Quantitation and Distribution of Epichloë-Derived Alkaloids in Perennial Ryegrass Tissues.

Perennial ryegrass (Lolium perenne L.), an economically important pasture and turf grass, is commonly infected with asexual Epichloë species endophytes. Endophytes provide enhanced bioprotection by producing alkaloids, and research often focusses on the negative impact on grazing animals. However, alkaloid distribution throughout the plant and their role in biocontrol of insect pests and diseases are less well understood. Additionally, intermediate compounds have not been investigated for their impacts on animal welfare and biological control in pasture-based scenarios. Here, a single liquid chromatography-mass spectrometry (LC-MS) method was used to measure seven alkaloids in different perennial ryegrass tissues infected with SE or NEA12 endophytes. High alkaloid recoveries and a clear plant matrix effect emphasize the importance of using matrix-matched standards for accurate quantitation. The method is sensitive, detecting alkaloids at low concentrations (nanogram levels), which is important for endophyte strains that produce compounds detrimental to livestock. Concentrations were generally highest in seeds, but distribution differed in the shoots/roots: peramine, terpendole E, terpendole C and lolitrem B were higher in shoots, whilst ergovaline, paxilline and epoxy-janthitrem I were more evenly distributed throughout the two tissues. Knowledge of alkaloid distribution may allow for concentrations to be predicted in roots based on concentrations in the shoots, thereby assisting future determinations of resistance to insects, especially subterranean root-feeding pests.

Single Seed Near-Infrared Hyperspectral Imaging for Classification of Perennial Ryegrass Seed.

The detection of beneficial microbes living within perennial ryegrass seed causing no apparent defects is challenging, even with the most sensitive and conventional methods, such as DNA genotyping. Using a near-infrared hyperspectral imaging system (NIR-HSI), we were able to discriminate not only the presence of the commercial NEA12 fungal endophyte strain but perennial ryegrass cultivars of diverse seed age and batch. A total of 288 wavebands were extracted for individual seeds from hyperspectral images. The optimal pre-processing methods investigated yielded the best partial least squares discriminant analysis (PLS-DA) classification model to discriminate NEA12 and without endophyte (WE) perennial ryegrass seed with a classification accuracy of 89%. Effective wavelength (EW) selection based on GA-PLS-DA resulted in the selection of 75 wavebands yielding 88.3% discrimination accuracy using PLS-DA. For cultivar identification, the artificial neural network discriminant analysis (ANN-DA) was the best-performing classification model, resulting in >90% classification accuracy for Trojan, Alto, Rohan, Governor and Bronsyn. EW selection using GA-PLS-DA resulted in 87 wavebands, and the PLS-DA model performed the best, with no extensive compromise in performance, resulting in >89.1% accuracy. The study demonstrates the use of NIR-HSI reflectance data to discriminate, for the first time, an associated beneficial fungal endophyte and five cultivars of perennial ryegrass seed, irrespective of seed age and batch. Furthermore, the negligible effects on the classification errors using EW selection improve the capability and deployment of optimized methods for real-time analysis, such as the use of low-cost multispectral sensors for single seed analysis and automated seed sorting devices.

Near-Infrared Hyperspectral Imaging Pipelines for Pasture Seed Quality Evaluation: An Overview.

Near-infrared (800-2500 nm; NIR) spectroscopy coupled to hyperspectral imaging (NIR-HSI) has greatly enhanced its capability and thus widened its application and use across various industries. This non-destructive technique that is sensitive to both physical and chemical attributes of virtually any material can be used for both qualitative and quantitative analyses. This review describes the advancement of NIR to NIR-HSI in agricultural applications with a focus on seed quality features for agronomically important seeds. NIR-HSI seed phenotyping, describing sample sizes used for building high-accuracy calibration and prediction models for full or selected wavelengths of the NIR region, is explored. The molecular interpretation of absorbance bands in the NIR region is difficult; hence, this review offers important NIR absorbance band assignments that have been reported in literature. Opportunities for NIR-HSI seed phenotyping in forage grass seed are described and a step-by-step data-acquisition and analysis pipeline for the determination of seed quality in perennial ryegrass seeds is also presented.

A Metabolomic Study of Epichloë Endophytes for Screening Antifungal Metabolites.

Epichloë endophytes, fungal endosymbionts of Pooidae grasses, are commonly utilized in forage and turf industries because they produce beneficial metabolites that enhance resistance against environmental stressors such as insect feeding and disease caused by phytopathogen infection. In pastoral agriculture, phytopathogenic diseases impact both pasture quality and animal production. Recently, bioactive endophyte strains have been reported to secrete compounds that significantly inhibit the growth of phytopathogenic fungi in vitro. A screen of previously described Epichloë-produced antifeedant and toxic alkaloids determined that the antifungal bioactivity observed is not due to the production of these known metabolites, and so there is a need for methods to identify new bioactive metabolites. The process described here is applicable more generally for the identification of antifungals in new endophytes. This study aims to characterize the fungicidal potential of novel, 'animal friendly' Epichloë endophyte strains NEA12 and NEA23 that exhibit strong antifungal activity using an in vitro assay. Bioassay-guided fractionation, followed by metabolite analysis, identified 61 metabolites that, either singly or in combination, are responsible for the observed bioactivity. Analysis of the perennial ryegrass-endophyte symbiota confirmed that NEA12 and NEA23 produce the prospective antifungal metabolites in symbiotic association and thus are candidates for compounds that promote disease resistance in planta. The "known unknown" suite of antifungal metabolites identified in this study are potential biomarkers for the selection of strains that enhance pasture and turf production through better disease control.

The Known Antimammalian and Insecticidal Alkaloids Are Not Responsible for the Antifungal Activity of Epichloë Endophytes.

Asexual Epichloë sp. endophytes in association with pasture grasses produce agronomically important alkaloids (e.g., lolitrem B, epoxy-janthitrems, ergovaline, peramine, and lolines) that exhibit toxicity to grazing mammals and/or insect pests. Novel strains are primarily characterised for the presence of these compounds to ensure they are beneficial in an agronomical setting. Previous work identified endophyte strains that exhibit enhanced antifungal activity, which have the potential to improve pasture and turf quality as well as animal welfare through phytopathogen disease control. The contribution of endophyte-derived alkaloids to improving pasture and turf grass disease resistance has not been closely examined. To assess antifungal bioactivity, nine Epichloë related compounds, namely peramine hemisulfate, n-formylloline-d3, n-acetylloline hydrochloride, lolitrem B, janthitrem A, paxilline, terpendole E, terpendole C, and ergovaline, and four Claviceps purpurea ergot alkaloids, namely ergotamine, ergocornine, ergocryptine, and ergotaminine, were tested at concentrations higher than observed in planta in glasshouse and field settings using in vitro agar well diffusion assays against three common pasture and turf phytopathogens, namely Ceratobasidium sp., Drechslera sp., and Fusarium sp. Visual characterisation of bioactivity using pathogen growth area, mycelial density, and direction of growth indicated no inhibition of pathogen growth. This was confirmed by statistical analysis. The compounds responsible for antifungal bioactivity of Epichloë endophytes hence remain unknown and require further investigation.

Global Changes in Asexual Epichloë Transcriptomes during the Early Stages, from Seed to Seedling, of Symbiotum Establishment.

Asexual Epichloë fungi are strictly seed-transmitted endophytic symbionts of cool-season grasses and spend their entire life cycle within the host plant. Endophyte infection can confer protective benefits to its host through the production of bioprotective compounds. Inversely, plants provide nourishment and shelter to the resident endophyte in return. Current understanding of the changes in global gene expression of asexual Epichloë endophytes during the early stages of host-endophyte symbiotum is limited. A time-course study using a deep RNA-sequencing approach was performed at six stages of germination, using seeds infected with one of three endophyte strains belonging to different representative taxa. Analysis of the most abundantly expressed endophyte genes identified that most were predicted to have a role in stress and defence responses. The number of differentially expressed genes observed at early time points was greater than those detected at later time points, suggesting an active transcriptional reprogramming of endophytes at the onset of seed germination. Gene ontology enrichment analysis revealed dynamic changes in global gene expression consistent with the developmental processes of symbiotic relationships. Expression of pathway genes for biosynthesis of key secondary metabolites was studied comprehensively and fuzzy clustering identified some unique expression patterns. Furthermore, comparisons of the transcriptomes from three endophyte strains in planta identified genes unique to each strain, including genes predicted to be associated with secondary metabolism. Findings from this study highlight the importance of better understanding the unique properties of individual endophyte strains and will serve as an excellent resource for future studies of host-endophyte interactions.

Metabolic Potential of Epichloë Endophytes for Host Grass Fungal Disease Resistance.

Asexual species of the genus Epichloë (Clavicipitaceae, Ascomycota) form endosymbiotic associations with Pooidae grasses. This association is important both ecologically and to the pasture and turf industries, as the endophytic fungi confer a multitude of benefits to their host plant that improve competitive ability and performance such as growth promotion, abiotic stress tolerance, pest deterrence and increased host disease resistance. Biotic stress tolerance conferred by the production of bioprotective metabolites has a critical role in an industry context. While the known antimammalian and insecticidal toxins are well characterized due to their impact on livestock welfare, antimicrobial metabolites are less studied. Both pasture and turf grasses are challenged by many phytopathogenic diseases that result in significant economic losses and impact livestock health. Further investigations of Epichloë endophytes as natural biocontrol agents can be conducted on strains that are safe for animals. With the additional benefits of possessing host disease resistance, these strains would increase their commercial importance. Field reports have indicated that pasture grasses associated with Epichloë endophytes are superior in resisting fungal pathogens. However, only a few antifungal compounds have been identified and chemically characterized, and these from sexual (pathogenic) Epichloë species, rather than those utilized to enhance performance in turf and pasture industries. This review provides insight into the various strategies reported in identifying antifungal activity from Epichloë endophytes and, where described, the associated antifungal metabolites responsible for the activity.

Novel Antifungal Activity of Lolium-Associated Epichloë Endophytes.

Asexual Epichloë spp. fungal endophytes have been extensively studied for their functional secondary metabolite production. Historically, research mostly focused on understanding toxicity of endophyte-derived compounds on grazing livestock. However, endophyte-derived compounds also provide protection against invertebrate pests, disease, and other environmental stresses, which is important for ensuring yield and persistence of pastures. A preliminary screen of 30 strains using an in vitro dual culture bioassay identified 18 endophyte strains with antifungal activity. The novel strains NEA12, NEA21, and NEA23 were selected for further investigation as they are also known to produce alkaloids associated with protection against insect pests. Antifungal activity of selected endophyte strains was confirmed against three grass pathogens, Ceratobasidium sp., Dreschlera sp., and Fusarium sp., using independent isolates in an in vitro bioassay. NEA21 and NEA23 showed potent activity against Ceratobasidium sp. and NEA12 showed moderate inhibition against all three pathogens. Crude extracts from liquid cultures of NEA12 and NEA23 also inhibited growth of the phytopathogens Ceratobasidium sp. and Fusarium sp. and provided evidence that the compounds of interest are stable, constitutively expressed, and secreted. Comparative analysis of the in vitro and in planta metabolome of NEA12 and NEA23 using LCMS profile data revealed individual metabolites unique to each strain that are present in vitro and in planta. These compounds are the best candidates for the differential bioactivity observed for each strain. Novel endophyte strains show promise for endophyte-mediated control of phytopathogens impacting Lolium spp. pasture production and animal welfare.

Ergot alkaloid mycotoxins: physiological effects, metabolism and distribution of the residual toxin in mice.

The complex ergot alkaloids, ergovaline and ergotamine, cause dysregulation of physiological functions, characterised by vasoconstriction as well as thermoregulatory and cardiovascular effects in grazing livestock. To assess the effect of the mycotoxins, blood pressure and heart rate of male mice were measured, and metabolite profiling undertaken to determine relative abundances of both ergotamine and its metabolic products in body and brain tissue. Ergotamine showed similar cardiovascular effects to ergovaline, causing elevations in blood pressure and reduced heart rate. Bradycardia was preserved at low-levels of ergovaline despite no changes in blood pressure. Ergotamine was identified in kidney, liver and brainstem but not in other regions of the brain, which indicates region-specific effects of the toxin. The structural configuration of two biotransformation products of ergotamine were determined and identified in the liver and kidney, but not the brain. Thus, the dysregulation in respiratory, thermoregulatory, cardiac and vasomotor function, evoked by ergot alkaloids in animals observed in various studies, could be partially explained by dysfunction in the autonomic nervous system, located in the brainstem.

A Simple LC-MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass.

The rapid identification and quantitation of alkaloids produced by Epichloë endophyte-infected pasture grass is important for the agricultural industry. Beneficial alkaloids, such as peramine, provide the grass with enhanced insect protection. Conversely, ergovaline and lolitrem B can negatively impact livestock. Currently, a single validated method to measure these combined alkaloids in planta does not exist. Here, a simple two-step extraction method was developed for Epichloë-infected perennial ryegrass (Lolium perenne L.). Peramine, ergovaline and lolitrem B were quantified using liquid chromatography-mass spectrometry (LC-MS). Alkaloid linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, selectivity, recovery, matrix effect and robustness were all established. The validated method was applied to eight different ryegrass-endophyte symbiota. Robustness was established by comparing quantitation results across two additional instruments; a triple quadruple mass spectrometer (QQQ MS) and by fluorescence detection (FLD). Quantitation results were similar across all three instruments, indicating good reproducibility. LOQ values ranged from 0.8 ng/mL to 6 ng/mL, approximately one hundred times lower than those established by previous work using FLD (for ergovaline and lolitrem B), and LC-MS (for peramine). This work provides the first highly sensitive quantitative LC-MS method for the accurate and reproducible quantitation of important endophyte-derived alkaloids.

Analysis of the Indole Diterpene Gene Cluster for Biosynthesis of the Epoxy-Janthitrems in Epichloë Endophytes.

Epoxy-janthitrems are a class of indole diterpenes with structural similarity to lolitrem B. Two taxa of asexual Epichloë endophytes have been reported to produce epoxy-janthitrems, LpTG-3 (Lolium perenne Taxonomic Group 3; e.g., NEA12) and LpTG-4 (e.g., E1). Epichloë epoxy-janthitrems are not well understood, the biosynthetic pathway and associated gene complement have not been described and while the literature suggests they are associated with superior protection against pasture insect pests and are tremorgenic in grazing mammals, these properties have not been confirmed using isolated and purified compounds. Whole genome sequence analysis was used to identify candidate genes for epoxy-janthitrem biosynthesis that are unique to epoxy-janthitrem producing strains of Epichloë. A gene, jtmD, was identified with homology to aromatic prenyl transferases involved in synthesis of indole diterpenes. The location of the epoxy-janthitrem biosynthesis gene cluster (JTM locus) was determined in the assembled nuclear genomes of NEA12 and E1. The JTM locus contains cluster 1 and cluster 2 of the lolitrem B biosynthesis gene cluster (LTM locus), as well as four genes jtmD, jtmO, jtm01, and jtm02 that are unique to Epichloë spp. that produce epoxy-janthitrems. Expression of each of the genes identified was confirmed using transcriptome analysis of perennial ryegrass-NEA12 and perennial ryegrass-E1 symbiota. Sequence analysis confirmed the genes are functionally similar to those involved in biosynthesis of related indole diterpene compounds. RNAi silencing of jtmD and in planta assessment in host-endophyte associations confirms the role of jtmD in epoxy-janthitrem production. Using LCMS/MS technologies, a biosynthetic pathway for the production of epoxy-janthitrems I-IV in Epichloë endophytes is proposed.

Generation of Epichloë Strains Expressing Fluorescent Proteins Suitable for Studying Host-Endophyte Interactions and Characterisation of a T-DNA Integration Event.

Methods for the identification and localisation of endophytic fungi are required to study the establishment, development, and progression of host-symbiont interactions, as visible reactions or disease symptoms are generally absent from host plants. Fluorescent proteins have proved valuable as reporter gene products, allowing non-invasive detection in living cells. This study reports the introduction of genes for two fluorescent proteins, green fluorescent protein (GFP) and red fluorescent protein, DsRed, into the genomes of two distinct perennial ryegrass (Lolium perenne L.)-associated Epichloë endophyte strains using A. tumefaciens-mediated transformation. Comprehensive characterisation of reporter gene-containing endophyte strains was performed using molecular genetic, phenotypic, and bioinformatic tools. A combination of long read and short read sequencing of a selected transformant identified a single complex T-DNA insert of 35,530 bp containing multiple T-DNAs linked together. This approach allowed for comprehensive characterisation of T-DNA integration to single-base resolution, while revealing the unanticipated nature of T-DNA integration in the transformant analysed. These reporter gene endophyte strains were able to establish and maintain stable symbiotum with the host. In addition, the same endophyte strain labelled with two different fluorescent proteins were able to cohabit the same plant. This knowledge can be used to provide the basis to develop strategies to gain new insights into the host-endophyte interaction through independent and simultaneous monitoring in planta throughout its life cycle in greater detail.

Genetic modification of asexual Epichloë endophytes with the perA gene for peramine biosynthesis.

Development of grass-endophyte associations with minimal or no detrimental effects in combination with beneficial characteristics is important for pastoral agriculture. The feasibility of enhancing production of an endophyte-derived beneficial alkaloid through introduction of an additional gene copy was assessed in a proof-of-concept study. Sexual and asexual Epichloë species that form symbiotic associations with cool-season grasses of the Poaceae sub-family Pooideae produce bioactive alkaloids that confer resistance to herbivory by a number of organisms. Of these, peramine is thought to be crucial for protection of perennial ryegrass (Lolium perenne L.) from the Argentinian stem weevil, an economically important exotic pest in New Zealand, contributing significantly to pasture persistence. A single gene (perA) has been identified as solely responsible for peramine biosynthesis and is distributed widely across Epichloë taxa. In the present study, a functional copy of the perA gene was introduced into three recipient endophyte genomes by Agrobacterium tumefaciens-mediated transformation. The target strains included some that do not produce peramine, and others containing different perA gene copies. Mitotically stable transformants generated from all three endophyte strains were able to produce peramine in culture and in planta at variable levels. In summary, this study provides an insight into the potential for artificial combinations of alkaloid biosynthesis in a single endophyte strain through transgenesis, as well as the possibility of using novel genome editing techniques to edit the perA gene of non-peramine producing strains.

miR-9a mediates the role of Lethal giant larvae as an epithelial growth inhibitor in Drosophila.

Drosophila lethal giant larvae (lgl) encodes a conserved tumor suppressor with established roles in cell polarity, asymmetric division, and proliferation control. Lgl's human orthologs, HUGL1 and HUGL2, are altered in human cancers, however, its mechanistic role as a tumor suppressor remains poorly understood. Based on a previously established connection between Lgl and Fragile X protein (FMRP), a miRNA-associated translational regulator, we hypothesized that Lgl may exert its role as a tumor suppressor by interacting with the miRNA pathway. Consistent with this model, we found that lgl is a dominant modifier of Argonaute1 overexpression in the eye neuroepithelium. Using microarray profiling we identified a core set of ten miRNAs that are altered throughout tumorigenesis in Drosophila lgl mutants. Among these are several miRNAs previously linked to human cancers including miR-9a, which we found to be downregulated in lgl neuroepithelial tissues. To determine whether miR-9a can act as an effector of Lgl in vivo, we overexpressed it in the context of lgl knock-down by RNAi and found it able to reduce the overgrowth phenotype caused by Lgl loss in epithelia. Furthermore, cross-comparisons between miRNA and mRNA profiling in lgl mutant tissues and human breast cancer cells identified thrombospondin (tsp) as a common factor altered in both fly and human breast cancer tumorigenesis models. Our work provides the first evidence of a functional connection between Lgl and the miRNA pathway, demonstrates that miR-9a mediates Lgl's role in restricting epithelial proliferation, and provides novel insights into pathways controlled by Lgl during tumor progression.

Genomic and metabolic characterisation of alkaloid biosynthesis by asexual Epichloë fungal endophytes of tall fescue pasture grasses.

Symbiotic associations between tall fescue grasses and asexual Epichloë fungal endophytes exhibit biosynthesis of alkaloid compounds causing both beneficial and detrimental effects. Candidate novel endophytes with favourable chemotypic profiles have been identified in germplasm collections by screening for genetic diversity, followed by metabolite profile analysis in endogenous genetic backgrounds. A subset of candidates was subjected to genome survey sequencing to detect the presence or absence and structural status of known genes for biosynthesis of the major alkaloid classes. The capacity to produce specific metabolites was directly predictable from metabolic data. In addition, study of duplicated gene structure in heteroploid genomic constitutions provided further evidence for the origin of such endophytes. Selected strains were inoculated into meristem-derived callus cultures from specific tall fescue genotypes to perform isogenic comparisons of alkaloid profile in different host backgrounds, revealing evidence for host-specific quantitative control of metabolite production, consistent with previous studies. Certain strains were capable of both inoculation and formation of longer-term associations with a nonhost species, perennial ryegrass (Lolium perenne L.). Discovery and primary characterisation of novel endophytes by DNA analysis, followed by confirmatory metabolic studies, offers improvements of speed and efficiency and hence accelerated deployment in pasture grass improvement programs.

A Review of Perennial Ryegrass Endophytes and Their Potential Use in the Management of African Black Beetle in Perennial Grazing Systems in Australia.

The major insect pest of Australian cool temperate pastures is the root-feeding insect Heteronychus arator (African black beetle, ABB). Significant pasture damage can occur even at low ABB densities (11 individuals per square meter), and often re-sowing of the whole paddock is required. Mitigation of the effects of pasture pests, and in particular subterranean species such as the larval form of ABB, can be challenging. Early detection is limited by the ability to visualize above-ground symptoms, and chemical control of insects in soil is often ineffective. This review takes a look at the historical events that molded the pastoral landscape in Australia. The importation route, changes in land management and pasture composition by European settlers may have aided the establishment of ABB in Australia. Perennial ryegrass Lolium perenne is discussed as it is one of the most important perennial agricultural grasses and is widely-sown in moderate-to-high-rainfall temperate zones of the world. Endophytic fungi from the genus Epichloë form symbiotic relationships with cool season grasses such as Lolium perenne (perennial ryegrass). They have been studied extensively and are well documented for enhancing persistence in pasture via a suite of bioactive secondary metabolites produced by the fungal symbionts. Several well-characterized secondary metabolites are discussed. Some can have negative effects on cattle (e.g., ergovaline and lolitrems) while others have been shown to benefit the host plant through deterrence of insect pests from feeding and by insecticidal activity (e.g., peramine, lolines, ergopeptines). Various control methods for ABB are also discussed, with a focus on the potential role of asexual Epichloë endophytes.

Phylogenomics of asexual Epichloë fungal endophytes forming associations with perennial ryegrass.

BACKGROUND: Perennial ryegrass (Lolium perenne L.) is one of the most important species for temperate pastoral agriculture, forming associations with genetically diverse groups of mutualistic fungal endophytes. However, only two taxonomic groups (E. festucae var. lolii and LpTG-2) have so far been described. In addition to these two well-characterised taxa, a third distinct group of previously unclassified perennial ryegrass-associated endophytes was identified as belonging to a putative novel taxon (or taxa) (PNT) in a previous analysis based on simple sequence repeat (SSR) marker diversity. As well as genotypic differences, distinctive alkaloid production profiles were observed for members of the PNT group. RESULTS: A detailed phylogenetic analysis of perennial ryegrass-associated endophytes using components of whole genome sequence data was performed using complete sequences of 7 nuclear protein-encoding genes. Three independently selected genes (encoding a DEAD/DEAH box helicase [Sbp4], a glycosyl hydrolase [family 92 protein] and a MEAB protein), none of which have been previously used for taxonomic studies of endophytes, were selected together with the frequently used 'house-keeping' genes tefA and tubB (encoding translation elongation factor 1-α and ß-tubulin, respectively). In addition, an endophyte-specific gene (perA for peramine biosynthesis) and the fungal-specific MT genes for mating-type control were included. The results supported previous phylogenomic inferences for the known species, but revealed distinctive patterns of diversity for the previously unclassified endophyte strains, which were further proposed to belong to not one but two distinct novel taxa. Potential progenitor genomes for the asexual endophytes among contemporary teleomorphic (sexual Epichloë) species were also identified from the phylogenetic analysis. CONCLUSIONS: Unique taxonomic status for the PNT was confirmed through comparison of multiple nuclear gene sequences, and also supported by evidence from chemotypic diversity. Analysis of MT gene idiomorphs further supported a predicted independent origin of two distinct perennial ryegrass-associated novel taxa, designated LpTG-3 and LpTG-4, from different members of a similar founder population related to contemporary E. festucae. The analysis also provided higher resolution to the known progenitor contributions of previously characterised perennial ryegrass-associated endophyte taxa.

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.

High throughput whole rumen metagenome profiling using untargeted massively parallel sequencing.

BACKGROUND: Variation of microorganism communities in the rumen of cattle (Bos taurus) is of great interest because of possible links to economically or environmentally important traits, such as feed conversion efficiency or methane emission levels. The resolution of studies investigating this variation may be improved by utilizing untargeted massively parallel sequencing (MPS), that is, sequencing without targeted amplification of genes. The objective of this study was to develop a method which used MPS to generate "rumen metagenome profiles", and to investigate if these profiles were repeatable among samples taken from the same cow. Given faecal samples are much easier to obtain than rumen fluid samples; we also investigated whether rumen metagenome profiles were predictive of faecal metagenome profiles. RESULTS: Rather than focusing on individual organisms within the rumen, our method used MPS data to generate quantitative rumen micro-biome profiles, regardless of taxonomic classifications. The method requires a previously assembled reference metagenome. A number of such reference metagenomes were considered, including two rumen derived metagenomes, a human faecal microflora metagenome and a reference metagenome made up of publically available prokaryote sequences. Sequence reads from each test sample were aligned to these references. The "rumen metagenome profile" was generated from the number of the reads that aligned to each contig in the database. We used this method to test the hypothesis that rumen fluid microbial community profiles vary more between cows than within multiple samples from the same cow. Rumen fluid samples were taken from three cows, at three locations within the rumen. DNA from the samples was sequenced on the Illumina GAIIx. When the reads were aligned to a rumen metagenome reference, the rumen metagenome profiles were repeatable (P < 0.00001) by cow regardless of location of sampling rumen fluid. The repeatability was estimated at 9%, albeit with a high standard error, reflecting the small number of animals in the study. Finally, we compared rumen microbial profiles to faecal microbial profiles. Our hypothesis, that there would be a stronger correlation between faeces and rumen fluid from the same cow than between faeces and rumen fluid from different cows, was not supported by our data (with much greater significance of rumen versus faeces effect than animal effect in mixed linear model). CONCLUSIONS: We have presented a simple and high throughput method of metagenome profiling to assess the similarity of whole metagenomes, and illustrated its use on two novel datasets. This method utilises widely used freeware. The method should be useful in the exploration and comparison of metagenomes.

Sequence Analysis of SSR-Flanking Regions Identifies Genome Affinities between Pasture Grass Fungal Endophyte Taxa.

Fungal species of the Neotyphodium and Epichloë genera are endophytes of pasture grasses showing complex differences of life-cycle and genetic architecture. Simple sequence repeat (SSR) markers have been developed from endophyte-derived expressed sequence tag (EST) collections. Although SSR array size polymorphisms are appropriate for phenetic analysis to distinguish between taxa, the capacity to resolve phylogenetic relationships is limited by both homoplasy and heteroploidy effects. In contrast, nonrepetitive sequence regions that flank SSRs have been effectively implemented in this study to demonstrate a common evolutionary origin of grass fungal endophytes. Consistent patterns of relationships between specific taxa were apparent across multiple target loci, confirming previous studies of genome evolution based on variation of individual genes. Evidence was obtained for the definition of endophyte taxa not only through genomic affinities but also by relative gene content. Results were compatible with the current view that some asexual Neotyphodium species arose following interspecific hybridisation between sexual Epichloë ancestors. Phylogenetic analysis of SSR-flanking regions, in combination with the results of previous studies with other EST-derived SSR markers, further permitted characterisation of Neotyphodium isolates that could not be assigned to known taxa on the basis of morphological characteristics.