Mycology An International Journal on Fungal Biology ISSN: 2150-1203 (Print) 2150-1211 (Online) Journal homepage: https://www.tandfonline.com/loi/tmyc20 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 Copyright 2012 Mycological Society of China Published online: 22 Aug 2012. Submit your article to this journal Article views: 557 View related articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tmyc20 Mycology Vol. 3, No. 3, September 2012, 201–209 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] ISSN 2150-1203 print/ISSN 2150-1211 online © 2012 Mycological Society of China http://dx.doi.org/10.1080/21501203.2012.718292 http://www.tandfonline.com Published online 06 Sep 2012 202 K. Han et al. 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