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Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China
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ABSTRACT |
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Epichloë species are fungal symbionts (endophytes) of grasses, six European and four North American biological species in genus Epichloë have been described in previous researches. In this study we describe a new Epichloë species, Epichloë yangzii Li et Wang, found in natural symbioses with Roegneria kamoji native to China. We investigated the host specificity, morphology, interfertility tests and molecular phylogenetic evidences of this new species. The results indicated that E. yangzii is host specific and seedborne. Most morphological characteristics of this new species are typical in the genus. However differences are evident in several features including size of perithecia, asci and ascospores. In mating tests E. yangzii was not interfertile with E. elymi isolates from related hosts in genera Elymus. Phylogenetic relationships based on sequences of ß-tubulin gene (tub2) introns and translation elongation factor 1-
gene (tef1) introns showed that members of the new species grouped into exclusive clades with high bootstrap value.
Key words: E. yangzii, host specificity, interfertility tests, morphology, phylogenetic relationship, Roegneria kamoji
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTAXONOMYRESULTSDISCUSSIONLITERATURE CITED |
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). Although the first Epichloë species was recorded in 1798 (as a Sphaeria species), members in this genus did not attract much attention from scientists for nearly 180 y. In 1977 Bacon et al (1977) found a clavicipitaceous endophyte, inhabiting Lolium arundinaceum (= Festuca arundinacea: tall fescue), a forage grass associated with fescue toxicosis of cattle and other livestock. That endophyte later was classified as Neotyphodium coenophialum (Glenn et al 1996) and characterized as an interspecific hybrid with Epichloë spp. ancestors (Tsai et al 1994). More researches have shown that Epichloë species and their asexual relatives (Neotyphodium species)—collectively called epichloé endophytes—can mediate enhanced plant growth, toxicity to herbivores, plant resistances to insects and pathogens, and increased tolerance of their hosts to drought and some other environmental stresses (Clay 1990, Schardl 1996, Stone et al 2000, Schardl 2001, Schardl et al 2004). Because of these beneficial properties epichloé endophytes have been investigated recently in many countries (Clay 1990, Leuchtmann et al 1994, Schardl and Leuchtmann 1999, Gentile et al 2005).
Epichloë and Neotyphodium species systemically infect their host plants and colonize all aerial parts of the plants (Stone et al 2000, Schardl et al 2004). The sexual cycle of Epichloë species is initiated by external stromata on shoots (specifically on the flag leaf sheath) of their hosts, preventing emergence of the inflorescences. This is commonly known as "choke" of grasses (Schardl 1996). Heterothallic matings occur on the stromata and produce reproductive structures. Effective horizontal transmission is common in some Epichloë species, whereas some species are capable of vertical transmission via plants seeds, as are the asexual Neotyphodium species (Schardl 1996, Schardl et al 2004).
Before 1993 all members of the genus Epichloë in cool season grasses were classified in the single species E. typhina (Pers.:Fr.) Tul., based on the characteristic sexual stromata (White 1993). Since phylogenetic analysis engaged in taxonomy, more Epichloë species were newly established (Leuchtmann and Schardl 1998, Schardl and Leuchtmann 1999). To date 10 Epichloë species have been described and this species are endophytic in tribes Aveneae, Brachypodieae, Bromeae, Brachyelytreae, Meliceae, Poeae and Triticeae (White 1993, White 1994, Leuchtmann et al 1994, Leuchtmann and Schardl 1998, Schardl and Leuchtmann 1999). Most of them are highly host specific, and the interfertility groups have restricted host ranges within the Poöideae (Clay and Leuchtmann 1989, Schardl et al 1997, Schardl et al 2004).
Of these 10 established species, nine mating populations (MP) so far have been identified. All these mating populations but MP-I (E. typhina from plants in numerous genera and E. clarkii from Holcus lanatus, in Europe) are specific at the level of host tribe, genus or species (White 1993, Leuchtmann and Schardl 1998, Schardl and Leuchtmann 1999).
Roegneria C. Koch is a large genus in Triticeae of Poaceae. Plants in this genus are spread widely in Eurasia, while several are found and referred as Elymus L. species in North America (Cai 1997, 2002). In China R. kamoji Ohwi is one of the most widespread weed grasses in the middle and lower basin of Yangtze River, and choke development has been briefly recorded on this plant (Fang 1996). However details on this causal fungus have not been reported previously. In this paper a new species E. yangzii Li & Wang obtained from R. kamoji, is recommend and described.
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MATERIALS AND METHODS |
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The stromata or culms bearing stroma were cut into 5–10 mm long segments and disinfected by double treatments of 75% alcohol and NaOCl (1% available Cl). After rinsing in sterile water the samples were placed on potato-dextrose agar (PDA) and incubated at ca. 28 C in dark. After 1–2 wk incubation colonies developed from the cut injuries of plant samples. Fungal isolates were obtained by single conidium isolation, and subcultured on PDA plates under the same conditions. Fungal isolates were stored at 4 C.
For mating tests, Elymus virginicus 184, harboring E. elymi E184 (ATCC 200850, mat-1) and El. virginicus 757, harboring E. elymi E757 (ATCC 201553, mat-2) were provided by Dr C.L. Schardl (University of Kentucky). Seeds of both El. virginicus 184 and 757 disinfected by double treatments of 75% alcohol and NaOCl (1% available Cl) were soaked in the sterilized water 2 d and planted in 20 cm pots containing paddy soil. El. virginicus seedlings were grown in a greenhouse. E. elymi E184 and E757 also were isolated and maintained respectively as described above.
Mating tests.— – Epichloë-infected R. kamoji were checked daily in Weigang (Rnj5201, Rnj5202) and Yueya Lake (Rnj5701, Rnj5702, Rnj5704) in Nanjing from early Mar 2005. When the first stroma formation on a culm was evident the plant was transplanted into a 20 cm pot with paddy soil and removed into the greenhouse before insect mediated mating could occur. In total 28 stromata that developed on R. kamoji and a single stroma on one of the El. virginicus 184 plants were used as the stromatal parents in the mating tests. Seven fresh fungal cultures (E184, E757, Rnj3302, Rnj3303, Rnj3304, Rnj4201 and Rnj4301 respectively) on PDA were the sources of spermatia. The spermatia were inoculated onto freshly and fully emerged stromata by rubbing culture suspensions with the stromata. Control tests were conducted by rubbing three stromata with sterile water. Every stroma was covered by a paper bag.
Stroma development was checked after 3 wk for perithecial formation. A small piece of the yellow stroma tissue with a few perithecia was removed, crushed in water, and viewed at 100x or 200x. When the mature asci and ascospores had been observed (approx. 4 wk), the stroma was cut and placed over water agar (2%) to check for the ejection and germination of ascospores.
Morphological examination.— – Small pieces of stromata were excised, crushed in water and examined by light microscopy. Mean lengths and widths of conidia and standard errors were reported based on at least 20 measurements each. The morphological characteristics of mature stromata and perithecia were observed from microtome sections, while asci and ascospores were observed from free-hand sections. The septa of ascospores were confirmed by phase contrast microscope observation.
Plant tissues (pith, leaf sheath, seed) of stroma-forming and nonstromal plants were stained by rose Bengal, and fungal hyphae were detected with a light microscope. To confirm the seed transmission of the endophyte, mature seeds collected from stroma-forming plants were soaked in the water in Petri dishes at 20 C for 2 d and planted in pots. At third leaf stage, the fungal hyphae within the leaf sheathes were checked as described above.
For characterization and identification of fungal isolates, morphology and growth rate of colonies were observed and recorded on PDA plates after 24 d incubation at 25 C in the dark. Mycological properties of hyphae, conidia and phialides were observed respectively under the light microscope by using 2 wk old colonies on the same plates as similarly determined.
Isolation of genomic DNA.— –
Total genomic DNA of each fungal isolate was isolated by the potassium acetate extraction method (George et al 1998), with slight modifications. Fungal isolates were cultured on liquid culture medium GY (per 1 L: 5 g glucose, 2 g yeast extract, 0.5 g MgSO4·7H2O, 3 g KH2PO4, 2 g K2HPO4·3H2O, pH 6.5). After 30 d of cultivation, mycelia were collected, freeze-dried and ground into fine powders in liquid nitrogen with a mortar and a pestle. Approx. 25 mg of powdered mycelium was suspended in 650 µL of extraction buffer (100 mM Tris-HCl, pH 8.0; 50 mM EDTA; 500 mM NaCl; and 1% sodium dodecylsulfate), incubated at 65 C for 30–45 min. Then 100 µL of 5 M potassium acetate was added and the sample mixed thoroughly. Cellular proteins were removed with chloroform-isoamyl alcohol (24:1 v/v) extraction, and DNA was precipitated in 100% ethanol and washed by chilled 70% ethanol. Finally the DNA was dissolved thoroughly in 100 µL TE buffer (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0) and stored at 4 C.
DNA amplification and sequencing.— –
DNA fragments including ß-tubulin gene (tub2) intron 1–3 and translation elongation factor 1- gene (tef1) intron 1–4 were amplified by PCR as described previously (Schardl et al 1997, Gentile et al 2005), with slight modifications. The primers used for amplification of tub2 were tub2-d 5'-gagaaaatgcgtgagattgt-3', tub2-u 5'-tggtcaaccagctcagcacc-3', and for tef1 were tef1-d 5'-gggtaaggacgaaaagactca-3' and tef1-u 5'-cggcagcgataatcaggatag-3' respectively. Reactions were performed in 25 µL volumes containing 2.5 µL 10xPCR buffer (100 mM KCl, 80 mM (NH4)2SO4, 100 mM Tris-HCl, pH 8.4), 5 mM MgCl2, 200 µM each of dATP, dGTP, dCTP and dTTP, 80 nM each primer, 1.25 U Taq DNA Polymerase (Shenergy Biocolor, Shanghai, PRC) and 1 µL fungal genomic DNA extract (approx. 10 ng). Reactions were carried out in a MyGeneTM Series Peltier thermal cycler (Long-Gene, Hangzhou, PRC) programmed for an initial incubation at 94 C for 3 min, followed by 35 cycles at 94 C 45 s, 58 C 45 s, 72 C 75 s, then a final extension reaction at 72 C 10 min. Amplified products were separated by electrophoresis in 1.5% agarose gels and purified with 3 S Spin DNA Agarose Gel Purification Kit (Shenergy Biocolor, Shanghai, PRC). PCR products were ligated with pUCm-T (Shenergy Biocolor, Shanghai, PRC) and amplified in Escherichia coli JM109. PCR products were sequenced by Shanghai Shenergy Bio-color BioScience & Technology Inc. (Shanghai, PRC). DNA fragments of tub2 and tef1 from six fungal isolates were sequenced.
Molecular phylogenetic analysis.— –
For phylogenetic analysis, we chose representative Epichloë isolates from different host species or geographic origins and out-group taxon, Claviceps purpurea (Fr.) Tul. (ATCC20102) (TABLE I). DNA sequences first were analyzed by DNAssist 2.0 (Patterton and Graves 2000) and aligned by Clustal X 1.81 (Thompson et al 1994). Alignments were checked by eye for ambiguities and adjusted if necessary. Alignment gaps were treated as missing information.
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), in which all nucleotide substitutions were unordered and reversible. Branch-and-bound as well as heuristic search methods for finding the optimal trees under the MP criterion were conducted. To assess the robustness of the topology 1000 bootstrap replicates were run by MP using heuristic search.
For the neighbor joining (NJ) gene tree, Kimura two-parameter distance matrices were calculated by DNADIST implemented in PHYLIP 3.63 (Felsenstein 2004). The transition to transversion ratio (ts/tv) was assumed to be 2.0. NJ gene trees were derived with NEIGHBOR implemented in PHYLIP. Bootstrap (1000) analysis employed SEOBOOT, DNADIST, NEIGHBOR and CONSENSE in PHYLIP 3.63.
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TAXONOMY |
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Stromata similar to that of Epichloë typhina 35.3–58.1 mm long, singly forming on the culm, preventing emergence of the inflorescence. In early stage the conidial stroma somewhat waxy, and then gradually turning white, smooth, moist, and covered with a dense layer of conidiogenous cells and conidia. Conidiogenous cells hyaline, unbranched, 25–45 µm long, 4–5 µm wide at the base, tapering from medial septum to 1.0 µm at the tip; conidia hyaline, aseptate, lunate to reniform, 5.3–.0 x 2.0–2.5 µm. After fertilization the stromata become bright yellow to orange, tuberculate, dry, and covered with perithecia. Perithecia pyriform, with short necks, 205–275 µm high, 90–140 µm wide, ca 25 per mm2. Abundant asci in each perithecium. Asci 189–252 x 5.0–5.3 µm, with apical thickening and pore, containing eight asco-spores. Ascospores filamentous, 183–250 x 1.9–2.1 µm, hyaline, ejected in chains, and developing up to 20-septa after germination.
Anamorph. – Neotyphodium sp.
Known distribution. – As an endophytic fungus inhabiting Roegneria kamoji Ohwi grown in five provinces in the middle and lower basin of Yangtze River, central part of China provinces Jiangsu, Zhejiang, Anhui, Hubei and Hunan.
Etymology. – Referring to the geographic origin where the Roegneria plants used in this study were grown, in the middle and lower basin of Yangtze River of China.
HOLOTYPE. – CHINA: Xuanwu Lake, Nanjing, Jiangsu province, fungal stroma on Roegneria kamoji Ohwi (synonyms: Roegneria tsukushiensis Honda = Agropyron kamoji Ohwi = Agropyron tsukushiense (Honda) Ohwi var. transiens (Hack.) Owhi = Elymus tsukushiensis var. transiens Honda). Jul 2003, leg. W. Li & Z. Wang. (Rnj3302)
Specimens examined. – Rnj3203 from R. kamoji, Nanjing, Weigang Apr 2003, leg. W. Li & Y. Ji, AS 5.870 (China General Microbiological Culture Collection Center); Rnj3302 to Rnj3304 from R. kamoji, Nanjing, Xuanwu Lake Jul 2003, leg. W. Li & Z. Wang, AS 5.876, AS 5.877, AS 5.878; Rnj4201 from R. kamoji, Nanjing, Weigang Apr 2004, leg. W. Li & Z. Wang. Rnj4301 from R. kamoji, Nanjing, Xuanwu Lake Apr 2004, leg. W. Li & Z. Wang; Rnj5201, Rnj5202 from R. kamoji, Nanjing, Weigang Apr 2005, leg. W. Li & Z. Wang; Rnj5701, Rnj5702, Rnj5704 from R. kamoji, Nanjing, Yueya Lake Apr 2005, leg. W. Li & Z. Wang.
Additional specimens examined. – DCP03158 to DCP03162 from Roegneria plants, CHINA. Suzhou, Jiangsu. 22 May 2000, leg. Z. Wang. DCP15261 to 15264 from Roegneria plants, CHINA. Nanjing, Jiangsu. 26 May 2001, leg. Z. Wang. DCP15265 to 15272 from Roegneria plants, CHINA. Hefei, Anhui. 30 May 2001, leg. H. Yu. DCP40424 to 40431 from Roegneria plants, CHINA. Hangzhou, Zhejiang. 20 May 2002, leg. Z. Wang. Rcs4101, Rcs4201 to Rcs4205 from R. kamoji, CHINA. Changsha, Hunan. 3 May 2004, leg. W. Li & K. Wu. Rwh4101 to Rwh4105 from R. kamoji, CHINA. Wuhan, Hubei. 3 May 2004, leg. W. Li & K. Wu.
Known distribution. – Central part of China, in the middle and lower basin of Yangtze River.
Characteristics in culture. – On PDA plates, colony diam 20–62 mm after 24 d at 25 C, white, cottony, smooth or slightly rough, the growth rate very or moderately slow variable with different sampling sites; aerial mycelium smooth, septated and ca. 2 µm wide; colony reverses pale brown and lighter toward the margins. Sporulation in culture abundant; conidiogenous cells discrete, arising solitary from the aerial mycelium, septated at the base, more or less cylindrical over the lower one-third, hyaline, smooth, 16.5–25.8 µm long, 2.2–2.9 µm at base, tapering to less than 1.0 µm at tip, septated at the base. Conidia hyaline, lunate to reniform, 4.7–5.2 x 2.0–2.9 µm.
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RESULTS |
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). Almost all other plants without stroma in the same population possessed endophytic fungal hyphae in culms, leaf sheaths and seeds.
Although stromata on R. kamoji occurred frequently, stroma development usually was observed only on a few tillers of each plant, other inflorescence developed and seed produced as usual. Seeds harvested from stromatized plants were also Epichloë-infected (FIG. 2). From the seedlings propagated from these seeds harboring the endophytic fungus, typical fungal hyphae of the endophyte were observed in the intercellular spaces in leaf sheathes. This indicated that the Epichloë species in R. kamoji plants could be transmitted vertically through seeds.
Interfertility groups.— – Because none of other Epichloë species originally was reported from Roegneria grasses, difference in mating population with E. elymi, the only species obtained from the same tribe Triticeae, was investigated.
Fungal isolates from R. kamoji were highly interfertile with each other when opposite mating types were paired. In such cases the perithecia were filled with abundant asci and ascospores, the ascospores ejected from the perithecia (FIG. 5). Eight ascospores were ejected end-to-end onto the water agar. Asco-spores germinated on the water agar resulted in production of phialides and conidia. If the same mating types were paired there was no reaction on the stromata and the white stromata would not change color over time. In contrast when the stroma of E184 was mated as female with strains from R. kamoji, or the stromata from R. kamoji as the female mated with E184 and E757, the perithecium-like structures were barren, with no discernible asci and ascospores. There was no conflict between all results of mating tests (TABLE II).
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MP trees based on tub2 and tef1 gene sequences were similar: Most members of a particular Epichloë species tended to cluster together. For example the isolates of E. brachyelytri, E. festucae, E. bromicola, E. clarkii, E. elymi and E. glyceriae grouped into exclusive clades with high bootstrap value (FIG. 7) and in accordance with previous reports (Schardl and Leuchtmann 1999, Craven et al 2001). Likewise the new group of E. yangzii clustered into distinct clades: the clade in the tub2 gene tree was supported in 89% of the bootstrap replicates; the clade in the tef1 gene tree was weaker, appearing in 73% of the bootstrap replicates. The topology of the two NJ gene trees was nearly identical to the MP trees (FIGS. 7, 8). Because NJ is relatively rapid it was feasible to include outgroup sequences. The same clade in NJ gene trees for tub2 and tef1 received respectively 83% and 87% bootstrap support (FIG. 8).
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DISCUSSION |
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Roegneria plants are natively grown in China, and R. kamoji is the native grass in the middle and lower basin of Yangtze River, (Cai 1997, 2002). In this area we found lots of grasses infected by endophytic fungi, stromata are produced only on Roegneria plants and most of them are R. kamoji, although choke development has been recorded on some other grasses in grassland in Inner Mongolia in China (White et al 2001). Hyphae of E. yangzii in R. kamoji easily were detected from leaf sheathes, culms and seeds (FIG. 2), and no other symptom were induced except the occasional stromatized culms. The fungal hyphae and plant cells co-exist benignly within the plant tissues and could be transmitted through seeds. In this way E. yangzii is similar to some other Epichloë species. For example, E. typhina and E. baconii are not seed transmissible in most of their hosts.
Most previously recorded Epichloë species are usually highly host specific, with the exception that E. typhina has a broad host range, inhabiting grass species within tribes of Aveneae, Brachypodieae and Poeae. E. baconii from Europe and E. amarillans from North America each infect two genera in Aveneae, E. festucae infects three genera in Poeae and Aveneae, and each of the remaining six Epichloë species has been reported from a single host grass species (Schardl et al 1997, Schardl and Leuchtmann 1999, Schardl et al 2004). Roegneria is closely related to genus Elymus L. also in tribe Triticeae (Baum 1991, Jensen and Chen 1992, Cai 1997). However morphological characteristics (TABLE III), mating tests (TABLE II) and phylogeny analysis (FIGS. 7, 8) indicated the differences between E. yangzii and E. elymi and which from Elymus species.
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). Mycological characteristics of the fungal culture on the PDA plates, including colony morphology, growth rate, the shapes and sizes of conidia and phialides, were fairly similar to those of other Epichloë species, indicating phylogenetic evidences are necessary for further taxonomic study.
The results of crossing experiments (TABLE II) indicated the differences in mating population between E. yangzii from Asia and E. elymi from North America. Studies have shown that interfertility groups have a strong tendency to follow both taxonomic and geographic positions of their host plants (Schardl 2001, Schardl et al 2004). Although E. amarillans (North American species, MP-IV) and E. baconii (European species, MP-V) both infect Agrostis spp., they are in different mating populations so their geographic separation seems to be important for their differentiation (White 1993, White 1994). The mating population of E. yangzii may be a new one, but the possibility of interfertility with other MPs should be tested in future studies.
In these four phylogeny trees (FIGS. 7, 8), the E. yangzii clade grouped most closely with E. bromicola, indicating the genetic similarities of these two species. However the host specific E. bromicola originally was obtained from Bromus erectus, B. benekenii and B. ramosus in tribe Bromeae in Europe. These hosts rarely were recorded in China (Cai 1997), and in our investigation we never found other grasses, except Roegneria plants, that were infected by Epichloë. Otherwise sexual structures of E. yangzii also were smaller than those of E. bromicola (TABLE III). These indicated that significant differences exist between these two species. Furthermore the possibility of interfertility between these two species would be interesting to investigate in further studies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: zwwang{at}njau.edu.cn
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LITERATURE CITED |
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