Non-Transgenic CRISPR-Mediated Knockout of Entire Ergot Alkaloid Gene Clusters in Slow-Growing Asexual Polyploid Fungi.

The Epichloë species of fungi include seed-borne symbionts (endophytes) of cool-season grasses that enhance plant fitness, although some also produce alkaloids that are toxic to livestock. Selected or mutated toxin-free endophytes can be introduced into forage cultivars for improved livestock performance. Long-read genome sequencing revealed clusters of ergot alkaloid biosynthesis (EAS) genes in Epichloë coenophiala strain e19 from tall fescue (Lolium arundinaceum) and Epichloë hybrida Lp1 from perennial ryegrass (Lolium perenne). The two homeologous clusters in E. coenophiala-a triploid hybrid species-were 196 kb (EAS1) and 75 kb (EAS2), and the E. hybrida EAS cluster was 83 kb. As a CRISPR-based approach to target these clusters, the fungi were transformed with ribonucleoprotein (RNP) complexes of modified Cas9 nuclease (Cas9-2NLS) and pairs of single guide RNAs (sgRNAs), plus a transiently selected plasmid. In E. coenophiala, the procedure generated deletions of EAS1 and EAS2 separately, as well as both clusters simultaneously. The technique also gave deletions of the EAS cluster in E. hybrida and of individual alkaloid biosynthesis genes (dmaW and lolC) that had previously proved difficult to delete in E. coenophiala. Thus, this facile CRISPR RNP approach readily generates non-transgenic endophytes without toxin genes for use in research and forage cultivar improvement.

Phosphatidic acid produced by phospholipase D is required for hyphal cell-cell fusion and fungal-plant symbiosis.

Although lipid signaling has been shown to serve crucial roles in mammals and plants, little is known about this process in filamentous fungi. Here we analyze the contribution of phospholipase D (PLD) and its product phosphatidic acid (PA) in hyphal morphogenesis and growth of Epichloë festucae and Neurospora crassa, and in the establishment of a symbiotic interaction between E. festucae and Lolium perenne. Growth of E. festucae and N. crassa PLD deletion strains in axenic culture, and for E. festucae in association with L. perenne, were analyzed by light-, confocal- and electron microscopy. Changes in PA distribution were analyzed in E. festucae using a PA biosensor and the impact of these changes on the endocytic recycling and superoxide production investigated. We found that E. festucae PldB, and the N. crassa ortholog, PLA-7, are required for polarized growth and cell fusion and contribute to ascospore development, whereas PldA/PLA-8 are dispensable for these functions. Exogenous addition of PA rescues the cell-fusion phenotype in E. festucae. PldB is also crucial for E. festucae to establish a symbiotic association with L. perenne. This study identifies a new component of the cell-cell communication and cell fusion signaling network for hyphal morphogenesis and growth of filamentous fungi.

Physiological consequences of gamma ray irradiation in tall fescue with elimination potential of Epichloë fungal endophyte.

Perennial plants and their associated microorganisms grow in the areas that may be contaminated with long-lived gamma-emitting radionuclides. This will induce gamma stress response in plants and their accompanying microorganisms. The present work investigated the growth and physiological responses of Epichloe endophyte infected tall fescue to gamma radiation, as well as whether the endophyte could persist and infect the host plant once exposed to gamma radiation. Seeds of Iranian native genotype of 75B+ of tall fescue were exposed to different doses, including 5.0, 10.0, 15.0, 20.0, 30.0 and 40.0 krad of gamma ray from a 60Co source. Irradiated and unirradiated seeds were sown in pots and grown under controlled conditions in the greenhouse. The growth and physiological parameters associated with plant tolerance to oxidative stress of host plants, as well as endophytic infection frequency (% of plants infected) and intensity (mean number of endophytic hyphae per the field of view), were examined in 3 months-old seedlings. The results indicated that all gamma radiation doses (except 5.0 kr) significantly reduced the height and survival percentage of the host plant. Days to the emergence of seedling increased gradually as gamma doses rose. A dose-rate dependent induction was seen for photosynthetic pigments and proline content. Malondialdehyde (MDA) content grew with elevation of irradiation doses. Depending on the dose and time, the activities of antioxidant enzymes in the host plant responded differently to gamma radiation. Gamma radiation altered the enzyme activities with sever decline in SOD and CAT activities. However, it had barely any effect on in APX and POD activities. The results also revealed that the persistence and intensity of endophyte were affected after gamma-ray irradiation. The initial percentage of tall fescue seeds infected with the endophyte was 91% in un-irradiated seeds. Presence of the viable endophyte started to decline significantly (23%) at 5.0 kr of gamma radiation. A dramatic reduction in the presence and intensity of endophyte occurred at 10.0 to 40.0 kr intensities. Gamma radiation × trait (GT)-biplot analysis indicated positive correlations between the endophyte symbiosis and antioxidant enzyme activities. Also, negative correlations were observed between the endophyte and MDA content in the host plant. Our results suggest that radiation stress (doses over 5.0 kr) caused reduction in the growth and antioxidant enzyme activities of the host plant that accompanied by a dramatic reduction in the persistence and intensity of endophyte fungi. Our findings have provided the basic information for future studies on the effect of gamma irradiation on the interaction between endophytic fungi and its host plant.

A homologue of the fungal tetraspanin Pls1 is required for Epichloë festucae expressorium formation and establishment of a mutualistic interaction with Lolium perenne.

Epichloë festucae is an endophytic fungus that forms a mutualistic symbiotic association with the grass host Lolium perenne. Endophytic hyphae exit the host by an appressorium-like structure known as an expressorium. In plant-pathogenic fungi, the tetraspanin Pls1 and the NADPH oxidase component Nox2 are required for appressorium development. Previously we showed that the homologue of Nox2, NoxB, is required for E. festucae expressorium development and establishment of a mutualistic symbiotic interaction with the grass host. Here we used a reverse genetics approach to functionally characterize the role of the E. festucae homologue of Pls1, PlsA. The morphology and growth of ΔplsA in axenic culture was comparable to wild-type. The tiller length of plants infected with ΔplsA was significantly reduced. Hyphae of ΔplsA had a proliferative pattern of growth within the leaves of L. perenne with increased colonization of the intercellular spaces and the vascular bundles. The ΔplsA mutant was also defective in expressorium development although the phenotype was not as severe as for ΔnoxB, highlighting potentially distinct roles for PlsA and NoxB in signalling through the NoxB complex. Hyphae of ΔplsA proliferate below the cuticle surface but still occasionally form an expressorium-like structure that enables the mutant hyphae to exit the leaf to grow on the surface. These expressoria still form a septin ring-like structure at the point of cuticle exit as found in the wild-type strain. These results establish that E. festucae PlsA has an important, but distinct, role to NoxB in expressorium development and plant symbiosis.

Complex epigenetic regulation of alkaloid biosynthesis and host interaction by heterochromatin protein I in a fungal endophyte-plant symbiosis.

Epichloë festucae forms mutualistic symbiotic interactions with grasses of the Lolium and Festuca genera. Protection from insect and mammalian herbivory are the best-documented host benefits of these associations. The two main classes of anti-mammalian alkaloids synthesized by E. festucae are the ergot alkaloids and indole diterpenes, of which ergovaline and lolitrems are the principal terminal products. Synthesis of both metabolites require multiple gene products encoded by clusters of 11 genes located at the subtelomeric regions of chromosomes I and III respectively. These loci are essentially unexpressed in axenic culture but among the most highly expressed genes in planta. We show here that heterochromatin 1 protein (HepA) is an important component of the regulatory machinery that maintains these loci in a silent state in culture. Deletion of this gene led to derepression of eas and ltm gene expression under non-symbiotic culture conditions. Although there was no obvious culture phenotype, RNAseq analysis revealed that around 1000 genes were differentially expressed in the ΔhepA mutant compared to wild type with just one-third upregulated. Inoculation of the ΔhepA mutants into seedlings of Lolium perenne led to a severe host interaction phenotype characterized by a reduction in tiller length but an increase in tiller number. Hyphae within the leaves of these associations were much more abundant in the intercellular spaces of the leaves and aberrantly colonized the vascular bundles. This physiological change was accompanied by a dramatic change in the transcriptome with around 900 genes differentially expressed, with two thirds of these upregulated. This major physiological change was accompanied by a decrease in ltm gene expression and loss of the ability to synthesize lolitrems. These results show that HepA has an important role in controlling the chromatin state of these sub-telomeric secondary metabolite genes, including their symbiosis-specific regulation.

Analysis of Epichloë festucae small secreted proteins in the interaction with Lolium perenne.

Epichloë festucae is an endophyte of the agriculturally important perennial ryegrass. This species systemically colonises the aerial tissues of this host where its growth is tightly regulated thereby maintaining a mutualistic symbiotic interaction. Recent studies have suggested that small secreted proteins, termed effectors, play a vital role in the suppression of host defence responses. To date only a few effectors with important roles in mutualistic interactions have been described. Here we make use of the fully assembled E. festucae genome and EffectorP to generate a suite of 141 effector candidates. These were analysed with respect to their genome location and expression profiles in planta and in several symbiosis-defective mutants. We found an association between effector candidates and a class of transposable elements known as MITEs, but no correlation with other dynamic features of the E. festucae genome, such as transposable element-rich regions. Three effector candidates and a small GPI-anchored protein were chosen for functional analysis based on their high expression in planta compared to in culture and their differential regulation in symbiosis defective E. festucae mutants. All three candidate effector proteins were shown to possess a functional signal peptide and two could be detected in the extracellular medium by western blotting. Localization of the effector candidates in planta suggests that they are not translocated into the plant cell, but rather, are localized in the apoplastic space or are attached to the cell wall. Deletion and overexpression of the effector candidates, as well as the putative GPI-anchored protein, did not affect the plant growth phenotype or restrict growth of E. festucae mutants in planta. These results indicate that these proteins are either not required for the interaction at the observed life stages or that there is redundancy between effectors expressed by E. festucae.

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.

Further investigation of equine fescue oedema induced by Mediterranean tall fescue (Lolium arundinaceum) infected with selected fungal endophytes (Epichloë coenophiala).

AIMS To determine if equine fescue oedema (EFO) induced by grazing Mediterranean-type tall fescue (Lolium arundinaceum) infected with selected endophytes (Epichloë coenophiala) could be prevented by treatment with the corticosteroid, methylprednisolone, and anti-histamine, cetirizine, and to determine concentrations of lolines, specifically N-acetyl norloline (NANL), in grasses grazed by horses that did and did not develop EFO. METHODS Four horses were grazed on AR542-infected Mediterranean tall fescue pasture (from Day 0) for 7 days prior to being subjected to euthanasia. Two of these horses were treated with 250 mg methylprednisolone and 300 mg cetirizine hydrochloride every 12 hours orally from Days 0-7. Two more horses grazed meadow fescue (Festuca pratensis) infected with the naturally-occurring, common endophyte (Epichloë uncinata) for 21 days before euthanasia. All horses were observed closely for signs of EFO, and blood samples were taken daily for measurement of concentrations of total protein (TP) in serum. Following euthanasia post-mortem examinations were conducted on all horses. Pasture samples of meadow fescue and Mediterranean tall fescue from the current study, and endophyte-infected Mediterranean tall fescue from a previous study that were associated with EFO, were analysed for concentrations of lolines using gas chromatography. RESULTS By Day 7, the treated and untreated horses grazing AR542-infected Mediterranean tall fescue all developed signs of EFO, and concentrations of TP in serum of all horses were <45 g/L. No signs of EFO were observed in horses grazing meadow fescue and concentrations of TP remained above 60 g/L. Necropsy showed marked oedema and eosinophilic inflammation in the intestines of all horses grazing Mediterranean tall fescue. In the sample of meadow fescue, concentrations of total lolines and N-acetyl norloline (NANL) were 2,402 and 543 mg/kg, respectively. In the three samples of Mediterranean tall fescue associated with EFO, concentrations of total lolines were 308, 629 and 679 mg/kg, and concentrations of NANL were 308, 614 and 305 mg/kg. CONCLUSIONS AND CLINICAL RELEVANCE In horses grazing Mediterranean tall fescue infected with the AR542 endophyte, treatment with methylprednisolone and cetirizine did not prevent development of EFO. Results of pasture analysis indicated that lolines or NANL are unlikely to be the causative agent of this disease.

Defining the pathways of symbiotic Epichloë colonization in grass embryos with confocal microscopy.

Asexual cool-season grass endophytes of the genus Epichloë (Ascomycota: Clavicipitaceae) are strictly vertically disseminated. The hosts of these mutualistic fungi express no symptoms during the fungal lifecycle that takes place entirely within the plant, while their hosts receive beneficial outcomes. These fungi are distributed in two major locations within the mature seeds of their hosts; namely, within the embryo (including the scutellum, coleoptile, plumule, radicle, and coleorhiza tissues) and between the aleurone and pericarp layers, with the latter hyphae playing no role in transmission of the fungus to the next plant generation. Conflicting evidence remains in the literature on the timing of embryo colonization. In a detailed investigation, utilizing confocal microscopy to observe the distribution of Epichloë coenophiala strain AR601 in tall fescue (Lolium arundinaceum), we tracked endophyte hyphal colonization in the ovary (pre-fertilization) through to the fully mature seed stage. Confocal microscopy images revealed that at the early and mature developmental stages of the embryo sac, before host grass fertilization, there were large quantities of endophyte mycelium present, especially around the antipodal cells, indicating that this endophyte enters the embryo sac before the fertilization stage. After host fertilization, fungal hyphae could be seen in the true embryo and early nonstarchy endosperm. Understanding the mechanisms of transmission to the seed is important for commercial seed producers and end users.

Leaf endophytic fungus interacts with precipitation to alter belowground microbial communities in primary successional dunes.

Understanding interactions between above- and belowground components of ecosystems is an important next step in community ecology. These interactions may be fundamental to predicting ecological responses to global change because indirect effects occurring through altered species interactions can outweigh or interact with the direct effects of environmental drivers. In a multiyear field experiment (2010-2015), we tested how experimental addition of a mutualistic leaf endophyte (Epichloë amarillans) associated with American beachgrass (Ammophila breviligulata) interacted with an altered precipitation regime (±30%) to affect the belowground microbial community. Epichloë addition increased host root biomass at the plot scale, but reduced the length of extraradical arbuscular mycorrhizal (AM) fungal hyphae in the soil. Under ambient precipitation alone, the addition of Epichloë increased root biomass per aboveground tiller and reduced the diversity of AM fungi in A. breviligulata roots. Furthermore, with Epichloë added, the diversity of root-associated bacteria declined with higher soil moisture, whereas in its absence, bacterial diversity increased with higher soil moisture. Thus, the aboveground fungal mutualist not only altered the abundance and composition of belowground microbial communities but also affected how belowground communities responded to climate, suggesting that aboveground microbes have potential for cascading influences on community dynamics and ecosystem processes that occur belowground.

Epichloë festucae endophytic growth in florets, seeds, and seedlings of perennial ryegrass (Lolium perenne).

Many symbiotic Epichloë species are seed-transmitted in their grass hosts. For a detailed investigation of Epichloë festucae colonization throughout the life cycle of its host, the authors transformed strain Fl1 with a fungal-active gene for enhanced cyan-fluorescent protein (eCFP), introduced it into perennial ryegrass (Lolium perenne), and used confocal microscopy to track its growth in the shoot apex, floral primordium, floral organs, seeds, and seedlings. Hyphae intercellularly colonized leaf sheaths, blades, true stems, and leaf primordia, and among floral primordia the endophyte exhibited different levels of colonization. In preanthesis florets, E. festucae colonized the pistil and stamen, but not pollen grains, and ramified throughout the ovule nucellus, but not the integument or embryo sac. Generally, only a single hypha was observed extended from the ovary placenta into the ovule. Within 4 d after anthesis, fungal hyphae had ramified throughout the developing seed and embryo. As the embryo matured, fungal hyphae became abundant between the testa and aleurone layer, and around the shoot apex and radical of the embryonic axis. During germination, hyphae accumulated in the mesocotyl and invaded the newly formed shoot apex near the meristem. In this host-fungus symbiosis, transmission to seedlings averaged 41% in 2010 and 76% in 2011. Each year, the frequency of ovary infection was similar to the frequency of infecting embryos and seedlings, indicating that colonization of the ovary and embryo was required for seed transmission.

Geographic Variation in Festuca rubra L. Ploidy Levels and Systemic Fungal Endophyte Frequencies.

Polyploidy and symbiotic Epichloë fungal endophytes are common and heritable characteristics that can facilitate environmental range expansion in grasses. Here we examined geographic patterns of polyploidy and the frequency of fungal endophyte colonized plants in 29 Festuca rubra L. populations from eight geographic sites across latitudes from Spain to northernmost Finland and Greenland. Ploidy seemed to be positively and negatively correlated with latitude and productivity, respectively. However, the correlations were nonlinear; 84% of the plants were hexaploids (2n = 6x = 42), and the positive correlation between ploidy level and latitude is the result of only four populations skewing the data. In the southernmost end of the gradient 86% of the plants were tetraploids (2n = 4x = 28), whereas in the northernmost end of the gradient one population had only octoploid plants (2n = 8x = 56). Endophytes were detected in 22 out of the 29 populations. Endophyte frequencies varied among geographic sites, and populations and habitats within geographic sites irrespective of ploidy, latitude or productivity. The highest overall endophyte frequencies were found in the southernmost end of the gradient, Spain, where 69% of plants harbored endophytes. In northern Finland, endophytes were detected in 30% of grasses but endophyte frequencies varied among populations from 0% to 75%, being higher in meadows compared to riverbanks. The endophytes were detected in 36%, 30% and 27% of the plants in Faroe Islands, Iceland and Switzerland, respectively. Practically all examined plants collected from southern Finland and Greenland were endophyte-free, whereas in other geographic sites endophyte frequencies were highly variable among populations. Common to all populations with high endophyte frequencies is heavy vertebrate grazing. We propose that the detected endophyte frequencies and ploidy levels mirror past distribution history of F. rubra after the last glaciation period, and local adaptations to past or prevailing selection forces such as vertebrate grazing.

The endophytic symbiont Epichloë festucae establishes an epiphyllous net on the surface of Lolium perenne leaves by development of an expressorium, an appressorium-like leaf exit structure.

Epichloë festucae forms a mutualistic symbiotic association with Lolium perenne. This biotrophic fungus systemically colonizes the intercellular spaces of aerial tissues to form an endophytic hyphal network. E. festucae also grows as an epiphyte, but the mechanism for leaf surface colonization is not known. Here we identify an appressorium-like structure, which we call an expressorium that allows endophytic hyphae to penetrate the cuticle from the inside of the leaf to establish an epiphytic hyphal net on the surface of the leaf. We used a combination of scanning electron, transmission electron and confocal laser scanning microscopy to characterize this novel fungal structure and determine the composition of the hyphal cell wall using aniline blue and wheat germ agglutinin labelled with Alexafluor-488. Expressoria differentiate immediately below the cuticle in the leaf blade and leaf sheath intercalary cell division zones where the hyphae grow by tip growth. Differentiation of this structure requires components of both the NoxA and NoxB NADPH oxidase complexes. Major remodelling of the hyphal cell wall occurs following exit from the leaf. These results establish that the symbiotic association of E. festucae with L. perenne involves an interconnected hyphal network of both endophytic and epiphytic hyphae.

Fungal endophyte infection of ryegrass reprograms host metabolism and alters development.

Beneficial associations between plants and microbes play an important role in both natural and agricultural ecosystems. For example, associations between fungi of the genus Epichloë, and cool-season grasses are known for their ability to increase resistance to insect pests, fungal pathogens and drought. However, little is known about the molecular changes induced by endophyte infection. To study the impact of endophyte infection, we compared the expression profiles, based on RNA sequencing, of perennial ryegrass infected with Epichloë festucae with noninfected plants. We show that infection causes dramatic changes in the expression of over one third of host genes. This is in stark contrast to mycorrhizal associations, where substantially fewer changes in host gene expression are observed, and is more similar to pathogenic interactions. We reveal that endophyte infection triggers reprogramming of host metabolism, favouring secondary metabolism at a cost to primary metabolism. Infection also induces changes in host development, particularly trichome formation and cell wall biogenesis. Importantly, this work sheds light on the mechanisms underlying enhanced resistance to drought and super-infection by fungal pathogens provided by fungal endophyte infection. Finally, our study reveals that not all beneficial plant-microbe associations behave the same in terms of their effects on the host.

Detection and Isolation of Epichloë Species, Fungal Endophytes of Grasses.

Epichloë species (including former Neotyphodium species) are endophytic fungi that significantly affect fitness of cool-season grass hosts, potentially by increasing nutrient uptake and resistance to drought, parasitism and herbivory. Epichloë species are obligately biotrophic, living in the intercellular spaces of their plant hosts, and spreading systemically throughout host aerial tissues. The reproduction of Epichloë species is versatile; some strains have both sexual and asexual modes of reproduction, but others are restricted to one or the other mode. The reproduction mode determines the dissemination mechanism, and the asexual species most important to agriculture are strictly seed-borne, cause no signs or symptoms, and are undetectable except by specialized microscopic, molecular or antigenic procedures. These procedures can be used to identify endophytes in a variety of plant tissues. Similar protocols can be modified to detect less common symbionts, such as the penicillate "p-endophytes," when they occur by themselves or together with Epichloë species.

The Fungal Cell-Wall Integrity MAPK Cascade Is Crucial for Hyphal Network Formation and Maintenance of Restrictive Growth of Epichloë festucae in Symbiosis With Lolium perenne.

Epichloë festucae is a mutualistic symbiont that systemically colonizes the intercellular spaces of Lolium perenne leaves to form a highly structured and interconnected hyphal network. In an Agrobacterium tumefaciens T-DNA forward genetic screen, we identified a mutant TM1066 that had a severe host interaction phenotype, causing stunting and premature senescence of the host. Molecular analysis revealed that the mutation responsible for this phenotype was in the cell-wall integrity (CWI) mitogen-activated protein kinase kinase (MAPKK), mkkA. Mutants generated by targeted deletion of the mkkA or the downstream mpkA kinase recapitulated the phenotypes observed for TM1066. Both mutants were defective in hyphal cell­cell fusion, formed intrahyphal hyphae, had enhanced conidiation, and showed microcyclic conidiation. Transmission electron microscopy and confocal microscopy analysis of leaf tissue showed that mutant hyphae were more abundant than the wild type in the intercellular spaces and colonized the vascular bundles. Hyphal branches failed to fuse but, instead, grew past one another to form bundles of convoluted hyphae. Mutant hyphae showed increased fluorescence with AF488-WGA, indicative of increased accessibility of chitin, a hypothesis supported by changes in the cell-wall ultrastructure. These results show that the CWI MAPK pathway is a key signaling pathway for controlling the mutualistic symbiotic interaction between E. festucae and L. perenne.

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

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

Formation of arthroconidia during regeneration and selection of transformed Epichloë festucae protoplasts.

Transformation is an essential tool for modern fungal research and has played a fundamental role in gaining insight into gene function. Polyethylene glycol (PEG)-mediated transformation of protoplasts is the most commonly used method for genetic transformation of filamentous fungi. Selectable marker genes, that confer resistance to antibiotics, are generally incorporated with the DNA of interest, allowing transformed cells to grow through the antibiotic overlay. Colonies arising from transformed fungal cells are sub-cultured and further analysed. However, the morphological state of the fungal cells during the transformation procedure has been largely overlooked. We investigated the morphological appearance of transformed fungal cells prior to their emergence through the antibiotic overlay. Hyphae appeared to segment and bulge, reminiscent of arthroconidia, an asexual spore typically produced by segmentation of pre-existing hyphae. Selective expression of eGFP under the control of a spore specific promoter, PcatA, in these cells confirmed their spore-like nature. Reducing the oxygen availability to surface-grown cultures partially recapitulated this morphological form. A GFP fusion to the cell wall integrity MAP kinase MpkA localised to the arthroconidia nuclei suggesting the cell wall integrity signalling pathway modulates cell wall stress responses in arthroconidia. This dramatic morphological change was also observed in transformed Magnaporthe oryzae cells suggesting it may be a more general phenomenon in filamentous fungi. Given the changes in cellular structure and spore-like appearance, these observations may have technical implications for deleting genes involved in these processes in Epichloë festucae and, more broadly, a range of fungal species.

Epichloë spp. associated with grasses: new insights on life cycles, dissemination and evolution.

Epichloë species with their asexual states are specialized fungi associated with cool-season grasses. they grow endophytically in tissues of aerial parts of host plants to form systemic and mostly asymptomatic associations. Their life cycles may involve vertical transmission through host seeds and/or horizontal transmission from one plant to other plants of the same species through fungal propagules. Vertical transmission has been well studied, but comparatively little research has been done on horizontal dissemination. The goal of this review is to provide new insights on modes of dissemination of systemic grass endophytes. The review addresses recent progress in research on (i) the process of growth of Epichloë endophytes in the host plant tissues, (ii) the types and development of reproductive structures of the endophyte, (iii) the role of the reproductive structures in endophyte dissemination and host plant infection processes and (iv) some ecological and evolutionary implications of their modes of dissemination. Research in the Epichloë grass endophytes has accelerated in the past 25 y and has demonstrated the enormous complexity in endophyte-grass symbioses. There still remain large gaps in our understanding of the role and functions of these fungi in agricultural systems and understanding the functions, ecology and evolution of these endophytes in natural grass populations.

Fertilization of Epichloe typhina stromata by mycophagous slugs.

Epichloë typhina, a fungal endophyte of cool season grasses, is heterothallic and an obligate out-crosser. In areas of endemism, its spermatia are moved between stromata of the two opposite mating types through egg-laying activities of Botanophila flies. In western Oregon, where the fungus was inadvertently introduced into seed-production fields of Dactylis glomerata (= orchardgrass, cocksfoot), flies do not appear to be the sole vectors for E. typhina fertilization. Here we examined the role of the common agricultural slug pest Deroceras reticulatum and mycophagous slug species Prophysaon andersoni and Arion subfuscus in E. typhina spermatia transfer. Frass from P. andersoni, A. subfuscus and D. reticulatum fed stromata of one mating type was transferred to stromata of the opposite mating type, resulting in 100%, 93% and 25% stromata fertilization respectively. An experiment designed to mimic field conditions examined stromata fertilization on E. typhina-infected plants of opposite mating type in the presence of slugs. Treatments with P. andersoni and D. reticulatum had greater stromata fertilization compared to the no-slug control, but the slug treatments were not different. This appears to be the first report of mollusks vectoring viable spermatia leading to the cross fertilization of stromata of different mating types.