RESEARCH ARTICLE

Mutualistic fungal endophytes in the Triticeae – survey and description Stuart D. Card, Marty J. Faville, Wayne R. Simpson, Richard D. Johnson, Christine R. Voisey, Anouck C. M. de Bonth & David E. Hume AgResearch Ltd, Grasslands Research Centre, Palmerston North, New Zealand

Correspondence: Stuart D. Card, AgResearch Ltd, Grasslands Research Centre, Private Bag 11008, Palmerston North 4442, New Zealand. Tel.:+64 6 351 8046; fax: +64 6 351 8032; e-mail: [email protected] Received 26 May 2013; revised 9 December 2013; accepted 14 December 2013. Final version published online 21 January 2014. DOI: 10.1111/1574-6941.12273

MICROBIOLOGY ECOLOGY

Editor: Wietse de Boer Keywords Epichloe€; Elymus; Hordeeae; Hordeum; Neotyphodium.

Abstract Grasses of the tribe Triticeae were screened to determine the presence of mutualistic epichloae fungal endophytes. Over 1500 accessions, from more than 250 species, encompassing 22 genera within the Triticeae were screened using immunodetection and direct staining/microscopy techniques. Only two genera, Elymus and Hordeum, were identified as harbouring epichloae endophytes with accessions native to a range of countries including Canada, China, Iran, Kazakhstan, Kyrgyzstan, Mongolia, Russia and the USA. Genetic analysis based on simple sequence repeat data revealed that the majority of endophytes cluster according to geographical regions rather than to host species; many strains isolated from Hordeum grouped with those derived from Elymus, and amongst the Elymus-derived strains, there was no clear correspondence between clustering topology and host species. This is the first detailed survey demonstrating the genetic diversity of epichloae endophytes within the Triticeae and highlights the importance of germplasm centres for not only preserving the genetic diversity of plant species but also the beneficial microorganisms they may contain.

Introduction Many members of the grass family Poaceae have co-evolved with endophytic fungal symbionts of the genus Epichlo€e, including asexual morphs formerly classified in the form genus Neotyphodium (Schardl et al., 2004). Collectively known as epichloae, these symbiotic fungi form permanent associations with their cool-season grass hosts, having relationships ranging from pathogenic to mutualistic, with the latter expressing no obvious host symptoms. Lineages of single fungal genotypes are transmitted maternally (vertically) through seeds (Freeman, 1902; Christensen et al., 2008) following colonisation of the ovule and subsequent embryo (McLennan, 1920; Sampson, 1933; Philipson & Christey, 1986). These endophytes remain confined to the intercellular plant spaces throughout their life cycle, and at the onset of seed germination, hyphae within the shoot apical meristem start to colonise leaf primordia and axillary buds, advancing systemically throughout the aboveground parts of the plant (Philipson & Christey, 1986). The fungus spreads via a process termed intercalary hyphal ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

extension, whereby the growth of these biotrophic endophytes is synchronised and strictly regulated with that of their grass hosts (Christensen et al., 2002, 2008). Many of the Epichlo€e, and all Neotyphodium species, are transmitted vertically through host seeds; however, many Epichlo€e are also transmitted horizontally through the dissemination of ascospores produced on stromata (Leyronas & Raynal, 2008). These structures are formed on host inflorescences and manifest as the disease known as choke in some instances. Within the grass family Poaceae, epichloid fungal endophytes are described only within the subfamily Pooideae. This subfamily is split into 13 tribes (USDA, 2013) with Poeae being the most intensively studied with respect to fungal endophytes (Easton, 2007). This tribe includes the cosmopolitan grass genera Festuca L. and Lolium L. that have been the focus for epichloid endophyte research due to the effect of the symbiosis on pasture-based agricultural systems. The ecological role of these fungi derives mainly from the production of different types of secondary metabolites that primarily confer protective effects to FEMS Microbiol Ecol 88 (2014) 94–106

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their grass hosts. Most notably are the ergot and indole diterpene alkaloids produced by ‘common-toxic’ endophyte strains that can cause livestock problems (Johnson et al., 2013). The endophytes of these pasture grasses have a great economic importance within the USA and New Zealand (NZ) and are utilised/exploited by intentionally infecting improved grass cultivars with selected fungal endophyte strains (Latch & Vaughn, 1995) that have either greatly reduced or no toxicity to livestock. These ‘novel associations’ are now seen as a necessary component of many grass pastures due to the biotic and abiotic enhancements they confer (Bouton et al., 2002; Easton, 2007). Many of these effects are due to the production of other endophyte-produced alkaloids such as peramine and lolines that provide significant protection against a number of major invertebrate pests (Prestidge et al., 1985; Rowan & Gaynor, 1986; Brimble & Rowan, 1988). Epichloae endophytes are also found within other tribes of the Pooideae, such as Aveneae Dumort. (former tribe), Brachypodieae P. Beauv., Brachyelytreae, Bromeae Scop., Meliceae Endl., Stipeae Dumort. and Triticeae Dumort. (White, 1987; Clay, 1990; Leuchtmann & Clay, 1993; White et al., 1993; Leuchtmann & Schardl, 1998; Meijer & Leuchtmann, 1999; Schardl, 2001; Vazquez de Aldana et al., 2003; Moon et al., 2004, 2007; Gentile et al., 2005; Zhang et al., 2010; Charlton et al., 2012). The tribe Triticeae is one of the most economically important, as it contains many major food crops, such as barley (Hordeum vulgare C.), rye (Secale cereale L.), wheat (Triticum spp.) and triticale (9 Triticosecale Wittm. ex A. Camus.). There are 30 genera in the tribe (according to Yen & Yang, 2009) comprising over 350 species (Barkworth & Von Bothmer, 2009), and epichloid endophytes have been reported from many genera including Agropyron spp. (Nan & Li, 2001; Yanagida et al., 2004, 2005), Elymus spp. (White, 1987; Leuchtmann & Clay, 1993; Saikkonen et al., 2000; Nan & Li, 2001; Vinton et al., 2001; Moon et al., 2004; Spooner & Kemp, 2005; Tajimi et al., 2005; Zhang & Nan, 2007a, b; Tintjer et al., 2008; Rudgers & Swafford, 2009; Charlton et al., 2012), Elytrigia sp. (Nan & Li, 2001), Hysterix sp. (Leuchtmann & Clay, 1993), Hordelymus sp. (Leyronas & Raynal, 2001; Moon et al., 2004; Oberhofer & Leuchtmann, 2012), Hordeum spp. (Wilson et al., 1991a, b, c; Mahmood et al., 1993; Clement et al., 1997; Youssef & Dugan, 2000; Nan & Li, 2001; Moon et al., 2004; Wilson, 2007), Leymus sp. (Nan & Li, 2001), Roegneria sp. (Nan & Li, 2001; Kang et al., 2011), Sitanion sp. (White, 1987) and Triticum spp. (Bishop et al., 1997; Marshall et al., 1999). However, associating these fungi with defined species within the host tribe is complicated due to the constant changing genetic and species concepts within the Triticeae (Barkworth & Von Bothmer, 2009). FEMS Microbiol Ecol 88 (2014) 94–106

The aim of this research was to comprehensively assess accessions of grass species within the tribe Triticeae for the presence of epichloae endophytes and to investigate these fungi with respect to their biogeography and diversity.

Materials and methods Sourcing Triticeae germplasm

Triticeae tribe seed accessions were obtained from international germplasm collections (Supporting Information, Table S1) and imported into NZ in compliance with the NZ Biosecurity Act 1993. Accessions provided by the Margot Forde Germplasm Centre (MFGC) included many sourced recently in situ in countries of origin, specifically for this project. They were then catalogued and stored, at 0 °C and 30% relative humidity, in the MFGC, NZ’s national gene bank of grassland plants. Screening Triticeae germplasm for epichloae endophytes

Triticeae germplasm was obtained from the MFGC and assessed for epichloae endophyte presence via a multiphase screening process. The initial screen was achieved by first staining seed tissues and inspecting for characteristic Epichlo€e hyphae using bright-field microscopy. As these staining methods are destructive and do not distinguish between viable and nonviable hyphae in the seed (Card et al., 2013), further screening was required using immunodetection to identify individual plants, within accessions, that harboured viable endophyte. However, the selection of suitable screening methods also depended on the amount and quality of seed available. For example, accessions with limited numbers of seed were only screened using immunodetection as this maximised the number of live plants. All fungi detected in this process were then assessed morphologically for additional characteristics common to epichloae endophytes. Staining of endophytic mycelium within seed As the endophyte/seed colonisation pathway has not been fully elucidated in these grass species (Johnson et al., 2013), two microscopy methods were used to detect endophytic hyphae in seed tissue. These were the routine seed squash (SS, Latch & Vaughn, 1995) and the infection layer (IL, Card et al., 2011) methods. The IL method identifies hyphae present on the scutellum surface, while the SS focuses on hyphal colonisation of the nucellus remnants found between the aleurone cells and the pericarp. ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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Tissue-print immunoblot and endophyte isolation The number of seed sown depended on availability and ranged from two seed to a maximum of 96 seed per accession. After 6 weeks growth in a glasshouse, seedlings were assayed for the presence of epichloid endophytes using an immunoblotting technique that detects specific epichloae mycelial proteins using a polyclonal antiserum (Simpson et al., 2012). Epichloid endophytes were subsequently isolated from immunoblot-positive plants using the method of Latch and Vaughn (1995). Fungal colonies identified as epichloid species were subcultured on potato dextrose agar (PDA) for further characterisation. For long-term storage of fungal endophytes, colonies were cultured on sterilised wheat or barley grain and stored in 20% glycerol at 80 °C (Card, 2005). Morphological examination of endophytic fungi

All endophytes isolated were then examined with respect to certain morphological features characteristic of epichloae. Conidiophore and conidial measurements were made after inoculating 4% water agar with endophytecolonised wheat or barley grain (see above). After 2–4 weeks incubation at 22 °C in the dark, the grain was removed and the water agar area containing the resulting endophyte colony was dissected from the Petri plate and placed on a glass slide. A drop of water was placed onto the colony before a cover slip was placed over the top. Endophyte mycelia were observed and imaged using a BX50 microscope at 4009 magnification and DP12 digital camera system (Olympus NZ Ltd). Measurements of 50 conidiophores and mature conidia were made using ANALYSISB 5.0 software (Soft Imaging System GmbH, Germany). Fungal colony morphology was examined, and the radial growth rates of colonies were measured following inoculation of PDA plates with colonised wheat or barley grain (see above). Colonies were incubated for 4 weeks at 22 °C in the dark, with radial dimensions measured using a digital calliper. An average measurement was calculated from four replicate colonies per endophyte strain. Scanning electron microscopy

Endophyte-colonised water agar plugs (c. 2 mm2) were fixed (3% glutaraldehyde, 2% formaldehyde, in 0.1 M phosphate buffer pH7.2) for 2 days at room temperature. Plugs were rinsed three times in phosphate buffer (15 min each) and subsequently dehydrated through a graded series of ethanol solutions (25%, 50%, 75%, 95% and 100%) for 15 min each. Plugs were then dehydrated ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

S.D. Card et al.

in 100% ethanol for 1 h and critical point dried using liquid CO2. Dried samples were mounted onto aluminium specimen support stubs using conductive silver paint, sputter coated with gold and observed under a FEITM QuantaTM 200 Environmental Scanning Electron Microscope (SEM). Simple sequence repeat analysis of endophyte strains

Plants with endophyte-infected tillers were sampled for DNA isolation by excising 100–200 mg basal tiller tissue. Total genomic (plant + endophyte) DNA was isolated from tiller tissue using the FastDNA kit (MP Biomedicals, Solon, OH) following the manufacturer’s instructions for fresh plant tissue. Total genomic DNA was diluted by a factor of 10 prior to use for simple sequence repeat (SSR) assay. A total of 25 SSRs (Table S2) were used for endophyte diversity characterisation, including B10 and B11 (Moon et al., 1999), a set of six Neotyphodium lolii expressed sequence tag-derived SSRs (prefix ‘ans’), two SSRs mined from an Epichlo€e festucae genome assembly (prefix ‘egs’; http://www.endophyte.uky.edu/ef/) and 15 SSRs mined from a Triticeae-derived Epichlo€e genome (endophyte strain AR3018 sourced from Elymus; R.D. Johnson, A.K. Khan & M.J. Faville, unpublished data). Briefly (except for B10 and B11) dinucleotide-through to pentanucleotide-based SSRs were identified from these sources using the SSR detection programme Sputnik (http://espressosoftware.com/sputnik/index.html). Primers flanking putative SSRs were designed using PRIMER 3.0 (http://primer3.sourceforge.net/) according to the following parameters: 18–24 nucleotides, optimal melting temperature 60 °C, G + C% of 20–60%, expected amplicon length 100–300 bp. Except for B10 and B11, forward primers were synthesised with a 21-nucleotide M13 tail sequence at the 5′-terminus (5′-TGTAAAACGA CGGCCAGT-3′), to facilitate universal labelling of PCR products by a 5′-fluorescein phosphoramidite (6-FAM)labelled M13 primer (Schuelke 2000), and reverse primers were synthesised with the sequence 5′-GTTTCTT-3′ at the 5′-terminus end to promote nontemplated adenylation at the 3′-terminus end of PCR product (Brownstein et al., 1996). All SSR primers were synthesised by Integrated DNA Technologies, Inc. (Coralville, IA). PCR using B10 and B11 was performed as described by Moon et al. (1999), except that a 10 lL reaction volume was used. For all other SSRs, PCR amplifications were conducted in a 10 lL reaction volume as per Faville et al. (2004) except that a final concentration of 2.5 mM magnesium chloride and 0.75 units of Platinum Taq DNA polymerase (Life Technologies, Carlsbad, CA) were used. PCR was FEMS Microbiol Ecol 88 (2014) 94–106

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performed on iCycler thermocyclers (Bio-Rad, Hercules, CA). Genotypic analysis followed Faville et al. (2004), except that capillary electrophoresis on an ABI 3100 Genetic Analyser (Life Technologies) used POP-7TM polymer. Electropherograms were analysed using ABI PRISM GENESCAN v3.7 (Life Technologies), and fragments were sized using GENEMARKER v1.75 (Softgenetics LLC, State College, PA). Polymorphism information content (PIC) was calculated (Botstein et al. 1980) for individual SSR markers. Each SSR fragment was scored as present (1) or absent (0) and entered into a binary matrix. The binary matrix was used to determine genetic relationships amongst the 73 genotyped endophyte strains by calculating Jaccard’s coefficient of similarity (Sneath & Sokal, 1973). A dendrogram derived from similarity coefficients was constructed using the unweighted pair group method with arithmetic averages (UPGMA; Sneath & Sokal, 1973). A cophenetic matrix was generated from the UPGMA dendrogram, and a Mantel test of correlation between cophenetic values and the Jaccard similarity coefficients was executed (n = 1000 permutations) to ascertain the reliability of the UPGMA clustering. These analyses were implemented in NTSYSPC v2.20x (Rohlf, 2008). A Mantel test for isolation by distance was computed using genetic similarity data and geographical distance for all analysed strains, implemented in the program IBD (Jensen et al., 2005) using default settings.

Results Screening Triticeae tribe germplasm for epichloae endophytes

Over 1500 Triticeae accessions were received from 17 international germplasm centres and botanical gardens (Table S1). The accessions included more than 250 species from 22 genera within the Triticeae tribe (Table 1) and were screened using multiple endophyte detection techniques. Only two genera contained epichloae endophytes, Elymus (16 species, subspecies and variants) and Hordeum (three species), with the majority of the 42 infected accessions originating in Asia (37 accessions), followed by North America (three accessions; Table 2). These accessions comprised species native to a range of countries including Canada, China, Iran, Kazakhstan, Kyrgyzstan, Mongolia, Russia and USA (Table S3). The multiphase detection and identification process is currently the most robust for screening the presence of epichloae endophytes. The accessions of Elymus and Hordeum infected with epichloid endophytes originated from a diverse range of locations including subalpine meadows and winter pastures at elevations of up to FEMS Microbiol Ecol 88 (2014) 94–106

Table 1. Triticeae genera assessed for the presence of epichloid endophytes

Genera*

Number of species†

Number of accessions

Aegilops Agropyron Amblyopyrum Australopyrum Dasypyrum Elymus Elytrigia Eremopyrum Henrardia Heteranthelium Hordelymus Hordeum‡ Kengyilia Leymus Pascopyrum Psathyrostachys Pseudoroegneria Roegneria Stenostachys Taeniatherum Thinopyrum Triticum* Total

16 32 3 1 2 118 8 5 1 2 1 41 7 5 1 1 2 1 1 3 1 9 261

195 198 20 4 16 584 56 26 4 4 19 285 10 12 6 1 2 3 1 24 1 77 1548

*Only species of Elymus and Hordeum were found to harbour epichloid endophytes. † Includes subspecies and variants. ‡ Only nondomesticated species screened.

2950 m to botanical gardens and farm villages as low as 145 m above sea level (Table S3). The oldest seed harbouring viable epichloae endophyte was an accession of Hordeum bogdanii (BZ 8168 harbouring AR3012) collected in Kazakhstan in 1966, previously deposited in the National Small Grains Collection, Idaho, USA (provided by NGRP, USDA; Table S3). No samples of Elymus and Hordeum were found to harbour nonviable epichloae endophytes. Morphological examination of endophytic fungi

Endophytic fungi resembling epichloid species were identified through morphological characteristics of hyphae emerging from either end of the excised plant tissue after 3–5 days incubation and subsequently producing conidia borne on individual conidiogenous cells (phialides) in the characteristic ‘T’ configuration (Christensen et al., 1993; Fig. S1). On PDA, average colony diameters 8.9–96.8 mm after 28 days at 22 °C (Table S4), white, cottony, dense, smooth; aerial mycelium rich and smooth, septated and c. 2 lm wide; colony reverses pale brown and with some strains lighter towards margin. No strains were sterile. ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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Table 2. Species of Triticeae containing epichloae endophytes Triticeae species, subspecies and variants

Number of accessions Examined

Infected

Continent originally collected from

Elymus canadensis

8

1

E. caninus E. ciliaris E. dahuricus E. dahuricus subsp. excelsus E. elymoides subsp. brevifolius E. gmelinii E. mutabilis E. mutabilis var. oschensis E. nevskii E. pendulinus subsp. brachypodioides E. sibiricus E. uralensis E. virginicus

53 10 33 14

3 2 7 3

2

1

4 13 1

1 3 1

North America Asia Asia Asia

6 9

2 1

Asia Asia

22 1 10

1 1 1

104 11

5 1

Asia Asia North America Asia Asia

32 15

7 1

Asia Asia

Elymus sp. Hordeum brevisubulatum subsp. violaceum H. bogdanii H. roshevitzii

North America Asia Asia Asia Asia

Sporulation in culture abundant at 1 week; conidiogenous cells discrete, arising solitarily from aerial mycelium, septate at the base, more or less cylindrical over the lower one-third, hyaline and smooth. Conidiogenous cells 12.7– 28 lm long, c. 2.3 lm wide at base, tapering to c. 1.1 lm at tip. Conidia hyaline, oval or reniform, 1.7–16.5 9 1.2– 9.1 lm. No relationships could be identified between morphological characteristics and host species. SSR analysis of endophyte strains

A total of 73 confirmed plant–endophyte associations, from 42 Elymus and Hordeum accessions, were assigned a

unique AR (AgResearch Ltd) code (Table S3), and their genetic diversity examined using 25 SSR markers. A total of 192 PCR products were amplified from 25 SSR loci. All SSRs detected polymorphism amongst the analysed endophytes (Table S2), with PIC values ranging from 0.002 to 0.756 (average of 0.449), although marker ans016 (PIC 0.002) was only marginally effective in this regard. Similarly, the number of amplicons generated per SSR ranged from 4 to 14 with an average of 7.7. Comparison of 15 SSRs generated a Triticeae endophyte source (Elymus-derived endophyte strain AR3018; mean PIC 0.455), with the 10 SSRs from non-Triticeae endophyte sources (mean PIC 0.438) indicated similar capacity of the two marker groups to detect polymorphism in endophytes from Triticeae grass hosts. However, amplification efficiency, as assessed by electrophoretic peak intensities, tended to be superior using Triticeae endophyte SSRs (data not presented). UPGMA cluster analysis, using Jaccard similarity coefficient data from multilocus SSR genotypes, classified the 73 endophyte strains into 39 unique genetic variants based on clustering at 1.00 similarity (Fig. 1). A Mantel test indicated a high correlation (r = 0.987) between the Jaccard similarity matrix and a cophenetic matrix derived from the UPGMA dendrogram, supporting the observed clustering. Coalescence from 73 to 39 endophyte strains was due in part to 100% similarity amongst strains sourced from the same population accession (Fig. 1, Table S3). However, amongst the 11 clusters within which multiple strains shared 100% similarity, four were composed of endophyte strains sourced from different population accessions (Fig. 1: AR3067–AR3068; AR3001– AR3045; AR3002–AR3042; AR3018–AR3037). Of the 42 population accessions examined, seven (BZ 2153, BZ 2155, BZ 2160, BZ 2162, BZ 2679, BZ 4455 and BZ 4820; Table S3) contained more than one endophyte genetic variant, a further two (BZ 2198 and BZ 4833) were initially assigned more than one endophyte strain, but these proved to be identical by SSR (Fig. 1), and the remainder were homogeneous for endophyte strain. Reliable geographical location data (GPS co-ordinates and/or local address information) were obtained for host accessions of 29 of the 39 unique endophyte strains. The remaining 10 could only be characterised by more general

Fig. 1. Dendrogram of genetic similarity amongst epichloid endophytes from 73 Elymus and Hordeum spp. plants sampled from 42 accessions, assessed by the unweighted pair group method of arithmetic averages. Genetic similarity, indicated by branch lengths between pairs of endophytes, was measured across 25 SSR loci using the Jaccard coefficient. Endophyte identity is indicated by AR code, and host plant species and country of origin were obtained via accession passport information, where available. Numbers at left of selected AR numbers indicate population accessions (1 = BZ2155; 2 = BZ2160; 3 = BZ2162; 4 = BZ4455; 5 = BZ4820; 6 = BZ2679; 7 = BZ2153) that contained more than one endophyte variant. Major clusters recognised in the dendrogram as having a geographical basis are as follows: I – Kazakhstan, Kyrgyzstan, Southern Russia and Northwest China Elymus and Hordeum; II – Northwest and Northeast China Elymus and Hordeum; IIIa – Northeast China Elymus: IIIb – Eastern and Central China and Eastern Russia Elymus; IV – Northeast China and Mongolia Elymus; V – North America Elymus.

ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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Fig. 2. Map of Kazakhstan, Kyrgyzstan, China, Mongolia and Southern Russia showing location of Elymus and Hordeum accessions that contained endophyte strains in Cluster I (green) and Cluster II (blue) as indicated on the SSR dendrogram (Fig. 1). Endophyte strains are listed by AR number together with their host species (EC, Elymus caninus; ED, Elymus dahuricus; EG, Elymus gmelinii; EM, Elymus mutabilis; EN, Elymus nevskii; ES, Elymus sibiricus; EU, Elymus uralensis; HB, Hordeum bogdanii).

location information (e.g. Northwest China). Although moderately biased by the predominance of host plant accessions sampled from China, geographical origin of the host clearly influenced endophyte genetic diversity, as revealed in the dendrogram topology (Fig. 1). The majority of the endophyte strains grouped into five main clusters (Fig. 1, I–V) that corresponded broadly to the geographical origins of their grass hosts. The most clearly differentiated groups are clusters I and II, containing 23 endophyte strains, of which detailed geographical location data were obtained for host accessions of 16 strains. These were originally collected from grass species present in the region where China, Kazakhstan, Kyrgyzstan, Russia and Mongolia meet (Figs 1 and 2). The topmost group of Elymus-sourced endophytes in Cluster I diverged at 0.36 similarity and was composed exclusively of strains from host plants collected from Russia, Kyrgyzstan and Kazakhstan. Grass accessions containing endophytes in Cluster I were found in two regions separated by great distance and a series of mountain ranges and lakes, ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

including the Dzungarian Alatau and the Tarbagatay Range. In turn, these subclusters would have been geographically separated from Cluster II by the Tian Shan, Altai and Borohoro Mountain ranges and the Dzungarian basin that may have represented harsh geographical barriers inhibiting the dispersal of seed from these grasses. Endophytes from Cluster II that did not conform to this pattern are strains AR3003, AR3010-11 that were isolated from accessions originally collected from Inner Mongolia and AR3001 from Chengde, China. These endophytes are geographically separated from the rest of the strains in Cluster II by the Gobi Desert and Taihang, L€ ulang and Yin Mountain ranges. Correspondingly, the most basal group on the dendrogram (Cluster V) was highly dissimilar to the other endophytes analysed (divergence at 0.04 similarity) and was exclusively comprised of strains from Elymus species sourced from North America. A Mantel test for isolation by distance yielded a moderate but statistically significant result (r = 0.507, P < 0.001), furnishing provisional support for an FEMS Microbiol Ecol 88 (2014) 94–106

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influence of geographical distance on genetic similarity amongst endophyte strains. A Mantel test considering only those endophyte strains from Elymus host plants yielded a slightly weaker correlation between genetic similarity and geographical distance (r = 0.490, P < 0.001). Further work using a larger number of populations that span a more continuous geographical distribution will be needed to provide a more robust picture pertaining to the extent to which these endophytes exhibit isolation by distance. General delineation of endophyte strains at host genus level was observed, with two Hordeum-exclusive groups of endophytes from H. bogdanii within clusters I and II and a single discrete Hordeum brevisubulatum branch. An exception was individual endophytes from Hordeum roshevitzii (AR3062) and H. bogdanii (AR3063) that grouped closely with Elymus-derived endophyte strains on a subbranch within Cluster II. However, within the Elymus genus, there was no clear correspondence between clustering of endophyte strains and host species. For example, the topmost group in Cluster I described endophytes sharing similarity ≥ 0.86, and these were sourced from at least four different Elymus host species (Fig. 1).

Discussion We report here on an extensive programme, screening Triticeae accessions for epichloid endophytes. A total of 42 accessions from two genera, Elymus and Hordeum, were found to harbour endophytes that conformed to morphological descriptions of Neotyphodium/Epichlo€e spp., including those previously isolated from Triticeae grasses (Wilson, 2007; Zhang & Nan, 2007a; Kang et al., 2011; Charlton et al., 2012). We report here for the first time three Triticeae species that have not before been recognised as hosts of these endophytic fungi, namely Elymus gmelinii, Elymus pendulinus subsp. brachypodioides and H. roshevitzii. However, for many of the grass genera, we could not acquire sufficient accessions to be completely convinced that these species do not contain epichloid endophytes. For example, while other authors have reported epichloid endophytes of Hordelymus (Leyronas & Raynal, 2001; Moon et al., 2004; Oberhofer & Leuchtmann, 2012), we screened 19 accessions and failed to identify endophyte-infected accessions using the SS and tissue-print immunoblot techniques. We included the two accessions screened by Leyronas and Raynal (2001) and found no evidence of epichloid endophyte. Leyronas and Raynal did not isolate the fungus (C. Leyronas, pers. commun.), and therefore, no formal identification was ever achieved. More recently, however, Oberhofer and Leuchtmann (2012) have described the genetic diversity of endophytes from FEMS Microbiol Ecol 88 (2014) 94–106

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Hordelymus europaeus after isolating a number of strains of the fungus. We are also aware of the report of an epichloid endophyte of the South American grass, H. comosum (Wilson et al., 1991c). We screened four accessions of this species using the SS technique and detected no endophyte. We were unable to screen a further three accessions of Hordeum comosum for viable endophyte (PI 264404, PI 264405 and PI 269648, provided by NGRP, USDA), previously identified as harbouring endophyte by Wilson, as none of the seed germinated. This is not surprising as the same authors commented on the low viability of this accession in 1991. Although variability in endophyte infection frequencies has been observed in grass populations from wild environments (Leyronas & Raynal, 2001; Rudgers et al., 2009; Oberhofer & Leuchtmann, 2012), many of the accessions screened in this study were found with a high level of endophyte infection (data not shown). This increased the chances of detecting endophyte in seed and subsequent seedlings and indicates that endophyte-infected plants may have a selective ecological advantage over endophyte-free plants. Unlike many other mutualistic microbial symbionts that facilitate the transfer of nutrients between them and their plant hosts, epichloae/grass associations are largely based on protection of the host from biotic and abiotic stresses (Clay & Schardl, 2002). This fundamentally involves the production of endophyte-produced secondary metabolites that deter herbivores, although there are reports of epichloae that confer other attributes, such as tolerance to low water availability (Zhang & Nan, 2007b, 2010; Oberhofer et al., 2013) and heavy-metal pollution to their Triticeae hosts (Zhang et al., 2012). There are reports of epichloae endophytes being detected in other species of Triticeae, although we suspect these are incorrect or there is disagreement on the taxonomy of the host. For instance, there are reports of Agropyron species being infected by Neotyphodium (Yanagida et al., 2004, 2005); however, the species reported as infected, Agropyron ciliare var. minus, is a synonym of Elymus ciliaris according to Tropicus (2012). Similarly, with respect to the findings of epichloae endophytes by Leuchtmann and Clay (1993), Hysterix patula has been treated as a synonym of Elymus hystrix (Zhang et al., 2009). The first documented report of infected Triticum by epichloid endophytes was by Bishop et al. (1997) who discuss the isolation of a Neotyphodium species from a cultivar of spring wheat (Triticum aestivum L.). However, the images and description of the isolate in their manuscript do not fit those of any documented Neotyphodium species, and this misidentification was later corrected and described as a species closely related to Fusarium proliferatum (Kwon & Anderson, 2001). ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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Marshall et al. (1999) later described Neotyphodium isolated from two wild Triticum species, namely T. dichasians Bowden [now known as Aegilops markgrafii (Greuter) K. Hammer] and T. tripsacoides (Jaub. & Spach) Bowden [now known as Amblyopyrum muticum (Boiss.) Eig]. The authors described 100% transmission of the endophyte to seed of both host species. We, however, found no evidence for the presence of epichloae endophytes in these species of Triticeae after screening 77 accessions from nine species of Triticum, 195 accessions from 16 species of Aegilops and 20 accessions of Amblyopyrum. We are certain that if epichloae endophytes were present in this material, we would have detected them, as many of these accessions were sourced from gene banks that limit the use of fungicides, which could negatively affect the development and survival of these fungi. Additionally, these gene banks also use conditions of storage, low temperature and relative humidity that are favourable for endophyte survival. As we also used a number of techniques to screen these accessions, we are confident that these genera do not harbour these fungi. As highlighted, associating these fungi with species of Triticeae is not only obscured by incorrect identification of the host but also the complex taxonomy of the Triticeae tribe itself. Barkworth (1992) states ‘There is little likelihood that there will ever be a single, universally accepted, taxonomic treatment of the Triticeae because of its highly reticulate evolutionary history and its low crossing barriers’. This complexity could also be interfering with our attempts to cluster endophyte strains with host species. From our molecular marker analysis, we show that the majority of endophytes cluster according to specific geographical regions rather than to host species, with many of the endophytes isolated from Hordeum, grouping with the Elymus-derived strains and no clear correspondence between clustering topology and host species within Elymus itself. This is perhaps not surprising as Elymus and Hordeum are known to have close genomic relationships with some Elymus species, such as Elymus canadensis and Elymus virginicus, known to be natural hybrids between the two genera (Dewey, 1971; Lu & Bothmer, 1990; Salomon et al., 1991). Evidence suggests that these grass/endophyte associations have existed during early grass evolution that spawned today’s Pooid grasses (Schardl et al., 2008) where phylogenetic studies indicate a preponderance of codivergence of epichloae species with their grass hosts. The anamorphic Neotyphodium species, however, probably evolved through a series of interspecific hybridisation events that provided evolutionary diversification (Schardl, 2010). Moon et al. (2004) have shown that the sexual species are haploid, whereas the majority of the asexual species are heteroploid and often close to diploid. There ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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is great host specificity amongst the epichloae with most species linked to individual grass species, genera or tribes, and this could be attributed to geographical isolation and the mode of transmission exhibited by these fungi (Karimi et al., 2012). We propose that the species isolated from Triticeae only exist in certain species of Elymus, Hordeum, Hordelymus and Roegneria (a probable synonym of Elymus according to GRIN Taxonomy of Plants; Schardl et al., 2008; USDA, 2013) with some having had a common ancestral host. Understanding how endophyte species diversity relates to taxonomic diversity of host grasses is crucial to understanding the ecology and evolution of these symbionts (Schardl & Leuchtmann, 2005). The majority of the infected accessions were originally collected in Asia reflecting the fact that the majority of the perennial Triticeae are found in this continent, specifically in western China (Barkworth et al., 2005). These accessions originated from a variety of locations including mountain slopes, central cities, meadows, forests, lakesides and from a range of altitudes (145–2476 m). General geographical patterns were identified that link genetic similarity, measured using SSR molecular markers, to geographical regions where the host material was sourced. Two major clusters (I and II) contained both Elymus and Hordeum hosts, including numerous Elymus species, indicating some host commonality. These two clusters would have been isolated from the rest of the endophyteinfected Elymus and Hordeum accessions in our collection by many geographical barriers including the Taklimikan Desert and Kunlun Mountains to the south, the Gobi Desert and Mu Us Desert and the Khangai Mountains to the east and the Garagum Desert and Qizilqum Desert to the west. An earlier, limited SSR analysis of a small number of Triticeae endophyte strains, alongside endophytes from Lolium and Festuca pasture grasses, indicates that the Elymus- and Hordeum-derived endophytes form a group that is genetically distinct from the pasture endophytes (M. Faville, unpublished data), an observation supported by the molecular phylogenetic evaluation reported by Moon et al. (2004). The next stage is to identify these endophyte strains to species level and investigate their chemotypic diversity, especially their potential to produce antiherbivore alkaloidal compounds that have been inferred as important selective factors in the endophyte evolutionary process (Schardl et al., 2012). Many researchers view these endophytes as interesting from purely a fungal viewpoint. We consider now that with our extensive screen of this particular tribe, this research could show insights into the relationships of the Pooideae grasses themselves, especially with respect to Triticeae taxonomy and ecology. Elymus L., known generally as wildrye or wheat grass, is the largest, most diverse and FEMS Microbiol Ecol 88 (2014) 94–106

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most complex genus within the perennial Triticeae and contains at least 150 allopolyploid species that are geographically widespread (Jensen & Chen, 1992; Okito, 2008). The genus includes multiple distinct genomic combinations with at least one set of Pseudoroegneria genomes that may be combined with genomes from one or more of several other Triticeae genera (Mason-Gamer et al., 2005). In contrast, Hordeum is a genus comprised of only 31 annual and perennial species distributed throughout the temperate and dry areas of the world (Blattner, 2006) with 2–4 recognised genome types or groups of species (Jacobsen & Von Bothmer, 1992; Jakob & Blattner, 2009). In economic terms, the Poaceae are by far the most valuable of all plant families (Bouchenak-Khelladi et al., 2010), and in terms of human survival, most of the world’s population depend on cereal production, with these crops producing three times as much edible dry matter as pulses, tubers, fruits, sugars, meat, milk and eggs combined (Harlan, 1992). These mutualistic endophytes have great potential in plant breeding programmes due to their seed-borne nature and the increased number of desirable traits they could contribute. Elite cultivated crop gene pools exhibit limited genetic diversity (Feuillet et al., 2008), and therefore, combining two mutualistic organisms together may offer an expanded genetic potential. This research highlights the importance of germplasm collections, especially collections of wild cereal species and landraces that were critical for launching many of the biggest biotechnological revolutions of the 20th century (Dugan et al., 2011). These collections are in essence insurance policies for the future (Wilkes, 1977) in terms of not only plant traits but also the microorganisms they harbour (Wilson et al., 1991b; Sachs, 2009). As our population increases and the climate of our planet changes, any advantages in producing stable food supplies should be a priority for us all.

Acknowledgements We wish to thank the New Zealand Ministry of Science and Innovation, The Grains Research and Development Corporation (Australia), The New Zealand Foundation for Arable Research and Grasslanz Technology Ltd for financial support. We thank Vicki Bradley, Western Regional Plant Introduction and Research Station, United States Department of Agriculture, Washington State University; Dallas Kessler, Agriculture and Agri-food Canada; Elizabeth Heighington, Toronto Zoo, Canada; Stephen Reader, John Innes Centre, UK; Attila Simon, Research Centre for Agrobiodiversity, Hungary; Simon Jeppson and Fredrik Ottosson, NordGen, Sweden and Ian Thomas; University of Aberystwyth, Wales for supply of germplasm. Jana Schmidt and Zo€e Erridge (AgResearch FEMS Microbiol Ecol 88 (2014) 94–106

Ltd) for SSR genotyping technical support; Anar Khan (AgResearch Ltd) for bioinformatics support; Kenyon Moore and Stephen Slack, Margot Forde Germplasm Centre (MFGC), for cataloguing of germplasm; Zane Webber, MFGC, for sourcing seed material from international collecting trips; Doug Hopcroft and Michael Low from the Manawatu Microscopy and Imaging Centre for preparation and assistance with SEM; Stephen Clement (now retired from Western Regional Plant Introduction and Research Station, United States Department of Agriculture) and Mike Christensen (AgResearch Ltd) for valuable discussion. Data used to develop SSR markers egs027 and egs030 were obtained from the Epichlo€e festucae Genome Project, supported by grant funds from the National Science Foundation (NSF EF-0523661), Department of Agriculture National Research Initiative (USDA-NRI 2005-35319-16141), and Kentucky Biomedical Research Infrastructure Network-Kentucky IDeA Networks for Biomedical Research Excellence (NIH 2 P20 RR-16481).

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Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. An example of an endophyte exhibiting typical epichloae morphological features including conidia borne on phialides in the characteristic ‘T’ configuration. Table S1. Germplasm collections accessed for Triticeae seed accessions. Table S2. Summary data from genotyping of endophyte strains from 73 plants of Elymus and Hordeum species by 25 simple sequence repeat markers. Table S3. Endophyte-infected accessions of Elymus and Hordeum. Table S4. Epichloid endophytes and their morphological characteristics.

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