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Department of Biological Resources Management, The University of Shiga Prefecture, 2500 Hassaka-cho, Hikone City, Shiga 522-8533, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Choke diseases were surveyed in two closely related grass species, Agropyron ciliare var. minus and Agropyron tsukushiense var. transiens, in Shiga Prefecture, Japan. Perithecia and ascospores were not observed in either case. Stromata on A. ciliare var. minus enclosed and sterilized young inflorescences, as in the typical choke symptoms by Epichloë typhina. On the other hand stromata on A. tsukusiense var. transiens thinly covered mature spikes with white epiphyllous hyphae, as in stromata of Ephelis spp. The fungal isolates produced typical Neotyphodium-type conidia. Molecular phylogenetic analyses using the beta-tubulin gene (tubB) indicated that the two Agropyron species are infected with the species grouping into a novel single clade among Epichloë species and they are closely related to a haploid of hybrid Neotyphodium species. The host plant features may be the cause of the differences between stromata of A. ciliare var. minus and A. tsukushiense var. transiens.
Key words: beta-tubulin, endophyte, Epichloë, Neotyphodium
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The stroma structure (choke) on which conidia are formed, encloses the young inflorescence and prevents seed production. Neotyphodium spp. are asexual close relatives of Epichloë (Siegel et al 1984). Neotyphodium spp. have not been reported to prevent flowering and are transmitted asexually through the seed. Epichloë and Neotyphodium spp. are called endophytes and have attracted attention because of the mutualistic nature of their association with hosts.
Many researchers, including our group, have been studying the distribution of grass species infected with endophytes in Japan, having covered more than 27 species from 13 genera so far. Choke diseases have been reported in four species, Poa sphondylodes, Festuca rubra, Phleum pratense and Brachypodium sylvaticum (Tsukiboshi et al 2002). We found choke diseases of two Agropyron species growing in Japan, Agropyron ciliare var. minus and Agropyron tsukushiense var. transiens. Both species are common in Japan. Agropyron ciliare var. minus is an indigenous Japanese plant, whereas A. tsukushiense var. transiens is introduced. They are perennial and grow along roadsides, on farmland and in cities (Osada 1993). The endophyte infection might confer an advantage to the Agropyron species, enhancing their persistence. We previously reported the distribution and variation in hyphal growth types in A. ciliare var. minus (Yanagida et al 2004).
In the current paper we report new choke diseases and their molecular phylogenetic analysis using a comparison of nucleotide sequences of beta-tubulin genes (tubB) in A. ciliare var. minus and A. tsukushiense var. transiens in Japan.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Isolation of endophytes.— –
The fragments were cut from interior tissues of the stromata regions under sterile conditions and directly rubbed against PDA media. After incubation 7–14 d at 25 C the edges of the hyphal colonies were inoculated onto a new PDA plate. In nonchoked plants, the leaves were sterilized by 1% NaClO and used for fungal isolation. Resultant isolates were used in subsequent experiments (TABLE I).
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PCR reaction and sequencing.— –
PCR amplification of tubB fragments was performed with the primers designed by Tsai et al (1994) and Ex Taq (Takara Bio Inc.). The PCR temperature program was 94 C for 7 min for 1 cycle, followed by 94 C for 1 min, 60 C for 1 min, 72 C for 1 min for 40 cycles and a final extension at 72 C for 7 min. The PCR product was purified using the QIAquick PCR Purification Kit (QIAGEN) and sequenced with PCR-primers and an ABI Prism genetic analyzer (Model 310).
Phylogenetic analysis.— –
The nucleotide sequence of a ca. 520 bp region on tubB was used in the phylogenetic analysis. Twenty-six corresponding sequences from another Epichloë/Neotyphodium species were obtained from the GenBank database (TABLE I
). These nucleotide sequences from 40 fungal data were aligned with Clustal W (Thompson et al 1994) and manually edited. This alignment was deposited in TreeBASE. A phylogenetic analysis of the data was done by using the maximum-likelihood criterion as implemented with PAUP*4.0b10 (Swofford 2002) using the K80+G (kimura 2-parameter +gamma distribution of rate substitutions) model as selected by hLRT and AIC in Modeltest v. 3.7 (Posada and Crandall 1988). The transition/transversion ratio was 1.6796, and the gamma distribution shape parameter (alpha) was 0.3798. Maximum likelihood analyses with heuristic search resulted in a single tree (score: –lnL = 1812.46394). To assess the confidence in each of the internal nodes of the constructed phylogeny 1000 bootstrap trees were generated under the maximum parsimony criteria using the heuristic search method (Felsenstein 1985).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). We subsequently examined 49 additional areas of A. tsukushiense var. transiens habitats in Shiga Prefecture for choked A. tsukushiense var. transiens. The plant was observed in all areas, but stromata were found only at one site, in which seven choked plants grew adjacently. All the choked plants showed incomplete, irregularly shaped stromata similar to those observed at the Usogawa River.
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) were used in further experiments. The fungal isolates produced typical Neotyphodium-type conidia.
Beta-tubulin gene (tubB) phylogeny.— –
PCR amplification of the tubB gene yielded a single product of approximately 720 bp fragments for eight isolates from A. ciliare var. minus and six isolates from A. tsukushiense var. transiens (TABLE I). Direct sequence analysis showed that there were no DNA sequence ambiguities and Southern blot analysis of the tubB indicated single signal in each sample (data not shown), which suggest that there was only one copy of the gene in each genome. A single copy of the tubB gene and ability to form stromata indicate that the Japanese fungi isolated from Agropyron spp. are nonhybrid.
To determine placement of the closely related fungi, phylogenetic analysis was carried out. The tubB nucleotide sequences in fungi isolated from A. ciliare var. minus and A. tsukushiense var. transiens were almost identical (FIG. 2). The maximum likelihood tree for the tubB region formed generally received clades with high bootstrap support. All Japanese isolates from Agropyron spp. grouped into a same clade with some asexual endophytes, namely Neotyphodium chisosum, HboTG -2(1), HbrTG-2(1), HbrTG-1 and HeuTG-2 (1). Previous known Epichloë species did not group into this clade. The bootstrap value for this clade was 86. Pairwise comparisons of the tubB sequences within this clade showed that the sequences of Japanese fungi isolated from Agropyron spp. are identical with or different only by one or two bases from those of AY137612
[GenBank]
(HbrTG-1), AF457483
[GenBank]
(HboTG-2) and AF457485
[GenBank]
(HbrTG-2). Except HbrTG-1, the other four isolates had two or three alleles of tubB (Moon 2004).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) (Christensen et al 2000). However, among pathogenic fungi in both plants, tubB sequences are almost identical and belong to the same clade in the phylogenetic tree (FIG. 2). This suggests that differences in host plant features resulted in the differences in choke symptoms between A. tsukushiense var. transiens and A. ciliare var. minus. Phylogenetic trees showed the Japanese Epichloë isolates from Agropyron spp. (tribe Triticeae) are novel Epichloë species grouping with Neotyphodium spp., hybrid isolates of HbrTG-2 and HboTG-2 and a haploid isolate of HbrTG-1. These Neotyphodium isolates originated in Eurasia (the ATCC record showed that HbrTG-2 and HboTG-2 were isolated respectively from Kazakhstan and Iran). The hosts of these isolates were Hordeum or Hordelymus (Tribe Triticeae). These similarities of hosts and geographical distribution also strongly suggested that the Japanese isolates from Agropyron spp. are closely related to the haploid isolate HbrTG-1 and more closely related to the presumed ancestral haploid of hybrid isolates HbrTG-2 and HboTG-2.
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FOOTNOTES |
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1 Corresponding author. E-mail: i14nyanagida{at}ec.usp.ac.jp
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Christensen MJ, Takahashi K, Kohno K, Tsykiboshi T, Kobayashi M. 2000. Occurrence of an Ephelis fungus on Ishigaki Island and observations on its Epiphytic Association with host grasses. JIRCAS J 8:49–59.
Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791.[CrossRef]
Moon CD, Craven KD, Leuchtmann A, Clements SL, Schardl CL. 2004. Prevalence of interspecific hybrids amongst asexual fungal endophytes of grasses. Mol Ecol 13:1455–1467.[CrossRef][Medline]
Osada T. 1993. Illustrated grasses of Japan (enlarged edition). [In Japanese]. Tokyo, Japan: Heibonsha Ltd. 777 p.
Posada D, Crandall KA. 1998. Modeltest: testing the model of DNA substitution. Bioinformatics 14:817–818.
Sampson K. 1933. The systemic infection of grasses by Epichloë typhina (Pers.). Trans Brit Mycol Soc 18:30–47.
Siegel MR, Johnson MC, Varney DR, Nesmith WC, Buckner RC, Bush LP, Burrus PB, Jones TA, Boling JA. 1984. A fungal endophyte in tall fescue: incidence and dissemination. Phytopathology 74:932–937.
Swofford DL. 2002. PAUP*: Phylogenetic analysis using parsimony (*and other methods), version 4.0. Illinois Natural History Survey: Champaign, Illinois.
Thompson JD, Higgins DG, Gibson TJ. 1994. Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-spechific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680.
Tsai H-F, Liu J-S, Staben C, Christensen MJ, Latch GCM, Siegel MR, Schardl CL. 1994. Evolutionary diversification of fungal endophytes of tall fescue grass by hybridization with Epichloë species. Proc Natl Acad Sci USA 91:2542–2546.
Tsukiboshi T, Yoshida S, Shinohara H, Tsushima S. 2002. Bibliography of parasitic and symbiotic microbes on wild plants in Japan. [In Japanese]. Ibaraki, Japan: The National Institute for Agro-environmental Sciences. 169 p.
Yanagida N, Baba N, Tajimi A, Ueda M, Noishiki Y, Mori K, Ishiguro T, Nakamura T. 2004. Variation and distribution of endophytic fungus, Neotyphodium typhinim, isolated from Agropyron ciliare var. minus (Aokamojigusa) around the coast land of Lake Biwa. [In Japanese]. Grassland Sci 50:174–179.
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