Mycologia, 101(5), 2009, pp. 681–685. DOI: 10.3852/08-181 # 2009 by The Mycological Society of America, Lawrence, KS 66044-8897
A new Neotyphodium species from Festuca parvigluma Steud. grown in China Yong-gan Chen Yan-ling Ji Han-shou Yu Zhi-wei Wang1
1-a gene (tefA) most of these species have been shown to have evolved from Epichloe¨ species hybridizations or directly from Epichloe¨/Neotyphodium lineages (Moon et al 2004, Tsai et al 1994). Several endophytes have been isolated from Festuca spp. plants grown in Europe and America (Schardl and Leuchtmann 2005). However to date none has been reported from Asian native Festuca plants. In this paper we describe a new Festuca endophyte of F. parvigluma, Neotyphodium sinofestucae, based on host specificity, morphology and molecular phylogeny.
Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China
Abstract: Neotyphodium species evolved from Epichloe¨ species and are asexual, seedborne endophytes in many cool-season grasses. Here we propose a new species inhabiting Festuca parvigluma native to China. Morphology, host specificity and molecular phylogenetic evidence supported recognition of this new species. Sequences of b-tubulin gene (tubB) introns and translation elongation factor 1-a gene (tefA) introns were present as two copies in all five isolates examined. In phylogenetic analyses copy 1 was closely related to E. yangzii in the EBY clade and copy 2 with E. typhina in the ETC clade, indicating this new species might have originated as a result of hybridization between members of these two clades. Referring to the distribution area of host plants, Neotyphodium sinofestucae is proposed for this new species. Key words: endophyte, hybridization, Neotyphodium sinofestucae, phylogeny
MATERIALS AND METHODS
Biological materials.—Gramineous plants were collected in Nanjing, China. Five isolates obtained from F. parvigluma together with representative isolates of Epichloe¨ and Neotyphodium species were selected for phylogenetic analysis (SUPPLEMENTARY TABLE I). Endophyte isolation and morphology.—Young culms of F. parvigluma were cut, surface sterilized and fungal isolates were obtained as described by Li et al (2006). Single conidium isolation was repeated three times on PDA by streaking. These isolates were stored on PDA slants. Five isolates were cultured at 25 C on PDA, growth rates of colonies were measured every 2 d with four repeats per isolate; fungal morphological characters were measured after 3 wk incubation (Li et al 2006). DNA extraction, amplification and sequencing.—Genomic DNA was extracted by potassium acetate and stored at 4 C (Li et al 2006). Amplification of tubB and tefA fragments was carried out with a MyGeneTM Series Peltier thermal cycler (LongGene, Hangzhou, PRC). Individual gene copies were amplified separately with selective primers CB-1U, KB-3U, CF-1D, KF-4D (SUPPLEMENTARY TABLE II). Selective primers were designed based on conserved sequences with 39 end polymorphic sites. PCR amplification, product separation, purification and cloning into E. coli DH 5a were carried out as described by Li et al (2006). Clones were sequenced by Genestar Biotechnology Co. Ltd., Tianjin, PRC.
INTRODUCTION
Epichloe¨ endophytes refers to a group of fungi inhabiting aerial tissues of cool-season grasses, comprising teleomorphic Epichloe¨ spp. and their asexual derivatives, Neotyphodium spp. (Schardl et al 2004). These fungi grow systemically within plant intercellular spaces without causing disease symptoms but occasionally develop stromata on the culms. Some endophytes grow into the ovule of inflorescences and are transmitted via seeds (Schardl and Phillips 1997). Grass-endophyte symbioses provide various benefits to agriculture (Schardl et al 2004). Seventeen species of Neotyphodium endophytes have been well characterized by morphology, host specificity and molecular data (Moon et al 2007, Schardl and Leuchtmann 2005). By analyses of btublin gene (tubB) and translation elongation factor
Phylogenetic analysis.—DNA sequences were analyzed by DNAssist 2.2 (Patterton and Graves 2000), Clustal X 1.81(Thompson et al 1994) then manually adjusted if necessary. Alignment gaps and ambiguous characters were treated as missing information. Maximum parsimony (MP) analyses were performed in MEGA 4 (Tamura et al 2007), in which all characters were unordered and reversible. We conducted close neighbor interchange search methods for finding the optimal trees under the MP criterion. To assess the robustness of the topology 1000 bootstrap replicates were run by MEGA 4. Alignments for tubB and tefA were submitted to TreeBASE (SN4358).
Accepted for publication 23 March 2009. 1 Corresponding author. E-mail:
[email protected]
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FIG. 1. Morphology of N. sinofestucae. A. Conidiogenous cell and conidia, bar 5 1.5 mm; B. Hyphae within F. parvigluma, bar 5 15 mm; C. Surface of colony (3 wk), bar 5 2.0 mm; D. Reverse of colony (3 wk), bar 5 2.0 mm.
TAXONOMY
Neotyphodium sinofestucae Y. Chen, Y. Ji et Z. Wang, sp. nov. FIG. 1 MycoBank: MB 513026 Coloniae 6.0–14.7 mm diam aetate 3 hebdomadum ad 25 C in PDA, elevatae, convolutae, albae, gossypinae. Coloniae reversae brunneae in centro, eburneae ad marginem. Hyphae vegetative hyalinae, 2.4–2.7 mm latae. Cellulae conidiogenae singulae, plerunque non septatae ad basim, cinlindracae, 19–28 mm longae, 2.4–2.7 mm latae ad basim, ad 1.3–1.5 mm gradatim angustatae. Conidia reniformia, hyaline, laevia, 5.2–6.3 3 2.6–3.1 mm.
Colony diam 6.0–14.7 mm after 3 wk at 25 C on PDA, colonies arising from agar surface, convoluted, white and cottony. Colony reverses brown centrally to light tan at margins. Vegetative hyphae hyaline, septate, 2.4–2.7 mm wide. Phialides solitary, arising from lateral orthotropic hyphae, cylindrical at base and tapering toward the apex, hyaline, determinate, TABLE I.
usually without septa at or near the base, 19–28 mm long, 2.4–2.7 mm wide at base, about 1.3–1.5 mm wide at apex. Conidia ellipsoid to reniform, hyaline, smooth, 5.2–6.3 3 2.6–3.1 mm. Etymology. Referring to the geographic origin (China) and genus of the host plant (Festuca). HOLOTYPE. CHINA. JIANGSU: Nanjing, fungal within Festuca parvigluma Steud. Apr 2004, leg. Y. Ji & Z. Wang. (NAU020001, Nanjing Agricultural University Herbarium, Nanjing, PRC)
Known host range. Festuca parvigluma Steud. Known distribution. As an endophytic fungus inhabiting F. parvigluma grown in Nanjing, Jiangsu province in China. Specimens examined. Fnj4602–Fnj4604 from F. parvigluma, Nanjing, Apr 2004, leg. Y. Ji & Z. Wang; Fnj6605 and Fnj6606 from F. parvigluma, Nanjing, Apr 2006, leg. Y. Ji. Additional specimens examined. Fnj6601 and Fnj6602 from F. parvigluma, Nanjing, Apr 2006, leg. Y. Ji. ZJ-1 and ZJ-3 from F. parvigluma, Nanjing, Apr 2008, leg. Y. Chen &
Morphological properties of some Neotyphodium species
Endophytes
Host
Growth (mm/wk)
Conidial shape
Conidiogenous cell (mm)
Conidia size (mm)
Length
Width
10–30 13–25 15–31
2–5 1.5–3.0 1.5–2.5
N. chisosuma N. huerfanumb N. starriic
Stipa eminens 1.8 on PDA at 20 C Festuca arizonia 7 on PDA at 22 C F. subulata 5 on PDA at 22 C
5–9 3 2.5–4 3–4 3 2–2.5 4–7 3 1.5–3
N. tembladeraed N. uncinatume N. sinicumf
F. agentina F. pratensis Roegneria spp.
N. sinofestucae
F. parvigluma
4–10 3 2–4 10–31 1.5–3.5 13 3 1–2 9–18 1.5–2 4.6–5.9 3 16.1–24.2 1.8–2.5 2.1–3.4 5.2–6.3 3 19–28 2.4–2.7 2.6–3.1
a
ellipsoid, obovate and pyriform short cylindrical to lunate holoblastic allantoid or narrowly reniform , 7 on PDA at 24 C lunate, reniform and sigmoid , 3 on PDA at 25 C uncinate 6.1–16.2 on PDA at reniform 25 C 2.0–4.9 on PDA at ellipse to reniform 25 C
White and Morgan-Jones 1987a. White et al 1987. c White and Morgan-Jones 1987b. d Cabral et al 1999. e Schardl and Leuchtmann 2005, Li et al 2004. f Kang et al 2009. b
CHEN ET AL: NEW SPECIES OF NEOTYPHODIUM
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FIG. 2. Neotyphodium spp. and Epichloe¨ spp. tubB and tefA phylograms based on maximum parsimony (MP). Numbers (. 50%) at branches are the percentage of trees containing the corresponding clade based on 1000 bootstrap replications. tubB MP tree shown: tree length 5 156 steps, consistency index 5 0.759, retention index 5 0.952, rescaled consistency index 5 0.757. tefA MP tree shown: tree length 5 157 steps, consistency index 5 0.866, retention index 5 0.969, rescaled consistency index 5 0.839. Circles represent N. sinofestucae; the boxes represent Neotyphodium endophytes obtained from Roegneria. Y. Ji; ZZQ-1–ZZQ-6 from F. parvigluma, Nanjing, Apr 2008, leg. Y. Chen & Y. Ji.
radial growth rates, conidiogenous cell and conidia dimensions are provided (TABLE I).
RESULTS
Phylogenetic relationships.—PCR amplification of tubB and tefA genes from endophyte genomic DNA yielded products of 725 bp and 844 bp respectively. Phylogenetic analysis revealed that some N. sinofestucae isolates clustered with E. yangzii in the E. yangzii and E. bromicola complex (EBY), while others clustered with E. typhina in the E. typhina complex (ETC). This result indicated the presence of multiple gene copies in genomic DNA. The individual gene copies were amplified separately by selective primers and sequenced (SUPPLEMENTARY TABLE II). Two
Characteristics of the endophyte.—F. parvigluma host plants were rarely found in China. Only 126 plants were investigated 2004–2008, of which 88 were found to be infected with endophytes. Within host tissue, endophyte hyphae are thin, long, little curved and run parallel to each other in the intercellular spaces of aerial plant tissues (FIG. 1B). In culture on PDA colonies are white, cottony, circular with a regular margin and bright yellow reverse (FIG. 1C, D). Conidia are reniform to ellipsoid (FIG. 1A). Colony
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copies of tubB and tefA were obtained from all five N. sinofestucae isolates (FIG. 2). Phylogenetic analyses were conducted to determine the origin of F. parvigluma. Phylogenetic trees were constructed based on tubB and tefA sequences for N. sinofestucae together with sequences of representative Epichloe¨ endophytes (FIG. 2). In the MP analysis copy one of the tubB and tefA genes of N. sinofestucae grouped with the EBY clade and copy two of the tubB and tefA genes of N. sinofestucae grouped with ETC. Members of N. sinofestucae clustered together in both clades. In the tubB phylogram N. siegelii and N. chisosum grouped respectively with copy 1 and copy 2 of N. sinofestucae. N. australiense occurred in the same clade as N. sinofestucae copy 2 (FIG. 2). DISCUSSION
The growth rate of N. sinofestucae was 2.0–4.9 mm/wk on PDA at 25 C, which is slower than other Neotyphodium strains symbiotic with Festuca plants (TABLE I). This characteristic could be used to distinguish N. sinofestucae from N. huerfanum, N. starrii and N. tembladerae. Sporulation by N. sinofestucae was poor on PDA plates with so few conidia produced that they were difficult to find. Slow growth and poor sporulation are distinguishing characteristics of N. sinofestucae. In the phylogenetic analysis N. sinofestucae presented two copies of tubB and tefA that clustered respectively with the ETC and EBY clades, indicating this new species possibly originated from hybridization between members of these clades (FIG. 2). Several Neotyphodium species have been shown to have originated through hybridization and possess two or three copies of tubB and tefA loci, with the exception of N. uncinatum (FIG. 2). N. uncinatum is presumed to have originated from a hybridization between E. typhina and E. bromicola but respectively lost a copy of tubB and tefA (Schardl and Leuchtmann 2005). N. sinofestucae showed similar origins with N. uncinatum and N. sinicum spp. (FIG. 2). However N. sinofestucae was distinct from N. uncinatum, based on morphological characters, phylogentic relationships and geographic distributions (Schardl and Leuchtmann 2005). Compared to the isolates obtained from Roegneria plants (Kang et al 2009), N. sinofestucae grows significantly more slowly and lacks septate conidiogenous cells (TABLE I). RAPD analysis, other gene fragments and microsatellite sequences also showed these two groups of isolates were quite different (Chen et al 2008, Chen and Wang unpubl). Based on host specificity, fungal morphology and phylogenetic evidence, we propose the endophyte found in F. parvigluma native to China as a new species, Neotyphodium sinofestucae. We think this
species is the first Neotyphodium endophyte reported from Chinese native Festuca plants (Li et al 2004, Moon et al 2007, Schardl and Leuchtmann 2005). ACKNOWLEDGMENTS
We acknowledge Prof. X. K. Qin for advice on species determination of plant samples. We thank two reviewers for critical reading and valuable advice. This work was fully supported by National Science Foundation of China (No. 30670008). LITERATURE CITED
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