Symbioses between cool-season grasses (Subfamily Pooideae) and endophytic fungi in the genera Epichlo and Neotyphodium straddle a continuum of interactions from antagonistic to highly mutualistic. Although these two genera of endophytes are closely related, Neotyphodium endophytes are strictly seed-transmitted and provide many physiological and defensive benefits to their hosts, while Epichlo spp. have an obligately sexual contagious stage wherein host inflorescences are replaced by fungal sexual structures (stromata), effectively sterilizing the plant. Between these two extremes of interactions are Epichlo spp. with a mixed strategy, where some grass tillers are sterilized while others develop normally and yield healthy endophyte-infected seeds. These symbioses offer a unique opportunity to dissect evolutionary mechanisms that may drive movement along this continuum. The research presented characterizes distinct hybridization processes in endophytes and grasses that result in the generation of astounding genetic diversity for the symbiosis. Interspecific hybridization via hyphal anatomosis is a common feature of Neotyphodium endophytes, and may promote mutualism by combining suites of defensive alkaloid genes and ameliorating the adverse evolutionary effects of an asexual lifestyle. My results demonstrate that several genetically distinct hybrid endophytes infect grass species in tribe Poeae. Further, I show that a highly mutualistic asexual endophyte infecting tall fescue (=Festuca arundinaceum Schreb.), Neotyphodium coenophialum, also infects two closely related and interfertile relatives of this host. My findings suggest that this seed-borne endophyte may have been introgressed into these grasses through sexual grass hybridization events. These findings highlight interspecific hybridization as a means of generating tremendous genetic variability in both endophytes and their hosts, thus magnifying the adaptive evolutionary potential of these symbioses. Further, I establish a phylogenetic framework for grasses naturally harboring Epichlo and Neotyphodium endophytes. I show that patterns of genetic divergence among grass lineages are emulated by those of their fungal symbionts. These results suggest that endophytes have co-evolved with grasses in subfamily Pooideae, and may have played a critical role in the evolutionary success and radiation of this group of grasses.

Epichloë species and their anamorphs, Neotyphodium species, are fungal endophytes that inhabit cool-season grasses and often produce bioprotective alkaloids. These alkaloids include lolines, which are insecticidal and insect feeding deterrents. Lolines are exo-1-aminopyrrolizidines with an oxygen bridge between carbons 2 and 7, and are usually methylated and formylated or acetylated on the 1-amine. In previously published studies lolines were shown to be derived from the amino acids L-proline and L-homoserine. In addition the gene cluster involved in loline-alkaloid biosynthesis has also been characterized. In this dissertation a survey of plant-endophyte symbioses revealed a phenotype with only N-acetylnorloline. This phenotype provided insights into loline alkaloid production. This dissertation focuses on determining the steps to loline biosynthesis after the amino acid precursors. The study involves feeding isotopically labeled potential precursors to loline-alkaloid-producing cultures of Neotyphodium uncinatum, as well as RNA interference (RNAi) of N. uncinatum genes for steps in the pathway. Synthesized deuterated compounds were fed to loline-alkaloid-producing cultures of N. uncinatum to test their possible roles as precursors or intermediates in the loline-alkaloid pathway. N-Formylloline was extracted from the cultures and assayed by GCMS for incorporation of the deuterium label. The results indicated that N-(3-amino, 3-carboxy)propylproline and exo-1-aminopyrrolizidine are intermediates in the loline-alkaloid biosynthetic pathway. Plasmids were also designed for expression of double-stranded RNA homologous to loline-alkaloid biosynthesis genes, and introduced by transformation into N. uncinatum. This RNAi strategy resulted in fungal transformants altered in loline-alkaloid profiles. The RNAi results indicated that N-acetyl-1-aminopyrrolizidine is the intermediate before oxygen bridge formation. Based on the results of this study and the likely roles of the loline-alkaloid biosynthesis genes inferred from signature sequences of their predicted protein products, I propose a pathway of bond formation steps in loline-alkaloid biosynthesis.

Les associations symbiotiques entre différentes espèces de champignon du genre Epichloë et des plantes fourragères sont à l’origine de la production de mycotoxines responsables, dans certains pays, de toxicoses du bétail et de pertes économiques. Le développement de d’E. coenophiala dans la fétuque, qui est associé à la production d’ergovaline, est responsable de « fescue foot disease » alors que la présence d’E. festucae var. lolii dans le ray-grass anglais, qui est accompagnée d’une production de lolitrem B, est responsable du « ryegrass staggers » et de la production d’ergovaline, dont les effets toxiques associés à la présence de lolitrème B sont mal documentés. Mon travail de thèse a consisté à explorer les effets de la distribution de différents fourrages de fétuque et ray-grass endophytés toxinogènes produits dans des conditions agricoles françaises, sur la santé et la production animale et les mécanismes d’action de l’ergovaline et du lolitrème B. / The symbiotic associations between different varieties of fungi of the Epichloë kind and forages are responsible for the production of mycotoxins which are responsible of toxicoses in livestock and economic losses in some countries. The development of E. coenophiala in tall fescue, which is associated with the production of ergovaline, is responsible for « fescue foot disease » while the presence of E. festucae var. lolii in ryegrass, which is accompanied by the production of lolitrem B, is responsible for the « ryegrass staggers ». and the production of ergovaline, whose the toxic effects associated in the presence of lolitrem B are poorly documented. My thesis was to explore the effects of the distribution of different toxic forages of endophyte-infected tall fescue or ryegrass produced in French agricultural conditions on health and animal production and the mechanisms of action of ergovaline and lolitrem B.

Les associations symbiotiques Epichloë- graminées fourragères les plus étudiées concernent le ray grass anglais (Lolium perenne) avec Epichloë festucae var. lolii et la fétuque élevée (L. arundinaceum) avec E. coenophiala. Ces champignons peuvent synthétiser des alcaloïdes toxiques pour le bétail tels que l’ergovaline et le lolitrème B. Mon travail de thèse a consisté à explorer les facteurs en lien avec la production de mycotoxines dans les graminées. Pour cela différentes études ont été conduites sur la fétuque et le ray grass afin de révéler i) la présence de souches endophytées toxinogènes dans le Sud Ouest de la France et ii) l’influence des conditions climatiques françaises sur la synthèse d’ergovaline et de lolitrème B dans des variétés fourragères connues pour être responsables de cas de toxicité sur d’autres continents. / The most studied symbiotic associations Epichloë- cool seasons grasses concern perennial ryegrass (Lolium perenne) with Epichloë festucae var. lolii and tall fescue (L. arundinaceum) with E. coenophiala. These fungi can synthesize alkaloids that could be toxic to livestock such as ergovaline and lolitrem B. My thesis work was to explore the factors related to the production of mycotoxins in grasses. Different studies have been conducted on tall fescue and perennial ryegrass to reveal i) the presence of endophyte-infected toxigenic strains in the South West of France and ii) the influence of environmental conditions of Saint-Affrique (Aveyron) on the synthesis of ergovaline and lolitrem B in forage varieties known to be responsible for toxicity on other continents.

Appendix content (raw data and statistics; sequences) unavailable online, but available with print copy / Epichloë/Neotyphodium endophytes are a group of clavicipitaceous fungi that form symbiotic associations with temperate grasses. The asexual N. lolii form asymptomatic mutualistic associations with ryegrass whereas the sexual E. typhina behaves similar to a mutualist during the vegetative phase of plant growth but switches to epiphytic growth and formation of an external stroma upon development of the floral inflorescence. The aim of this project was to study the metabolic interaction between these endophytes and their perennial ryegrass host. The role of endophyte thiamine biosynthesis in host colonisation and stroma development was chosen, because of the key role this coenzyme plays in primary cellular metabolism and because thiamine biosynthetic genes are induced in several fungal-plant interactions. The orthologue (thil) of Saccharomyces cerevisiae THI4 was isolated from N. lolii and E. typhina by PCR using degenerate primers designed to conserved regions of known thiazole biosynthetic genes. This gene is expressed in planta and in culture, and is alternatively spliced, with distinct patterns of the isoforms expressed under different nutritional conditions. Mutant with a deletion in the E. typhina thil gene was constructed and shown to have reduced hyphal density and branching compared to the wild-type on defined media lacking thiamine. Both thiamine and thiazole complemented this defect. Artificial inoculation of the mutants into plants showed that the thil mutant retained the ability to colonise the perennial ryegrass host and form stromata. However, the mutant had some differences in host colonisation and growth, including reduced hyphal branching and reduced detrimental effects on the host. In addition, glycogen-like deposits, which were abundant in the wild-type hyphae, were not evident in the mutants. Unexpectedly, both the thil mutant and wild-type strains formed some stromata on vegetative tissue. Electron microscopic examination revealed that the cells of epiphytic hyphae found on the vegetative tillers typically were enlarged, lacking in cytoplasm and highly vacuolated, an ultrastructure similar to that found for hyphae growing in reproductive tillers. The mutants retained the ability to form conidia on the outer layer of the stromata. Extensive vascular colonisation and hyphal ramification in the mesophyll were common characteristics of stromata bearing regions. Although the morphology and ultrastructure of stromata formed on vegetative tillers is very similar to those on reproductive tillers, one significant difference was the presence of abundant glycogen-like deposits in hyphae of vegetative tillers. Furthermore, there were dramatic differences in the levels of glycogen-like deposits in hyphae in different regions of the vegetative tillers, indicating that the energy demand changes during stroma development. This is the first report of E. typhina forming stromata on non-inflorescence tillers.

Abstract Most plants form symbiotic associations with various fungi in natural ecosystems. Traditionally, many of these associations are viewed as mutually beneficial, but recent studies show that symbiotic interactions can be complex and labile. I studied the variability of interactions between grasses and fungi, using root-associated fungi and foliar endophytes in grasses as study systems. First, I studied experimentally how root-associated fungi colonize their host grasses in different natural and artificial conditions. I then investigated experimentally how foliar endophytes affect their host grasses, and whether the endophyte contributes to the host’s adaptation to local and/or foreign habitats. Finally, I reviewed the current use of foliar endophytes in agriculture, with a particular focus on Europe, and critically considered their potential for practices beyond agriculture. I found root fungi to be common, but different types of root fungi had contrasting colonization patterns in natural environments. However, I found that grasses lose almost all of their root fungi in controlled and more favourable conditions, indicating that the associations are fairly loose and conditional to environmental context. My results also showed that foliar endophytes affected the host’s performance in varying ways, depending on plant origin and experimental country, indicating that the associations were context dependent and could represent conditional mutualism. I also found that endophytes contribute to plant adaptation only weakly, but that the grasses were clearly locally adapted to their sites of origin, especially in regard to reproduction. However, the grasses of subalpine origin performed well vegetatively also in Northern Finland, suggesting that they may have high adaptive potential under changing climates. The literary review showed that foliar endophytes are successfully used in agriculture, e.g. in the USA and New Zealand, and that they possess the potential for several practical applications. However, the intentional use of endophyte-enhanced grasses is non-existent in Europe, although many European grass cultivars have great potential for improvement via endophytes. Taken together, these results show that plant–fungal interactions are highly variable along sites, environmental contexts and origins of the symbiotum, making predictions for these interactions difficult. / Tiivistelmä Luonnollisissa ekosysteemeissä melkein kaikilla kasveilla on symbionttisia sienikumppaneita. Perinteisesti monien näiden vuorovaikutusten on oletettu olevan molemmille hyödyllisiä, mutta viimeaikaiset tutkimukset osoittavat symbionttisten yhteyksien olevan vaihtelevia. Väitöskirjassani tutkin tätä heinien ja sienien välisten yhteyksien vaihtelevuutta, käyttäen heinissä esiintyviä juurisieniä ja lehtiendofyyttejä tutkimuskohteinani. Ensiksi tutkin kokeellisesti, kuinka juurisienet kolonisoivat heiniä erilaisissa luonnon- ja kasvihuoneolosuhteissa. Seuraavaksi tutkin kokeellisesti, kuinka lehtiendofyytit vaikuttivat heinien menestymiseen ja edesauttoivatko endofyytit heinien sopeutumista paikallisiin ja/tai vieraisiin elinympäristöihin. Viimeiseksi selvitin kirjallisuuskatsauksen avulla, kuinka lehtiendofyyttejä hyödynnetään maataloudessa ja arvioin endofyyttien potentiaalista käyttöarvoa maatalouden ulkopuolella, erityisesti keskittyen niiden hyödyntämiseen Euroopassa. Tutkimukseni osoitti, että heinien juurisienet ovat yleisiä, mutta niiden keskinäiset runsaussuhteet vaihtelivat luonnollisissa ympäristöissä. Kasvihuoneolosuhteissa heinät kuitenkin menettivät juurisienensä, viitaten siihen, että kyseinen yhteys on melko löyhä ja riippuvainen ympäristöstä. Tutkimukseni osoitti myös, että lehtiendofyytit vaikuttivat heinien menestykseen vaihtelevasti riippuen heinien alkuperästä sekä koemaasta, viitaten siihen, että nämä yhteydet ovat niin ikään olosuhteista riippuvaisia. Endofyytti vaikutti vain heikosti heinien sopeutumiseen, mutta heinät olivat selkeästi paikallisesti sopeutuneita niiden alkuperäisiin elinympäristöihin, erityisesti heinien lisääntymisen suhteen. Alppien heinät kuitenkin menestyivät vegetatiivisesti myös Pohjois-Lapissa, mikä viittaa siihen, että näillä heinillä saattaa olla korkea sopeutumispotentiaali muuttuvissa olosuhteissa. Kirjallisuuskatsaukseni osoitti, että lehtiendofyyttejä käytetään menestyksekkäästi mm. USA:n ja Uudessa-Seelannin maataloudessa. Euroopassa niiden käyttö on kuitenkin lähes olematonta, vaikka endofyyteillä voisi olla monia käyttömahdollisuuksia, sekä maataloudessa että ympäristön hoidossa. Kaiken kaikkiaan tutkimukseni osoittavat, että heinien ja sienten väliset vuorovaikutukset ovat hyvin vaihtelevia riippuen ympäristöstä ja heinien alkuperästä, minkä vuoksi näiden sienien vaikutuksia heiniin on vaikea ennustaa.

The relationship between biodiversity and ecosystem functioning is important in maintaining agroecosystem sustainability. Plant-microbe symbioses, such as exists between the grass tall fescue (Schedonorus arundinaceum) and the asexual fungal endophyte Epichloë coenophiala, can be utilized to enhance agroecosystem functions, such as herbivore resistance. “Novel” E. coenophiala strains that vary in the production of mammal- and insect-toxic compounds have been identified, inserted into tall fescue cultivars, and are planted in pastures globally. Novel fungal endophyte-tall fescue associations may have divergent ecosystem function effects. This study assessed effects of different fescue-endophyte symbiotic combinations on pasture ecosystem function, including aboveground (fescue biomass, plant species richness, alkaloid synthesis, arthropod abundance) and belowground (soil microbial biomass, soil enzyme activity, trace gas fluxes) parameters. Results showed no significant effects of increasing symbiotic diversity within a fescue stand on aboveground measurements, bar arthropod abundance and alkaloid synthesis. Most soil parameters quantified had significant symbiotic diversity effects. For example, soil microbial biomass decreased whereas soil enzyme activity increased with increasing symbiotic diversity. Overall, our results suggested that increasing symbiotic diversity had weak to moderate effects on aboveground processes and stronger effects on certain belowground processes, indicating that symbiotic diversity can impact ecosystem functions and warrants further research.