Festuca grasses from various natural habitats are often infected with Neotyphodium endophytes.
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(102 KB)Epichloë species usually depend on flies of genus Botanophila which transfer gametes for sexual reproduction. Sequence analysis of COII detected six distinct Botanophila taxa that were associated with Epichloë in Europe and North America. Three were restricted to samples from Europe, two to samples from North America, and one was present on both continents. In North America the common taxon (identified as B. lobata) was found on Epichloë hosts of native grasses as well as on E. typhina and E. festucae of introduced grasses. In Switzerland, up to four different Botanophila taxa occurred at a single location. There was no strict specificity of flies for Epichloë host species, although several taxa appeared to prefer some hosts over others. Comparison of the Botanophila phylogeny with that of the associated Epichloë hosts did not suggest co-evolution of fungus and fly. Thus, associations between Botanophila flies and Epichloë hosts may have arisen independently more than once. Keywords: Anthomyiidae, Diptera, Clavicipitaceae, choke disease, cytochrome oxydase, grass endophytes, molecular phylogeny, mutualism
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(283 KB)We have investigated community and ecosystem consequences of endophyte symbiosis with tall fescue over the past 13 years. Lolium arundinaceum is the most abundant plant in the eastern USA, and most is infected by the wild-type endophyte Neotyphodium coenophialum in Kentucky 31. We established two large experimental grasslands (in 1994 and in 2000) with E+ and E- seed sown in each on recently ploughed herbaceous vegetation. Other plant species established naturally by seed or vegetative fragments. No other treatments were applied and plots were subject to natural biotic and abiotic variation. A third experiment examined ecological influences on endophyte infection dynamics starting from an intermediate infection frequency. We found wide-ranging consequences of the endophyte from significant effects on soil feedback and decomposition rates, to plant-plant competition, diversity, productivity, invasibility and succession, to plant-herbivore interactions and energy flow to higher trophic levels. Further, we found that herbivore pressure caused rapid increases in infection frequency. Our results suggest that endophyte symbiosis in tall fescue can have a transforming effect on ecological systems. Keywords: Lolium arundinaceum, Neotyphodium coenophialum, soil, competition, herbivory, trophc interactions, predators, MaxQ endophyte
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(909 KB)The naturally occurring mutualistic symbiosis of Neotyphodium gansuense and drunken horse grass (Achnatherum inebrians) was studied previously in China. In this paper, new data on the interactions of endophyte, host and pathogenic fungi, mite and insect pests are presented. Fungal diseases and pests were examined when test plants were grown in pots in a climate chamber or in the field. There were usually no significant (P>0.05) differences in the levels of powdery mildew infection (caused by Blumeria graminis) under climate chamber conditions; the only exception was that E+ plants had significantly less powdery mildew infection at 50% soil water holding capacity (WHC) than at 30% WHC. There was no significant difference (P<0.05) in the levels of infection of E- and E+ plants with leaf spot disease, caused by Bipolaris sorokiniana, under field conditions. Regardless of whether grown under growth chamber or field conditions, densities of the mite Tetranychus cinnabarinus on E+ grass were significantly (P<0.05) lower than that on E- grass. Densities of the bird-cherry-oat aphid (Rhopalosiphum padi) on E+ grass were significantly (P<0.05) lower than that on E- grass. Keywords: Neotyphodium gansuense, Achnatherum inebrians, diseases, pests, resistance, endophytes
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(332 KB)Introduced Lolium grasses are utilised in Japan for forage, turf and soil conservation. Many of these grasses have become naturalised in disturbed ecosystems. Neotyphodium endophytes in the tissues of grasses can increase host fitness by increasing tolerance to both biotic and abiotic stresses. Consequently, endophyte infection may be a factor enhancing invasion success of exotic grasses in the Japanese islands. We detected infection by Neotyphodium endophytes in 162 of 173 naturalised ryegrass populations in Japan. Italian ryegrass (L. multiflorum) had a higher infection rate than perennial ryegrass (L. perenne). Italian ryegrass also exhibited geographic variation in infection rate. It is unlikely that the infected Italian ryegrasses found in this study have come from cultivars used in meadows, since the use of forage ryegrass cultivars infected with Neotyphodium endophytes has been restricted in Japan to prevent toxicity problems. Possible sources of the ryegrasses may be turf seeds, soil conservation materials and/or contaminants in imported plant materials. Keywords: alien species, invasive plants, Italian ryegrass, Lolium multiflorum, Neotyphodium endophyte, perennial ryegrass, Lolium perenne
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(429 KB)Fungal endophytes associated with pasture grasses can have community-wide effects on insect consumers. Here we asked the question to what degree endophyte infection, simulated nitrogen deposition and grass cultivar influence the abundance of colonising herbivores and their natural enemies. In a fully randomised field experiment, consisting of four Lolium perenne monocultures of known endophyte infection status and a nitrogen addition treatment, we determined the abundance of colonising aphids, their parasitoids and predators, and other grass herbivores. The three colonising cereal aphid species did not respond to endophyte infection, possibly because peramine concentrations were relatively low (3.9 μg/g). There was a significant interaction between nitrogen addition and plant cultivar on the abundance of Sitobion avenae, suggesting a cultivar-specific response to nitrogen addition. Aphid predators were affected by an interaction between endophyte and plant cultivar, but abundance of aphid parasitoids and other grass herbivores was not affected by any treatment. The fungus Claviceps purpurea naturally infected our experimental plants and infection rates differed among cultivars and were more likely to occur on endophyte-infected plants, in particular on wild-type Samson. We conclude that strong effects of endophytes on insect abundance may not occur in systems built upon L. perenne because overall peramine levels rarely reach threshold levels for insect toxicity. Keywords: fungal endosymbionts, multitrophic interactions, field experiment, insect food webs, alkaloids
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(739 KB)This research quantified frequencies of Neotyphodium infected (E+) tillers and mature seed from field-grown E+ plants of two wild tall fescue accessions from Morocco and Sardinia, Italy. Tiller infection rates were 100% (n = 50 from 10 E+ plants/accession) for each accession and over 99% of the seed (n = 2394) from E+ plants of both accessions harboured viable Neotyphodium endophyte. Germination rates for E+ seed were 93.8% (Morocco accession) and 97.8% (Sardinia). These results indicate that E+ wild tall fescue plants are capable of near perfect vertical transmission of viable endophyte into seed. They also suggest that viable endophyte is retained with current seedregeneration protocols at the USDA-ARS Western Regional Plant Introduction Station, Pullman, Washington USA. Keywords: wild tall fescue, Neotyphodium infection frequencies, vertical transmission
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(291 KB)About 28% of agricultural land in Bulgaria is grassland that is mainly confined to foothills, intermediate mountain areas and alpine pastures and meadows
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(144 KB)Ruminants consuming toxic endophyte-infected (E+) tall fescue are exposed to ergot alkaloids that usually result in a malady of problems identified as fescue toxicosis. Grazing studies investigating the effects of E+ tall fescue on the prevalence of E. coli O157:H7 from naturally-infected cattle have not been consistent. Further, in vitro studies with selected ergot alkaloids did not affect growth of E. coli O157:H7. Dietary stressors usually associated with E+ tall fescue consumption did result in increased faecal shedding of E. coli O157:H7 from sheep experimentally-inoculated. Animal species, animal model (naturally vs experimentally-infected), environmental conditions, type of diet, and length of exposure to E+ tall fescue may influence the association between grazing E+ tall fescue and E. coli O157:H7 shedding. A better understanding of how E+ tall fescue influences shedding of E. coli O157:H7 from ruminants will aid in the development of management strategies for onfarm pathogen control. Keywords: cattle, E. coli O157:H7, sheep, tall fescue
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(327 KB)There is a growing recognition that endophyte effects on host plant traits may be propagated through food chains. We studied Neotyphodium occultans effects on soil nematode communities mediated by current and past patch occupancy by endophyteinfected Lolium multiflorum populations. A microcosm experiment was performed to evaluate whether abundance and diversity of nematodes at different trophic levels were affected by endophyte infection through rhizosphere-mediated or littermediated effects. We found that presence of endophyte-infected plants and their aerial litter both triggered a bottom-up trophic cascade enhancing the abundance of herbivorous and predaceous nematode taxa. Endophyte infection also increased overall nematode richness, mostly through changes induced at the highest trophic level in this soil food web. Our results suggest that fungal endophytes can modify the linkages between aboveand belowground community compartments, with potential consequences on plant patch dynamics. Keywords: soil food webs, Lolium multiflorum, Neotyphodium occultans, plant-soil feedback, after-life effects, indirect interactions, trophic cascades
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(318 KB)The agronomic importance of the grass-endophyte association became apparent after it was linked to livestock diseases. This paper reports the incidence of endophytes in natural grasslands in Argentina from data collected through grass seed collections, tillers and culms from natural grasslands. A total of 159 samples were analysed: 144 grass collections from 30 species were obtained from accessions (germplasm bank of EEA Balcarce- INTA), and 15 samples were collected in the field in the province of Buenos Aires (Balcarce and Ayacucho). The samples were analysed according to Saha et al. (1988) or Belanger (1996). Endophytes were found in only nine of the 30 species of accessions analysed; accessions of Lolium multiflorum showed the highest incidence of endophyte infection, ranging from 57 to 100%. Of the 15 field populations collected: Festuca arundinacea and L. multiflorum samples were infected with infection ranging from 89 to 100%; populations of Bothriochloa, Deyeuxia, Piptochaetium, Sporobolus, Stipa megapotamia, S. papposa, were endophyte free. The economic losses to the livestock industry and the potential benefits of endophytes as biological agents to improve plant fitness and pest resistance of forage grasses increases the importance of determining the incidence of fungal endophytes in grass germplasm collections and natural grasslands. Keywords: native grasses, endophytes, naturalised grasses, Argentina
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(657 KB)In the systemic grass endophyte associations only one fungal genotype is transmitted vertically to seed progeny, promoting stable interactions between the fungal genotype and the host lineage.
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(132 KB)Neotyphodium endophytes infect the natural grass populations of Lolium perenne and Festuca sp. L. perenne samples were collected from 62 different locations in Denmark and were analysed for the presence of Neotyphodium lolii.
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(164 KB)Fungal endophytes of grasses affect ecosystem processes through mutualistic interactions with host plants, but how grass endophytes affect litter decomposition remains unclear. In this study, previously published data on litter quality and decomposition of grasses are summarised and effects of fungal endophytes of grasses on litter quality and decomposition are reviewed to discuss possible roles of endophytes in decomposition. Aboveground litters of grasses have relatively low nitrogen (N) and lignin contents and show slow increase of N and lignin contents during decomposition. Endophyte-infected litter have slightly lower N contents than non-infected litter, but the differences so far reported were not statistically significant. A negative effect of grass endophytes on litter decomposition rates was demonstrated, but the effect of endophyte infection on decomposition was not as strong as the effects of other biological and environmental factors. This suggests that grass endophytes have a relatively minor effect of on litter quality and decomposition, but more studies are necessary to verify this tentative conclusion. Keywords: decomposition, lignin, litter, nitrogen
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(292 KB)We examined the occurrence of Neotyphodium endophytes in native grasses from Argentina using both herbarium material and plants collected from the field. Neotyphodium endophytes were detected in 34 out of more than 150 host species examined throughout the country. The incidence of infection was highly variable within many of the species. We examined the relationship between incidence of endophyte and environmental conditions in four host species, Bromus setifolius from Santa Cruz, Phleum alpinum and Poa rigidifolia from Tierra del Fuego and Poa bonariensis from the Pampean region. The results suggest that each endophyte-host association seems to be affected in different ways by environmental factors. B. setifolius only contained endophyte in moist rather than dry regions, while in contrast, Ph. alpinum showed the highest endophyte incidence in the driest sites. Endophyte incidence in P. rigidifolia was highest where soil fertility was high, and P. bonariensis contained the most endophyte in high pH rather than low pH soils. Field observations suggest that in poor growing conditions, such as those of the extra-Andean Patagonia, or highly acidic soils, the association is not favoured leading to a decrease in the endophyte frequency of infection or even to the complete loss of the association. Keywords: Bromus setifolius, native grasses, Phleum alpinum, Poa bonariensis, Poa rigidifolia, South America
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(367 KB)Morphological and molecular methods were used to identify the endophytic mycobiota of the grass Dactylis glomerata. Fungal endophytes belonging to 109 different species were isolated from asymptomatic plants sampled in different ecosystems in Spain. Species accumulation curves showed that most species commonly infecting this grass have been identified, but the number of singleton species occasionally infecting the plants is likely to increase with more sampling effort. A large endophytic assemblage consisting of fungi with diverse ecological roles, and potentially unknown species was found in a small number of plants. Keywords: endophytes, Dactylis glomerata, diversity, abundance
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(321 KB)Infection of tall fescue and ryegrass pastures with Neotyphodium endophytes are of much greater interest in New World pastures than in Europe where they have been present as long as agriculture itself. This paper presents an overview of the importance of endophyte infection in pastures, enhancing the productivity and persistence of their hosts, both directly and through protection from invertebrate pressure, the biological factors behind this importance and the measures available to profit from the advantages of endophyte infection while palliating the negative effects. Keywords: tall fescue, ryegrass, livestock, edaphic stress, invertebrate pests
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(324 KB)We have been collaborating on a multidisciplinary research programme with undergraduate (baccalaureate) students for the past 3 years, funded by the National Science Foundation in the USA. The project focuses on the ecological interaction between endophytic fungi and the grasses they inhabit and involves students (10-15 annually) and faculty (5 and 1 post-doc) from the departments of Biology, Chemistry and Mathematics. We briefly present some of our results from ongoing projects on stomatal conductance and photosynthetic rates in tall fescue, wound-induced responses at the molecular level, and modelling tri-trophic level interactions. We also discuss how we came together to initiate the project; challenges, pitfalls, and advantages of doing multidisciplinary, multi-investigator research with undergraduates. We will present our assessment results, which are focused on how students feel their involvement in multidisciplinary research differs from what it would be in disciplinary research. Keywords: Neotyphodium coenophialum, tall fescue, woundinduced response, tri-trophic interactions, water use efficicency, stomatal conductance, photosynthesis
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(707 KB)Studies of systemic endophyte in grasses have skyrocketed in the past two decades. However, the vast majority of these studies still occur in agroecosystems. We show that ecological and evolutionary concepts derived from these systems may be misleading because they fail to incorporate the enormous variability found in endophyte-host grass interactions in wild grass populations. This variability stems from 1) genetic differences in host plants and endophytes, 2) environmental factors such as light and soil nutrients and moisture, and 3) the tangled web of interacting species in natural communities, such as conspecific and interspecific plants, multiple generalist and specialist herbivore species, and the third trophic level, predators and parasites. Studies of natural populations and communities continue to lag far behind those involving agronomic grass systems. However, we argue that additional studies of infected wild grass populations and communities are essential to advance ecological and evolutionary concepts of endophytegrass interactions. Keywords: endophytes, herbivore resistance, grasses, natural populations and communities, natural enemies, Neotyphodium, pathogens, plant defenses, variability
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(1338 KB)Defensive mutualism in many grass endophytes involves herbivore deterrence, enhanced disease suppression and drought tolerance. According to our current understanding of the mechanisms, these defensive effects are unique features of the fungi and specific fungal qualities are responsible for each. We examined a selection of species of family Clavicipitaceae for defensive traits including ergot alkaloids and loline production and development of endophytic and epiphytic mycelial states. When features responsible for defensive effects are mapped onto phylogenetic trees showing relationships in the family, it is apparent that all defensive features of endophytic fungi pre-existed evolution of endophytism. We suggest that once endophytism evolved, the alkaloids and other fungal–mediated defensive features switched from previous functions to serve host defense functions. Defensive mutualism in the Clavicipitaceae endophytes naturally evolved through use of pre-existing fungal features after the step to endophytism was made. Keywords: Clavicipitaceae, defensive mutualism, endophytes, ergot alkaloids, loline alkaloids
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(385 KB)In a random survey of 107 alpaca producers across Australia, 23% had observed staggers over the previous 3 years with signs usually persisting 3-4 months
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(343 KB)
We have identified a novel siderophore from the mutualistic grass symbiont Epichloë festucae. Targeted gene replacement of a non-ribosomal peptide synthetase (termed sidF) from E. festucae eliminated biosynthesis of this extracellular siderophore. Structural characterisation, based on extensive mass spectrometry showed similarities to fusarinine-type siderophores. Plants inoculated with strains carrying a targeted deletion in sidF have a dramatic phenotype. Siderophore loss changed a mutualistic symbiont into an antagonistic fungus, causing de-regulation and proliferation of fungal hyphae, plant stunting and sometimes even tiller death. We hypothesise that loss of the biosynthesis of the siderophore disrupts iron homeostasis within the whole symbiotum, which is a critical factor for maintenance of mutualistic endophyte-grass symbioses. Keywords: siderophore, iron homeostasis, NRPS
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(779 KB)Conspicuous white mycelial growth was noted on inflorescences of a small percentage of perennial ryegrass plants (cultivar ‘Extreme’) that were being managed for seed production.
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(137 KB)The stability of endophyte alkaloids was investigated in ryegrass herbage cut and then dried in the field under a simulated hay drying regime in early and mid-summer. Ryegrass (Lolium perenne) infected with typical ‘wild-type’ and the ‘AR37’ strain of endophyte (Neotyphodium lolii) was measured for peramine, ergovaline and lolitrem B for wild-type, and epoxy-janthitrems for AR37. Despite very contrasting weather conditions, relative decline in alkaloid concentrations was similar in both early and mid-summer. Concentrations had halved within 5 to 7 days, except for lolitrem B in mid-summer which had no decline until after 4 days of drying. Ergovaline showed quite variable and erratic responses over time possibly due to the low concentrations in the cut herbage. At least in the mid-summer experiment, if herbage had been conserved at the time of normal haying making, alkaloid concentrations would have been similar to those of the herbage at the time of cutting. Keywords: endophyte, Neotyphodium lolii, Lolium perenne, peramine, lolitrem, ergovaline, janthitrems
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(260 KB)Tall fescue (Lolium arundinaceum) drought tolerance is often enhanced by endophyte infection; however, the physiological mechanisms are not clearly understood. Endophyte-infected (E+) and endophyte-free (E-) clones of two contrasting tall fescue genotypes were drought stressed to investigate differences in endophyte-mediated dehydrin expression. Both genotypes had shown enhanced drought survival due to their endophyte in previous trials. As drought stress intensified, E+ and E- plants of both genotypes produced dehydrins; however, quantitative and qualitative differences were noted. Genotype 60, a mesic type, accumulated more dehydrins in E+ than in E- plants, whereas genotype 330, a xeric type, displayed low levels of dehydrin and no differences between E+ and E- plants in accumulation patterns. Lack of dehydrin response in genotype 330 suggests that putative dehydrin involvement in endophyte-enhanced tiller survival may not be a universal mechanism in tall fescue. In the absence of endophyte, genotype 330 still expressed much less dehydrin than E- genotype 60, indicating lesser importance of dehydrin in a xeric plant for dehydration tolerance, perhaps benefiting more from constitutive adaptive traits in the host. Keywords: dehydrin, drought, tall fescue
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(647 KB)Twenty half-sibling families of perennial ryegrass inoculated with a strain of endophyte isolated from tall fescue were analysed for loline concentrations. Inoculation success varied between families. Frequency of loline expression of different families varied moderately. Family effect on loline concentrations was significant. In another set of material, derived from plants exposed to field selection, endophyte infection frequency in seed harvested from plants verified as infected was close to 100%. Loline concentrations varied significantly between families. For nine of 23 families, none of the three plants sampled showed any measurable loline. As in other data sets, the results show variation in concentrations of endophyte alkaloids controlled by the genetics of the host plant. Keywords: Neotyphodium, host variation, adaptation, symbiosis, lolines
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(249 KB)In our search for novel small molecule bioactive agents from endophytic fungi of the Sonoran desert, extracts derived from cultures of some selected endophytic fungal strains have been screened in assays for inhibition of heat shock protein 90 (Hsp90) activity, cancer cell proliferation and migration. Chaetomium chiversii, Chaetomium globosum, Fusarium acuminatum and Fusarium oxysporum producing metabolites active in these assays were cultured on large-scale and the derived extracts were subjected to bioactivity-guided fractionation to obtain a variety of natural products with diverse structures, and potential applications in cancer chemotherapeutics. This paper discusses the isolation, characterisation, and biological activity of some selected small molecule metabolites. Keywords: endophytic fungi, small molecule natural products, isolation, structure elucidation biological activity, cytotoxic agents, cell migration inhibitors; Hsp90 inhibitors
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(724 KB)Seed transmitted Neotyphodium endophytes colonise the apical meristem (growing point) of young seedlings and later spread into mature leaves and flowers of host plants. However, their location and orientation in the meristems, and the process of their spread into other parts of the plant, are not fully understood. To elucidate this process, the morphology and orientation of mycelia of three Neotyphodium endophytes (N. coenophialum, N. lolii and N. occultans) and their sexual relative, Epichloë typhina were observed using dissected meristem tissues with differential interference contrast microscopy, after chemical clearing. Mycelia of Neotyphodium species more or less ramified in pith or nodal tissue beneath the apical meristem, but were sparsely branched within apical meristems and inflorescences developing from the zone. The distribution of E. typhina mycelia was similar to the Neotyphodium species, but they sometimes appeared on the surface of the merstematic tissues. Keywords: apical meristem, differential interference contrast microscopy, hyphal growth
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(689 KB)Seed transmitted Neotyphodium endophytes colonise the apical meristem (growing point) of young seedlings and later spread into mature leaves and flowers of host plants. However, their location and orientation in the meristems, and the process of their spread into other parts of the plant, are not fully understood. To elucidate this process, the morphology and orientation of mycelia of three Neotyphodium endophytes (N. coenophialum, N. lolii and N. occultans) and their sexual relative, Epichloë typhina were observed using dissected meristem tissues with differential interference contrast microscopy, after chemical clearing. Mycelia of Neotyphodium species more or less ramified in pith or nodal tissue beneath the apical meristem, but were sparsely branched within apical meristems and inflorescences developing from the zone. The distribution of E. typhina mycelia was similar to the Neotyphodium species, but they sometimes appeared on the surface of the merstematic tissues. Keywords: apical meristem, differential interference contrast microscopy, hyphal growth
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(689 KB)Some isolates of Epichloë festucae are asymptomatically infected by Epichloë festucae virus 1 (EfV1), a member of the family Totiviridae. This virus has a genome composed by a 5109 bp molecule of double stranded RNA (dsRNA). In addition, a 3 kbp dsRNA molecule which could be the genome of another virus (EfV2) was frequently found in isolates of the fungal endophyte. In a survey of two populations of E. festucae it was found that 73% of the isolates were infected by one or both viruses. Although both viruses are very efficiently transmitted to conidia, a barrier to virus transmission occurs during ascospore formation. Keywords: Epichloë festucae, virus, dsRNA, Totivirus
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(335 KB)Experiments were conducted to examine aspects of the ecology of the epiphyllous stage of a Neotyphodium endophyte on leaves of Poa ampla (big bluegrass) plants. Conidia were found to be readily water dispersed and to survive for several weeks on leaf surfaces. Neotyphodium was found to colonise excised grass leaves and grow on growing meristems of seedlings. The significance of epiphyllous stages of endophytes was also evaluated and discussed. Keywords: Clavicipitaceae, Neotyphodium, conidia, dissemination, water dispersal, endophytes, epiphytes, epiphyllous, big bluegrass, Poa ampla
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(971 KB)Perennial ryegrass, tall fescue and meadow fescue infected with a noxA mutant defective in the ability to produce reactive oxygen species (ROS), had a stunted phenotype and showed premature senescence. Microscopic observations of leaf tissue from perennial ryegrass and tall fescue showed a lack of regulated growth of mutant hyphae in these tissues with increasedv branching, growth and vascular colonisation. However, the fungal phenotype of noxA-infected meadow fescue was very similar to the wild-type phenotype suggesting that an increase in fungal biomass and vascular bundle colonisation is not necessarily a requirement for host stunting. An alternative hypothesis to explain the stunting phenotype is a breakdown in normal signaling between endophyte and host that is ROS dependent. Keywords: Epichloë festucae, Lolium perenne, NoxA, NADPH oxidase, reactive oxygen species (ROS)
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(3360 KB)Sex is considerably more costly for an organism than clonal reproduction. How it conveys sufficient benefits to outweigh this cost remains unclear. One of the main arguments that such benefits exist is the wide distribution of sex and the short evolutionary lifespan of asexual lineages. However, too little is known about the reproductive biology of microorganisms to be certain that sex is wide-spread among them. Among the epichloë endophytes the ability to carry out sexual reproduction is frequently lost, and closely related sexual and asexual lineages can be readily collected. This offers an opportunity to test if asexual endophyte lineages have a shorter lifespan than sexual lineages, which would indicate that sex conveys advantages to epichloë endophytes. Using a novel phylogeny-based statistical approach, we found strong evidence that asexual endophyte lineages are short-lived compared to sexual lineages. Keywords: sex, clonal reproduction, evolutionary biology, ecology
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(763 KB)The association between perennial ryegrass (PRG) and fungal endophyte is an important factor in the protection of New Zealand pastures from insect pests, and the genetic background of the host plant significantly affects endophyte performance. Quantitative trait locus (QTL) analysis of phenotypic data from herbage harvested from a mapping population in autumn 2005 identified loci in the PRG genome influencing three traits: (1) endophyte mycelial mass; (2) ergovaline level; (3) peramine level. Three QTL were identified for each trait. For mycelial mass two QTL accounted for more than 75% of the variation (PV) for in the population and have considerable potential for development as pre-inoculation marker-assisted selection (MAS) tools for endophyte colonisation in PRG breeding programmes. The largest-effect QTL for peramine level (18% PV) coincided with a major mycelial mass QTL, implying peramine concentration is partly determined by endophyte colonisation. All other alkaloid QTL, however, were independent of mycelial mass, indicating it may be possible to develop MAS that will facilitate breeding for alkaloid phenotype, independent of the degree of endophyte colonisation. Keywords: DIMS, ELISA, Lolium, map, metabolic profiling, Neotyphodium, SSR
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(1098 KB)The association between perennial ryegrass (PRG) and fungal endophyte is an important factor in the protection of New Zealand pastures from insect pests, and the genetic background of the host plant significantly affects endophyte performance. Quantitative trait locus (QTL) analysis of phenotypic data from herbage harvested from a mapping population in autumn 2005 identified loci in the PRG genome influencing three traits: (1) endophyte mycelial mass; (2) ergovaline level; (3) peramine level. Three QTL were identified for each trait. For mycelial mass two QTL accounted for more than 75% of the variation (PV) for in the population and have considerable potential for development as pre-inoculation marker-assisted selection (MAS) tools for endophyte colonisation in PRG breeding programmes. The largest-effect QTL for peramine level (18% PV) coincided with a major mycelial mass QTL, implying peramine concentration is partly determined by endophyte colonisation. All other alkaloid QTL, however, were independent of mycelial mass, indicating it may be possible to develop MAS that will facilitate breeding for alkaloid phenotype, independent of the degree of endophyte colonisation. Keywords: DIMS, ELISA, Lolium, map, metabolic profiling, Neotyphodium, SSR
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(1098 KB)Ergot alkaloids are produced by ergot fungi (Claviceps spp.) that parasitise ears of grain, and related epichloë endophytes (Epichloë and Neotyphodium spp.) that systemically persist in grasses as protective mutualists.
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(200 KB)Two controlled environment experiments were performed to test the effects of nitrogen, phosphorus and carbohydrates on endophyte (Neotyphodium lolii) and alkaloid concentrations in ryegrass (Lolium perenne). Three perennial ryegrass cultivars (‘high sugar grasses’ AberDove and AberDart; control Fennema) that differ in carbohydrate content were infected with three strains of N. lolii (common strain, CS; AR1; AR37). Infected and uninfected plants were grown under high (9 mM) and low (2.25 mM) nitrogen (AberDove, Fennema; CS, AR1, AR37) or under high (2 mM KH2PO4) and low (0.05 mM KH2PO4) phosphorus (AberDart, Fennema; CS, AR1). Quantitative realtime Polymerase Chain Reaction (qPCR) was used to estimate endophyte concentrations in harvested leaf tissues. High N and P supply as well as high carbohydrate content of the host grass reduced endophyte concentrations. Alkaloid production was also reduced under both increased N supply and in the high sugar cultivar, and was linearly related to endophyte concentration (except ergovaline). The results stress the need for wider quantification of fungal endophytes in the grassland/ foliar endophyte context, and have implications for how introducing new cultivars, novel endophytes, or increasing nutrient inputs, affect the role of endophytes in grassland ecosystems. Keywords: Neotyphodium lolii, foliar endophyte, Lolium perenne, perennial ryegrass, qPCR, high sugar ryegrass, nitrogen, phosphate, carbohydrate, AR1, AR37, alkaloids
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(407 KB)Plants have evolved a range of responses to herbivory ranging from tolerance to both inducible and constitutive chemical defenses. Previous research has shown lolines produced by Neotyphodium coenophialum are inducible by host damage. In this paper, we examined whether this inducibility can also be caused by damage from an invertebrate herbivore and whether this phenomenon also occurs in N. uncinatum. We also investigated whether changes in gene expression for lolC, a gene in the loline biosynthesis pathway, accompanies changes in defense response. For N. coenophialum, both mechanical and herbivore damage had a negative effect on subsequent aphid herbivores. For N. uncinatum, damage did not cause an increase in defense, although there was an increase in endophyte-free hosts. Relative expression of lolC varied significantly over time, and between damage types for N. uncinatum with herbivore damaged plants having a lower relative lolC -1 expression. Keywords: Neotyphodium, gene expression, inducible defense, loline, lolC
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(475 KB)Fungal endophytes (Neotyphodium lolii) in perennial ryegrass (Lolium perenne) produce a range of bioactive alkaloids which are implicated in both toxicity to grazing animals and resistance to insects. The understanding of regulatory and biochemical mechanisms of the symbiosis will provide clues for the genetic manipulation of beneficial alkaloid production. This paper presents approaches to analyse data from high-throughput microarray experiments and targeted metabolomic analyses. Combined with bioinformatics analyses, potential genes were found associated with the accumulation of alkaloids and other metabolites. The advantages and limitations of our approach to address the molecular mechanisms of the symbiosis will be discussed. Keywords: Lolium perenne, Neotyphodium lolii, metabolomics, microarray
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(644 KB)A pot experiment investigated interactions between foliar endophytic (EP, Neotyphodium lolii) and root arbuscular mycorrhizal (AM, Glomus mosseae) fungi in Lolium perenne. The effects of three phosphorus (P) forms (KH2PO4-P, phosphate rock-P (PR) and phytate-P) on EP and AM colonisation and plant growth parameters were also assessed. AM arbuscular and vesicular structures were estimated by microscopic counting and fungal concentrations were quantified by qPCR of G. mosseae and N. lolii specific genes. Significantly more AM structures were formed in roots of EP-free compared to EP-infected plants, indicating that AM development is suppressed by the presence of foliar EPs. Total AM concentrations were also generally higher in EP-free plant roots, but not significantly. EP concentrations were significantly reduced by higher plant-available P in the growth medium, but mycorrhizal infection did not significant affect EP concentrations. Mycorrhizal inoculation increased shoot dry matter (DM) at low plant-available P in the PR treatment. Infection with the endophytic strain AR37 also significantly increased shoot DM and fungal concentrations were higher compared to CS (common or wild-type strain)- and AR1-infected plants. Insoluble phytate-P produced the same amount of biomass as soluble-P (K-Pi) did, suggesting that L. perenne has the ability to use organic P. Keywords: Neotyphodium lolii, Glomus mosseae, foliar endophyte, arbuscular mycorrhizae, Lolium perenne, qPCR, phosphate, AR1, AR37
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(308 KB)Metabolic profiling using gas-chromatography mass spectrometry was performed for endophyte-infected (E+) and corresponding endophyte-free (E-) clones of two ryegrass genotypes cultivated under sufficient water supply and drought stress. In total, 243 metabolites representing both known and unknown compounds were analysed for samples taken at the end of the drought stress period and after rewatering (n=10 replications per level of genotype, endophyte infection, and water supply).
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(147 KB)Neotyphodium is an asexual, vertically transmitted, obligate fungal endosymbiont infecting cool-season grasses such as Arizona fescue. The relationship between Neotyphodium and several native grass hosts ranges from antagonistic to mutualistic. One theory that may explain how Neotyphodium infection is maintained despite inconsistent mutualistic benefit to the host is the bounded hybrid superiority hypothesis. This hypothesis argues that hybrids are more fit than non-hybrids in response to some environmental stresses. Neotyphodium infects hosts in both hybrid and non-hybrid forms. We tested the possibility of hybrid superiority in depauperate habitats (low soil water and nitrate) by quantifying the types and frequency of host infections (uninfected, hybrid-infected and non-hybrid-infected), and the quality of resources available between three host populations. A second theory, the geographic mosaic theory of coevolution, may also explain different symbiotic outcomes at the population level in response to variation in abiotic and biotic population characters. We provide cursory support for both hypotheses. Keywords: geographic mosaic theory of coevolution, hybrid, Festuca, Neotyphodium, symbiosis, mutualism, bounded hybrid superiority
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(445 KB)We examined the effect of endophyte infection, endophyte and host plant maternal genotype on traditional growth parameters. We also measured leaf water potential, leaf rolling, and stomatal density to provide explanations for differences in biomass production and relative growth rates. Our general findings show that Neotyphodium infection, Neotyphodium haplotype and its interaction with host maternal genotype influence Arizona fescue growth, and biomass production. Endophyte haplotype and its interaction with host maternal genotype is the most critical and consistent factor in influencing growth and physiological outcomes. Endophyte-host interactions are likely to be enormously complex because of the genetic and environmental variation that exists in natural populations. The outcome of these interactions in natural grass-endophyte systems is exceedingly difficult to predict based simply on the presence or absence of the endophyte. Keywords: growth rate, haplotype, leaf rolling, plant biomass, water potential, stomatal density
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(535 KB)We have identified three proteins specifically expressed during the interaction between N. lolii and L. perenne in natural compatible associations. Two are pathogenesis-related PR-10 proteins, part of a ubiquitous plant response to pathogens. We have identified seven different L. perenne PR-10 genes and found that endophyte infection increases PR-10 transcript levels, but only mildly. Furthermore, PR-10 mRNA levels also increase in a dysfunctional symbiosis, but the two identified PR-10 proteins are absent. This suggests that part of the regulation of protein expression is at the level of translation and that PR-10 proteins could be required for the symbiosis to be functional. A third symbiosis-specific protein is a very highly expressed N. lolii superoxide dismutase. Such enzymes neutralise superoxide formed by plants as a defence mechanism. Our results suggest that N. lolii elicits a limited host defence, comparable to that elicited by arbuscular mycorrhiza. Keywords: Neotyphodium lolii, Lolium perenne, host response, proteomics, gene expresion, pathogenesis related protein PR-10, fungal superoxide dismutase
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(658 KB)Neotyphodium endophytes in the pooid grasses perennial ryegrass and tall fescue (Lolium spp.) are important ecologically and agriculturally by virtue of their ability to produce several alkaloids and their effects on host growth and responses to biotic and abiotic stresses. Such responses can be positive, neutral, or negative, and depend on the interaction of host genetics x endophyte genetics x environment (and management), which is complex and difficult to predict. Understanding the interactions is key to elucidating the ecological role of endophytes in natural and impacted ecosystems and to optimally select plant and endophyte combinations that support profitable livestock production with stable plant populations. High phenotypic variability among individual plants in an endophyte-infected population can promote population fitness across a wide range of stressful environments. Strong interactions between environmental conditions and host-endophyte combination reveal the importance of testing new grass-endophyte associations in the range of environments targeted for eventual use to determine stability of the desired traits. Care must also be taken when creating new symbiota that an important fitness trait is not lost by selecting endophytes that lack a particular alkaloid. Genetic variations within perennial ryegrass and tall fescue provide opportunities for plant breeders to perform further selections with novel symbiota to exploit host control over growth and physiology and thus overcome some of performance loss. This paper reviews the challenges of understanding grass-endophyte interactions, with particular attention to strategies of improving the performance of novel symbiota for agricultural purposes. Keywords: Lolium, Festuca, Neotyphodium, plant-endophyte interactions, symbiosis
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(288 KB)Neotyphodium endophytes in the pooid grasses perennial ryegrass and tall fescue (Lolium spp.) are important ecologically and agriculturally by virtue of their ability to produce several alkaloids and their effects on host growth and responses to biotic and abiotic stresses. Such responses can be positive, neutral, or negative, and depend on the interaction of host genetics x endophyte genetics x environment (and management), which is complex and difficult to predict. Understanding the interactions is key to elucidating the ecological role of endophytes in natural and impacted ecosystems and to optimally select plant and endophyte combinations that support profitable livestock production with stable plant populations. High phenotypic variability among individual plants in an endophyte-infected population can promote population fitness across a wide range of stressful environments. Strong interactions between environmental conditions and host-endophyte combination reveal the importance of testing new grass-endophyte associations in the range of environments targeted for eventual use to determine stability of the desired traits. Care must also be taken when creating new symbiota that an important fitness trait is not lost by selecting endophytes that lack a particular alkaloid. Genetic variations within perennial ryegrass and tall fescue provide opportunities for plant breeders to perform further selections with novel symbiota to exploit host control over growth and physiology and thus overcome some of performance loss. This paper reviews the challenges of understanding grass-endophyte interactions, with particular attention to strategies of improving the performance of novel symbiota for agricultural purposes. Keywords: Lolium, Festuca, Neotyphodium, plant-endophyte interactions, symbiosis
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(288 KB)The symbiosis between the asexual Ascomycetous fungi of the genus Neotyphodium and host grasses is considered to be symptomless. Symbioses involving Epichloë can show symptoms but this is restricted to the formation of stroma on floral structures. Fungal mycelium of both Epichloë and Neotyphodium endophytes occupy the intercellular space of plant tissues with no breach of host cell walls. Research involving these endophytes and their host grass symbioses relies on staining of host tissues and microscopic examination, immuno-detection or in-vitro isolation to determine infection status. This work reports on the observation of two independent instances of spontaneous change in the endophyte fungus that manifests as changes in the host grass vegetative morphology; one involving a genetically modified Epichloë festucae inoculated into a perennial ryegrass (Lolium perenne) population and the other a resident wild type Neotyphodium lolii. The relationship between wild type and variant fungus is confirmed as is the connection between variant fungus and host morphology. This work demonstrates that spontaneous in-planta changes in fungal endophytes occur and that these can impact on fungus/host grass symbioses. Keywords: Neotyphodium, Epichloë, symbiosis, endophyte, perennial ryegrass
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(803 KB)Early investigations into ryegrass staggers were directed towards the characterisation of tremorgen-producing Penicillium species (Gallagher et al. 1977).
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(362 KB)We used constraint based stoichiometric modelling of metabolic fluxes in Epichloё festucae (FL1), a targeted gene replacement of a non-ribosomal peptide synthetase (termed sidF) from E. festucae, and the symbiotic association of these endophytic fungi and their host Lolium perenne. SidF encodes an excreted ironchelating siderophore and the sidF knockouts (KO) are impaired in their ability to take up iron. After constructing the metabolic network at a genome scale, we applied constraints on enzymatic reactions that require iron as co-factor to study the variations in metabolic network capabilities of the siderophore mutant versus wildtype, in culture and in planta. We compared fluxes calculated for the production of amino acids with observed concentrations of these amino acids in planta. We report a counter-intuitive result from considering metabolism on a systems level in our models. Keywords: stoichiometric metabolic network modelling, flux balance analysis, symbiosis, Neotyphodium lolii, Lolium perenne, Epichloё festucae.
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(270 KB)Many grasses live in association with fungi (Neotyphodium spp. endophytes) growing in the intercellular spaces of the grass. The endophytes produce a range of alkaloids that protect the grass against grazing animals and insects. There has been speculation about the mobilisation and translocation of different alkaloids. We aimed to determine if metabolites produced by endophytes are mobilised and translocated by the plant. We developed a versatile method for the analysis plant fluids, using a linear ion trap mass spectrometer. Cut leaf fluid and guttation fluid of different grass endophyte associations in ryegrass and tall fescue were analysed. Peramine was detected in the cut leaf and guttation fluid of all ryegrass-endophyte associations that are able to produce peramine, but were found only in the cut leaf fluid of the tall fescue association. Lolines and ergot peptides were also detected in some associations. Keywords: Neotyphodium, ryegrass, tall fescue, guttation, peramine, ergovaline, loline
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(260 KB)The association of Epichloë and Neotyphodium endophytes with host grasses is one of balance between both partners. The host plants are symptomless with the exception of stromata that can form on reproductive tillers of grasses infected with Epichloë spp. The hyphae of the endophytes, spread systemically throughout the above ground parts of plants and in leaves, are aligned parallel with the longitudinal leaf axis and are seldom branched. In this review, the reader is guided through the interaction of host grass and endophytes as revealed by microscopy and then given a model to explain the unique association that these endophytic fungi have with host grasses. This model proposes that hyphae within meristematic tissue branch profusely, providing hyphal strands that extend by intercalary growth amongst enlarging plant cells. Hyphae cease branching and elongating when amongst stationary cells but remain metabolically active, producing a range of bioactive products that enhance the persistence of host grasses. By this process the synchronisation of endophyte and plant growth is achieved, providing a generally mutualistic relationship. Keywords: Neotyphodium, Epichloë, intercalary hyphal extension
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(5903 KB)
The AR37 endophyte strain in ryegrass produces epoxyjanthitrems but no peramine, lolitrem B or ergovaline, associated with the wild-type (HE) endophyte. An experiment commenced in 2005 to measure dairy cow health and production responses to AR37 in ryegrass. Four herds of 15 cows grazed on each of three endophyte treatments (AR37, AR1, HE and endophytefree (Nil) control) for 12 days in spring 2005 and autumn 2006, and in summer 2006 four groups of 10 cows were fed indoors to accurately assess cow dry matter and alkaloid intakes. Milk yields (kg/cow/day) were similar in spring but in autumn were lower (P<0.001) for cows fed ryegrass containing HE (11.8) than for those fed AR37 (14.1), AR1 (14.9), and Nil endophyte (14.2) ryegrass. Mild clinical signs of ryegrass staggers were noted in 67% of cows fed HE ryegrass in autumn. In summer, ryegrass staggers occurred in all cows fed HE ryegrass and average milk production was lower from these cows (10.9 kg/cow/day) than from those fed AR1 (12.9) and nil (13.5), but not significantly different from cows fed AR37-infected ryegrass (11.6 kg/cow/ day). No deleterious effects of AR37 on animal health were observed in any season. This experiment is ongoing and a more definitive evaluation of the AR37 endophyte is anticipated. Keywords: novel endophytes, milk production, cow health, cow intake, season
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(239 KB)Tall fescue use has been limited in the south central USA because of insufficient persistence of endophyte free cultivars and poor animal performance from tall fescue infected with wild-type endophyte. Here we report on the development of a tall fescue – novel endophyte combination with improved adaptation to the south central USA. A tall fescue ecotype from Oklahoma was infected with several novel endophytes. Small plot trials were conducted in Oklahoma and Texas to evaluate grazing tolerance, forage yield, and animal safety. One of the ecotypenovel endophyte combinations (AGRFA 144) had greater persistence and forage yield than Jesup MaxQ® in a number of environments. Lamb weight gains from the new tall fescuenovel endophyte combination were comparable to that of Jesup MaxQ and substantially greater than KY-31 infected with a toxic endophyte. Cultivar release of the new tall fescue-novel endophyte combination in 2008 is anticipated. Keywords: novel endophyte, tall fescue, cultivar
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(219 KB)Little research has evaluated possible endophyte benefits to adaptation and production of grasses in the irrigated pastures of the semiarid, cold-desert environments of the western USA. Severe irrigation shortages are common; however, production demands are increasing, necessitating maximizing tall fescue’s (Festuca arundinacea Schreb.) productivity when grown in sub-optimal conditions including drought, salinity, and cold temperatures. In a field study under irrigation in the Intermountain Western USA, the yield advantage to Kentucky 31 tall fescue infected with wild-type Neotyphodium over endophyte-free Kentucky 31 was greatest (over 15%) when irrigation was severely limited to natural precipitation. In an evaluation of salinity tolerance there were no significant differences in plant LD50 values due to endophyte infection. In a recent study Jesup MaxQ recovered better from winter injury than Jesup E– when grown in a high-elevation colddesert, irrigated environment. These studies suggest the need for additional research to elucidate the potential advantages of wildtype and novel endophytes for tall fescue production in irrigated environments typical of the semiarid western USA. Keywords: tall fescue, Festuca arundinacea Schreb., Neotyphodium, drought tolerance, salinity tolerance, winterhardiness
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(233 KB)Italian (Lolium multiflorum) and short-term hybrid ryegrasses (L. boucheanum) are utilised by farmers for their reliable establishment and yield potential for winter feeding, but lack summer production and persistence. To improve the performance of these ryegrasses, endophyte (Neotyphodium lolii) strains from perennial ryegrass were inoculated into several cultivars and evaluated for agronomic performance in the Northland region of New Zealand. ‘Moata’ tetraploid Italian ryegrass families usually showed a reduction in yield when infected with the AR5 strain compared with endophyte-free Moata. Infection of the Italian ryegrass cultivars ‘Corvette’ and ‘Status’ with the N. lolii endophytes strains, AR1 and more particularly AR37, usually increased plant production and persistence. Natural infection with the N. occultans endophyte also improved plant performance. Improved plant performance was most closely associated with reduced damage from African black beetle. Interactions between plant line/cultivar and endophyte strain offer the potential for plant breeders to provide enhanced endophyte-infected cultivars of Italian ryegrasses to farmers. Keywords: endophyte, Neotyphodium lolii, Neotyphodium occultans, Italian ryegrass, Lolium multiflorum, dry matter yields, plant persistence, insect damage
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(477 KB)Angus-cross steers grazed wild type endophyte-infected (E+), nonergot alkaloid-producing endophyte-infected (AR542), or endophyte-free (E-) tall fescue pastures at two locations. Twenty-four animals per location were transported to a research feedlot after the completion of the grazing phase and fed for 100 d. Grazing average daily gain (ADG) was 0.47 kg/d greater (P=0.001) for cattle grazing AR542 than those grazing E+ pastures. In the feedlot, overall ADG did not differ among AR542 and E+ (P=0.40) or AR542 and E- (P=0.19), and averaged 2.09 + 0.15 kg/d across all treatments. Feed efficiency was greater (P=0.02) for E+ than AR542. Hot carcass weight was 40 kg greater (P=0.01) for AR542 than E+. Other carcass traits did not differ (P≥0.10) among treatments. Net present values (NPV) were $177.08/ha for E- and $336.76/ha for AR542 compared to E+. Keywords: beef, endophyte, carcass
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(231 KB)Tests are required to monitor contamination of AR1-infected seed with ryegrass seed infected with the common toxic endophyte to ensure contamination is maintained below 5% for First Generation seed and below 2% for Breeders and Basic seed. To achieve this AR1 seed is tested for the presence of lolitrem toxins that are produced by the endophytic fungus in toxic seeds. This was done by HPLC analysis until 2006 when an ELISA method, more suited to processing large sample numbers, was successfully established as a replacement. The ELISA is specific for lolitrems and lolitriol, provides rapid sample turnaround and analytical costs have been reduced. It is anticipated that in the future the test will be transferred to a commercial testing laboratory and will be extended to monitor contamination of AR37 seed with common toxic ryegrass seed. Keywords: AR1 ryegrass, wild-type endophyte, lolitrem, ELISA, quality assurance
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(269 KB)Perennial ryegrass containing a high level of standard (wild) endophyte is a significant component of the feed base for the grazing industries in the high rainfall and irrigated regions of Australia. The AR1 selected endophyte was first commercialised in Australia by Heritage Seeds in autumn 2002 into a market that had low awareness, and a limited understanding, of perennial ryegrass endophyte. The early uptake of AR1 was slow, mainly due to difficulties with delivering a very technical message with limited resources and justifying a significant price increase with no independent supporting data from Australia. Changing the marketing message to focus on the potential benefits from AR1, along with support from the developers of AR1 (AgResearch) and an overall increase in awareness of ryegrass endophyte, saw a dramatic increase in the uptake of AR1 varieties in 2005 and 2006. It is expected that this trend toward selected endophytes will continue into the future. Keywords: perennial ryegrass, endophyte, AR1, commercialisation
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(252 KB)Quality assurance and quality control is the foundation of any diagnostic test. The two laboratories in the United States that use HPLC to quantitate endophyte toxins in tall fescue (Festuca arundinacea) and perennial ryegrass (Lolium perenne) are Oregon State University (OSU) and University of Missouri (MU). Japan, the major importer of grass straw has six new laboratories that will test agricultural imports for endophyte toxins. A quality assurance program was set up between the Japanese Ministry of Agriculture and the State of Oregon. The latter includes both OSU and the MU. All units are using an accurate crystalline standard and have exchanged “check” samples among themselves. To date OSU and MU have values that differ by 10%. OSU has identified a contaminating and coeluting peak as the cause of the differences. Both laboratories are changing to a Gemini column to rectify the differences. Japanese laboratories are in the process of evaluating their split check samples. Keywords: quality assurance, quality control, endophyte, tall fescue, perennial ryegrass
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(745 KB)Seed Certification and Seed Testing Schemes provide the rules and guidelines to maintain both varietal genetic purity and physical purity during field production and seed processing. However the Seed Certification agencies are not involved in ensuring that the quality of the endophytic component of seed is maintained. The delivery of quality endophytic seed to end users therefore requires that the commercial seed entity that is producing and marketing a novel endophyte product develops and maintains strict production and quality assurance guidelines to ensure that the benefits of novel endophyte technology are realised. The paper uses the New Zealand (NZ) experience with AR1 endophyte in ryegrass and the USA experience with MaxQ® Jesup tall fescue to highlight the quality control steps required to deliver a quality product to end users. Keywords: novel endophyte, tall fescue, ryegrass, Neotyphodium, MaxQ, AR1, seed production
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(297 KB)Neotyphodium endophytes are obligate vertically-transmitted endo-symbionts of Lolium multiflorum grass. Thus, the success of endophyte is tied with the success of the seed. Here we present results from experiments aiming to test the effect of endophyte infection on seed viability and germination vigour. Endophyteinfected and non-infected seeds of three L. multiflorum accessions were compared under accelerated ageing conditions (40ºC and ≈15% moisture content). Endophyte negatively affected seed viability and seed germination vigour in only one of the evaluated accessions. The effect of endophyte presence on seed quality in pasture grasses could depend on the population genetic background, and be related to direct or indirect effects of fungal infection on seed morphological or physiological characteristics. Keywords: Lolium multiflorum, Neotyphodium endophyte, seed quality, accelerated ageing
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(317 KB)Pasture growth, botanical composition and sheep grazing preference were measured over 20 months in tall fescue (cultivar Advance), without endophyte (Nil) or infected with AR542 (MaxPTM) endophyte, and clover pastures sown into a dryland soil, Canterbury, New Zealand. Pastures were rotationally grazed with sheep, with grazing preference for the two endophyte treatments measured in late autumn and early spring. Annual dry matter production from April 2004 to April 2005 was not significantly different between AR542 (6293 kg DM/ha) and Nil (5864 kg DM/ha) tall fescue. The number of tall fescue plants/m2 and their basal diameter was greater for AR542 (35 plants/m2, 7.5 cm diameter) than Nil endophyte tall fescue (28 plants/m2, 6.8 cm diameter). AR542 endophyte tall fescue pastures had fewer weeds, mainly annual grasses, than Nil endophyte pastures throughout the trial. Grazing preference, measured by the number of sheep grazing each plot, and decline in pasture height did not differ between Nil and AR542 tall fescue. Keywords: tall fescue, novel endophyte, AR542, botanical composition, weed, grazing preference
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(427 KB)Baling of perennial ryegrass (Lolium perenne) and tall fescue (Festuca arundinacea) straw residue following seed harvest has become a common practice in Oregon’s grass seed industry. In 2005, almost 670 000 tons [607 000 Mg (tonne)] of grass straw was exported to Japan and Korea for use as livestock feed. Most of the grass seed varieties grown today are turf-type, which in recent years have been developed with high levels of fungal endophyte infection. Currently, over 73% and 43% of perennial ryegrass and tall fescue varieties, respectively, are ranked as having a “High” (>61% infected) infection level. Concern for the safe use of these grass straw residues had led to their routine analysis for toxin content before shipment to consuming markets. These chemical analyses provide assurances against deleterious effects to livestock fed Oregon grass straw. Keywords: straw, endophyte-infected, perennial ryegrass, tall fescue
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(458 KB)Commercial seed lots of Flecha/AR542, Jesup/AR542, Jesup/ AR584, and Grasslands Advance/AR542 were stored at 25oC for 18 months
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(80 KB)Forage grass seed quality is influenced by plant genetics as well as seed density, storage conditions, age of seed, and endophyte infection status. We used eight seed lots of tall fescue (three cultivars, with natural endophytes, endophyte-free, or novel endophyte) to test the effect of seed density on germination and seedling vigour. Seed lots were separated into nine density fractions using a cylinder air column. Thousand seed weight values were determined. Three runs of four replications of plots consisting of 25 seeds of each entry were seeded in greenhouse experiments to determine seedling mass at 8 weeks after planting. Early germination counts as well as laboratory germination tests indicated significant differences among weight classes in speed of germination as well as seedling vigour. The lightest fraction of seed clearly contained inferior seed, but higher density seed lots tended to have better germination and seedling vigour than lower density ones. Keywords: seed density, seedling vigour, germination, tall fescue, endophyte
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(232 KB)Recent commercial development of tall fescue containing a nontoxic endophyte has renewed producer interest in replacing stands of toxic tall fescue
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(156 KB)Release of the cultivar ‘Kentucky 31’ in 1943 led to widespread planting of tall fescue in the USA. An endophytic fungus (Neotyphodium coenophialum) usually present in this grass produces compounds that enhance competitiveness and survival of tall fescue in many environments, but it also produces ergot alkaloids implicated in the cause of animal disorders. Screening of hundreds of fungus strains identified some that do not produce high levels of ergot alkaloids. A “novel” endophyte strain was inserted into University of Georgia-developed cultivars ‘Georgia 5’ and ‘Jesup’ and became commercially available under the name MaxQTM in 1999. It is estimated that at present there are some 121,000 hectares of novel endophyte tall fescue in at least 35 USA states. Persistence has been good and animal performance has been excellent. It is expected that novel endophyte tall fescue area will continue to increase in the USA in the future. Keywords: novel endophyte, tall fescue, Neotyphodium, MaxQTM
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(227 KB)The on-farm impacts of non-toxic tall fescue endophyte technology have only been partially realised in the United States. Based on current tall fescue acreage, the potential for non-toxic endophyte-infected tall fescue products is immense. However, 6 years after the introduction of the first non-toxic endophyte tall fescue technology to the U.S. market, less than 1% of the total tall fescue acres in the U.S. have been renovated using this technology. Success with leading producers that influence other producers, educational partnerships, and strong product advertising are factors favouring adoption of this technology. Factors slowing this technology adoption include key markets lacking adequate marketing and technical support, lack of producer awareness and inaccurate perceptions about the technology, and acreage moving out of pasture and into alternative enterprises including urban development. Rapidly changing social, political, and economic conditions have the potential to drive profit-minded producers to give more consideration to forage technology adoption. Keywords: farm level, technology application, technology adoption
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(553 KB)To investigate the interaction between Argentine stem weevil (Listronotus bonariensis), its parasitoid Microctonus hyperodae, fungal endophyte (Neotyphodium lolii) and its grass host, four endophyte-ryegrass (Lolium perenne) treatments were established on four farms in Wairarapa in autumn 2003. A diploid ryegrass contained either wild-type or AR1 endophyte or was endophytefree, while a tetraploid ryegrass contained AR1 endophyte. Wild-type, AR1 and endophyte-free ryegrasses average 52%, 91% and 2% endophyte-infected tillers for the 3 years of measurement. Moderate increases over time in the proportion of wild-type infected tillers indicated selection pressures favoured endophyte-infected tillers. Endophyte-free tiller populations were 11% lower than AR1 or wild-type but not significantly so. L. bonariensis populations were highest in endophyte-free and wild-type pastures and 80% lower in AR1 pastures. Mean rates of parasitism in L. bonariensis were lower in spring (9%) than autumn (35%), tended to be higher in wild-type than in AR1 diploid ryegrass, and only had a weak relationship with percent endophyte-infection. Keywords: endophyte, Neotyphodium lolii, Lolium perenne, dry matter yields, plant persistence, Listronotus bonariensis, Microctonus hyperodae
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(498 KB)The adoption of novel endophyte ryegrass in New Zealand has been successful and rapid, with AR1 novel endophyte seed representing 80% of the total ryegrass seed infected with endophyte within 3 years of release. Success was underpinned by a large amount of research into endophytes and the animal production benefits of novel endophytes. Awareness amongst farmers and seed retailers was created by scientists and seed company staff, often with an organised and collaborative campaign. The domination of the market with novel endophytes is likely to increase further with the on-farm success of AR1, and the recent introduction of third-generation endophytes. Keywords: endophyte, ryegrass, technology, transfer, adoption, knowledge, awareness, benefits, validation, research
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(213 KB)‘Grasslands Flecha’ tall fescue exhibits a partial degree of endogenous summer dormancy, a trait that contributes to plant survival during the long, dry summers of Mediterranean regions. Little is understood of the physiological mechanisms involved in summer survival of summer-dormant fescues and the possible role of endophytes. The influence of infection by endophyte strain AR542 in enhancing summer survival in ungrazed field plots and dehydration survival in greenhouse pots was investigated. Tiller population counts from mid-summer drought and autumn recovery showed no significant endophyte effect on survival. Results from the pot trial showed no differences between endophyte-free and endophyte-infected populations for rate of leaf dry-down, tiller-base water content, membrane leakage, or percentage tiller survival. Endophyte-free Flecha exhibited excellent summer survival, suggesting that endophyte infection may not be as critical for stand survival in summer dormant fescues as it is in summer active types. Infecting Flecha with novel endophytes may provide additional insurance against biotic stresses, thereby offering greater overall fitness across a range of environments. Keywords: drought, tall fescue, tiller survival, membrane damage, senescence
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(370 KB)Bacon and others discovered the tall fescue endophyte in 1977. Since that time, North American researchers have developed several endophyte-based technologies to reduce fescue toxicosis. The most recent has been the release of MaxQ, a novel endophyte that enhances persistence but does not cause fescue toxicosis. Despite its advantages, MaxQ has been adopted slowly across the fescue belt of the US. This paper presents possible social and managerial reasons for this slow adoption. It closes by offering some possible solutions. Keywords: MaxQ, novel endophyte, tall fescue toxicosis, pasture renovation, educational efforts
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Tall fescue (Festuca arundinacea) and perennial ryegrass (Lolium perenne) are perennial cool-season grasses which are infected with the endophytic fungi, Neotyphodium coenophialum and N. lolii, respectively. These endophytes have been increasingly selected for, as they confer benefits such as pest resistance and drought tolerance to the plant. However, livestock grazing endophyte-infected (E+) grasses are negatively impacted by fungal ergot and lolitrem alkaloids, which are responsible for a variety of mammalian diseases including fescue toxicosis (summer syndrome, fescue foot and fat necrosis) and ryegrass staggers. Most likely, not all of the ergot alkaloids involved in fescue toxicosis have been identified to date. During sample processing for the diagnostic analysis of the endophyte toxin ergovaline, other unidentified peaks occasionally elute that appear to coincide with clinical disease. Analysis of unidentified chromatographic peaks was performed on feed samples by LCMS/ MS to determine their chemical structures and identities. Ergotamine, ergovaline, ergocornine, ergonovine, ergocryptine, ergocrystine and lysergol appeared in various samples and matched controls. Newly identified compounds included ergosine, ergostine and ergoptine. In addition, several samples showed one or more of fourteen new ergots ranging in size from 381-611 mw, with key mass spectral characteristics of ergot alkaloidsspecifically, m/z 223 and 208 corresponding to the ergoline ring system and its demethylated variant, respectively. Once ingested, ergot alkaloids are thought to be metabolised in the rumen and/ or liver of livestock species. Ruminal metabolism of ergovaline was studied in sheep offered Neotyphodium coenophialuminfected tall fescue straw at two ergovaline levels (<10 ppb and 500 ppb) for 28 days. Ergovaline concentration in rumen fluid expressed as a percent of intake increased over sampling time and sampling day (P<0.05). Lysergic acid concentration in rumen fluid expressed as a percent of intake increased over time from day 0 to day 3 (P<0.05) but was not different between day 3 and day 28 at any time point (P>0.10). The faeces contained an average of 0.41 μmol/day ergovaline and 0.87 μmol/day lysergic acid. Urine contained no detectable ergovaline; lysergic acid concentration was 0.213 μmol/day. The appearance of lysergic acid in the faeces, urine and rumen fluid is most likely due to the degradation of ergovaline in the rumen from microbial degradation and further break down in the lower digestive tract. Hepatic metabolism was studied using a mouse model, where the in vitro metabolism of ergotamine in mouse liver microsomes was characterised by LC-MS/MS. Microsomal incubations produced nine predominate peaks which were confirmed to be ergotamine, ergotamine epimer, monohydroxylated metabolites (M1, M2, M1e, M2e) and dihydroxylated metabolites (M3, M4, M5). Keywords: tall fescue, perennial ryegrass, endophyte, ergot alkaloids, metabolism, livestock
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(897 KB)A defensive role for several of the major alkaloids of Neotyphodium spp. endophytes in forage grasses has been confirmed in genetic experiments. However, many aspects of the activity of endophyte-infected grasses towards herbivores remain unaccounted for, suggesting there are further anti-herbivore factors yet to be uncovered. The development of metabolomics has provided new tools for the discovery of novel metabolites without preconceptions of the likely chemical structural class. Prospects for the application of new methods from metabolomics to further elucidate the defensive chemistry of associations of Neotyphodium spp. endophytes with their grass hosts are reviewed. Keywords: Neotyphodium, Epichloë, defensive chemistry, invertebrate, vertebrate, gene deletion
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(875 KB)Vasoconstriction is one response associated with consumption of toxic endophyte-infected tall fescue. Because it is not known if endophyte-produced alkaloids act alone or collectively, the objective of this study was to examine the vasoconstrictive potentials of D-lysergic acid, ergovaline, and N-acetylloline individually or in paired combinations using bovine lateral saphenous veins biopsied from fescue naïve cattle. Segments (2- 3 cm) of vein were surgically biopsied from healthy Brahmaninfluenced yearling cattle (n=22; 330 ± 8 kg). The data indicated that ergovaline is a more potent vascular toxicant than lysergic acid and N-acetylloline. Further, the presence of N-acetylloline did not inhibit or potentiate the effects of the ergot alkaloids on vascular activity. The contractile responses of the remaining combinations did appear to differ from the individual concentration responses. This supports the possibility that a combinatorial or repetitive alkaloid exposure effect may exist in vivo and should be considered during in vitro evaluations of ergot alkaloids. Keywords: fescue-naïve cattle, fescue toxicosis, vasoconstriction
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(282 KB)Tall fescue is the primary pasture forage offered to goats in the southeastern United States. Data on how tall fescue endophyte (Neotyphodium coenophialum) affects meat goat performance are lacking. In three summers, yearling does were fed diets containing tall fescue seed to assess the effect of endophyte infection on goat growth and feed intake. In 2004 and 2005, does were fed endophyte-infected (EI) or endophyte-free (EF) tall fescue seeds added to the diets for 10 wk. Feed refusals were weighed daily. The EI diet reduced (P <0.05) weight gain and increased (P<0.05) feed refusals during each year. In 2006, feed refusal values of EI pens were used to adjust feed offering to EF pens daily. When feed intake was equalised daily during treatment, weight gain was still reduced by the EI diet. These data suggest that endophyte infection in tall fescue can adversely affect meat goat performance. Keywords: goats, tall fescue, endophyte, weight gain, feed intake
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(322 KB)Plant-endophyte associations can have major impacts on the dynamics of consumer interaction-webs but long-term effects of mycotoxins and the ability of herbivores to adapt to these toxins have not been studied. To understand the potential of aphids to cope with mycotoxins, we compared the life-history parameters for aphids conditioned for several generations on endophyte-infected plants with those of endophyte-naïve aphids on both endophyteinfected and endophyte-free grasses. Aphids conditioned on endophyte-infected plants produced more offspring during the first days of adulthood than endophyte-naïve aphids independent of the endophyte infection of the test environment. However, the endophyte-conditioned aphids tended to have a shorter lifespan, which resulted in similar numbers of total offspring produced for endophyte-conditioned and endophyte-naïve aphids. The difference in life-history parameters caused by the conditioning environment suggests that the effects of endophytes on herbivore life-history traits may represent an adaptive change that should be considered in future studies of endophyte-herbivore interactions. Keywords: Rhopalosiphum padi, Neotyphodium lolii, Lolium perenne, common strain, adaptation, long-term effects, microbes, endosymbionts, reproductive strategy, life-history traits
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(475 KB)Cereal leaf beetle (CLB), Oulema melanopus (L.) (Coleoptera: Chrysomelidae), a long known pest of small grains in Europe, was first recorded in North America (Michigan) in 1962.
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(167 KB)There are multiple vehicles for endophyte toxicosis in animals including exposure from pasture, straw residues and seed screenings. This report discusses the clinical cases typically seen with tall fescue and perennial ryegrass toxicosis in Oregon and Japan. Case I involves a herd of 330 Black Angus cattle. Before the March calving season the owner wished to increase the protein content of the feed ration by feeding pellets made of seed screenings and grass hay. Forty two animals were lost to tall fescue toxicosis and dry gangrene of the feet and legs. Case II involves 1300 beef cows in Eastern Oregon fed grass straw; 485 animals were lost due to dry gangrene characteristic of tall fescue toxicosis. Case III describes 4 of 15 cases of both tall fescue and perennial ryegrass toxicosis in Japanese black cattle. Case IV involves llamas and alpacas on pasture and lawn paddocks where some animals were affected by tall fescue and some by perennial ryegrass. Keywords: tall fescue, Festuca arundinacea L., perennial ryegrass, Lolium perenne, endophyte, Neotyphodium coenophialum, Neotyphodium lolii
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(553 KB)Perennial ryegrass (Lolium perenne) straw is used as a feed for livestock and horses. Some straw is infected with the endophyte, Neotyphodium lolii, which produces lolitrem tremorgens. Ingesting of the toxin produces clinical “ryegrass staggers.” A 28 day feeding trail was conducted using 61 Black Angus, pure and crossbred cattle. The cattle were fed one of three levels of lolitrem B in chopped perennial ryegrass straw ranging from 0 ppb up to 3058 ppb. Four Japanese Wagyu crossbred cattle were fed chopped ryegrass straw containing 1400 ppb lolitrem B. The cattle were evaluated and scored twice a day for clinical signs of gait difficulties. Cattle consuming 1400 ppb lolitrem B did not show any overt clinical signs. After 14 days, 7 out of 15 cattle in the 1954 ppb lolitrem B group showed clinical signs of ataxia, stiffness, and tremors as seen in ryegrass staggers. All of the cattle in the positive control group of up to 3058 ppb lolitrem B with a 4 week mean of 2574 ppb developed clinical signs of staggers. None of the Wagyu cattle at 1400 ppb lolitrem B developed clinical signs. A dose response curve was developed based on the results of this study coupled with a prior study for use in the testing laboratory. Keywords: lolitrem B, perennial ryegrass, Lolium perenne, endophyte fungus, Neotyphodium lolii, cattle, threshold levels
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(412 KB)Fescue toxicosis in cattle is characterised by a reduction in feed intake and hyperthermia during the summer in Southeast to Midwest regions of the United States. Several studies were performed using these endpoints to evaluate different administration routines that might produce a reliable model for this condition. Endophyte-infected (E+) or uninfected (E- ) fescue seed was administered in the standard diet as ground or whole seed during thermoneutral and heat stress periods to determine which seed form produced the greater response in different thermal environments. Time of administration was also tested by comparing response to whole seed administered either before or during heat stress. In each study, a high and low level of ergovaline in the diet was evaluated to identify potential dose differences. Ground E+ seed at either level produced a rapid decrease in feed intake in both environments, with no effect on core body temperature. Whole E+ seed treatment prior to heat stress resulted in a greater daily rise in core body temperature than E- with no effect on feed intake. In contrast, whole E+ seed administration during heat stress produced a dose-dependant upward shift in body temperature and reduction in daily feed intake to suggest that this is the more reliable routine for generating these characteristics of fescue toxicosis in cattle. Keywords: fescue toxicosis, cattle, heat stress
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(738 KB)Feed intake of rats may serve as a sensitive respondent to toxins found in endophyte-infected fescue. Unfortunately, there have been few controlled studies of feed intake to create a reliable model. In the present study, rats were fed diets containing different amounts of endophyte-infected fescue (E+) and ergovaline (EV; a primary toxin associated with fescue toxicosis) to develop a dose-response curve for feed intake. Dose-dependent reduction in feed intake was observed, with a decrease even at the lowest concentration of EV. Involvement of ergotamine (i.e. another toxin in E+) in fescue toxicosis-induced reduction in intake was evaluated by adding it to diets. In contrast to EV treatment, there was only a small reduction in feed intake with ergotamine, followed by rapid recovery to pretreatment levels. A more effective paradigm for evaluating the effect of different toxins on feed intake and potential treatments may be a restricted feed regimen. Rats placed on a 2-h restricted-feed program of ground commercial chow followed by E+ diet show a decrease in both feed intake and growth. Future studies will refine this routine to identify specific effects of toxins found in endophyte-infected fescue on feed intake. Keywords: rat, fescue toxicosis, feed intake
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(747 KB)Fescue toxicosis and ergot alkaloids are estimated by the United States Department of Agriculture (USDA) to cost the U.S. ranchers $860 million per year. The endophyte fungi Neotyphodium coenophialum is present in tall fescue (Festuca arundinacea). This fungus produces toxic alkaloids (principally ergovaline) that cause deleterious effects in cattle and other livestock when infected grasses are eaten. Previous studies in sheep and cattle have yielded no culturable microbes able to detoxify the ergot molecules, leading to the search for novel sources of anaerobic organisms capable of degrading ergot alkaloids. Anaerobic incubation of earthworm (Eisenea fetida) homogenate with pure ergovaline has exhibited a 60% decrease in ergovaline concentration. Digestive tracks were dissected from worms obtained from an endophyte-fed digester. Cloning and partial sequencing of bacterial 16S rDNA obtained from these samples revealed a diverse microbiota, including bacteria sequences which classified into the phyla Bacteroidetes, Firmicutes, Proteobacteria, and Planktomycetes. Future work is focused on refining our understanding of the contribution towards possible ergot alkaloids detoxification made by the bacteria of the worm-gut microbiota. Keywords: ergovaline, microbial degradation, endophyte, tall fescue, perennial ryegrass, Eisenea fetida
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(404 KB)Doppler ultrasonography was used to measure blood flow dynamics in the caudal artery of heifers that were fed diets with either endophyte-infected (E+) or non-infected (E+) tall fescue (Festuca arundinacea Schreb.) seed. Heifers were assigned to six pens and during an adjustment period were fed alfalfa hay for 5 d, and then the hay plus a concentrate with E- tall fescue seed for 9 d. An 11-d experimental period followed with three pens being changed to hay plus concentrate with E+ seed. Caudal artery area in E+ heifers had declined by 4 h from the initial feeding of the E+ diet before stabilising at the lower level between 4 and 28 h. Heart rates followed a similar trend. Blood flow rate was lower in E+ heifers from 28 h onwards. Results indicated that vasoconstriction can occur in cattle within 28 h from initial consumption of toxic fescue. Keywords: tall fescue, fescue toxicosis, cattle, vasoconstriction
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(280 KB)Young merino ewes were offered a pelleted ration containing approx 15% perennial ryegrass seed, either infected or not infected with wild endophyte. Each group was split so that 10 ewes received the toxin deactivation product, Elitox®. Groups were fed indoors on treatment for 4 weeks in winter (average daily temperature, 11.5ºC). Analysis for endophyte toxins indicated intakes of 63 μg/kg LW/day ergovaline and 23 μg/kg LW/day lolitrem B. Increases in respiration rate and rectal temperature were obvious after 1 week. Respiration rate more than doubled for ewes ingesting toxin-containing wild endophyte. Differences were greater with observations taken at 9 pm, compared with those taken at 9 am. The intake and liveweight gain of ewes ingesting endophyte increased relative to those on the endophyte-free diet. Administration of Elitox®, resulted in a significant interaction: respiration rate and intake increased in the absence of endophyte, but decreased in its presence. Keywords: Merino sheep, perennial ryegrass, lolitrem B, ergovaline, Elitox®, intake, respiration rate, faecal moisture
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(357 KB)Tall fescue, meadow fescue and ryegrass seeds with and without endophyte infection were fed to third-instar grass grub to determine the relative effects of different endophytes on consumption. Treatments were: tall fescue without endophyte or infected with four novel endophytes (AR514, AR542, AR584, ES), meadow fescue without endophyte or infected with Neotyphodium uncinatum and perennial ryegrass infected with a novel endophyte AR37. Grass grub larvae were initially given hard seed but when they failed to eat this, moist soil was added to allow seed to soften and germinate. After 7 days, all endophytes had reduced feeding compared to their endophytefree counterparts. At the completion of the trial, 15 days after adding moist soil, only AR37 in perennial ryegrass had no effect on damage to the seed. Of the endophytes in tall fescue, seeds containing AR542 were significantly more damaged than other endophyte treatments. Composition of loline alkaloids may be important in reducing feeding. Keywords: tall fescue, meadow fescue, ryegrass, endophytes, loline alkaloids, seed damage, grass grub, Costelytra zealandica
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(225 KB)The nature of plant-endophyte relationships may be affected by the addition of interacting species. Firstly, we show how the root hemiparasitic plant (Rhinanthus serotinus) can steal defending mycotoxins produced by the symbiotic endophytic fungus (Neotyphodium uncinatum) living within their shared host grass (Lolium pratense). The uptake of defensive mycotoxins from the endophyte infected host increases resistance of the hemiparasitic plant to the aphid (Aulacorthum solani). Endophyte infection increased performance of the hemiparasite but reduced the growth of the host grass, changing the mutualistic endophytic fungus to parasitic. Secondly, we present the interactions among endophyte, host plant, aphid herbivore (Rhopalosiphum padi) and aphid transmitted grass virus (BYDV). Endophyte infection lowered the frequency of BYDV in L. pratense. The reproduction of R. padi aphids was decreased on endophyte-infected plants compared to uninfected. With these results we would like to emphasise the importance of experiments with several community members to shed light on the complexity of endophyte-mediated community interactions. Keywords: Lolium pratense, Neotyphodium uncinatum, Rhinanthus serotinus, BYDV, aphid, multitrophic interactions
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(269 KB)Endophytes are known to increase the resistance of their host plant to voles directly through reduced palatability
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(134 KB)The objective of this study was to relate chemical components of pastures containing tall fescue suspected to be causative to reduced conception and early embryonic death in the mare. Pasture sampling was conducted on 143 pastures and over 500 sites during mare breeding. Tall fescue was 16- 42% of the forage in most pastures and about 65% of the plants were infected with Neotyphodium coenophialum. Ergovaline content ranged from 0 to 2220 ng/g. Lysergic acid was present in equal or lesser amounts than ergovaline. Approximately 10% of the mares did not conceive or had early foetal loss. Because of other components in the diet, a direct cause/effect relationship cannot be made between mare response and N. coenophialum related toxins. These results suggest that a large number of animals will be required when designing an experiment to show how tall fescue might affect reproduction in mares. Keywords: horse pastures, alkaloids, minerals
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(318 KB)How does diversity in plant cultivars and endophyte infection affect higher trophic levels? We manipulated the number of cultivars (1 or 4) and the endophyte infection (-E, +E, and both, –E and +E) of potted Lolium perenne plants and left aphids and their parasitoids to assemble naturally. Aphid number and plant biomass were not influenced by our treatments, while the number of parasitised aphids (mummies) was significantly higher on mixed plant stands than on monocultures. The effect of endophytes was stronger in mixed plant stands than in monocultures with the most mummies found in endophyte-free mixed plant stands. Although number of mummies did not differ among cultivars, the rate of parasitism varied with cultivar and showed an endophyte x cultivar interaction. The number of successfully emerging parasitoids was also higher on high diversity treatments than on monocultures, indicating that increased diversity at resource levels translates to increased abundance at consumer levels. Key words: fungal endosymbionts, biodiversity, genetic diversity, multitrophic interactions, insect food webs, insect density, Neotyphodium lolii
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(273 KB)The objective was to determine whether leptin played a role in abnormal reproductive function in ruminant females fed endophyte-infected (EI) tall fescue. Cow-calf pairs grazed endophyte-free (EF) or EI tall fescue starting in mid-April (early spring) for 2 years and yearling ewes were fed an EF or EI fescue seed diet for up to 37 days to examine signs of fescue toxicosis and measure serum concentrations of leptin. Signs of fescue toxicosis were present in EI fed cows and ewes. Serum concentrations of leptin were reduced in cows grazing EI compared with EF fescue from mid- to late May in the first year grazing trial, but not the second and were similar between EF and EI-fed ewes. Leptin concentrations also were reduced in cows which grazed EI fescue in the first year and lost a pregnancy before 126 days of gestation compared with cows that maintained. The mechanisms of increased embryonic losses in cows experiencing fescue toxicosis is not known, but may be associated with reduced nutritional status of the pregnant animal. Keywords: cows, ewes, fescue, leptin, reproduction
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(255 KB)The objective was to determine whether leptin played a role in abnormal reproductive function in ruminant females fed endophyte-infected (EI) tall fescue. Cow-calf pairs grazed endophyte-free (EF) or EI tall fescue starting in mid-April (early spring) for 2 years and yearling ewes were fed an EF or EI fescue seed diet for up to 37 days to examine signs of fescue toxicosis and measure serum concentrations of leptin. Signs of fescue toxicosis were present in EI fed cows and ewes. Serum concentrations of leptin were reduced in cows grazing EI compared with EF fescue from mid- to late May in the first year grazing trial, but not the second and were similar between EF and EI-fed ewes. Leptin concentrations also were reduced in cows which grazed EI fescue in the first year and lost a pregnancy before 126 days of gestation compared with cows that maintained. The mechanisms of increased embryonic losses in cows experiencing fescue toxicosis is not known, but may be associated with reduced nutritional status of the pregnant animal. Keywords: cows, ewes, fescue, leptin, reproduction
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(255 KB)Neotyphodium coenophialum (Ascomycota: Clavicipitaceae) is an endophytic fungus of grasses that produces alkaloids which can have detrimental effects on some insect herbivores, like fall armyworm caterpillars (Spodoptera frugiperda). Alkaloid production can be influenced by fungal genotype. We investigated if endophyte-produced alkaloids might also have effects at the next trophic level. We tested if variation in fungal isolate (three isolates: CS, AR502, AR542; plus a control lacking fungal infection) affected the growth and survival of Euplectrus comstockii (Eulophidae) parasitoids of S. frugiperda. Euplectrus comstockii is an ectoparasitoid of caterpillars of many noctuid species. Parasitoid pupal mass was not influenced by fungal isolate (F3,241=0.61, P=0.61, and F3,190=0.74, P=0.53 for female and male pupae, respectively). In contrast, fungal isolate influenced parasitoid survival (X2=58.3, df=3, P<0.0001). Survival was higher than expected for parasitoids reared from hosts fed plants lacking fungal infection, but was lower than expected for those reared from hosts fed plants infected with AR542 and CS fungal isolates. In contrast, parasitoids reared from hosts fed plants infected with fungal isolate AR502 did not experience higher mortality than expected by chance. Parasitoid mortality mainly occurred during the egg stage. Our results suggest N. coenophialum can influence the tritrophic interaction in ways that could be detrimental to the host plant and that this effect is influenced by fungal genotype. Keywords: fall armyworm, Euplectrus, Neotyphodium, novel endophytes, tall fescue
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(252 KB)This study examined how differences in the behavioural response of two sod webworm species to Neotyphodium endophytes mediated their susceptibility to an entomopathogenic nematode. In choice and non-choice assays, Parapediasia teterrella (Zincken) clearly preferred endophyte-free tall fescue over endophyte infected tall fescue whereas Fissicrambus mutabilis (Clemens) displayed no clear preference for either. P. teterrella spent more time searching for suitable host material than F. mutabilis and committed to the first host encountered only 37% of the time whereas F. mutabilis did so 90% of the time. Increased foraging times associated with P. teterrella host selection translated into greater rates of infection by the nematode Steinernema carpocapsae (Wieser). Results demonstrate the potential importance of insect behavioural response to fungal endophytes in determining herbivore vulnerability to natural enemies and support the more general idea that the form of insect response to plant defenses may, in part, predict the outcome of insect-natural enemy interactions. Keywords: tall fescue, sod webworm, entomopathogenic nematode, trophic interactions
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(371 KB)Ryegrass staggers is a neurotoxic disorder of livestock grazing perennial ryegrass (Lolium perenne L.) infected with the endophytic fungus Neotyphodium lolii. Ryegrass staggers was first reported in 1880 (Anonymous 1880) although at this stage the cause of the observed tremorgenic disorder was unknown. The presence of an endophyte within perennial ryegrass was first recognised in 1935 (Neill 1941) but it was not until 1981 that the correlation between endophyte infection and the incidence of ryegrass staggers was established (Fletcher & Harvey 1981; Mortimer et al. 1982). The tremorgenic compound, lolitrem B, produced by the endophyte was then isolated and implicated in the disease (Gallagher et al. 1981, 1982, 1984). The future then seemed clear: eradicate the endophyte and solve the ryegrass staggers problem. Endophyte-free plots, however, showed little growth and suffered severe damage from the larvae of Argentine Stem Weevil (Mortimer et al. 1982). The correlation between endophyte levels, weevil numbers and tiller damage was then made (Prestidge et al. 1982) and a few years later the beneficial effects of endophyte were shown to be due to peramine, an antifeedant produced by the fungus (Gaynor & Rowan 1986; Rowan et al. 1986).
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(199 KB)Biotypes of the Neotyphodium coenophialum-tall fescue grass symbiota are provided with enhanced protection from grazing vertebrate herbivores due to the production of toxic secondary metabolites. However, considerable controversy exists concerning this symbiotum and its toxicity to nematode species. A sterile in vitro system was developed to determine the interactive nature of known toxins specific to this mutualistic association and compounds within grass extracts known to be nematotoxic. The in vitro assay used Pratylenchus scribneri, the lesion nematode, as the target organism to determine the interactive nature of ergot alkaloids, the pyrrolizidine alkaloid (the lolines), total phenolic fractions, and specific phenolic compounds. The in vitro assay is described along with methods for testing toxicity. The results indicate that only two of three ergot alkaloids were toxic to P. scribneri, and there were possible potentiating or synergistic effects with other alkaloids and water soluble polyphenolics. HPLC analysis and UV mass spectrometry of root extracts revealed the presence of two major polyphenolics, chlorogenic and di-caffeoylquinic acids, both of which are natural constituents of this and other plants and have known toxicity to several species of nematodes. Further, it was determined that there were quantitative differences between the total phenolic and specific phenolic contents in roots of endophyte infected and noninfected tall fescue, cultivar Jesup. This in vitro assay offers a rapid and routine screen for acute testing chemical components of the tall fescue-endophyte symbiotum for toxicity to this nematode species. Keywords: Chlorogenic acid, di-caffeoylquinic acids, ergot alkaloid, lolines, nematode, polyphenolics, Pratylenchus scribneri, pyrrolizidine alkaloid
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(615 KB)Ryegrass Staggers (RGS) is predominantly a summer/autumn metabolic disorder of ruminants, caused by ingestion of the toxin lolitrem B. The toxin is produced by an endophyte (Neotyphodium lolii) found in perennial ryegrass (Lolium perenne). In clinical cases of RGS, animals experience neuromuscular uncoordination when under stress, e.g. when mustered by sheep dogs. Experiments with a standardised RGS scoring system show that resistance/susceptibility in sheep has a moderate/high heritability (0.37 ± 0.05). An AgResearch flock was established in 1993, with two lines bred for resistance (R) or susceptibility (S) to RGS. Annually, lambs are scored for susceptibility, when both lines graze together on high endophyte pastures, and are mustered by sheep dogs. In 2005 and 2006, 2% and 6.5% of R-line lambs, respectively, showed clinical staggers, compared with 94% and 91% of S-line lambs (P<0.001). Other results of genetic progress are also described. Keywords: sheep; breeding; resistance; ryegrass staggers, lolitrem B
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(261 KB)In two separate pot trials, tall fescue plants infected with the endophyte strain AR542, which is non-toxic to livestock, were shown to have lower numbers of root aphid per plant compared with uninfected plants, but more aphids than plants infected with other non-toxic endophytes (AR502, AR510 and AR514). These differences were not always significant when aphid numbers per gram of root were analysed. A Petri dish trial investigated the mechanism of resistance. Aphid survival was lower on endophyte-free fescue than on AR514- and AR542-infected fescue. Compared with endophyte-free, fewer aphids on roots of AR514- and AR542-infected tall fescue showed signs of feeding, as measured by production of wax and honey dew, and more were mobile on and around roots. Keywords: Aploneura lentisci, Festuca arundinacea, Neotyphodium
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(368 KB)A series of experiment were conducted to examine the toxicities of Neotyphodium endophyte in laboratory animals. Groups of mice and hamsters were fed endophytic perennial ryegrass seeds. The hamsters presented ergotism-like symptoms while mice hardly indicated the toxicosis. Goups of mice and hamsters were fed endophytic perennial ryegrass seeds for 8 days and total excreta collections were made to evaluate apparent absorptions of endophytic alkaloids. Accumulations of endophyte toxins in the tissues and organs were also analysed. Although approximately 50% of lolitrem B and 90% of ergovaline were absorbed by both species, and lolitrem B accumulation in the liver and fat tissues, only the hamsters showed mild tremors. A condensed lolitrem B fraction was orally administrated to mice and hamsters and total excreta collections were made. The mice were not affected by even large amounts of lolitrem B dosages with high absorptions (>80%) when orally administrated. Keywords: ergovaline, lolitrem B, toxicosis, mouse, hamster, endophyte
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(647 KB)Lolitrem B and epoxy-janthitrem residues were found in the fat of sheep grazing wild-type and AR37 endophyte-infected pastures, respectively. Epoxy-janthitrems were also detected in the milk of cows grazing AR37 pastures. In order to give preliminary information on the possibility that these endophyte toxins could pose a threat to human health, a 3-week toxicological trial has been conducted in mice. The results of this experiment suggest that, at the levels found in food, the endophyte toxins are unlikely to cause any safety concern. Keywords: lolitrem, epoxy-janthitrem, endophyte, AR37, fat, milk, toxin residues, toxicology
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(229 KB)Perennial ryegrass (Lolium perenne L.) and tall fescue (Festuca arundinacea Schreb.) are two of the most productive pasture species grown to feed livestock in Australia. The larvae of redheaded (RHC) (Adoryphorus couloni Burmiester) and blackheaded (BHC) (Accrosidius tasmaniae Hope) cockchafer cause significant damage to these pastures in Australia. If alkaloids produced by endophyte associations could reduce this impact, economic gains would be made. A pot experiment and then a proof of concept seed feeding experiment were undertaken to test the resistance of perennial ryegrass and tall fescue to cockchafer larvae. The objective was to allow larvae to feed on endophyte infected plants, assuming insecticidal alkaloids would be produced and on nil endophyte plants as controls. The plants in the pot experiment were tested for presence or absence of alkaloid endophyte but not for concentration or location in plant parts prior to larvae being introduced. In the seed feeding experiment seed fed to larvae was tested for all known alkaloid concentrations and profiles. The weight difference and mortality of third instar larvae and weight difference of plant root and shoot were measured in the pot experiment. The weight difference and mortality of the third instar larvae and the number and weight of seeds consumed were measured in the seed feeding experiment. There was no significant effect of endophyte treatments on the cockchafer weight change or mortality in either the perennial ryegrass or tall fescue pot or seed feeding experiments. There was a significant difference between cockchafer species (P<0.001) with the larger redheaded larvae consuming more than the blackheaded larvae. It is concluded in both experiments that red and blackheaded cockchafers are not sensitive to endophyte in perennial ryegrass and tall fescue. Endophyte infection can be harmful to livestock and manipulation in plant cultivars can provide insect resistance and drought tolerance which is still a valuable attribute in all areas where cockchafers are not a problem. However, even when cockchafers damage plants, endophyte infection may enhance the ability of these cultivars to recover. Keywords: endophytes, cockchafers, perennial ryegrass, tall fescue
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(270 KB)Two studies were performed to determine if Doppler ultrasound can detect vascular changes in cattle exposed to hot and cold conditions. Three calves per study (320±38 kg) were acclimated to 17.5oC, and then exposed to 32oC and 8oC. Animals were maintained under these conditions for 24 h and then returned to the original thermoneutral level. Three replicated ultrasound measurements of the median caudal artery were performed. Scans were repeated at -2, 5, 23, 24 and 28 h. Blood flow rates of 32oC cattle ranged from 27.84 (-2 h) to 43.42 ml/min at 23 h (P<0.05). Heart rate was unchanged, but respiration increased from 82 to120 breaths/min at 32oC (P<0.05). Cattle exposed to 8°C showed lower flow rates after 23 h of exposure (P<0.10) (29.29 at -2 h to 13.79 ml/min at 23 h). Blood flow rates of these cattle initially increased after the challenge. At 8°C, the heart rate was unaffected. Respiration rate decreased from 62 at -2h to 36 breaths/min at 23h (P<0.05) when housed under cool conditions. Arterial cross-sectional area increased from 0.058 to 0.076 cm2 at 5 h (P<0.05) exposure to 8°C then decreased to 0.043 cm2 at 23 h. The ability of the Doppler-image ultrasound to detect vascular changes demonstrates its use as a tool for assessing environmental stress effects on haemodynamics. Keywords: tall fescue, Doppler-ultrasound, cattle
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(224 KB)Experimental feeds containing 0, 500, 1000, 1500 or 2000 ppb of lolitrem B derived from perennial ryegrass straw were fed to 9 month-old Japanese Black steers (average liveweight 180 kg) to determine their threshold level of lolitrem B. Steers received feeds containing 1000, 1500 and 2000 ppb of lolitrem B showed signs of ryegrass staggers after 2 to 7 weeks of feeding. The lowest observed adverse effect level (LOAEL) of lolitrem B was approximately 24 μg/kg body weight per day. Only small amounts of lolitrem B residue were detected in muscle, liver, kidney and cerebrum. In contrast, approximately 100 ng/g of lolitrem B was detected in perirenal fat of steers with ryegrass staggers. Keywords: Lolitrem B, Japanese Black cattle, ryegrass staggers, muscle, fat
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(335 KB)The extent of Neotyphodium based resistance in wild fescue to bird cherry-oat aphid (Rhopalosiphum padi) was determined by quantifying densities of this aphid on a series of Neotyphodiuminfected (E+) and uninfected (E-) tall fescue entries. Little or no aphid survival was observed on E+ ‘Kentucky 31’ tall fescue and three E+ wild tall fescue accessions (16079, 15978, 16075) from Tunisia; however, three E+ Tunisia accessions (16036, 16044, 16085) supported populations of R. padi. These results suggest that wild tall fescue from Tunisia harbour diverse Neotyphodium endophytes. They also support earlier observations by entomologists that the magnitude of insect (including aphids) resistance in E+ grasses varies with the host genotype and Neotyphodium strain involved in the interaction. Keywords: Neotyphodium endophyte, tall fescue, Rhopalosiphum padi, host plant resistance
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(258 KB)
Some strains of Epichloë and Neotyphodium endophytes produce the ergot alkaloid ergovaline, which is implicated in livestock toxicoses caused by ingestion of endophyte-infected grasses.
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(133 KB)Endophytes, belonging to the genus Neotyphodium, live symptomlessly within the intercellular spaces of cool-season grasses, and confer a number of biotic and abiotic advantages to their hosts. We identified a novel endophyte gene (designated Nc25) that is expressed preferentially in planta, is one of the most abundant fungal transcripts in endopyte-infected grasses and which is distributed and highly expressed in a wide range of endophyte/ grass associations. Nc25 is novel and shows no homology to sequence databases or fungal genome initiatives. Characterisation indicates that it encodes a small secreted protein. Re-introduction of a Nc25 deletion strain into perennial ryegrass showed no visible effect on the symbiosis but an unknown oligopeptide, detected only in infected plants, was eliminated. Surprisingly, the oligopeptide is unrelated to the predicted peptide product of Nc25. We hypothesise that Nc25 may regulate the oligopeptide biosynthetic pathway and are investigating this using Affymetrix gene chips to determine how Nc25 affects global gene expression. In addition we are interested in the biological function of this secondary metabolite during symbiosis and in particular whether it has bioactivity that may confer abiotic or biotic advantages to the host plant. Keywords: Neotyphodium, in planta expressed gene, oligopeptide, EGFP
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(1195 KB)Although much is known about the signals and mechanisms that lead to pathogenic interactions between plants and fungi comparatively little is known about fungal-plant mutualistic symbiotic interactions. The association between Epichloë festucae and perennial ryegrass is a powerful experimental system to understand the molecular basis for fungal-plant mutualistic interactions. We have adopted a forward genetics approach to dissect this symbiosis. Previously we described the use of plasmid mutagenesis to isolate a symbiotic mutant. We describe here the development of an Agrobacterium tumefaciens mediated T-DNA transformation system for E. festucae. Keywords: Epichloë festucae, Agrobacterium tumefaciens, TDNA, transformation
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(820 KB)As a first step towards a functional genomics approach to gain a greater understanding of this important symbiosis, we have generated, sequenced and analysed two EST libraries from cultures of N. lolii and six in planta subtracted EST libraries enriched for differentially expressed genes. A total of 12871 ESTs were sequenced which, after filtering for quality, clustered into 1066 contigs and 3230 singletons to give a set of 4296 unique sequences or unigenes. BLASTX analysis revealed that 60% of fungal sequences derived from cultures were of unknown function with a sub-set of these corresponding to orphans. For the in planta-derived ESTs, most of the sequences with homologs in the public databases (98%) were of ryegrass origin. Comparisons made against fully sequenced genomes revealed that most fungal ESTs were homologous to genes present in both pathogenic and non-pathogenic ascomycete filamentous fungi, whereas the subtracted libraries comprised mostly plant genes. A range of sequences having significant homology to demonstrated pathogenicity/virulence genes in other fungal pathosystems were also identified, as well as some ESTs with proven roles in endophyte secondary metabolism. Keywords: ESTs, cDNA, Neotyphodium lolii, Lolium perenne, symbiosis, mutualism, suppression subtractive hybridisation
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(353 KB)Epichloë endophytes are protective mutualists of cool-season grasses.
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(209 KB)Symbiotic grass associations with fungal endophytes (genera Neotyphodium and Epichloë) display enhanced fitness as well as prolonged field persistence over their endophyte free equivalents. Perennial ryegrass, an important agronomic grass, is typically associated with the N. lolii endophyte. The endophyte lives within the intercellular spaces without inducing any symptoms in the plant. The aim of this study is to elucidate the biosynthetic function of fungal secondary metabolite gene clusters. Non-ribosomal peptide synthetase genes (NRPSs) of unknown function were targeted, as these genes are commonly associated with the production of bioactive peptides some of which are ecologically important. Some novel endophyte NRPS genes have been identified using a degenerate PCR screen; one of these, NRPS5 will be discussed here. Clones were obtained by screening a fosmid Epichloë festucae genomic DNA library and we are currently determining gene function by using targeted gene replacement followed by an assessment in vitro and in planta using metabolomics and appropriate bioassay screens. Keywords: endophyte, NRPS, secondary metabolism
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(338 KB)Elymus canadensis (Canada wildrye - CWR) is a native perennial cool season bunch grass tolerant to a range of soils, winter hardy and able to grow across the United Sates and as far North as Southern Alaska. Canada wildrye is often used for prairie restoration, conservation and erosion stabilisation. Young CWR plant tissue is palatable and nutritious to grazing animals. CWR has been reported to harbour a sexual endophytic fungus, Epichloë elymi, but some accessions have been identified that have not produced stroma. We isolated and characterised the epichloë endophytes from three endophyte-infected CWR accessions collected from Mexico and Texas. We established that the endophytes present in these CWR accessions are of hybrid origin, with E. elymi and E. amarillans ancestral genomes, and are therefore considered to be asexual isolates. The endophytes were examined for their alkaloid potential, particularly the detrimental ergot alkaloids, with inconclusive results. Keywords: Elymus canadensis, Canada wildrye, hybrid, Epichloë elymi, Epichloë amarillans
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(353 KB)The aim of this project was to undertake large scale transcript profiling of endophyte and plant genes during symbiosis, and to determine the impact of targeted endophyte gene deletions on expression of plant and endophyte genes. We have designed and developed an Affymetrix NimbleExpress™ GeneChip® representing expressed sequence tags (ESTs) of the fungal endophyte Neotyphodium lolii Lp19 and its ryegrass host, Lolium perenne. In total, 8511 genes were represented on the microarrays with approximately eleven 25 base pair oligonucleotides per gene. Experiments were conducted to analyse differential expression of genes from endophyte-infected and endophyte-free plant material, and from endophytes grown in culture. In some symbioses, endophytes had targeted mutations in genes involved in signalling, synthesis of secondary metabolites or in genes of unknown function. Here we describe the processes which guided design of the GeneChip®, the results of quality control assessments of hybridised arrays and considerations concerning statistical analyses of gene expression. Keywords: Affymetrix, GeneChip®, NimbleExpress, Neotyphodium lolii, Epichloë festucae, ryegrass, Lolium perenne, endophyte, symbiosis.
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(295 KB)Grass associations with Epichloë/Neotyphodium endophytes display enhanced fitness as well as prolonged field persistence over their endophyte free equivalents. To gain a comprehensive understanding of the complex biological interactions that occur between a plant host and fungal symbiont, a transcriptomics approach using custom designed Affymetrix GeneChip®s was employed. We are currently comparing and analysing symbiotic interactions of perennial ryegrass (Lolium perenne) with endophytes N. lolii and E. festucae as well as comparing it against endophyte-free perennial ryegrass. Both N. lolii and E. festucae have been grown in culture in order to compare in planta versus in vitro gene expression. Additionally, targeted gene replacements in E. festucae have been performed and a comparative analysis of the knock-outs with wild-type E. festucae infected plants is in progress. These comparative analyses have revealed changes in gene expression which may lead to the identification of gene pathways/networks and the roles of these genes in symbiosis. Keywords: Neotyphodium, Epichloë, transcriptomics, symbiosis
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(501 KB)We investigated on the genetic diversity of endophytes in native populations of tetraploid, hexaploid and octaploid tall fescue (Lolium arundinaceum) in Europe and North Africa
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(257 KB)Although much is known about the beneficial and detrimental effects of the interaction between Neotyphodium lolii and Lolium perenne in the field comparatively little is known about the molecular and cellular events that underlie these effects and how mutualism is maintained. Because N. lolii is quite intractable to genetic analysis we have developed the synthetic association between Epichloë festucae and perennial ryegrass as our model experimental system to study the epichloë endophytegrass symbiotic interaction. Using this system we have recently shown that reactive oxygen species (ROS) produced by a specific endophyte NADPH oxidase (NoxA) have a critical role in regulating hyphal growth and development in perennial ryegrass. We have also shown that two additional components, NoxR and RacA, are required to regulate ROS production to maintain a mutualistic interaction. Using a combined molecular and genetic approach we have recently cloned and characterised genes for peramine and lolitrem biosynthesis. An overview of these advances and the opportunities now available to better understand and exploit this important fungal-grass interaction is presented. Keywords: Epichloë festucae, Neotyphodium lolii, Lolium perenne, peramine, lolitrems, reactive oxygen species
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(1861 KB)Neotyphodium lolii is a fungal endophyte that lives entirely within the intercellular spaces of its grass host, perennial ryegrass (Lolium perenne, L.). Infection is symptomless and the endophyte relies on the host plant for dissemination via the seed. The association is mutually beneficial since the endophyte confers a number of biotic and abiotic advantages to the host. This paper presents an overview of the functional genomics approaches we are using at AgResearch to dissect the molecular basis of this symbiosis and will broadly describe the fields of genomics, transcriptomics, proteomics and metabolomics, as applied to this system. We have used isogenic ryegrass lines infected or uninfected with endophyte in combination with a suite of molecular biology tools, including Expressed Sequence Tags (ESTs), cDNA and Affymetrix GeneChip® microarray analysis, 2D-gel electrophoresis (to identify novel proteins associated with symbiosis), and metabolic profiling. By using a multidisciplinary approach we aim to identify genes which are important in both the establishment and maintenance of symbiosis, as well as elucidate how endophyte confers enhancements to its host. Keywords: Neotyphodium, Epichloë, symbiosis, functional genomics
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(1187 KB)Endophytic fungi of temperate grasses form stable symbiotic associations that provide mutual benefits for both species.
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(232 KB)Neotyphodium and Epichloë species are closely related asexual and sexual endophytic fungi, respectively, that form symbiotic associations with cool season grasses of the sub family Pooideae, including several important forage and turf grass species. The endophytes confer a number of advantages to their hosts, but also can cause animal toxicosis. These positive and negative effects are, in many cases, due to the production of fungal secondary metabolites. In filamentous fungi secondary metabolite genes are commonly clustered and are well documented to often contain non-ribosomal peptide synthetases (NRPSs) at their core. Members of this gene family encode large multifunctional proteins that synthesise a diverse range of bioactive compounds, many of which have been shown to serve as pathogenicity or virulence factors, in addition to suggested roles in niche adaptation. We have used a degenerate PCR approach to identify members of the NRPS gene family from Neotyphodium and Epichloë species, and have shown that at least 13 NRPS genes exist among these genomes. The distribution of these genes among different Neotyphodium/Epichloë lineages suggests that a common ancestor contributed most of the complement of NRPS genes, which have been either retained or lost during the evolution of these fungi. Keywords: Neotyphodium, Epichloë, non-ribosomal peptide synthetase, NRPS
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(351 KB)Genes for the synthesis of the bioprotective metabolites, peramine and indole-diterpenes, were recently cloned from Neotyphodium lolii and Epichloë festucae (Tanaka et al. 2005; Young et al. 2005; Young et al. 2006).
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(171 KB)Epichloë typhina comprises interfertile strains associated with several grass species in tribes Poeae, Aveneae and Brachypodieae. Molecular phylogenetics indicate that E. typhina is paraphyletic to E. sylvatica and E. clarkii. Although interfertility barriers are evident between E. typhina and E. sylvatica, E. clarkii is interfertile with E. typhina. However, E. clarkii is associated specifically with Holcus lanatus (Aveneae). We addressed the possibility that E. typhina is a complex of genetically isolated populations distinguishable by host specificity. Populations of E. typhina were sampled from three Cantons on a transect from southwest to northeast Switzerland, and from 1–3 host species in each Canton. No fewer than 20 isolates were obtained from each host at each location. The intron-rich 5’-end of the beta-tubulin gene was sequenced from each isolate, and all of the sequences were aligned for phylogenetic and coalescence analyses. There was no indication of geographic isolation of the populations, but no haplotypes (sequence variants) were shared between isolates from different hosts. Therefore, E. typhina appears to be a complex of genetically isolated sympatric populations that are specific for different hosts. Keywords: Brachypodium species, Clavicipitaceae, Dactylis glomerata, Epichloë, epichloë endophytes, grasses, host specificity, molecular phylogenetics, Poa species, Poaceae, population genetics
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(768 KB)Epichloë festucae systemically colonises the intercellular spaces of perennial ryegrass (Lolium perenne) aerial tissues forming a mutually beneficial association between the fungus and host plant.
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(177 KB)AgResearch is a world leader in the commercial utilisation of endophytes in forage grasses. These fungal endophytes have a symbiotic relationship with their host grass and and have traditionally been an agricultural double edged sword. On one hand, they increase plant yield, provide resistance to abiotic stress, and produce insecticidal secondary metabolites. However, they also produce toxic mammalian alkaloids that seriously affect the health of the grazing livestock. AgResearch has identified and commercialised endophyte strains (AR1, AR37 and MaxQ/ MaxP) that lack the capacity to produce two of the main classes of toxic mammalian alkaloids. In addition to the successful commercial strains, AgResearch has characterised a number of strains with potentially beneficial traits that were not placed into a commercial development pipeline because they would lead to negative effects on livestock (e.g. lolitremB producers). As part of our strategy for developing the next generation of endophytes, we are looking for strains with novel activities against some of the insect pests that are not deterred by the current commercial strains. It is likely that some of these strains will be lolitrem B or ergovaline producers and therefore some means of eliminating these pathways is desirable.
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(195 KB)The ascomycete Epichloë festucae is a model endophyte that 1) switches between mutualistic and antagonistic states, 2) is seed transmissible, 3) has a sexual state amenable to genetic analysis, and 4) is rich in bioprotective alkaloids. This fungus grows systemically and intercellularly throughout the life of its host plant. On each reproductive tiller the fungus either infects benignly and transmits clonally in seeds, or produces its sexual state (stroma) and chokes inflorescence development. The E. festucae genome was estimated at 29 Mb in six chromosomes. The genome sequence was assembled from cloned insert end reads (4.2 x coverage) and preassembled pyrosequencing reads (454-sequencing: 20 x raw, 1.7 x assembled), giving 3967 supercontigs, of which 1004 were larger than 2 kb and covered 92% of the genome. Gene prediction with FGENESH identified ~10,000 putative genes. We also sequenced 25,000 ESTs from each of two normalised libraries — one of choked inflorescences, the other of benignly infected inflorescences — yielding 5077 E. festucae unigenes, annotated by BLAST and InterPro. Sequence data and annotations are stored in a database for visualisation and inspection with the GBrowse browser. The genomic sequences can be queried by BLAST at http://www.genome.ou.edu/blast/ ef_blastall.html. Keywords: bioinformatics, DNA sequence, Epichloë festucae, expressed sequence tags, Festucae pratensis, fungal genomics, Lolium pratense
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(379 KB)In fungal pathogenesis the cAMP signalling cascade is usually essential for virulence. Deletion of the adenylate cyclase gene, the enzyme that synthesises cAMP, often results in an attenuated or avirulent phenotype. Our aim was to identify the signalling mechanisms regulating colonisation of perennial ryegrass (Lolium perenne) by the fungal symbiont Epichloë festucae Fl1. We have identified genes from several signalling networks, and here report on the outcomes of targeted disruption of the E. festucae Fl1 adenylate cyclase gene (acyA). A dual genome (endophyte/ ryegrass) Affymetrix GeneChip® has been synthesised and we are undertaking large scale transcript profiling of the L. perenne/ E. festucae ΔacyA symbiotum to identify target genes regulated by the endophyte cAMP signalling network. Keywords: cAMP, adenylate cyclase, acyA, Neotyphodium lolii, Epichloë festucae, symbiosis, Affymetrix GeneChip
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(403 KB)Neotyphodium coenophialum strain e19 from tall fescue cv. Kentucky 31 carries dmaW1 and dmaW2, two gene homologues that encode dimethylallyltryptophan synthase, the enzyme for the first step in ergot-alkaloid biosynthesis. In our effort to disrupt both homologues and ultimately obtain marker-free mutants, we are using a marker-exchange strategy employing the Cre/ loxP site-specific recombination system. Of 1522 transformants obtained and screened, three were likely dmaW2 disruptants because they gave no PCR product from the wild-type locus, but yielded the larger PCR fragment from the disruption construct. The putative dmaW2-knockouts were also transformed with pKAES186, a plasmid with a cassette containing the cre and ble genes in between loxP sequences. The transformants obtained were screened for the presence of hph, cre and ble genes. The preliminary results indicate a loop-out of the hph gene. The transformants inoculated into endophyte-free tall fescue preserved their compatibility with the plant. The fungus grown from these plants will be further analysed for the presence of hph, cre and ble genes. Keywords: Cre/LoxP, dimethylallyltryptophan synthase, dmaW, Epichloë, ergot alkaloids, Festuca arundinacea, gene knockouts, Lolium arundinaceum, Neotyphodium coenophialum, tall fescue
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(406 KB)
The viability of this international symposium on grass/fungal endophytes is a direct measure of the nature and strength of the underlying science itself. The symposium’s past was dominated by a multidiscipline approach and a geographic focus in New Zealand, USA, and Australia, due mainly to the economic impact of tall fescue (Festuca arundinacea Schreb.) and perennial ryegrass (Lolium perenne L.) when infected with Neotyphodium endophytes. There have been many research and technological findings reported during the past five symposia, with novel endophytes as an example of one having positive on-farm impact. In the future, it is anticipated that 1) the multidisciplinary approach will continue; 2) patenting issues will cause disruptions (hopefully small) in information sharing; 3) funding will drive the underlying science thereby continuing a concentration of work in New Zealand, Australia, and the USA, but opportunities will arise in these major countries for collaborative research with scientists in other geographies; 4) commercialisation and on-farm use of novel endophytes will expand; 5) sequencing and gene expression data will be available for the main fungal genomes, and microarray, genomics, and transciptome approaches will grow in importance; and 6) development of a “mind map” will help identify knowledge gaps for determining endophyte effects on animals. However, it was the inspiration, leadership, confidence, and even boldness of individual scientists in the field that pushed both this symposium and the science to the level it is today. Therefore, as the symposium moves forward, younger scientists need to assume leadership roles, and then be bold and confident in pushing forward; especially with continuing the symposium. Keywords: Neotyphodium, fungal endophytes, symposium, grasses, tall fescue, perennial ryegrass, biotechnologies
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(416 KB)
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