A Neotyphodium Endophyte from Festuca Myuros L

Mycology An International Journal on Fungal Biology

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A Neotyphodium endophyte from Festuca myuros L. in Nanjing, China

Kui Han , Yan-Ling Ji , Yong Wang & Zhi-Wei Wang

To cite this article: Kui Han , Yan-Ling Ji , Yong Wang & Zhi-Wei Wang (2012) A Neotyphodium endophyte from Festucamyuros L. in Nanjing, China, Mycology, 3:3, 201-209, DOI: 10.1080/21501203.2012.718292 To link to this article: https://doi.org/10.1080/21501203.2012.718292

Published online: 22 Aug 2012.

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A Neotyphodium endophyte from Festuca myuros L. in Nanjing, China Kui Han, Yan-Ling Ji, Yong Wang and Zhi-Wei Wang* Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing, China (Received 25 January 2012; ﬁnal version received 27 July 2012)

Some Festuca species are infected by seed-transmitted Neotyphodium fungi without signiﬁcant pathogenic symptoms. Fungal isolates were obtained from Festuca myuros L. growing in Nanjing. The endophyte infection level was estimated as high as 91.4% in 148 plant samples from different sites, indicating that endophyte infection is common in F. myuros in Nanjing. Morphological characteristics of all 11 isolates were identical with each other. Phylogenetic analysis based on tefA and tubB loci indicated that these fungal isolates clustered with Neotyphodium sinofestucae, a previously reported species harbored in Festuca parvigluma. These fungal endophytes from F. myuros in China were then identiﬁed as N. sinofestucae.Small differences in morphological features and close evolutionary relationship in tefA and tubB genes were demonstrated among isolates from these two different host species. This exhibit species diversity of grass endophytes in Festuca spp. Keywords: Festuca myuros; Neotyphodium sinofestucae; morphological properties; phylogenetics

Introduction in more than 14 Festuca species. Except for heterozygotic The epichloae (Epichloë/Neotyphodium) (Hypocreales, N. typhinum and N. tembladerae, most of these species Clavicipitaceae) are referred to as grass-specific and mutu- were limited to few host species (Moon et al. 2002; Schardl alistic fungal endophytes inhabiting cool season grasses and Leuchtmann 2005; Iannone et al. 2011). Relatively (Shen et al. 2009; Schardl 2010). Recently, Epichloë strict host specificity was observed in the relationships spp. and Neotyphodium spp. were reported as the major between Festuca species and their fungal endophytes. members of epichloae, and the name was given due to In China, grass endophytes were poorly studied in the the evolutionary relationships between Epichloë spp. and last century. Recently, two sexual species and four asexual Neotyphodium spp. (Glenn et al. 1996). species were reported from caespitose southern grasses and Both Epichloë spp. and Neotyphodium spp. are typ- natural grasses growing in semi-arid northern grasslands ically mutualistic to grasses belonging to the subfamily (Wang et al. 2010; Kang et al. 2011). In eastern China, a Poöideae (Clay and Schardl 2002). These endophytes con- recently reported Neotyphodium species, N. sinofestucae, fer many well-documented benefits to their host grasses, isolated from Festuca parvigluma Steud., has a putative ranging from protection against herbivory by both insects hybrid origin between Epichloë yangzii and E. typhina and mammals to resistance to biotic and abiotic stress fac- (Chen et al. 2009). Until now, F. parvigluma grown in tors (Schardl et al. 2004, 2009). Neotyphodium endophytes Nanjing, China, was known as the only host for this new live in the apoplastic space of host grasses, establishing a species. perennial symbiosis (Christensen et al. 2008). There are In this study, we obtained endophytic fungal isolates 23 recorded Neotyphodium species isolated from plants from Festuca myuros L. for the first time. Their tax- of tribes Poeae, Aveneae, Bromeae and Triticeae. The onomic identity was established by morphological and / species of genus Festuca (Poeae) harbor many described phylogenetic characteristics, and a new host endophyte and unknown Neotyphodium species (Moon et al. 2004; combination was reported in this study. Li et al. 2006; Moon et al. 2007; Chen et al. 2009), and are known as the most famous host complex for clavicipi- Materials and methods taceous endophytes. Among all the reported Neotyphodium species, at least Biological materials seven species, including N. coenophialum, N. uncinatum, Gramineous plants were collected in Nanjing, China, in N. typhinum, N. huerfanum, N. starrii, N. tembladerae and the spring from 2006 to 2008. During the mid of April to a Chinese native species – N. sinofestucae – were found June, we collected 148 Festuca plants from Zhenzhuquan

*Corresponding author. Email: [email protected]

Recreation Center, Nanjing. These grasses were identified two alleles of each gene. PCR amplification, product sepa- based on the characteristics of the flowering tillers depend- ration, purification and cloning in Escherichia coli DH5α ing on the morphological characteristics illustrated and were practiced as described (Chen et al. 2009). DNA documented in Flora of China, vol. 22, Poaceae (Wu et al. fragments were sequenced by Beijing Genomics Institute 2006). Plants were transplanted into pots and maintained (Beijing, PRC). in a greenhouse in Nanjing Agricultural University. Fresh ◦ plants and seeds were stored at 4 C for use. Phylogenetic analysis Closely related representative Epichloë and Neotyphodium Detection, isolation and morphological examination isolates of different host species and geographic ori- of endophytes gins were selected by comparisons with BLASTN for Plant culms or leaf sheaths were subjected to endophyte phylogenetic analysis. DNA sequences were analyzed detections. When plant samples were collected after head- by DNAssist 2.2 (Patterton and Graves 2000), cutting ing and plant culms were available, culms were cut into two the sequence that was longer than most of others, and longitudinal parts and pith tissues were scraped and stained aligned by ClustalX 2.01 (Larkin et al. 2007), then with alkaline Rose Bengal (1% in 1 mol/L NaOH) for sev- followed by eye check for ambiguities and adjusted if eral minutes and observed under the light microscope after necessary. Representative fungal isolates from Epichloë a rinse with water. Endophyte detection was judged by the and Neotyphodium species for phylogenetic analysis existence of fungal hyphae running parallel to the longitu- were selected from GenBank based on typical host and dinal axis of the plant tissue. The inner epidermis tissues its distribution (see Supplementary Table S1 available of leaf sheaths were examined when plant culms were not online at http://informahealthcare.com/doi/suppl/10.1080/ available. 21501203.2012.718292). Alignments for tefA and tubB The youngest culms or leaf sheath tissues of the plants were deposited in TreeBASE under accession nos. involving endophytic fungus were cut, surface-sterilized M11306 and M11301, respectively. and the fungal isolates were obtained as described else- Reconstructions of phylogenetic trees were generated where (Kang et al. 2011). These isolates were cultured on by MrBayes v3.12 (Ronquist and Huelsenbeck 2003) with potato dextrose agar (PDA) slants after three repeats of sin- the SYM model with a proportion of invariable sites and a ◦ + gle colony reisolation and then stored at 4 C for temporal gamma-distributed rate variation among sites (SYM G). preservations. This model was selected by MrModeltest v3.7 (Nylander Growth rates of colonies were measured everyday on 2004). Due to the lack of historical dates for interior plates with 20 mL of PDA, and morphological charac- nodes, MrBayes used a fixed substitution rate to reconstruct teristics of each isolate were measured and recorded as the trees. This results in branch length (and tree height) described elsewhere after 6 weeks cultivation on the same being measured in substitutions per site. The Markov chain PDA plates (Li et al. 2006). Monte Carlo (MCMC) method used by MrBayes was allowed to run for 5,000,000 generations. Every 1000th step was recorded and analyzed for height, tree likelihood DNA extraction, amplification and sequencing and many other components. Preceding these recordings is Total genomic DNA was extracted following the meth- a burn-in period equal to 25% of the MCMC chain. All ods described by Li et al. (2006) and stored at −20◦C data from the burn-in period are discarded and the oper- for use. DNA fragments used in this study including ators are not optimized during this time, thus preventing tubB intron 1–3 and tefA intron 1–4 were amplified operators from optimizing incorrectly on trees that are still using a LongGene Thermal cycler. Individual gene alle- considered random at the beginning of each run. This pro- les were amplified separately with selective primers listed cess was done in two independent runs from different tree in Table 1. Selective primers were designed based on con- topologies in order to avoid stacking at a local optimum, served sequences of 3-end polymorphic sites to amplify and resulted in a sample of 2000 trees.

Table 1. PCR primers used in this study.

Primer Sequence (5–3) Origins

tubB 1214 TGG TCA ACC AGC TCA GCA CC Gentile et al. (2005); Schardl and Leuchtmann (1999) tefA 1u CGG CAG CGA TAA TCA GGA TAG Gentile et al. (2005) tefA 6d GGG TAA GGA CGA AAA GAC TCA Gentile et al. (2005) tubB ESC GGA CCG AGA CAA CAT CAT This study tefA ESC CGA GAA GGT GAG ATA TAT C This study tefA EBY GGA GAA GGT AAG ACA TTC T This study Mycology 203

Results In the phylogenetic analysis based on tubB sequences, Sample collection and detection of fungal endophytes both the allele-1 and allele-2 of all fungal isolates in this study were closely related to N. sinofestucae iso- In 148 grass samples from Zhenzhuquan and Jiangjunshan lates Fnj4602 and Fnj6606, the fungal endophyte from in Nanjing, Jiangsu Province, 91.2% harbor fungal hyphae F. parvigluma, indicating a close evolutionary origin and similar to those of grass endophytes. Fungal hyphae were relationship between these two taxa (Figure 2). observed in intercellular spaces in plant culms and leaf In the phylogenetic analysis based on tefA sequences, sheaths of each infected sample. All the plant samples were the allele-1 of all isolates showed a similar result as that identified as F.myuros. of tubB (posterior probability = 0.99), while allele-2 of all isolates clustered in a branch largely separated from those Isolation and cultivation of endophytic fungus of N. sinofestucae isolates branch between F. parvigluma from Chinese native F. myuros and F.myuros isolates, partially suggesting a not so similar From plant culms, nine isolates with nearly the same cul- evolutionary pathway with a high posterior probability of ture features on PDA plates were obtained from indepen- 0.97 (Figure 3). dent F. myuros samples. The colonies reached 15–20 mm in diameter after 6 weeks at 28◦C in PDA; they appeared Discussion raised and strongly convoluted, white to slightly tan at margins and felted with abundant aerial hyphae, with Typical morphological features of Neotyphodium species colony margins clear; colony reverse tan (Figures 1A were observed from the fungal isolates obtained from and B). Hyphae were septated at the branching junction F.myuros plants (Table 2), and phylogenetic analysis shows and were 2.3–2.5 μm wide. Sporulation in culture was common ETC (E. typhina clades) and EBY (E. bromicola sparse; conidiogenous cells were 25.6–31.7 μm long, ca. and E. yangzii) origins of native Neotyphodium species in 2.2–3.1 μm wide at the base, tapering to ca. 0.8–1.1 μmat China (Figures 2 and 3) (Kang et al. 2009). This indi- the tip, the basal septum was observed fairly often; conidia cated that the fungal isolates should be considered as were hyaline, nephroid to lunate, 5.4–7.8 × 2.9–4.1 μm, N. sinofestucae reported previously from F. parvigluma in and a single conidium was observed per conidiogenous 2009 (Chen et al. 2009). cell (Figure 1C). For each isolate, typical morphology and Festuca species are known to have originated in the colony growth rate as typical Neotyphodium species were Mediterranean area in Europe, and many species natu- demonstrated besides the sparse and big conidia. The cul- rally grow in those area. At least seven Neotyphodium ture of strain Fnj7403 (CGMCC 3.13630) was deposited species had been recorded from Festuca species. Their evo- in the China General Microbiological Culture Collection lutionary origins were suspected by molecular phylogenetic Center. analyses using DNA fragments such as tefA, tubB and actG except N. typhinum. In the remaining six species, only N. uncinatum was proposed to be generated by hybridiza- Phylogenetic analysis tion between a member of ETC and EBY. In this study, Sequences of two alleles of tubB and tefA fragments were it was confirmed that all isolates of N. sinofestucae from about 439 and 572 bp, respectively. In phylogenetic analy- F. myuros have double alleles of tefA and tubB frag- ses based on the tubB and tefA sequences, fungal isolates ments, indicating the evolutionary origin of the species from F. myuros were clustered together (Figures 2 and 3). from ETC and EBY. This species is significantly different Both tefA and tubB phylogeny provide the support of a from N. typhinum in heterogeneity, from N. uncinatum in hybrid origin of these isolates, implicating a hybridization allele numbers of tefA and tubB fragments and from other between E. yangzii and E. typhina. Neotyphodium species from Festuca plants in evolutionary origins. The host plant investigated in this study was once identified as Bromus remotiflorus. Later, it was corrected as F. myuros L. This species was also recorded as B. remotiflorus or Festuca remotiflorus in some previous papers (Liu et al. 2005). Regarding the strict host specificity of fungal endophytes in Festuca spp., only two of them (N. tembladerae and N. typhinum) infect host plants belonging to different genera. This is another record showing one species of Neotyphodium endophyte infecting more Figure 1. Colony, phialides and conidia of fungal isolates from F.myuros grown on PDA. (A) Obverse and (B) reverse of colonies than one species of Festuca as reported by Iannone et al. after 6 weeks incubation; (C) phialide and conidium after 10 days. (2011). It is likely that host specificity of N. sinofestucae (Incubation on PDA at 25◦C.) Bar = 5 μm. is lesser than that of species originally found in Europe 204 K. Han et al.

N. australiense 937(1) 0.64N. coenophialum e19(2) N. melicicola 827(1) N. lolli 135 0.82 N. siegelii e915(2) E. festucae ATCC201550 E. festucae ATCC90661 N. tembladerae 4055(1) 0.76 N. tembladerae 1169(1) 1N. stromatolongum Cnj6611 N. stromatolongum Cnj6617 Epichloe sp. 9924 0.84 N. occultans 992(1) 0.93N. coenophialum e19(13) E. baconii ATCC76552 E. baconii ATCC200745 0.67 E. amarillans ATCC200743 0.99 N. chisosum 134(2) 0.77 E. amarillans 906 E. glyceriae ATCC200747 1E. glyceriae ATCC200755 E. bromicola 9633 0.99 E. bromicola 9630 Fnj7409(1) 0.93 Fnj7402(1) N.sinofestucae Fnj6606(1) Fnj7405(1) 0.62 N.siegelii e915(1) 0.56 E. yangzii Rnj4201 E. yangzii Rnj3302 E. yangzii Rnj4301 N. sinofestucae Fnj4602(1) 0.73 N. occultans 992(2) E. liyangensis Ply9101(1) E. liyangensis Ply9102(1) 0.71 1 N. sinicum Rts2102(1) 1 N. sinicum Rxy6106(1) 0.52 N. chisosum 134(1) E. elymi ATCC200850 1E. elymi ATCC201554 0.96E. brachyelytri ATCC200752 E. brachyelytri ATCC201561 N. melicicola 827(2) 1N. aotearoae 829 E. clarkii ATCC200741 1E. typhina E432 0.91E. clarkii ATCC200742 1E. typhina ATCC200851 E. typhina ATCC201666 0.96 E. typhina ATCC200738 0.99N. guerinii CBS 112036(1) 0.93N. guerinii ATCC MYA1235(1) E. typhina ATCC200740 0.57 0.94 N. sinicum Rxy6106(2) 0.93E. sylvatica ATCC200748 E. sylvatica ATCC200751 E. typhina ATCC200739 N. tembladerae 1169(2) 1 0.92 N. tembladerae 4055(2) 0.94N. huerfanumATCC 64040 E. liyangensis Ply9101(2) E. liyangensis Ply9102(2) N. uncinatum e167 0.97 0.89 N. coenophialum e19(1) N. typhinum var. canariense 989 E. typhina ATCC201667 Fnj7409(2) Fnj7405(2) Fnj7402(2) 0.64 N. australiense 937(2) N. chisosum 134(3) E. typhina ATCC201669 N. sinofestucae Fnj6606(2) N. sinofestucae Fnj4602(2) N. sinicum Rts2102(2) N. gansuense E7080 0.9 N. gansuense E7082 0.53N. gansuense var. inebrians 817 0.98 N. gansuense var. inebrians 818 N. guerinii CBS 112036(2) 0.99 N. guerinii ATCC MYA1235(2) Claviceps purpurea ATCC20102

5.0

Figure 2. Phylogenetic tree of Neotyphodium and Epichloë species, based on trimmed tubB sequences. The isolates obtained in this study are indicated in red. Mycology 205

Fnj7405(2) 0.97Fnj7409(2) Fnj7402(2) N. australiense 937(2) N. chisosum 134(3) 0.91 E. typhina ATCC201669 N. sinicum Rts2102(2) N. sinicum Rxy6106(2) N. sinofestucae Fnj4602(2) N. sinofestucae Fnj6606(2) 0.99 N. tembladerae 1169(2) 0.99 N. tembladerae 4055(2) E. liyangensis Ply9101(1) E. liyangensis Ply9102(2) 0.98 0.98 N. huerfanum ATCC64040 E. typhina ATCC201667 N. typhinum var. canariense 989 E. typhina ATCC200740 0.99E. typhina ATCC200738 1 1 E. typhina E432 1E. typhina ATCC200851 0.92 E. typhina ATCC201666 E. clarkii ATCC200742 1 0.99 E. clarkii ATCC200741 E. sylvatica ATCC200748 1 E. typhina ATCC200739 E. sylvatica ATCC200751 N. melicicola 827 1N. aotearoae 829 N. australiense 937(1) 0.51N. siegelii e915(2) E. festucae ATCC201550 0.81 E. festucae ATCC90661 1 N. lolli 135 N. coenophialum e19(1) N. tembladerae 1169(1) 0.84 N. tembladerae 4055(1) N. coenophialum e19(2) 1 N. occultans 992(2) N. stromatolongum Cnj6611 1 0.87 N. stromatolongum Cnj6617 1 Epichloe sp. 9924 E. baconii ATCC76552 E. amarillans 906 0.99 1 E. amarillans ATCC200743 N. chisosum 134(2) 0.58 E. baconii ATCC200745 E. bromicola 9633 0.53 N. occultans 992(1) E. bromicola 9630 0.69 E. yangzii Rnj3302 0.52 N. uncinatum e167 N. siegelii e915(1) 0.96 E. yangzii Rnj4201 E. yangzii Rnj4301 N. sinofestucae Fnj4602(1) N. sinofestucae Fnj6606(1) 0.99Fnj7405(1) Fnj7402(1) 0.96 Fnj7409(1) E. liyangensis Ply9101(1) E. liyangensis Ply9102(1) 1 0.6 N. sinicum Rxy6106(1) N. sinicum Rts2102(1) N. chisosum 134(1) 0.63 E. elymi ATCC200850 0.94 E. elymi ATCC201554 E. brachyelytri ATCC200752 0.99 E. brachyelytri ATCC201561 N. gansuense var. inebriuans 817 1N. gansuense var. inebriuans 818 E. glyceriae ATCC200747 1 E. glyceriae ATCC200755 Claviceps purpurea ATCC20102

5.0

Figure 3. Phylogenetic tree of Neotyphodium and Epichloë species, based on trimmed tefA sequences. The isolates obtained in this study are indicated in red. 206 K. Han et al.

Table 2. Morphological characteristics of partial Chinese indigenous Neotyphodium species.

Conidium size Conidiogenous Species Hosts Pedigree Colony diameter (mm) (μm) cell length (μm)

N. sinicum Roegneria spp. ETC, EBY 6.1–16.2/3 weeks, 25◦C 4.6–5.9 × 2.1–3.4 16.1–24.2 N. sinofestucae F.parvigluma ETC, EBY 6.0–14.7/3 weeks, 28◦C 5.2–6.3 × 2.6–3.1 19–28 Isolates in this F.myuros ETC, EBY 15–20/6 weeks, 28◦C 6.9 ± 0.8 × 3.3 ± 0.4 29.1 ± 6.6 study

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GenBank tubB tefA AF457472L06951 AF457510 X56847 AF457512 AF323381 AF323402 HM475160 HM475164 HM475161 HM475165 Poeae Japan L06964 AF457511 Aveneae Australia AF323379 AF323400 Aveneae New Zealand, Australia AF323370 AF323391 AgrostideaeAgrostideaeBrachypodieae Georgia, USA Switzerland USA L06958 L78270 AF231191 AF231196 L78271 AF231197 Triticeae China DQ134036 DQ134030 AgrostideaeAgrostideae Britain AF457467 L06961 AF457506 AF231193 Brachypodieae Japan L78278 AF231218 PoeaeAgrostideaePoeaePoeaePoeae SwitzerlandPoeae Switzerland Switzerland Switzerland Switzerland L78280 France L78274 L78285 AF231226 AF062429 AF231225 L78284 AF231229 AF231228 AF250752 AF231231 AF231221 Poeae USA AF457469 AF457507 Brachypodieae New York, USA AF062427 AF231200 Poeae Europe L06957 AF231211 Poeae China HM475156 HM475168 Poeae China HM475157 HM475169 Aveneae Switzerland L78288 AF231222 TriticeaeTriticeae China China DQ134039 DQ134040 DQ134033 DQ134034 Triticeae Missouri, USA L78273 AF231208 BromeaeBromeaePoeae Switzerland SwitzerlandPoeaeMeliceae AY033382 AY033385 New AY033356 York, USA AY033359 L78275 AF250738 AF231207 AF231216 L06955 AF231210 BrachypodieaeBrachypodieae Switzerland Switzerland L78291 L78292 AF231219 AF231223 Stipeae N. America AF457471 AF457509 PoeaeTriticeae Switzerland Kentucky, USA AF250742 L78281 AF457503 AF231206 Meliceae Indiana, USA L78276 AF231217 gene sequences. tefA and commutata tubB pratensis pratensis = ssp. rubra ssp. ssp. ) AF457470 AF457508 var. eminens L. arundinaceum Stipa eminens( Ec. ovatus Echinopogon ovatus Sphenopholis obtusata Calamagrostis villosa A. stolonifera Brachyelytrum erectum Phleum pratense Poa silvicola P.nemoralis P.pratense Lolium perenne Roegneria kamoji R. kamoji R. kamoji Holcus mollis Achnatherum Agrostis perennans B. erectum Bromus erectus Holcus lanatus Festuca rubra Br. erectus Glyceria striata G. striata P.pratensis P.pratensis Brachypodium sylvaticum Bra. sylvaticum Anthoxanthum odoratum Bra. pinnatum Dactylis glomerata Elymus virginicus El. villosus H. lanatus F.rubra e19 937 / 134 / / ATCC90664 ATCC6403 E432 Rnj3302 Rnj4201 Rnj4301 CBS109344 CBS109346 906 ATCC200743 ATCC200745 ATCC76552 ATCC200752 ATCC200851 ATCC201666 ATCC201667 ATCC201669 ATCC201561 9630 9633 ATCC201550 ATCC200741 ATCC200747 ATCC200755 Ply9101 Ply9102 ATCC200748 ATCC200751 ATCC200738 ATCC200739 ATCC200740 ATCC200850 ATCC201554 ATCC200742 ATCC90661 sp. 9924 N. coenophialum N. chisosum E. typhina E. yangzii E. yangzii E. yangzii Epichloë N. aotearoae N. australiense Supplementary Table S1. Endophyte infected grasses and GenBank accessions for SpeciesE. amarillans Isolate Host Species Host Tribe Geographic origin E. amarillans E. baconii E. baconii E. brachyelytri E. typhina E. typhina E. typhina E. typhina E. brachyelytri E. bromicola E. bromicola E. clarkii E. elymi E. elymi E. festucae E. clarkii E. glyceriae E. glyceriae E. liyangensis E. liyangensis E. sylvatica E. sylvatica E. typhina E. typhina E. typhina E. festucae Mycology 209 AF308139 AF308133 AF176275 AF457541 EU409308FJ189479 FJ189478 AF323390 FJ189476 AF323408 AF323386 FJ211220JQ309046 FJ211210 JQ309047 JQ309052 JQ309048 JQ309053 JQ309054 AF457497 AF457544 EF422755 FJ211212 FJ211202 EF422747 Agrostideae China EU526823 EU526818 Stipeae Eurasia AF457495 AF457539 Stipeae Eurasia AF457494 AF457538 PoeaePoeae Spain Europe AF176266 L06946 AF457543 AF308131 Poeae N. America AF323389 AF323407 MeliceaeMeliceae SwitzerlandPoeae S. Africa EF422746 Europe AF323384 AF323406 AF308138 AF308132 Poeae China FJ211219 FJ211209 PoeaePoeaePoeae China– China China America JQ309043 JQ309044 JQ309049 JQ309045 JQ309050 AF06246 JQ309051 AF276508 Poeae N. America AF457496 AF457545 TriticeaeTriticeae China China EU409305 EU409307 EU409303 EU409301 Meliceae France EF422754 Poeae N. America AF457493 AF457537 AgrostideaePoeae China China EU526824 EU526820 FJ211211 FJ211201 Stipeae China EF422757 Poeae Europe, New Zealand AY865628 AF457540 Stipeae China EF422756 perenne var. Poeae New Zealand AF176271 AF457542 ssp. Calamagrostis. epigeios A. inebrians A. inebrians – L. edwardii L. pratense P.huecu F.myuros F.myuros F.myuros R. kamoji R. kamoji M. transsilvanica M. decumbens L. pratense F.parvigluma F.arizonica Melica ciliata L. perenne Festuca arizonia C. epigeios F.parvigluma Achnatherum inebrians A. inebrians rigidum L. perenne 818 817 / / 8 / e915 989 Cnj6611 (AS5.904) ATCC20102 CBS102646 ATCC200844 ATCCMYA-1228 Fnj7402 Fnj7405 Fnj7409 4055 Rxy6106 Rts2102 992 ATCCMYA-1187 ATCC74483 Fnj6606 CBS 112036 ATCCMYA-1235 ATCC64040 CBS109342 / Cnj6617 Fnj4602 1169 E7082 E7080 27 135 inebrians inebrians canariense var. var. var. N. uncinatum N. stromatolongum N. stromatolongum N. sinicum N. sinicum N. tembladerae N. siegelii N. sinofestucae N. sinofestucae N. sinofestucae Claviceps purpurea N. tembladerae N. typhinum N. guerinii N. guerinii N. occultans N. sinofestucae N. gansuense N. huerfanum N. lolli N. melicicola N. sinofestucae N. gansuense N. gansuense N. gansuense