

Phylogenetic trees constraining monophyletic groups of taxa were constructed as follows based on four major characters: (i) two based on spore ornamentation types (reticulate and echinulate/tuberculate/verrucose); (ii) four based on host subfamily; (iii) two based on type of germination (conjugating primary basidiospores or nonconjugating); and (iv) two based on local- or systemic-infecting. Maximum parsimony analyses were run for each of the 10 resulting constraints (TABLE II) using the heuristic search option (1000 random sequence additions, TBR and multrees off). The trees with the best –ln likelihood score resulting from each constrained analysis and all three Bayesian trees were compared with the MP tree with the best –ln likelihood score, using the Shimodaira-Hasegawa test (Shimodaira and Hasegawa 1999). The range of –ln likelihood scores of trees from each constraint topology is shown (TABLE II). Likelihood settings were as determined by Model-test as previously described.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

View larger version (30K): [in this window] [in a new window]   FIG. 3. MP tree resulting from analysis of 1345 bp from the nLSU for the species in the Tilletiales. Numbers above the branches indicate MP bootstrap support percentages (>50%) from 1000 pseudoreplicates with 10 random taxon addition replicates per pseudoreplicate for major lineages only. Four major lineages are identified by Roman numerals I-IV and host subfamily when limited to a single subfamily. PAC refers to Panicoidae, Arundinoideae and Chloridoideae. Representatives from segregate genera are indicated in bold type as is the type species of Tilletia.

View larger version (30K): [in this window] [in a new window]   FIG. 4. Phylogenetic tree resulting from Bayesian analysis of 1345 bp of the nLSU of species in the Tilletiales. Thickened branches indicate >90% pooled posterior probabilities obtained from three independent Bayesian analyses, each consisting of 500 000 Markov chain Monte Carlo generations (GTR+G+I model), with a burn-in of 100 000 generations. Lineages identified in FIG. 3 are indicated with host subfamily association. Morphological characters from TABLE III are labeled as follows: Ret = reticulate spore, Tub = tuberculate/verrucose spores, Ridg = ridged spores, Fov = Foveolate, Conj = conjugating primary basidiospores, Nonconj = nonconjugating primary basidiospores, <30 = <30 primary basidiospores, >30 = >30 primary basidiospores, Local = local-infecting, and Syst = systemic-infecting. When a species in a group differs from the labeled characters, the difference for that species is indicated in bold with underlining.

Lineage III includes species infecting chloridoid hosts (85% in all analyses), including T. asperifolia, T. lycuroides, T. aegopogonis and T. obscurareticulata. These are the only four taxa in the analysis with reticulate spores that infect hosts other than Pooideae. With the exception of T. asperifolia, which has uninucleate, conjugating basidiospores, all form multinucleate, nonconjugating basidiospores. Lineage IV contains three panicoid-infecting species and includes C. ayresii, C. verruculosus and T. vittata. Conidiosporomyces species have open sori and Y-shaped conidia (either in sori or formed in culture). Tilletia vittata causes hypertrophy of the infected ovary so that it forms a conspicuous, spur-like outgrowth. Basidiospores of the three species in this lineage are uninucleate, and conjugation was observed (but rarely) only in C. ayersii. A few species do not fall into any of the four lineages described above. The relationships of T. setariae (panicoid host), T. ehrhartae (ehrhartoid host), T. rugispora (panicoid host) and T. horrida (ehrhartoid host) to other species remain unresolved. Morphological characters (TABLE III) for lineages are labeled (FIG. 4). For taxa with differing character states for a given character, differences are indicated in bold underlined text inside the brackets

Teliospore germination and growth in culture.— – Teliospore germination data for species in the analysis are provided (TABLE III). Nuclear condition of primary basidiospores could not be determined for seven species that had limited teliospore germination. The teliospore germination pattern in the type species T. tritici involves rapid conjugation of adjacent primary basidiospores. Teliospores germinate at 5–15 C, but no germination occurs at room temperature. The fungus infects the host at the seedling stage, forming a systemic infection and growing to the developing host ovaries, where the fungus proliferates and forms teliospores. This pattern of dikaryon formation, systemic infection and low temperature requirement occurs in all species closely related to T. tritici, with the exception of T. sterilis and T. cerebrina, which form multinucleate, nonconjugating primar y basidiospores. Zogg (1967) reported conjugation in T. olida, but it was was not observed in the T. olida specimen germinated in this study. Infection by T. olida and T. sterilis is systemic, but teliospores form in sori in host leaves rather than in the ovaries. Ingoldiomyces hyalosporus, T. polypogonis, T. indica and T. walkeri infect hosts in subfamily Pooideae, but teliospores of these species germinate at room temperature. Of these species, only T. polypogonis has a germination pattern similar to that of T. tritici.

Tilletia asperifolia, host Muhlenbergia asperifolia (subfamily Chloridoideae, Lineage III) is the only species outside the pooid-infecting clade (Lineage I) that exhibits the same type of germination pattern, systemic infection and temperature requirement as T. tritici. Tilletia aegopogonis and T. lycuroides, which form a well supported group with T. asperifolia, differ in having teliospores that germinate at room temperature to form multinucleate, nonconjugating basidiospores. Erratomyces patelii, host Vigna mungo (Fabaceae), also germinates at room temperature and produces conjugating basidiospores (Piepenbring and Bauer 1997). The infection type was not reported for this species but is probably local based on the isolated leaf spots that are formed.

In most of the taxa studied with hosts outside subfamily Pooideae, primary basidiospores germinated directly through formation of hyphae or indirectly through formation of ballistospores and did not conjugate under axenic conditions. Multinucleate and uninucleate nonconjugating primary basidiospores (FIG. 2F) germinate in a similar manner. Nonconjugating primary basidiospores were shown to be multinucleate in nine species, with hosts in Pooideae (I. hyalosporus, T. cerebrina, T. sterilis), Chloridoideae (T. aegopogonis, T. lycuroides, T. savilei) and Panicoideae (T. opaca, T. trachypogonis) ranging across Lineages I, II and III.

All species studied in culture produced allantoid ballistospores (FIG. 2C) and filiform to fusiform blastospores (FIG. 2A, D, E), although the two spore types were not produced in equal abundance in all isolates studied. Isolates of some taxa grew in a mycelial manner with relatively few secondary basidiospores. Ballistospores formed from sterigma-like structures on primary basidiospores, other ballistospores, or hyphae (FIG. 2C). Blastospores were aseptate, filiform, curved to coiled, and resembled primary basidiospores and were more abundant than ballistospores in cultures of most taxa in this study. Blastospores formed from other blastospores (FIG. 2E), and from hyphae, either singly on undifferentiated sporogenous cells (FIG. 2D), or from sporogenous cells with multiple denticles (FIG. 2B). Blastospores were not reported in Erratomyces (Piepenbring and Bauer 1997). In addition to the two types of secondary basidiospores just described, C. verruculosus also produced abundant Y-shaped blastospores in culture (FIG. 2G), similar in shape to the conidia formed in sori of C. ayresii. The Y-shaped spores germinated readily. Y-shaped conidia were not present in the sori of C. verruculosus, and this type of spore was not observed in cultures of C. ayresii or other species included in this study. All species included in this study, except E. patelii, had sterile cells intermingled with teliospores in the sorus.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
A strict generic concept of Tilletia as characterized by the reticulate teliospore ornamentation and pattern of germination and infection exhibited by the type species, T. tritici, is not supported based on the results of the analyses of nLSU data. The T. tritici pattern of teliospore germination, with a relatively small number of rapidly conjugating primary basidiospores and systemic host infection resulting in most or all of the host ovaries replaced by fungal sori, is restricted mostly to species in the pooid-infecting clade. However some members of this clade produce nonconjugating primary basidiospores, including T. cerebrina and T. sterilis. Several species that have been studied extensively, including T. bromi, T. fusca and T. togwateei, form mostly uninucleate, conjugating basidiospores, but a small percent of spores may be multinucleate. Boyd and Carris (1998) showed evidence that up to 12% of primary basidiospores produced by T. fusca are dikaryotic based on the formation of teliospores in cultures derived from single basidiospores. Multinucleate primary basidiospores may result either from migration of multiple nuclei from the basidium into developing basidiospores or from mitotic division in basidiospores as shown by Goates and Hoffmann (1987). The T. tritici germination pattern also is found in T. asperifolia (Lineage III), which has a chloridoid host and falls outside the pooid-infecting clade. Erratomyces patelii, which is strongly supported as a basal group to Tilletia and infects dicotyledonous hosts, exhibits this germination pattern as well. Similarly, the reticulate teliospore ornamentation exhibited by T. tritici, is restricted mostly to species in the pooid-infecting clade (Lineage I) but also occurs in T. aegopogonis, T. asperifolia, T. obscurareticulata and T. lycuroides in Lineage III.

The pathogens responsible for Karnal bunt of wheat, T. indica, and kernel smut of rice, T. horrida, were placed in Neovossia by some authors (Singh and Pavgi 1972, Vánky 1994, Whitney 1989) and in Tilletia by others (Durán 1987, Levy et al 2001, Pimentel et al 1998). Both species produce sterile cells in the sorus and numerous nonconjugating primary basidiospores (Castlebury and Carris 1999, Durán 1987). Durán and Fischer (1961) dismissed the value of number of primary basidiospores to delimit genera and our analysis supports their conclusion. Absence of sterile cells and production of numerous basidiospores were two characters used to distinguish Neovossia from Tilletia (Vánky 2002). The type species N. moliniae was shown by Brefeld (1895) to form 30–50 nonconjugating primary basidiospores. However, examination of specimens of N. moliniae, on Molinia (WSP 34463) and N. iowensis on Phragmites (V 573) revealed the presence of sterile cells in the sorus.

Two species of Neovossia infecting Phragmites communis have been described: N. iowensis from the USA (Hodson 1900) and N. danubialis T. Svulescu from Europe (Svulescu 1955). Neovossia danubialis and N. iowensis were merged with N. molinae by Vánky (1990) based on their similar teliospore morphology and germination patterns. Svulescu and Hulea (1955) showed that N. danubialis germinated to produce 10–15 nonconjugating primary basidiospores, similar to what was shown in this study for N. iowensis. Based on the morphological similarity and occurrence on the same host species, N. danubialis and N. iowensis are considered to be synonymous. Because of the differences in numbers of primary basidiospores and host genus between N. moliniae and N. iowensis, we are maintaining the two as distinct species. The results of this study suggest that there is no basis for recognizing Neovossia as a genus distinct from Tilletia and we consider N. iowensis to be a species of Tilletia. However we were not able to study viable collections of N. moliniae and therefore the status of Neovossia itself remains uncertain.

Our analyses place I. hyalosporus, with ridged teliospores and production of ballistosporic primary basidiospores, within the well supported clade of pooid-infecting species containing T. tritici and allied species, T. indica and T. walkeri (Lineage I). Two species of Conidiosporomyces, C. ayresii and C. verruculosus, were included in this analysis and were closely related to T. vittata (Lineage IV). Conidiosporomyces is distinguished from Tilletia based on the formation of a sac-like, apically open sorus and the presence of Y-shaped conidia (Vánky and Bauer 1992). The unusual Y-shaped conidia are formed in the sorus in C. ayresii and are formed in C. verruculosus in culture. Based on the results of the nLSU analyses the characters that have been used to segregate Ingoldiomyces or Conidiosporomyces from Tilletia cannot be considered generic level characters and at this point we consider both genera synonyms of Tilletia.

The phylogeny of Tilletiales appears to reflect that of the hosts, with a well supported group of closely related species evolving on hosts in the subfamily Pooideae and a poorly resolved group of more diverse species infecting hosts in Chloridoideae, Ehrhartoideae, Arundinoideae and Panicoideae. The relationships elucidated by the phylogenetic analyses in this study suggest a more rapid radiation of Tilletia species on pooid hosts than on hosts in other subfamilies. Phylogenetic studies in the grass family (Poaceae) show two well supported clades comprising six monophyletic subfamilies, the Bambusoideae plus Ehrhartoideae and Pooideae (BEP) clade, and the Panicoideae, Arundinoideae, Centothecoideae and Chloridoideae (PACC) clade (Kellogg 2001). The relationships among subfamilies in the PACC clade are not well resolved in existing phylogenies (Kellogg 2001). Host specificity for individual species of Tilletia remains problematic and species concepts vary from author to author. Genetically distinct lineages can be associated with specific hosts in nature (Boyd and Carris 1997, Boyd et al 1998). However some species of Tilletia, while apparently host specific in nature, have retained the ability to infect other hosts under artificial conditions (Royer and Rytter 1988). More variable gene regions will be required to investigate issues of host specificity and morphological species complexes in this group of fungi.

	ACKNOWLEDGMENTS

 
The authors thank Aimee S. Hyten and Douglas Linn for technical assistance and Amy Rossman for her comments on the manuscript. We express gratitude to Mary Palm for providing a viable collection of Neovossia iowensis.

	FOOTNOTES

 
Accepted for publication March 23, 2005.

Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

Corresponding author. E-mail: castlebury{at}nt.ars-grin.gov

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