Origin, divergence, and phylogeny of epichloe endophytes of native Argentine grasses

The epichlo? endophytes are systemic, constitutive, and often vertically transmitted fungal symbionts of grass species in subfamily Po?ideae. Prior studies indicate that several asexual epichlo? endophytes (Neotyphodium species) have evolved directly from sexual (Epichlo?) species, whereas others evolved by hybridization between two or more endophyte species. In this paper, we investigate the phylogenies of 27 Neotyphodium spp. isolates from 10 native grass species (in 4 tribes) in 22 populations throughout Argentina. Relationships among these fungi and a worldwide collection of epichlo? endophytes were estimated by phylogenetic analysis of sequences from variable portions (mainly introns) of genes for beta-tubulin (tub2) and translation elongation factor 1-alpha (tef1). Most of the Argentine endophyte isolates were interspecific hybrids of Epichlo? festucae and E. typhina. Only one isolate was a hybrid of a different ancestry, and three isolates were apparently non-hybrid endophytes. These results indicate that interspecific hybridization, which promotes genetic variation, was common during the evolution of the endophytes of Argentine grasses.     

Epichlo? festucae and related mutualistic symbionts of grasses.

Epichlo? and Neotyphodium species (Ascomycota) are mutualistic symbionts (endophytes) of temperate grasses, to which they impart numerous and profound fitness benefits. Epichlo? festucae, a common symbiont of Festuca, Lolium,and Koeleria spp., is a model for endophyte research that is amenable to Mendelian and molecular genetic analysis. Characteristics of E. festucae include: (i) production of the anti-insect alkaloids peramine and lolines, (ii) production of the anti-vertebrate alkaloids lolitrem B and ergovaline, (iii) efficient vertical transmission via host seeds, (iv) a mildly pathogenic state associated with the E. festucae sexual cycle, and (v) a clear role in enhancing survival of host plants. Genetic analysis of alkaloid production has recently begun. Also, physiological and ultrastructural studies suggest that signals communicated between E. festucae and host plants ensure an exquisitely balanced interaction to the mutual benefit of both partners. Several mutualistic Neotyphodium species are hybrids between E. festucae and other endophyte species.     

Biological and physiological characteristics of Neotyphodium gansuense symbiotic with Achnatherum inebrians

Biological and physiological characteristics of Neotyphodium gansuense were compared with Neotyphodium coenophialum and Epichlo? festucae at a range of temperatures and pH values, and on carbon and nitrogen amended media. N. gansuense was able to grow at 10-30 degrees C, but not at 5 degrees C, and slowly at 35 degrees C. The optimal temperature for both N. gansuense and N. coenophialum was 25 degrees C, but that of E. festucae was 20-25 degrees C. The optimal pH ranges for mycelial growth of N. gansuense, N. coenophialum and E. festucae were 5-9, 5-9 and 5-7, respectively. The Neotyphodium and Epichlo? endophytes varied in their ability to grow on media containing different carbon and nitrogen nutrients. The preference of N. gansuense for carbon source was sucrose>glucose, lactose, sorbitol, inulin, maltose, mannitol, starch, fructose>xylose. Growth of all three endophytes tested was significantly improved by peptone, tryptone, casein, yeast extract and l-proline. Yeast extract, peptone, casein, tryptone, l-proline, potassium nitrate, ammonium oxalic acid and l-leucine significantly improved growth of N. gansuense. However, ammonium nitrite was not utilized at all by any tested endophyte. N. gansuense grew significantly better on potato dextrose agar (PDA) and oat meal agar (OMA) than on corn meal agar (CMA) and drunken-horse-grass agar (DA), and most slowly on water agar (WA) and saltwater nutrient agar (SNA).     

Endophytic fungal beta-1,6-glucanase expression in the infected host grass

Mutualistic fungal endophytes infect many grass species and often confer benefits to the hosts such as reduced herbivory by insects and animals. The physiological interactions between the endophytes and their hosts have not been well characterized. Fungal-secreted proteins are likely to be important components of the interaction. In the interaction between Poa ampla and the endophyte Neotyphodium sp., a fungal beta-1,6-glucanase is secreted into the apoplast, and activity of the enzyme is detectable in endophyte-infected plants. Sequence analysis indicates the beta-1,6-glucanase is homologous to enzymes secreted by the mycoparasitic fungi Trichoderma harzianum and Trichoderma virens. DNA gel-blot analysis indicated the beta-1,6-glucanase was encoded by a single gene. As a secreted protein, the beta-1,6-glucanase may have a nutritional role for the fungus. In culture, beta-1,6-glucanase activity was induced in the presence of beta-1,6-glucans. From RNA gel blots, similar beta-1,6-glucanases were expressed in tall fescue (Festuca arundinacea Schreb.) and Chewings fescue (Festuca rubra L. subsp. fallax [Thuill] Nyman) infected with the endophyte species Neotyphodium coenophialum and Epichlo? festucae, respectively.     

Contribution of fungal loline alkaloids to protection from aphids in a grass-endophyte mutualism

Fungal endophytes provide grasses with enhanced protection from herbivory, drought, and pathogens. The loline alkaloids (saturated 1-aminopyrrolizidines with an oxygen bridge) are fungal metabolites often present in grasses with fungal endophytes of the genera Epichlo? or Neotyphodium. We conducted a Mendelian genetic analysis to test for activity of lolines produced in plants against aphids feeding on those plants. Though most loline-producing endophytes are asexual, we found that a recently described sexual endophyte, Epichlo? festucae, had heritable variation for loline alkaloid expression (Lol+) or nonexpression (Lol-). By analyzing segregation of these phenotypes and of linked DNA polymorphisms in crosses, we identified a single genetic locus controlling loline alkaloid expression in those E. festucae parents. We then tested segregating Lol+ and Lol- full-sibling fungal progeny for their ability to protect host plants from two aphid species, and observed that alkaloid expression cosegregated with activity against these insects. The in planta loline alkaloid levels correlated with levels of anti-aphid activity. These results suggested a key role of the loline alkaloids in protection of host plants from certain aphids, and represent, to our knowledge, the first Mendelian analysis demonstrating how a fungal factor contributes protection to plant-fungus mutualism.     

Direct classification of related species of fungal endophytes (Epichlo? spp.) using visible and near-infrared spectroscopy and multivariate analysis.

The aim of this work was to investigate the potential of visible and near-infrared (Vis-NIR) reflectance spectroscopy for the classification of three morphologically similar species of fungal endophytes of grasses. Vis-NIR spectra (400-2498 nm) from 34 isolates of Epichlo? sylvatica, 32 of Epichlo? typhina and 38 of Epichlo? festucae were recorded directly from fresh mycelium growing in potato dextrose agar plates. Multivariate procedures applied to the spectral data were discriminant modified partial least squares regression, soft independent modelling of class analogy and discriminant partial least squares regressions (PLS1, PLS2). Several types of data pretreatments were tested to develop the classification models. The best predictive models were achieved with PLS2 analysis; with this method, 90% of E. typhina and 100% of E. festucae and E. sylvatica external validation samples were successfully classified. These results show the potential of Vis-NIR spectroscopy combined with multivariate analysis as a rapid method for classifying morphologically similar species of filamentous fungi.     

A new Epichloe species with interspecific hybrid origins from Poa pratensis ssp. pratensis in Liyang, China

We describe a new Epichlo? species found in symbiosis with Poa pratensis ssp. pratensis in Liyang, China. Stromata characteristic of Epichlo? spp. were present on some of the reproductive tillers of individual host grasses. Only three of the 98 stromata observed on field plants became orange and produced perithecia. Phylogenetic analyses based on sequences of tubB and tefA indicated that this Epichlo? sp. was an interspecific hybrid related to both E. yangzii and members in the E. typhina complex clade (ETC). Allele-1 of tefA and tubB grouped in the E. bromicola/E. yangzii clade; allele-2 of these two genes clustered in a distinct subclade in the ETC. This is the first report of an Epichlo? species that has interspecific hybrid origins. We propose the name Epichlo? liyangensis Z. Wang, Y. Kang et H. Miao, sp. nov. for this species.     

Origin of a Fungal Symbiont of Perennial Ryegrass by Interspecific Hybridization of a Mutualist with the Ryegrass Choke Pathogen, Epichloe Typhina

Seed-borne fungal symbionts (endophytes) provide many cool-season grass species with biological protection from biotic and abiotic stresses. The endophytes are asexual, whereas closely related sexual species of genus Epichloe (Clavicipitales) cause grass choke disease. Perennial ryegrass (Lolium perenne) is a host of two endophyte taxa, LpTG-1 (L. perenne endophyte taxonomic grouping one = Acremonium lolii) and LpTG-2, as well as the choke pathogen, Epichloe typhina (represented by isolate E8). Relationships among these fungi and other Epichloe species were investigated by analysis of gene sequences, DNA polymorphisms and allozymes. The results indicate that LpTG-2 is a heteroploid derived from an interspecific hybrid. The LpTG-2 isolates had two copies each of nine out of ten genes analyzed (the exception being the rRNA gene locus), and the profiles for seven of these were composites of those from E. typhina E8 and A. lolii isolate Lp5. Molecular phylogenetic analysis grouped the two β-tubulin genes of LpTG-2 into separate clades. One (tub2-1) was related to that of E. typhina E8, and the other (tub2-2) to that of A. lolii. The mitochondrial DNA profile of LpTG-2 was similar to that of A. lolii, but its rRNA gene sequence grouped it with E. typhina E8. A proposed model for the evolution of LpTG-2 involves infection of a L. perenne-A. lolii symbiotum by E. typhina, followed by hybridization of the two fungi. Such interspecific hybridization may be a common and important mechanism for genetic variation in Epichloe endophytes.     

Development and characterization of EST-derived simple sequence repeat (SSR) markers for pasture grass endophytes.

Fungal endophytes of the genus Neotyphodium are common in temperate pasture grass species and confer both beneficial and deleterious agronomic characteristics to their hosts. The aim of this study was to develop molecular markers based on simple sequence repeat (SSR) loci for the identification and assessment of genetic diversity among Neotyphodium endophytes in grasses. Expressed sequence tags (ESTs) from both Neptyphodium coenophialum and Neotyphodium lolii were examined, and unique SSR loci were identified in 9.7% of the N. coenophialum sequences and 6.3% of the N. lolii sequences. A variety of SSRs were present, although perfect trinucleotide repeat arrays were the most common. Primers were designed to 50 SSR loci from N. coenophialum and 57 SSR loci from N. lolii and were evaluated using 20 Neotyphodium and Epichlo? isolates. A high proportion of the N. coenophialum and N. lolii primers produced amplification products from the majority of isolates and most of these primers detected genetic variation. SSR markers from both N. coenophialum and N. lolii detected high levels of polymorphism between Neotyphodium and Epichlo? species, and low levels of polymorphism within N. coenophialum and N. lolii. SSR markers may be used in appropriate combinations to discriminate between species. Comparison with amplified fragment length polymorphism (AFLP) data demonstrated that the SSR markers were informative for the assessment of genetic variation within and between endophyte species. These markers may be used to identify endophyte taxa and to evaluate intraspecific population diversity, which may be correlated with variation for endophyte-derived agronomic traits.     

Extraordinary ribosomal spacer length heterogeneity in a neotyphodium endophyte hybrid: implications for concerted evolution.

An extraordinary level of length heterogeneity was found in the ribosomal DNA (rDNA) of an asexual hybrid Neotyphodium grass endophyte, isolate Lp1. This hybrid Neotyphodium endophyte is an interspecific hybrid between two grass endophytes, Neotyphodium lolii, and a sexual form, Epichlöe typhina, and the length heterogeneity was not found in either of these progenitor species. The length heterogeneity in the hybrid is localized to the intergenic spacer (IGS) and is the result of copy-number variation of a tandemly repeated subrepeat class within the IGS, the 111-/119-bp subrepeats. Copy number variation of this subrepeat class appears to be a consequence of mitotic unequal crossing over that occurs between these subrepeats. This implies that unequal crossing over plays a role in the concerted evolution of the whole rDNA. Changes in the pattern of IGS length variants occurred in just two rounds of single-spore purification. Analysis of the IGS length heterogeneity revealed features that are unexpected in a simple model of unequal crossing over. Potential refinements of the molecular details of unequal crossing over are presented, and we also discuss evidence for a combination of homogenization mechanisms that drive the concerted evolution of the Lp1 rDNA.     

Evolutionary origins and ecological consequences of endophyte symbiosis with grasses

Over the past 20 yr much has been learned about a unique symbiotic interaction between fungal endophytes and grasses. The fungi (Clavicipitaceae, Ascomycota) grow intercellularly and systemically in aboveground plant parts. Vertically transmitted asexual endophytes forming asymptomatic infections of cool-season grasses have been repeatedly derived from sexual species that abort host inflorescences. The phylogenetic distribution of seed-transmitted endophytes is strongly suggestive of cocladogenesis with their hosts. Molecular evidence indicates that many seed-transmitted endophytes are interspecific hybrids. Superinfection may result in hyphal fusion and parasexual recombination. Most endophytes produce one or more alkaloid classes that likely play some role in defending the host plant against pests. Hybridization may have led to the proliferation of alkaloid-production genes among asexual endophytes, favoring hybrids. The ergot alkaloid ergovaline, lolitrems, and lolines are produced by only a single sexual species, Epichlo? festucae, but they are common in seed-transmitted endophytes, suggesting that E. festucae contributed genes for their synthesis. Asexual hybrids may also be favored by the counteracting of the accumulation of deleterious mutations (Muller's rachet). Endophyte infection can provide other benefits, such as enhanced drought tolerance, photosynthetic rate, and growth. Estimates of infection frequency have revealed variable levels of infection with especially high prevalence in the subfamily Pooideae. Longitudinal studies suggest that the prevalence of seed-transmitted endophytes can increase rapidly over time. In field experiments, infected tall fescue suppressed other grasses and forbs relative to uninfected fescue and supported lower consumer populations. Unlike other widespread plant/microbial symbioses based on the acquisition of mineral resources, grass/endophyte associations are based primarily on protection of the host from biotic and abiotic stresses.     

Expression of a novel chitinase by the fungal endophyte in Poa ampla

Many wild and cultivated cool-season grass species are naturally infected with fungal endophytes of the genera Neotyphodium and Epichlo?. These associations generally are considered mutualistic with the plants benefiting from reduced herbivory and the fungi benefiting from nutrients supplied by the plants. The fungi secrete proteins that might have a role in the interspecies symbiosis. In the interaction between Poa ampla Merr. and the endophyte Neotyphodium sp., a fungal chitinase was detected in the apoplastic protein fraction. The chitinase was also the major protein secreted in culture. Sequence analysis of the chitinase revealed it has a low level of amino acid sequence identity to other fungal chitinases and one of the conserved active site residues is altered. DNA gel-blot analysis indicated the chitinase was encoded by a single gene. Expression of similar chitinases also was detected in endophyte-infected tall fescue (Festuca arundinacea Schreb.), perennial ryegrass (Lolium perenne L.) and Chewings fescue (Festuca rubra L. subsp. fallax [Thuill] Nyman). This is the first report of an endophyte chitinase expressed in the infected host grass. As a secreted hydrolytic enzyme, the chitinase might have roles in the nutrition, growth or defense of the endophyte.     

Epichloe canadensis, a new interspecific epichloid hybrid symbiotic with Canada wildrye (Elymus canadensis)

Many Epichlo? endophytes found in cool-season grasses are interspecific hybrids possessing much or all of the genomes of two or three progenitors. Here we characterize Epichlo? canadensis sp. nov., a hybrid species inhabiting the grass species Elymus canadensis native to North America. Three distinct morphotypes were identified that were separated into two groups by molecular phylogenetic analysis. Sequence analysis of the translation elongation factor 1-α (tefA) and β-tubulin (tubB) genes revealed two copies in all isolates examined. Phylogenetic analyses indicated that allele 1 of each gene was derived from Epichlo? amarillans and allele 2 from Epichlo? elymi. This is the first documentation of an interspecific hybrid endophyte derived from parents of strictly North American origins. Alkaloid gene profiling using primers specific to genes in the peramine, loline, indole-diterpene and ergot alkaloid pathways may indicate chemotypic variation in the ergot alkaloid and loline pathways between the assigned morphotypes. All isolates have the gene enabling the production of peramine but lack genes in the indole-diterpene biosynthesis pathway. Morphology and phylogenetic evidence support the designation of isolates from El. canadensis as a new interspecific hybrid species.     

Geographic distribution patterns of vertically transmitted endophytes in two native grasses in Argentina

The incidence of epichloid endophytes in populations of wild grasses is usually variable, and the knowledge about distribution patterns and how environmental factors affect such an incidence is limited. Here we performed a broad scale survey data to study whether the distribution patterns and the incidence of vertically-transmitted endophytes in populations of two native grasses from South-America, Poa lanuginosa Poir. and Poa bonariensis (Lam.) Kunth., are associated with environmental characteristics. We also characterized the endophytes from different populations to establish if the genotype of the endophytes is also correlated with environmental variables. The incidence of endophytes ranged from 0 to 100 % in both host species. In P. lanuginosa, endophytes were only found in populations on sandy coastal dunes and their incidence was positively associated with winter regime rainfall and soil water availability in the growing season. In P. bonariensis, endophytes were only found in populations in xerophytic forests and their incidence was highly associated with plant community. The distributions of infested populations suggested that the endophytes are not found in those areas with the most favorable or most stressing growth conditions accordingly to climatic or edaphical characteristics. Only the vertically transmitted hybrid endophyte species Neotyphodium tembladerae was detected in both host species. Under the hypothesis of vertical transmission, these results suggested that the endophyte should have been lost in endophyte free populations but is maintained in populations established in environments presenting moderate stress as salinity or short drought periods.     

Broad-scale geographic patterns in the distribution of vertically-transmitted, asexual endophytes in four naturally-occurring grasses in Sweden

The presence and distribution of vertically-transmitted, asexual Neotyphodium/Epichloë endophytes in four species of widely distributed grasses, all members of the sub-family Pooideae, were determined at 57 spatially-distinct sites across Sweden in naturally-occurring grass populations. While these endophytes are well-studied in agricultural systems, much less is known about their abundance and distribution in non-agricultural systems at larger scales across landscapes. We used two screening methods to detect endophytes: 1) visual inspection of fixed, stained grass shoots, and 2) immunoblot cards with monoclonal antibodies specific for Neotyphodium / Epichloë endophytes. Infection frequency (i.e. the proportion of individuals) varied widely among species and among sites, a finding common to most studies performed in non-agricultural ecosystems. Festuca rubra had the highest overall infection frequency (23% visual screening, 27% immunoblot screening), while Deschampsia flexuosa (4%) and Poa trivialis (4%) had low frequencies of infection. None of the sampled Agrostis capillaris populations were infected. In F. rubra , the within-site infection frequency varied from 0 to 100%. A significant altitudinal gradient was found for F. rubra , with the infection frequency of the vertically-transmitted endophyte Epichloë festucae decreasing with increasing altitude above sea level. The high variation across and within grass species at different geographical locations that ours and others' studies reveal, agree with suggestions that agronomic model systems cannot be readily extrapolated to understand the ecological role of endophytes in native grasses in the wild. Further investigation into the underlying cause of the observed altitudinal gradient is required to understand broader-scale patterns in the distribution of endophyte-infected grasses.     

The effects of Epichloë endophytes on the growth and competitiveness of Achnatherum sibiricum are mediated by soil microbe diversity

季节性干旱驱动亚热带森林的碳积累 本研究旨在表明处于南亚热带的鼎湖山生物圈保护区的干旱频率和强度正在增加,并说明季节性干旱对亚热带森林碳积累的影响。这是为了应对全球气候变化导致的干旱加剧所带来的威胁开展的一项研究。我们使干旱指数(标准化降水指数、标准降水蒸散发指数、降水距平百分率及自校准帕尔默干旱指数)准确确定干旱期和降水量增加期。此后,将2003至2014年(12年)监测采集的实测涡动通量和土壤含水量数据在干旱期和湿润期之间进行比较,以确定干旱对生态系统碳积累的影响。在本研究所选择的12年期间,干旱的发生时间约占比20%,最强干旱事件和严重程度发生于2012至2013年。研究期间的年平均降水量和气温分别为1404.57 ± 43.2 mm和22.65 ± 0.1 °C,与30年记录(1990–2020)相比较,年降水量减少量可达523 mm,而气温则增加了2.55 °C。与全球针对大多数森林生态系统研究所发表的数据呈相反趋势,处于中国南亚热带区域的鼎湖山生物圈保护区在60%的干旱期内所监测的森林生态系统记录到显著的碳积累趋势,说明季节性干旱驱动了森林的碳积累。     

Epichlo? endophytes grow by intercalary hyphal extension in elongating grass leaves.

A fundamental hallmark of fungal growth is that vegetative hyphae grow exclusively by extension at the hyphal tip. However, this model of apical growth is incompatible with endophyte colonization of grasses by the symbiotic Neotyphodium and Epichlo? species. These fungi are transmitted through host seed, and colonize aerial tissues that develop from infected shoot apical meristems of the seedling and tillers. We present evidence that vegetative hyphae of Neotyphodium and Epichlo? species infect grass leaves via a novel mechanism of growth, intercalary division and extension. Hyphae are attached to enlarging host cells, and cumulative growth along the length of the filament enables the fungus to extend at the same rate as the host. This is the first evidence of intercalary growth in fungi and directly challenges the centuries-old model that fungi grow exclusively at hyphal tips. A new model describing the colonization of grasses by clavicipitaceous endophytes is described.     

Epichloë endophytes grow by intercalary hyphal extension in elongating grass leaves

A fundamental hallmark of fungal growth is that vegetative hyphae grow exclusively by extension at the hyphal tip. However, this model of apical growth is incompatible with endophyte colonization of grasses by the symbiotic Neotyphodium and Epichlo? species. These fungi are transmitted through host seed, and colonize aerial tissues that develop from infected shoot apical meristems of the seedling and tillers. We present evidence that vegetative hyphae of Neotyphodium and Epichlo? species infect grass leaves via a novel mechanism of growth, intercalary division and extension. Hyphae are attached to enlarging host cells, and cumulative growth along the length of the filament enables the fungus to extend at the same rate as the host. This is the first evidence of intercalary growth in fungi and directly challenges the centuries-old model that fungi grow exclusively at hyphal tips. A new model describing the colonization of grasses by clavicipitaceous endophytes is described.