[Phylogeny of Neotyphodium endophyte from western Chinese Elymus species based on act sequences].

OBJECTIVE: We analyzed phylogenetic relationships between asexual endophytes isolated from western Chinese Elymus species. METHODS: Fifteen act sequences were cloned from asexual endophytes of the western Chinese Elymus species. The phylogenetic tree and network topology structure were constructed using act sequences obtained and Genbank. RESULTS: The act sequences from asexual endophyte were single-copy gene. The western Chinese asexual endophye (Neotyphodium spp.) has a different origination from the North American sexual endophyte ( Epichloë elymi) from Elymus species, but there is a closer relationship between Neotyphodium spp. and Neotyphodium chisosum from North American Achnatherum eminens. Further, endophyte gene-flow exists between sexual Neotyphodium chisosum from North American Achnatherum eminens ( Hap 8) and asexual Neotyphodium sp. from western Chinese Elymus species (Hap 3).

Species diversity of Epichloë symbiotic with two grasses from southern Argentinean Patagonia.

In this work we performed morphological and molecular phylogenetic analyses (based on sequences of calmodulin M [calM], translation-elongation factor 1-α [tefA] and ß-tubulin [tubB] genes) to characterize the diversity of Epichloë endophytes in Bromus setifolius and Phleum alpinum. The phylogenies obtained from the three genes were congruent and allowed differentiation of three lineages of endophytes that also presented morphological differences. One lineage corresponds to the previously described species Epichloë tembladerae, which is present in a wide range of native grasses from Argentina including B. setifolius and P. alpinum. Another genotype isolated only from B. setifolius is a non-hybrid endophyte, a rare condition for the South American Epichloë endophytes. Isolates of this genotype, described herein as a new variety, Epichloë typhina var. aonikenkana, presented waxy colonies at maturity and a low production of conidia. The third lineage, exclusively found in isolates from P. alpinum, is a hybrid between E. typhina and a common ancestor of E. amarillans and E. baconii. Isolates of this lineage produce abundant conidia that are variable in shape and size. Based on its unique phylogenetic position and morphology, we propose the new species, Epichloë cabralii for this lineage. The new combinations Epichloë tembladerae and E. pampeana also are proposed for the previously described Neotyphodium tembladerae and Neotyphodium pampeanum species.

Detection and quantification of three distinct Neotyphodium lolii endophytes in Lolium perenne by real time PCR of secondary metabolite genes.

Perennial ryegrass (Lolium perenne) is a widely used pasture grass, which is frequently infected by Neotyphodium lolii endophytes that enhance grass performance but can produce alkaloids inducing toxicosis in livestock. Several selected endophyte strains with reduced livestock toxicity, but that confer insect resistance, are now in common use. Little is known regarding the survival and persistence of these endophytes when in competition with common toxic endophytes. This is mainly because there are currently no assays available to easily and reliably quantify different endophytes in pastures or in batches of seeds infected with multiple strains. We developed real time PCR assays, based on secondary metabolite genes known to differ between N. lolii endophyte strains, to quantify two selected endophytes, AR1 and AR37, and a common toxic ecotype used in New Zealand. A duplex PCR allowed assessment of endophyte:grass DNA ratios with high sensitivity, specificity and precision. Endophyte specific primers/probes could detect contamination of AR37 seeds with other endophytes down to a level of 3-25%. We demonstrated that it is possible to quantify different endophyte strains simultaneously using multiplex PCR. This method has potential applications in management of endophytes in pastures and in fundamental research into this important plant-microbe symbiosis.

Nomenclatural realignment of Neotyphodium species with genus Epicholë.

Nomenclatural rule changes in the International Code of Nomenclature for algae, fungi and plants, adopted at the 18th International Botanical Congress in Melbourne, Australia, in 2011, provide for a single name to be used for each fungal species. The anamorphs of Epichloë species have been classified in genus Neotyphodium, the form genus that also includes most asexual Epichloë descendants. A nomenclatural realignment of this monophyletic group into one genus would enhance a broader understanding of the relationships and common features of these grass endophytes. Based on the principle of priority of publication we propose to classify all members of this clade in the genus Epichloë. We have reexamined classification of several described Epichloë and Neotyphodium species and varieties and propose new combinations and states. In this treatment we have accepted 43 unique taxa in Epichloë, including distinct species, subspecies, and varieties. We exclude from Epichloë the two taxa Neotyphodium starrii, as nomen dubium, and Neotyphodium chilense, as an unrelated taxon.

Phylogenomics of fescue grass-derived fungal endophytes based on selected nuclear genes and the mitochondrial gene complement.

BACKGROUND: Tall fescue and meadow fescue are important as temperate pasture grasses, forming mutualistic associations with asexual Neotyphodium endophytes. The most frequently identified endophyte of Continental allohexaploid tall fescue is Neotyphodium coenophialum, while representatives of two other taxa (FaTG-2 and FaTG-3) have been described as colonising decaploid and Mediterranean hexaploid tall fescue, respectively. In addition, a recent study identified two other putatively novel endophyte taxa from Mediterranean hexaploid and decaploid tall fescue accessions, which were designated as uncharacterised Neotyphodium species (UNS) and FaTG-3-like respectively. In contrast, diploid meadow fescue mainly forms associations with the endophyte taxon Neotyphodium uncinatum, although a second endophyte taxon, termed N. siegelii, has also been described. RESULTS: Multiple copies of the translation elongation factor 1-a (tefA) and ß-tubulin (tub2) 'house-keeping' genes, as well as the endophyte-specific perA gene, were identified for each fescue-derived endophyte taxon from whole genome sequence data. The assembled gene sequences were used to reconstruct evolutionary relationships between the heteroploid fescue-derived endophytes and putative ancestral sub-genomes derived from known sexual Epichloë species. In addition to the nuclear genome-derived genes, the complete mitochondrial genome (mt genome) sequence was obtained for each of the sequenced endophyte, and phylogenetic relationships between the mt genome protein coding gene complements were also reconstructed. CONCLUSIONS: Complex and highly reticulated evolutionary relationships between Epichloë-Neotyphodium endophytes have been predicted on the basis of multiple nuclear genes and entire mitochondrial protein-coding gene complements, derived from independent assembly of whole genome sequence reads. The results are consistent with previous studies while also providing novel phylogenetic insights, particularly through inclusion of data from the endophyte lineage-specific gene, as well as affording evidence for the origin of cytoplasmic genomes. In particular, the results obtained from the present study imply the possible occurrence of at least two distinct E. typhina progenitors for heteropoid taxa, as well the ancestral contribution of an endophyte species distinct from (although related to) contemporary E. baconii to the extant hybrid species. Furthermore, the present study confirmed the distinct taxonomic status of the newly identified fescue endophyte taxa, FaTG-3-like and UNS, which are consequently proposed to be renamed FaTG4 and FaTG5, respectively.

The Epichloe endophytes associated with the woodland grass Hordelymus europaeus including four new taxa.

Epichloë endophytes (Clavicipitaceae, Ascomycota), including asexual forms placed in Neotyphodium, are common in cool-season grasses. Here we characterize the endophytes of the European woodland grass Hordelymus europaeus based on growth characteristics, morphology of conidiophores and conidia and phylogenetic relationships. Of the six different taxa found on H. europaeus, four are new, for which we propose the species names E. hordelymi, E. disjuncta, E. danica and subspecies E. sylvatica subsp. pollinensis. The other two are assigned to previously described E. bromicola and E. sylvatica. E. hordelymi, E. disjuncta and E. danica are asexual interspecific hybrids, while the other taxa are haploid. Only E. sylvatica subsp. pollinensis was found to reproduce sexually on H. europaeus. The high diversity of endophytes may be explained by repeated host jumps to H. europaeus with and without subsequent interspecific hybridizations.

Genetic diversity and structure of Neotyphodium species and their host Achnatherum sibiricum in a natural grass-endophyte system.

Achnatherum sibiricum (Poaceae) is a perennial bunchgrass native to the Inner Mongolia Steppe of China. This grass is commonly infected by epichloë endophytes with high-infection frequencies. Previously, we identified two predominant Neotyphodium spp., N. sibiricum and N. gansuense. In the present study, genetic diversity and structure were analyzed for the two predominant Neotyphodium spp. as well as the host grass. We obtained 103 fungal isolates from five populations; 33 were identified as N. sibiricum and 61 as N. gansuense. All populations hosted both endophytic species, but genetic variation was much higher for N. gansuense than for N. sibiricum. The majority of fungal isolates were haploid, and 13% of them were heterozygous at one SSR locus, suggesting hybrid origins of those isolates. Significant linkage disequilibrium of fungal SSR loci suggested that both fungal species primarily propagate by clonal growth through plant seeds, whereas variation in genetic diversity and the presence of hybrids in both endophytic species revealed that although clonal propagation was prevalent, occasional recombination might also occur. By comparing genetic differentiation among populations, we found around 4-7-fold greater differentiation of endophyte populations than host populations, implying more restricted gene flow of endophytes than hosts. We proposed that endophyte infection of A. sibiricum might confer the host some selective advantages under certain conditions, which could help to maintain high-endophyte-infection frequencies in host populations, even when their gene flows do not match each other. Furthermore, we suggested that the same genotype of endophyte as well as host should be confirmed if the objective of the study is to know the influence of endophyte or host genotype on their symbiotic relationship, instead of just considering whether the plant is infected by an endophyte or not, since endophytes from the same host species could exhibit high levels of genetic diversity, which is likely to influence the outcome of their symbiotic relationship.

Taxonomy, diversity and origins of symbiotic endophytes of Achnatherum sibiricum in the Inner Mongolia Steppe of China.

We sampled five Achnatherum sibiricum populations and documented the distribution and abundance of endophytes. In total, 438 fungal isolates were obtained. They were classified into 11 morphotypes based on growth rate and morphological characters including colonial morphology, the production of conidiogenous cells, conidial size and dimension on potato dextrose agar medium. Both tub2 and actG sequences identified only a single product in all representative isolates from three dominant morphotypes. All sequences fell in the same main clade, including Neotyphodium gansuense and Neotyphodium gansuense var. inebrians from another Achnatherum species in North China, Achnatherum inebrians, and Neotyphodium guerinii from European grasses Melica ciliate and Melica transsilvanica. Based on both morphological and phylogenetic analyses, isolates in morphotype I were described as representing a novel Neotyphodium species, Neotyphodium sibiricum sp. nov. Isolates in both morphotype II and III were identified as representing N. gansuense. Both tub2 and actG sequences suggested that N. sibiricum and N. guerinii were probably derived from a common Epichloë ancestor.

A new Neotyphodium species from Festuca parvigluma Steud. grown in China.

Neotyphodium species evolved from Epichloë species and are asexual, seedborne endophytes in many cool-season grasses. Here we propose a new species inhabiting Festuca parvigluma native to China. Morphology, host specificity and molecular phylogenetic evidence supported recognition of this new species. Sequences of beta-tubulin gene (tubB) introns and translation elongation factor 1-alpha gene (tefA) introns were present as two copies in all five isolates examined. In phylogenetic analyses copy 1 was closely related to E. yangzii in the EBY clade and copy 2 with E. typhina in the ETC clade, indicating this new species might have originated as a result of hybridization between members of these two clades. Referring to the distribution area of host plants, Neotyphodium sinofestucae is proposed for this new species.

Phylogenetic divergence, morphological and physiological differences distinguish a new Neotyphodium endophyte species in the grass Bromus auleticus from South America.

The fungi of genus Neotyphodium are systemic, constitutive, symbionts of grasses of subfamily Pooideae. In the southern hemisphere most of these asexual endophytes are the result of the hybridization between two sexual species, Epichlo" festucae and E. typhina, from the northern hemisphere. However the ancestral sexual species have not been detected in this region. Several grasses from Argentina are infected by Neotyphodium species. These endophytes are in general very similar macro- and micromorphologically and phylogenetically conform to species N. tembladerae. However the Neotyphodium spp. endophytes of some hosts, Bromus auleticus and Poa spicifomis var. spiciformis, have not been included in this species. In this work we studied the incidence and characterized the diversity of Neotyphodium species in populations of the native grass Bromus auleticus from Argentina. The incidence of endophytes was 100% in all populations investigated. Two groups of endophytes were differentiated by their morphologies, growth rates, conidial ontogenies and by relative resistance to the fungicide benomyl. Phylogenetic trees inferred from tefA and tubB intron sequences indicated that both N. tembladerae and the novel morphotype were hybrids of E. festucae and E. typhina, but the ancestral E. typhina genotype distinguished them. Isolates from plants that inhabit coastal dunes, xerophytic forests, savannahs and hills were similar morphologically and phylogenetically to N. tembladerae, whereas the endophytes from the humid pampa plains conformed to the novel group. We propose the endophyte of Bromus auleticus from humid pampas as a new species, Neotyphodium pampeanum.

A new stromata-producing Neotyphodium species symbiotic with clonal grass Calamagrostis epigeios (L.) Roth. grown in China.

We describe a new stromata-producing Neotyphodium species symbiotic with clonal Calamagrostis epigeios (L.) Roth. Stromata on the grass, 47.5-186 mm long, occurred frequently, but neither perithecium nor mature ascus was observed. Morphology of fungal isolates obtained from symptomatic and asymptomatic tillers were identical to each other and similar to those of epichloë endophytes. In phylogenetic analysis all selected five fungal isolates clustered into a significantly distinct clade based on sequences of beta-tubulin gene (tubB) introns and translation elongation factor 1-alpha gene (tefA) introns with bootstrap values of 99%, supporting erection of a new species. Concerning the production of extremely long stromata on the host plants and absence of sexual spores, we propose the name Neotyphodium stromatolongum Y. Ji, L. Zhan et Z. Wang, sp. nov.

Taxonomy of Neotyphodium endophytes of Chinese native Roegneria plants.

In this study we describe a new Neotyphodium species, Neotyphodium sinicum, found in natural symbiosis with asymptomatic Roegneria spp. (Elymus spp.) native to China. We investigated the host specificity, morphology, mating compatibility and molecular phylogenetic evidence for recognition of this new species. N. sinicum is host specific and seedborne and widely distributed in China. Most morphological characteristics of this new species are typical of other Neotyphodium species, but the growth on PDA plates tends to be more rapid. Three of five N. sinicum strains examined had double alleles of tefA and two of the five strains had double alleles of both tubB and tefA. Phylogenetic relationships based on sequences of tubB introns and tefA introns revealed that the allele-1 of tefA and tubB clustered together in a distinct subclade in the Epichloe bromicola/E. yangzii clade. The allele-2 grouped in the Epichlok" typhina clade (ETC). N. sinicum therefore appears to be an interspecific hybrid related to both E. yangzii and members of ETC. In mating tests N. sinicum failed to hybridize with E. yangzii.