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Kathryn Guthridge
Agriculture Victoria, AgriBio, Centre for AgriBioscience, Bundoora, VIC 3083, Australia

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Journal article
Published: 04 May 2021 in Microorganisms
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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.

ACS Style

Inoka Hettiarachchige; Christy Vander Jagt; Ross Mann; Timothy Sawbridge; German Spangenberg; Kathryn Guthridge. Global Changes in Asexual Epichloë Transcriptomes during the Early Stages, from Seed to Seedling, of Symbiotum Establishment. Microorganisms 2021, 9, 991 .

AMA Style

Inoka Hettiarachchige, Christy Vander Jagt, Ross Mann, Timothy Sawbridge, German Spangenberg, Kathryn Guthridge. Global Changes in Asexual Epichloë Transcriptomes during the Early Stages, from Seed to Seedling, of Symbiotum Establishment. Microorganisms. 2021; 9 (5):991.

Chicago/Turabian Style

Inoka Hettiarachchige; Christy Vander Jagt; Ross Mann; Timothy Sawbridge; German Spangenberg; Kathryn Guthridge. 2021. "Global Changes in Asexual Epichloë Transcriptomes during the Early Stages, from Seed to Seedling, of Symbiotum Establishment." Microorganisms 9, no. 5: 991.

Journal article
Published: 24 June 2020 in Microorganisms
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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.

ACS Style

Krishni Fernando; Priyanka Reddy; Inoka K. Hettiarachchige; German C. Spangenberg; Simone J. Rochfort; Kathryn M. Guthridge. Novel Antifungal Activity of Lolium-Associated Epichloë Endophytes. Microorganisms 2020, 8, 955 .

AMA Style

Krishni Fernando, Priyanka Reddy, Inoka K. Hettiarachchige, German C. Spangenberg, Simone J. Rochfort, Kathryn M. Guthridge. Novel Antifungal Activity of Lolium-Associated Epichloë Endophytes. Microorganisms. 2020; 8 (6):955.

Chicago/Turabian Style

Krishni Fernando; Priyanka Reddy; Inoka K. Hettiarachchige; German C. Spangenberg; Simone J. Rochfort; Kathryn M. Guthridge. 2020. "Novel Antifungal Activity of Lolium-Associated Epichloë Endophytes." Microorganisms 8, no. 6: 955.

Journal article
Published: 16 January 2020 in Molecules
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Lolitrem B is the most potent indole-diterpene mycotoxin produced by Epichloë festucae var. lolii (termed LpTG-1), with severe intoxication cases reported in livestock. To date, there are no in vivo metabolism studies conducted for the mycotoxin. A mouse model assay established for assessing toxicity of indole-diterpenes was used to investigate metabolic products of lolitrem B. Mice were administered lolitrem B at 0.5 and 2.0 mg/kg body weight (b.wt) intraperitoneally before body and brain tissues were collected at 6 h and 24 h post-treatment. Samples were cryoground and subjected to a biphasic or monophasic extraction. The aqueous and lipophilic phases were analysed using liquid chromatography high-resolution mass spectrometry (LC–HRMS); data analysis was performed with Compound Discoverer™ software. A total of 10 novel phase I metabolic products were identified in the lipophilic phase and their distribution in the liver, kidney and various brain regions are described. The biotransformation products of lolitrem B were found to be present in low levels in the brain. Based on structure–activity postulations, six of these may contribute towards the protracted tremors exhibited by lolitrem B-exposed animals.

ACS Style

Priyanka Reddy; Aaron Elkins; Joanne Hemsworth; Kathryn Guthridge; Simone Vassiliadis; Elizabeth Read; German Spangenberg; Simone Rochfort. Identification and Distribution of Novel Metabolites of Lolitrem B in Mice by High-Resolution Mass Spectrometry. Molecules 2020, 25, 372 .

AMA Style

Priyanka Reddy, Aaron Elkins, Joanne Hemsworth, Kathryn Guthridge, Simone Vassiliadis, Elizabeth Read, German Spangenberg, Simone Rochfort. Identification and Distribution of Novel Metabolites of Lolitrem B in Mice by High-Resolution Mass Spectrometry. Molecules. 2020; 25 (2):372.

Chicago/Turabian Style

Priyanka Reddy; Aaron Elkins; Joanne Hemsworth; Kathryn Guthridge; Simone Vassiliadis; Elizabeth Read; German Spangenberg; Simone Rochfort. 2020. "Identification and Distribution of Novel Metabolites of Lolitrem B in Mice by High-Resolution Mass Spectrometry." Molecules 25, no. 2: 372.

Journal article
Published: 27 December 2019 in Microorganisms
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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.

ACS Style

Inoka K. Hettiarachchige; Emma J. Ludlow; Piyumi N. Ekanayake; NaTasha D. Brohier; Sareena Sahab; Timothy I. Sawbridge; German C. Spangenberg; Kathryn M. Guthridge. Generation of Epichloë Strains Expressing Fluorescent Proteins Suitable for Studying Host-Endophyte Interactions and Characterisation of a T-DNA Integration Event. Microorganisms 2019, 8, 54 .

AMA Style

Inoka K. Hettiarachchige, Emma J. Ludlow, Piyumi N. Ekanayake, NaTasha D. Brohier, Sareena Sahab, Timothy I. Sawbridge, German C. Spangenberg, Kathryn M. Guthridge. Generation of Epichloë Strains Expressing Fluorescent Proteins Suitable for Studying Host-Endophyte Interactions and Characterisation of a T-DNA Integration Event. Microorganisms. 2019; 8 (1):54.

Chicago/Turabian Style

Inoka K. Hettiarachchige; Emma J. Ludlow; Piyumi N. Ekanayake; NaTasha D. Brohier; Sareena Sahab; Timothy I. Sawbridge; German C. Spangenberg; Kathryn M. Guthridge. 2019. "Generation of Epichloë Strains Expressing Fluorescent Proteins Suitable for Studying Host-Endophyte Interactions and Characterisation of a T-DNA Integration Event." Microorganisms 8, no. 1: 54.

Journal article
Published: 13 November 2019 in Microorganisms
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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.

ACS Style

Emma J. Ludlow; Simone Vassiliadis; Piyumi N. Ekanayake; Inoka K. Hettiarachchige; Priyanka Reddy; Tim I. Sawbridge; Simone J. Rochfort; German C. Spangenberg; Kathryn M. Guthridge. Analysis of the Indole Diterpene Gene Cluster for Biosynthesis of the Epoxy-Janthitrems in Epichloë Endophytes. Microorganisms 2019, 7, 560 .

AMA Style

Emma J. Ludlow, Simone Vassiliadis, Piyumi N. Ekanayake, Inoka K. Hettiarachchige, Priyanka Reddy, Tim I. Sawbridge, Simone J. Rochfort, German C. Spangenberg, Kathryn M. Guthridge. Analysis of the Indole Diterpene Gene Cluster for Biosynthesis of the Epoxy-Janthitrems in Epichloë Endophytes. Microorganisms. 2019; 7 (11):560.

Chicago/Turabian Style

Emma J. Ludlow; Simone Vassiliadis; Piyumi N. Ekanayake; Inoka K. Hettiarachchige; Priyanka Reddy; Tim I. Sawbridge; Simone J. Rochfort; German C. Spangenberg; Kathryn M. Guthridge. 2019. "Analysis of the Indole Diterpene Gene Cluster for Biosynthesis of the Epoxy-Janthitrems in Epichloë Endophytes." Microorganisms 7, no. 11: 560.

Journal article
Published: 07 November 2019 in Toxins
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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.

ACS Style

Simone Vassiliadis; Aaron C. Elkins; Priyanka Reddy; Kathryn M. Guthridge; German C. Spangenberg; Simone J. Rochfort. A Simple LC–MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass. Toxins 2019, 11, 649 .

AMA Style

Simone Vassiliadis, Aaron C. Elkins, Priyanka Reddy, Kathryn M. Guthridge, German C. Spangenberg, Simone J. Rochfort. A Simple LC–MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass. Toxins. 2019; 11 (11):649.

Chicago/Turabian Style

Simone Vassiliadis; Aaron C. Elkins; Priyanka Reddy; Kathryn M. Guthridge; German C. Spangenberg; Simone J. Rochfort. 2019. "A Simple LC–MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass." Toxins 11, no. 11: 649.

Review
Published: 27 May 2019 in Toxins
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Indole-diterpenes are an important class of chemical compounds which can be unique to different fungal species. The highly complex lolitrem compounds are confined to Epichloë species, whilst penitrem production is confined to Penicillium spp. and Aspergillus spp. These fungal species are often present in association with pasture grasses, and the indole-diterpenes produced may cause toxicity in grazing animals. In this review, we highlight the unique structural variations of indole-diterpenes that are characterised into subgroups, including paspaline, paxilline, shearinines, paspalitrems, terpendoles, penitrems, lolitrems, janthitrems, and sulpinines. A detailed description of the unique biological activities has been documented where even structurally related compounds have displayed unique biological activities. Indole-diterpene production has been reported in two classes of ascomycete fungi, namely Eurotiomycetes (e.g., Aspergillus and Penicillium) and Sordariomycetes (e.g., Claviceps and Epichloë). These compounds all have a common structural core comprised of a cyclic diterpene skeleton derived from geranylgeranyl diphosphate (GGPP) and an indole moiety derived from tryptophan. Structure diversity is generated from the enzymatic conversion of different sites on the basic indole-diterpene structure. This review highlights the wide-ranging biological versatility presented by the indole-diterpene group of compounds and their role in an agricultural and pharmaceutical setting.

ACS Style

Priyanka Reddy; Kathryn Guthridge; Simone Vassiliadis; Joanne Hemsworth; Inoka Hettiarachchige; German Spangenberg; Simone Rochfort. Tremorgenic Mycotoxins: Structure Diversity and Biological Activity. Toxins 2019, 11, 302 .

AMA Style

Priyanka Reddy, Kathryn Guthridge, Simone Vassiliadis, Joanne Hemsworth, Inoka Hettiarachchige, German Spangenberg, Simone Rochfort. Tremorgenic Mycotoxins: Structure Diversity and Biological Activity. Toxins. 2019; 11 (5):302.

Chicago/Turabian Style

Priyanka Reddy; Kathryn Guthridge; Simone Vassiliadis; Joanne Hemsworth; Inoka Hettiarachchige; German Spangenberg; Simone Rochfort. 2019. "Tremorgenic Mycotoxins: Structure Diversity and Biological Activity." Toxins 11, no. 5: 302.

Journal article
Published: 03 January 2019 in Toxins
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The most potent of the indole diterpenes, lolitrem B, is found in perennial ryegrass (Lolium perenne L.) infected with the endophyte Epichloë festucae var. lolii (also termed LpTG-1). Ingestion causes a neurological syndrome in grazing livestock called ryegrass staggers disease. To enable the rapid development of new forage varieties, the toxicity of lolitrem B and its biosynthetic intermediates needs to be established. However, most of these indole diterpenes are not commercially available; thus, isolation of these compounds is paramount. A concentrated endophyte-infected perennial ryegrass seed extract was subjected to silica flash chromatography followed by preparative HPLC and purification by crystallization resulting in lolitrem B and the intermediate compounds lolitrem E, paspaline and terpendole B. The four-step isolation and purification method resulted in a 25% yield of lolitrem B. After isolation, lolitrem B readily degraded to its biosynthetic intermediate, lolitriol. We also found that lolitrem B can readily degrade depending on the solvent and storage conditions. The facile method which takes into consideration the associated instability of lolitrem B, led to the purification of indole diterpenes in quantities sufficient for use as analytical standards for identification in pastures, and/or for toxicity testing in pasture development programs.

ACS Style

Priyanka Reddy; Myrna A. Deseo; Vilnis Ezernieks; Kathryn Guthridge; German Spangenberg; Simone Rochfort. Toxic Indole Diterpenes from Endophyte-Infected Perennial Ryegrass Lolium perenne L.: Isolation and Stability. Toxins 2019, 11, 16 .

AMA Style

Priyanka Reddy, Myrna A. Deseo, Vilnis Ezernieks, Kathryn Guthridge, German Spangenberg, Simone Rochfort. Toxic Indole Diterpenes from Endophyte-Infected Perennial Ryegrass Lolium perenne L.: Isolation and Stability. Toxins. 2019; 11 (1):16.

Chicago/Turabian Style

Priyanka Reddy; Myrna A. Deseo; Vilnis Ezernieks; Kathryn Guthridge; German Spangenberg; Simone Rochfort. 2019. "Toxic Indole Diterpenes from Endophyte-Infected Perennial Ryegrass Lolium perenne L.: Isolation and Stability." Toxins 11, no. 1: 16.

Original article
Published: 15 November 2018 in Molecular Genetics and Genomics
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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.

ACS Style

Inoka K. Hettiarachchige; Aaron C. Elkins; Priyanka Reddy; Ross C. Mann; Kathryn M. Guthridge; Timothy I. Sawbridge; John W. Forster; German C. Spangenberg. Genetic modification of asexual Epichloë endophytes with the perA gene for peramine biosynthesis. Molecular Genetics and Genomics 2018, 294, 315 -328.

AMA Style

Inoka K. Hettiarachchige, Aaron C. Elkins, Priyanka Reddy, Ross C. Mann, Kathryn M. Guthridge, Timothy I. Sawbridge, John W. Forster, German C. Spangenberg. Genetic modification of asexual Epichloë endophytes with the perA gene for peramine biosynthesis. Molecular Genetics and Genomics. 2018; 294 (2):315-328.

Chicago/Turabian Style

Inoka K. Hettiarachchige; Aaron C. Elkins; Priyanka Reddy; Ross C. Mann; Kathryn M. Guthridge; Timothy I. Sawbridge; John W. Forster; German C. Spangenberg. 2018. "Genetic modification of asexual Epichloë endophytes with the perA gene for peramine biosynthesis." Molecular Genetics and Genomics 294, no. 2: 315-328.

Journal article
Published: 01 June 2017 in Genome
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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.

ACS Style

Piyumi N. Ekanayake; Jatinder Kaur; Pei Tian; Simone J. Rochfort; Kathryn M. Guthridge; Timothy I. Sawbridge; German C. Spangenberg; John W. Forster. Genomic and metabolic characterisation of alkaloid biosynthesis by asexual Epichloë fungal endophytes of tall fescue pasture grasses. Genome 2017, 60, 496 -509.

AMA Style

Piyumi N. Ekanayake, Jatinder Kaur, Pei Tian, Simone J. Rochfort, Kathryn M. Guthridge, Timothy I. Sawbridge, German C. Spangenberg, John W. Forster. Genomic and metabolic characterisation of alkaloid biosynthesis by asexual Epichloë fungal endophytes of tall fescue pasture grasses. Genome. 2017; 60 (6):496-509.

Chicago/Turabian Style

Piyumi N. Ekanayake; Jatinder Kaur; Pei Tian; Simone J. Rochfort; Kathryn M. Guthridge; Timothy I. Sawbridge; German C. Spangenberg; John W. Forster. 2017. "Genomic and metabolic characterisation of alkaloid biosynthesis by asexual Epichloë fungal endophytes of tall fescue pasture grasses." Genome 60, no. 6: 496-509.

Journal article
Published: 24 April 2015 in BMC Evolutionary Biology
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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. 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 β-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. 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.

ACS Style

Inoka K Hettiarachchige; Piyumi N Ekanayake; Ross C Mann; Kathryn M Guthridge; Timothy I Sawbridge; German C Spangenberg; John W Forster. Phylogenomics of asexual Epichloë fungal endophytes forming associations with perennial ryegrass. BMC Evolutionary Biology 2015, 15, 1 -14.

AMA Style

Inoka K Hettiarachchige, Piyumi N Ekanayake, Ross C Mann, Kathryn M Guthridge, Timothy I Sawbridge, German C Spangenberg, John W Forster. Phylogenomics of asexual Epichloë fungal endophytes forming associations with perennial ryegrass. BMC Evolutionary Biology. 2015; 15 (1):1-14.

Chicago/Turabian Style

Inoka K Hettiarachchige; Piyumi N Ekanayake; Ross C Mann; Kathryn M Guthridge; Timothy I Sawbridge; German C Spangenberg; John W Forster. 2015. "Phylogenomics of asexual Epichloë fungal endophytes forming associations with perennial ryegrass." BMC Evolutionary Biology 15, no. 1: 1-14.

Journal article
Published: 01 January 2015 in Crop and Pasture Science
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Asexual fungal endophytes of the genus Epichloë form mutually beneficial associations with cool-season pasture grasses such as ryegrasses (Lolium spp.). Alkaloid production by the fungus confers both beneficial (deterrence of invertebrate herbivory) and detrimental (toxicity to mammalian livestock) attributes. A few novel strains with desirable metabolite profiles have been advanced into commercial production by inoculation of perennial ryegrass cultivars. In the present study, an integrated process for discovery of novel endophytes based on exploitation of genotypic information has been designed and implemented. A survey of genetic diversity was performed on a large-scale, customised germplasm collection (containing 244 accessions) in order to identify previously uncharacterised endophyte genotypes. Preliminary qualitative metabolic profiling in the endogenous genetic background permitted elimination of undesirable combinations, and definition of a subset of priority candidates. A novel method was developed for inoculation of endophytes into meristem-culture-derived callus tissue of single genotypes from multiple perennial ryegrass cultivars, in order to allow isogenic comparisons with respect to both host and endophyte genotype. Beneficial toxin profiles were confirmed for associations formed with the grass genotypic panel, and semi-quantitative metabolite analysis provided evidence for genotype-specific effects of both host and genotype on levels of alkaloid production. Vegetative stability was also assessed over both shorter and longer terms. A final set of three prioritised candidates was obtained, two of which (belonging to the known taxa E. festucae var. lolii and LpTG-2) produce the alkaloids ergovaline and peramine. The third candidate endophyte, belonging to a putative novel taxon, solely produces representatives of an additional alkaloid class, the epoxy-janthitrems.

ACS Style

Jatinder Kaur; Piyumi N. Ekanayake; Pei Tian; Eline Van Zijll De Jong; Mark Dobrowolski; Simone J. Rochfort; Ross C. Mann; Kevin F. Smith; John W. Forster; Kathryn M. Guthridge; German C. Spangenberg. Discovery and characterisation of novel asexual Epichloë endophytes from perennial ryegrass (Lolium perenne L.). Crop and Pasture Science 2015, 66, 1058 -1070.

AMA Style

Jatinder Kaur, Piyumi N. Ekanayake, Pei Tian, Eline Van Zijll De Jong, Mark Dobrowolski, Simone J. Rochfort, Ross C. Mann, Kevin F. Smith, John W. Forster, Kathryn M. Guthridge, German C. Spangenberg. Discovery and characterisation of novel asexual Epichloë endophytes from perennial ryegrass (Lolium perenne L.). Crop and Pasture Science. 2015; 66 (10):1058-1070.

Chicago/Turabian Style

Jatinder Kaur; Piyumi N. Ekanayake; Pei Tian; Eline Van Zijll De Jong; Mark Dobrowolski; Simone J. Rochfort; Ross C. Mann; Kevin F. Smith; John W. Forster; Kathryn M. Guthridge; German C. Spangenberg. 2015. "Discovery and characterisation of novel asexual Epichloë endophytes from perennial ryegrass (Lolium perenne L.)." Crop and Pasture Science 66, no. 10: 1058-1070.

Journal article
Published: 01 September 2012 in Crop Science
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ACS Style

Piyumi N. Ekanayake; Melanie L. Hand; German C. Spangenberg; John W. Forster; Kathryn M. Guthridge. Genetic Diversity and Host Specificity of Fungal Endophyte Taxa in Fescue Pasture Grasses. Crop Science 2012, 52, 2243 -2252.

AMA Style

Piyumi N. Ekanayake, Melanie L. Hand, German C. Spangenberg, John W. Forster, Kathryn M. Guthridge. Genetic Diversity and Host Specificity of Fungal Endophyte Taxa in Fescue Pasture Grasses. Crop Science. 2012; 52 (5):2243-2252.

Chicago/Turabian Style

Piyumi N. Ekanayake; Melanie L. Hand; German C. Spangenberg; John W. Forster; Kathryn M. Guthridge. 2012. "Genetic Diversity and Host Specificity of Fungal Endophyte Taxa in Fescue Pasture Grasses." Crop Science 52, no. 5: 2243-2252.