NEW ZEALAND JOURNAL OF BOTANY https://doi.org/10.1080/0028825X.2019.1651344 RESEARCH ARTICLE Epichloe novae-zelandiae, a new endophyte from the endemic New Zealand grass Poa matthewsii Adrian Leuchtmann a, Carolyn A. Young b, Alan V. Stewartc, Wayne R. Simpsond, David E. Humed and Barry Scott e aPlant Ecological Genetics, Institute of Integrative Biology, ETH Zurich, Zürich, Switzerland; bNoble Research Institute, LLC., Ardmore, OK, USA; cPGG Wrightson Seeds Ltd., Lincoln, New Zealand; dGrasslands Research Centre, AgResearch Ltd., Palmerston North, New Zealand; eGenetics Group, School of Fundamental Sciences, Massey University, Palmerston North, New Zealand ABSTRACT ARTICLE HISTORY Epichloe endophytes (Clavicipitaceae) infect pooid grass genera Received 10 June 2019 worldwide but predominantly in the Northern Hemisphere, but Accepted 29 July 2019 appear to be rare in native grasses of the Southern Hemisphere. First published online 15 Because of benefits that hosts may receive from the symbiosis, August 2019 Epichloe endophytes have been extensively studied and are HANDLING EDITOR considered important components of sustainable agriculture. Eric McKenzie There are only a few studies available on the incidence of endophyte infection in grasses of the Southern Hemisphere and KEYWORDS most grass species have never been examined. Here we report on Alkaloid genotype; a survey of native grasses of New Zealand including 25 endemic Dichelachne micrantha; or indigenous species. We sampled up to 10 plants per species at endemic New Zealand different sites from both the North and South Island of New grasses; endophyte infection; Epichloe novae-zelandiae; Zealand and examined tissues microscopically for endophyte interspecific hybrid; Poa infection. Overall, only two species were found to be infected, Poa matthewsii matthewsii (Matthew’s bluegrass) and Dichelachne micrantha (short-hair plume grass). Based on analyses of tefA and tubB genes, both endophytes were found to be interspecific hybrids. The endophyte of the new host D. micrantha was previously described as Epichloe australiensis, while the endophyte of P. matthewsii is a new species named here E. novae-zelandiae. The new species is a hybrid derived from E. amarillans, E. bromicola and E. typhina subsp. poae. Alkaloid analyses in planta suggested that E. novae-zelandiae can produce small amounts of peramine, early pathway indole-diterpenes and ergot alkaloids, but no lolines or lolitrems. Target specific primers suggested the presence of genes for ergot alkaloids and peramine, but genes of only early pathway steps for the other alkaloids. Furthermore, genes for both mating type idiomorphs (MTA and MTB) were present, a single copy of MTA and two copies of MTB. Endophytes of native grasses may provide a genetic resource that could be exploited for developing pasture grass cultivars with improved performance. CONTACT Adrian Leuchtmann [email protected] Supplemental data for this article can be accessed at https://doi.org/10.1080/0028825X.2019.1651344 © 2019 The Royal Society of New Zealand 2 A. LEUCHTMANN ET AL. Introduction Epichloe endophytes form natural associations with many pooid grass genera predomi- nantly in the Northern Hemisphere (Schardl 2010). They systemically infect above ground parts of the grasses and mostly remain symptomless. Some species, however, form external fruiting structures thus causing choke disease, and impairing flowering and seed set. Diversification of symptomless, asexual species has frequently occurred by interspecific hybridisation resulting in allopolyploid species with genomes from two or three parental species (Moon et al. 2004; Campbell et al. 2017). The more widespread asexual endophytes are extensively studied because hosts may receive substantial benefits from the symbiosis including increased resistance to drought, herbivores and pathogens (West 1994; Bazely et al. 1997; Clarke et al. 2006), and are therefore considered important components of sustainable agriculture (Kauppinen et al. 2016). Since the early twentieth century, Epichloe endophytes have been also reported from grasses native to South America, South Africa and Australia (Gibbs Russel and Ellis 1982; Miles et al. 1998; Cabral et al. 1999). Several of these grasses including species of Festuca, Poa, Melica and Echinopogon have been associated with toxicoses in grazing animals causing staggers or drunken-like behaviour (Everist 1981; Pomilio et al. 1989). However, com- pared to the Northern Hemisphere, endophyte infection appears to be much less common in native grasses of the Southern Hemisphere, although introduced grasses are frequently infected in agricultural settings of Southern Hemisphere countries. After separation of New Zealand from Australia with the opening of the Tasman Sea some 80 million years ago, a very rich endemic flora developed from descendants of the Gondwana biota (Molnar et al. 1975; Fleming 1979). According to the ‘Flora of New Zealand’ (Edgar and Connor 2000), 157 grass species are endemic to New Zealand and 31 additional species are indigenous with a shared distribution in Australia and other Pacific islands. There is little knowledge on incidence of endophyte infection in native New Zealand grasses since few of these species have been examined (Davis and Guy 2001; Rolston et al. 2002). So far, two grass species have been found to be infected by Epi- chloe endophytes, namely Echinopogon ovatus (G. Forst.) P. Beauv. (Miles et al. 1998) and Poa matthewsii Petrie (Stewart et al. 2004). Echinopogon ovatus is indigenous to Australia and New Zealand and host of two endophytes. Epichloe aotearoae (C.D. Moon, C.O. Miles &Schardl)Leuchtm.&Schardl,representingaunique,apparentlynon-hybridlineage within the Epichloe genus was isolated from Australian and New Zealand populations. The other species, E. australiensis (C.D. Moon & Schardl) Leuchtm., an interspecific hybrid that involved ancestral lineages closely related to extant E. festucae Leuchtm., Schardl & M.R. Siegel and E. typhina (Pers.) Tul. & C. Tul., was identified from Australian populations only (Moon et al. 2002). The endophyte of the endemic P. matthewsii is unde- scribed, although its morphology and cultural characteristics clearly allowed identification as Epichloe (Stewart et al. 2004). In a previous survey, Rolston et al. (2002) collected grass seeds from 24 native New Zealand grasses (19 of which were endemic), and examined seedlings for endophyte infec- tion using an immunoblot technique and morphological examination. No endophyte infections were detected in any of the grasses analysed, including samples from seven native species of Poa and two native species of Festuca. Likewise, incidental examination of 18 endemic grasses showed no endophyte infection (Davis and Guy 2001). This NEW ZEALAND JOURNAL OF BOTANY 3 contrasts with the common occurrence of endophytes in other species of Poa and Festuca in the Northern Hemisphere and South America. Rolston et al. (2002) suggest that colo- nisation of these grasses by endophytes occurred more recently after the isolation of New Zealand biota from continental populations involving Epichloe endophytes that have evolved in the Northern Hemisphere. However, there is one endophyte that diverged early in the evolution of Epichloe and appears to be unique to the Southern Hemisphere (Moon et al. 2002). Furthermore, introduced species of Festuca and Lolium, including the agriculturally important species Lolium perenne L. (perennial rygrass) and Schedonorus arundinaceus (Schreb.) Dumort. (tall fescue), are found to be commonly infected at road- sides in New Zealand (Rolston et al. 2002). Since most native grasses of New Zealand are unexamined for the presence of Epichloe (approximately 120 species), and as endophytes have been detected in some New Zealand grasses previously, it seemed likely that more grass species may harbour Epichloe endophytes. In the present study, we report results from a recent survey of native New Zealand grasses for detecting endophyte infections. We describe a new species of Epichloe from the endemic New Zealand grass, P. matthewsii, and report a new host of the previously described E. australiensis. In addition, we conducted PCR-based genetic analyses of mating type genes and genes involved in alkaloid synthesis from the new endophyte of P. matthewsii and from E. australiensis, which can predict potential alkaloid profiles of infected host grasses. Materials and methods Collection and screening for endophyte infection Collection of plant material was conducted from October 2017 to January 2018 at sites on the North Island and South Island of New Zealand (Table 1). One tiller per plant was sampled from up to 10 different plants at a site, stored in plastic bags and taken to the laboratory. Endophyte infection was evaluated microscopically in leaf sheath tissues stained for fungal hyphae with aniline blue (Clark et al. 1983). From all plants that tested positive, endophytes were isolated in pure culture for morphological confirmation and DNA sequencing. Plant species were identified from pressed voucher specimens col- lected in the field using the keys provided by Edgar and Connor (2000). Further samples from P. matthewsii had been collected in previous years from Port Hills near Christchurch and from 25 natural populations occurring in native forest rem- nants on Banks Peninsula. These populations were screened for endophyte infection by immunoblotting (Simpson et al. 2012). Endophyte isolation Isolations were made from surface-disinfected