Background Forage perennial ryegrass (Lolium perenne L.) has been extensively researched regarding vegetative growth and quality, with its seed being relatively unexplored. The declining viability of Epichlo & euml; fungal endophytes in ryegrass seed during storage underscores the need for dedicated biochemical research.Methods We conducted a lipidomic investigation of seeds from five fungal endophyte-perennial ryegrass associations: tetraploid ryegrass cultivars KLp1102, KLp1103 and KLp903 infected with the endophyte strain AR501 and cultivar KLp1102 independently infected with strain AR1 or AR37.Results Storage lipids in perennial ryegrass seeds were predominantly triacylglycerols (TGs) containing C18:2, C18:1, C18:3 and C16:0 fatty acyl chains. Notably, TGs and diacylglycerols (DGs) containing medium-chain fatty acids (C8:0, C12:0 and C14:0) showed differential accumulations among the seeds of the three cultivars infected with AR501, as well as among the seeds of KLp1102 infected with AR1, AR37 and AR501. The distinct responses of typical storage glycerolipids (C18 and C16 fatty acyl chains) and medium-chain glycerolipids (C8, C12 and C14) among KLp1102-AR1, KLp1102-AR37 and KLp1102-AR501 samples highlighted the influence of endophyte strains on host biochemistry. These findings suggest that medium-chain TGs and DGs play critical roles in maintaining effective ryegrass-endophyte associations.Conclusions This study identifies numerous structural and signalling lipid species, providing a foundational understanding of lipid composition in perennial ryegrass seed. The results offer new opportunities to investigate the mechanisms underlying endophyte viability during storage and to elucidate the genetic regulation of differentially accumulated signalling lipids across different ryegrass cultivars.

The relationship between Epichloë fungal endophytes found in some temperate grasses spans the continuum from antagonistic to mutualistic. The predominant focus here is on those that form mutualistic associations with those temperate grasses of the New World due to their substantial economic impact. This review describes the evolutionary origin, discovery, lifestyle and life cycle of Epichloë as background to understand the close association between plant host and fungal endophyte. Many asexual Epichloë fungal endophytes are unique in being obligate mutualists, lacking a free-living stage in nature, and being maternally inherited through host seed. The compatibility between Epichloë and its natural host plant is so complete and specific that manually transferring an Epichloë endophyte strain to other grasses, even some that are closely related genetically, though possible, often results in endophyte and/or plant death or poorly performing plant genotypes. The stability of the association is facilitated through the biotic and abiotic protection of the host grass through the endophyte promoting endogenous defense responses in the host, or directly through the production of secondary metabolites. Epichloë endophytes are renowned for producing a diverse range of alkaloids that can negatively affect invertebrate and vertebrate herbivores, either through toxicity or feeding deterrence, thereby enhancing host survival. However, Epichloë chemistry can significantly impact the health and welfare of grazing animals resulting in large economic impacts. This includes effects known as ryegrass staggers, heat stress and fescue toxicosis. In some temperate areas of the world, notably New Zealand, Australia and USA, grasses such as perennial ryegrass and tall fescue need to have the Epichloë-derived protection to ensure persistence against invertebrate pests and drought but without the downsides of clinical and subclinical losses for grazing animals. Extensive screening has identified strains of Epichloë which have been successfully commercialised for ryegrasses and fescues that ensure persistence of the grass host without causing serious health and welfare impacts on the grazing animal. The development and deployment of selected Epichloë strains therefore represent a critical intersection of plant protection, animal welfare, and agricultural productivity, a theme explored throughout this review.

In this chapter, the authors describe the biology of the Epichloë-grass association, the secondary metabolites that are linked to the bioactivity, control of invertebrate pests, occurrence of mammalian toxicity globally, toxic responses in terms of animal productivity, health and welfare, utilisation of Epichloë strain variation in commerce, along with flow-on effects to animal product quality and food safety. There is a particular focus on the most well-studied, mutualistic tall fescue and perennial ryegrass endophyte associations due to their significant economic impacts and prospects to advance future sustainable grassland farming.

The inoculation of Epichloë endophytes into modern cereals, resulting in systemic infection, depends on the genetics of both the host and the endophyte strain deployed. Until very recently, the only modern cereal to have been infected with Epichloë, in which normal phenotype seed-transmitted associations were achieved, is rye (Secale cereale). Whilst minor in-roads have been achieved in infecting hexaploid wheat (Triticum aestivum), the phenotypes of these associations have all been extremely poor, including host death and stunting. To identify host genetic factors that may impact the compatibility of Epichloë infection in wheat, wheat–alien chromosome addition/substitution lines were inoculated with Epichloë, and the phenotypes of infected plants were assessed. Symbioses were identified whereby infected wheat plants were phenotypically like uninfected controls. These plants completed their full lifecycle, including the vertical transmission of Epichloë into the next generation of grain, and represent the first ever compatible wheat–Epichloë associations to be created.

Epichloe is a genus of filamentous fungal endophytes that have co-evolved with cool-season grasses with which they form long-term, symbiotic associations. In natural ecosystems Epichloe have implications for species diversity, food web structures and fundamental ecological processes. Epichloe produce a range of secondary metabolites that can have negative effects on herbivores, be they invertebrates or vertebrates. In many managed pastoral systems, selected asexual Epichloe strains are purposefully associated with grass cultivars (termed novel endophyte-grass associations) as they confer beneficial traits that protect the host from abiotic stresses (e.g. drought) and biotic stresses (e.g. herbivory from invertebrates) while having minimal impact on the health and welfare of ruminant livestock. As well as conferring beneficial traits, agriculturally selected strains of Epichloe can also increase plant biomass and seed yield. Epichloe endophyte-technology has and continues to be a scientific, agricultural, and marketing success with selected asexual strains of Epichloe now essential components of many pasture ecosystems, within New Zealand and abroad. An estimated 90% of proprietary ryegrass now sold in New Zealand contains a selected endophyte strain while the economic impact in New Zealand of AR37 alone has been estimated at NZ$3.6 billion over 20 years. With a changing climate, agriculture will continue to experience increasing temperatures, elevated atmospheric CO2 levels and changing precipitation patterns that will likely increase the geographic range and overwintering survival rates of pests. Furthermore, plant disease outbreaks are expected to intensify due to increases in the severity of existing phytopathogens and/or invasions by new phytopathogens. Functional symbioses, such as those between temperate grasses and Epichloe, are among the most successful mechanisms by which plants can improve their ability to tolerate stress. Future research is likely to utilise targeted genetic manipulation techniques to broaden the biocontrol ability of Epichloe strains, particularly to mitigate climate related stresses.

Context Data are lacking on the effects of selected endophytes of perennial ryegrass (Lolium perenne L.) on ryegrass persistence. Aim We aimed to determine the impact of Epichloë endophyte on the persistence of mid- and late-heading perennial ryegrass cultivars. Method Two mid-heading (Samson, Bronsyn) and two late-heading (One50, Rohan) cultivars, infected with selected endophytes (AR37, nea2/6) or with standard toxic endophyte, were established in a replicated plot study grazed by cattle in a subtropical environment of the upper North Island of New Zealand. Persistence characteristics were quantified at least five times per annum, over 4 years. Key results Endophyte strain had an effect on persistence; infection with standard endophyte resulted in higher ryegrass ground cover percentage, ryegrass content in pasture dry matter and autumn yield than infection with nea2/6 on many occasions, and with AR37 on some occasions. There were negligible impacts on ryegrass tiller density or nutritive value. Trends were dominated by the main effect of endophyte; interactions with heading date were inconsistent. Conclusions Cultivars were more persistent when infected with standard endophyte than with selected endophytes, although persistence declined over 4 years for all cultivars and irrespective of heading date. Implications Reliance on selected endophyte is unlikely to prevent persistence decline of perennial ryegrass in a subtropical environment. Other strategies will be required to maintain the persistence of high-quality pastures based on perennial ryegrass.

AbstractBackgroundGrazing approaches are needed to increase the resilience of perennial ryegrass (Lolium perenne L.)‐based pastures subject to increasing drought stress. One opportunity has focused on seedhead management in late spring. Paddock‐level studies demonstrated increased pasture resilience when ryegrass seedheads are allowed to mature, but knowledge is lacking on how defoliation management affects plant carbohydrate status and hence resilience in the sward.MethodsA glasshouse study was conducted from spring to autumn using 1 m deep root tubes. Plant growth and water‐soluble carbohydrate (WSC) reserves were measured every 4–6 weeks. Defoliation treatments comprised “VEGETATIVE”—regular defoliation based on leaf stage and trimmed to 4 cm; “FLOWERING”—no defoliation spring to anthesis; and “SENESCENT”—no defoliation spring to reproductive tiller senescence. Thereafter, regular defoliation was carried out for all treatments until the end of the study. From spring to the end of summer, plants were watered daily in WET (no drought, well watered) and on four occasions in DRY (drought) treatments, with daily watering thereafter.ResultsHerbage mass, tillering, root depth, root mass, and WSC were generally higher in SENESCENT than VEGETATIVE with FLOWERING intermediate (p < 0.05). Nutritive values were similar in VEGETATIVE and FLOWERING, but in SENESCENT, metabolizable energy and crude protein declined and neutral detergent fiber increased (p < 0.05). Soil moisture effects were small, with the DRY treatment resulting in moderate suppression of herbage growth and a minor reduction in WSC reserves (p < 0.05).ConclusionsResults were consistent with field studies and recommendations to allow perennial ryegrass tillers to set seed to improve pasture resilience.

AbstractBackgroundPerennial ryegrass (Lolium perenne) in New Zealand pastures is typically infected with the mutualist Epichloë fungal endophyte. This endophyte assists the plant in resisting biotic and abiotic stresses, but the standard strain of endophyte is toxic to livestock. Elite ryegrasses with selected endophytes have been developed to provide protective properties to the grass plant and lessen or eliminate the negative impacts on livestock.MethodsUsing immunology and molecular techniques, the presence of endophyte infection and endophyte strain in ryegrass tillers was determined for 24 dairy pastures sampled for up to 7 years in regions of the North and South Islands.ResultsIn general, infection levels were high and showed small increases over time. Some pastures failed to reach 70% infection. The sown, selected endophytes were the dominant endophyte strains present and these were stable over time. Standard endophyte was the primary nonsown endophyte, and while generally low and so of little importance, it increased over time and for some pastures, this would have been detrimental to livestock. Pasture establishment technique influenced the level of contamination.ConclusionsResults reinforce the importance of following best practice procedures in the seed industry and on‐farm. Researchers should monitor trials for contaminating nonsown standard endophyte.

Context Data are lacking on the effects of heading date of perennial ryegrass (Lolium perenne L.) on ryegrass persistence. Aim We aimed to determine the impact of heading date on perennial ryegrass persistence. Method Two mid-heading (Samson, Bronsyn) and two late-heading (One50, Rohan) cultivars, infected with Epichloë endophytes (AR37, nea2/6 or standard toxic), were established in a replicated plot study grazed by cattle in a subtropical environment of the upper North Island of New Zealand. Persistence characteristics were quantified at least five times per annum, over 4 years. Key results Late-heading cultivars had higher yield, nutritive value, perennial ryegrass content in pasture dry matter, ground cover and tiller density than mid-heading cultivars. There were large seasonal impacts on all ryegrass characteristics, with a major decline over summer and recovery during late autumn into early spring, with less recovery in the final year. Overall, there was a strong linear decline in ryegrass content measured during spring (2018–21). Conclusions The two late-heading cultivars were more persistent than the two mid-heading cultivars, although persistence declined over 4 years for all cultivars. Implications Results support industry recommendations of choosing late-heading cultivars for improved pasture quality and persistence.

Epichloë spp. often form mutualistic interactions with cool-season grasses, such as Lolium perenne. However, the molecular mechanisms underlying this interaction remain poorly understood. In this study, we employed reduced representation bisulfite sequencing method (epiGBS) to investigate the impact of the Epichloë sp. LpTG-3 strain AR37 on the methylome of L. perenne across multiple grass generations and under drought stress conditions. Our results showed that the presence of the endophyte leads to a decrease in DNA methylation across genomic features, with differentially methylated regions primarily located in intergenic regions and CHH contexts. The presence of the endophyte was consistently associated with hypomethylation in plants across generations. This research sheds new light on the molecular mechanisms governing the mutualistic interaction between Epichloë sp. LpTG-3 strain AR37 and L. perenne. It underscores the role of methylation changes associated with endophyte infection and suggests that the observed global DNA hypomethylation in L. perenne may be influenced by factors such as the duration of the endophyte-plant association and the accumulation of genetic and epigenetic changes over time.

Perennial ryegrass staggers (staggers) is a neurotoxic condition in livestock that is caused by consumption of ryegrass (Lolium perenne) infected with specific strains of Epichloë fungal endophytes. These grass-endophyte associations produce toxins that can adversely affect animals and can in some cases lead to death. In sheep, symptoms typically include head shaking, changes in gait, stiffness and falling. Affected sheep can recover after removing them from pastures containing toxic strains of endophyte. A pilot case study was conducted in Lincoln, New Zealand to determine if ryegrass staggers could be identified with data collected through GPS tracking and accelerometers. Fourteen sheep per treatment grazed in either a toxic endophyte-infected ryegrass paddock or an endophyte-free control paddock for 17 days in late March and early April 2017. Randomly selected sheep were fitted with collars containing a 3-axis accelerometer recording movements at 12 Hz (10 collars in endophyte infected paddock and 6 in the control paddock). Three sheep per treatment were also tracked at 3-minute intervals with GPS receivers. Sheep were scored by an experienced observer for symptoms of staggers weekly and at the end of the study using a 0 to 5 scale. Control sheep did not display any symptoms of staggers and 10 sheep in the infected pasture displayed little or no symptoms (0 or 1 score). The other 4 sheep in infected pasture had scores from 2 to 4 at the end of the study. Sheep grazing in the infected pasture (2.91 m/min ± 0.04 SE) moved slower (P=0.04) than sheep in the control pasture (3.12 m/min ± 0.05 SE). Distance travelled varied among days, but there did not appear to be any temporal trends. Machine learning analyses of accelerometer data showed that the behavior of affected sheep changed during the study. Activity of sheep displaying symptoms (scores ≥ 2) increased more in the morning and midday during the latter part of the study than control sheep and sheep with few or no symptoms (score < 2). However, behavior of individual sheep at night remained relatively consistent during the study. Accelerometers may be useful for remotely detecting perennial ryegrass staggers.

The dairy industry provides an important contribution to the Australian economy, but its productivity relies on grass pastures that suffer significant damage from invertebrate pests. Managing these pests remains a challenge as information on their abundance and impact is only available for a handful of taxa in a few Australian dairy regions. In this study, we undertook an extensive survey of above‐ and below‐ground pest communities across seven dairy regions in south‐eastern Australia by repeatedly sampling 57 paddocks in 2017 and 2018. We then applied energetic models to estimate the amount of metabolisable energy produced by pastures that are potentially consumed by pests. Our survey indicates that dairy farmers encounter a similar composition of above‐ground pests in most sampled regions, with a few pests, especially Sminthurus viridis (lucerne flea) and Rhopalosiphum padi (bird cherry‐oat aphid), dominating pest communities. Below‐ground pests were more variable between regions and are more likely to require region‐specific control strategies. Our energetic modelling suggests that pests consume a threefold greater percentage of metabolisable energy produced by pastures during autumn than spring. S. viridis and R. padi were among the most economically important pests across all regions and seasons, while other pests, including several species of scarabs and the small pointed snail, Prietocella barbara , were predicted to be particularly damaging in specific regions. Together, our field survey and energetic modelling provide baseline information to assist dairy farmers manage invertebrate pests and help guide future research in the Australian dairy industry.

Asexual Epichloë are obligate fungal mutualists that form symbiosis with many temperate grass species, providing several advantages to the host. These advantages include protection against vertebrate and invertebrate herbivores (i.e., grazing livestock and invertebrate pests, respectively), improved resistance to phytopathogens, increased adaptation to drought stress, nutrient deficiency, and heavy metal-containing soils. Selected Epichloë strains are utilised in agriculture mainly for their pest resistance traits, which are moderated via the production of Epichloë-derived secondary metabolites. For pastoral agriculture, the use of these endophyte infected grasses requires the balancing of protection against insect pests with reduced impacts on animal health and welfare.

The aphid Aploneura lentisci is widespread in Australia and New Zealand, living all year round on roots of its secondary grass hosts. The fungal endophyte (Epichloë festucae var. lolii), strain AR37 in Lolium perenne is known to greatly reduce populations and was a likely reason for the superior growth and persistence of this association previously observed in the field. Aphid populations were quantified in a field trial near Ballarat, comparing yields of perennial ryegrass infected with eight different endophyte strains and an endophyte-free (Nil) control in a common ryegrass background (Grasslands Samson (G. Samson)). AR37 and another endophyte strain, AR5, had significantly fewer aphids than all other endophytes. These differences were significantly related to yield increases taken before and after sampling that persisted until the end of the trial. In a pot trial comparing commercially available ryegrass-endophyte combinations with equivalent Nil controls, aphid numbers were lower on G. Samson AR37 and Banquet II with AR5 (Endo®5) than on all other cultivar-endophyte combinations. Compared with Nil controls, the common toxic strain in G. Samson, and two strains in Trojan also reduced aphid numbers. The AR5 endophyte produces the alkaloid ergovaline but high concentrations of this in roots of potted plants could not account for differences in root aphid numbers. Root concentrations of epoxyjanthitrems, the only known alkaloids produced by AR37, were low and unlikely to be the cause of resistance to A. lentisci.

Epichloe fungal endophytes that systemically colonise cool-season grasses have become an internationally well-researched symbiosis. While sexual species of Epichloe can cause choke disease of grass seed heads, asexual forms are symptomless endophytes that colonise embryos to become seed transmitted. In mutualistic associations, the grass benefits through greater tolerance of abiotic and biotic stresses. In New Zealand, at least 12 species of naturalised and native grasses have been documented as infected with a range of asexual or sexual Epichloe. The impact of endophyte-infected ryegrass and tall fescue has been significant for pastoral farmers, with Epichloe enhancing pasture performance. Production of bioactive secondary metabolites reduces herbivory by invertebrate pests but some are also toxic to grazing animals. Selected strains of Epichloe have been utilised to reduce livestock problems while retaining improved pasture performance. Documented Epichloe-infected native grasses are rare, justifying greater efforts of discovery, description and conservation as taonga (a treasure) for Maori and all New Zealanders. Given their ecological success in New Zealand ecosystems, along with economic and environmental benefits, there are good prospects to further develop both endemic and naturalised Epichloe-grass associations that may benefit other cool-season grasses and potentially deliver new beneficial properties.

ABSTRACT In identifying endophytes for use in pastures in New Zealand there have been two strategies used, either exclusion of ergopeptine and lolitrem alkaloids or, retaining some ergopeptine expression to enhance the stability of natural defences against invertebrate pests. Both have their strengths and weaknesses. It is a matter of balancing these to ensure the end-user has access to a product that satisfies their needs – a persistent pasture with low to nil animal toxicity in terms of animal production and welfare. The range and intensity of ergot alkaloids in grazed pasture on both pasture and ruminants is reviewed, with emphasis on New Zealand. Ergot alkaloids associated with pasture are produced by associations between certain Epichloë endophyte strains and temperate grasses. Ergot alkaloids have been shown to improve persistence of pasture through providing resistance/deterrence to insect pests as well as deterring grazing animals. However, ergovaline is toxic to grazing animals. Some commercially available ryegrass-endophyte associations can produce ergovaline concentrations close to those found in associations between ryegrass and the standard endophyte. It is feasible to eliminate ergot alkaloids from pasture grasses in New Zealand as endophyte strains are commercially available that do not express ergovaline and yet still provide excellent pest resistance.

(2020). Epichloë fungal endophytes play a fundamental role in New Zealand grasslands. Journal of the Royal Society of New Zealand: Vol. 50, Ngā Kete: The 2020 Annual Collection of Reviews, pp. 279-298.

Infection of the pasture grass Lolium perenne with the seed-transmitted fungal endophyte Epichloë festucae enhances its resilience to biotic and abiotic stress. Agricultural benefits of endophyte infection can be increased by generating novel symbiotic associations through inoculating L. perenne with selected Epichloë strains. Natural symbioses have coevolved over long periods. Thus, artificial symbioses will probably not have static properties, but symbionts will coadapt over time improving the fitness of the association. Here we report for the first time on temporal changes in a novel association of Epichloë strain AR37 and the L. perenne cultivar Grasslands Samson. Over nine generations, a seed maintenance program had increased the endophyte seed transmission rates to > 95% (from an initial 76%). We observed an approximately fivefold decline in endophyte biomass concentration in vegetative tissues over time (between generations 2 and 9). This indicates strong selection pressure toward reducing endophyte-related fitness costs by reducing endophyte biomass, without compromising the frequency of endophyte transmission to seed. We observed no obvious changes in tillering and only minor transcriptomic changes in infected plants over time. Functional analysis of 40 plant genes, showing continuously decreasing expression over time, suggests that adaptation of host metabolism and defense mechanisms are important for increasing the fitness of this association, and possibly fitness of such symbioses in general. Our results indicate that fitness of novel associations is likely to improve over time and that monitoring changes in novel associations can assist in identifying key features of endophyte-mediated enhancement of host fitness.

Epichloe endophytes (Clavicipitaceae) infect pooid grass genera worldwide but predominantly in the Northern Hemisphere, but appear to be rare in native grasses of the Southern Hemisphere. Because of benefits that hosts may receive from the symbiosis, Epichloe endophytes have been extensively studied and are considered important components of sustainable agriculture. There are only a few studies available on the incidence of endophyte infection in grasses of the Southern Hemisphere and most grass species have never been examined. Here we report on a survey of native grasses of New Zealand including 25 endemic or indigenous species. We sampled up to 10 plants per species at different sites from both the North and South Island of New Zealand and examined tissues microscopically for endophyte infection. Overall, only two species were found to be infected, Poa matthewsii (Matthew's bluegrass) and Dichelachne micrantha (short-hair plume grass). Based on analyses of tefA and tubB genes, both endophytes were found to be interspecific hybrids. The endophyte of the new host D. micrantha was previously described as Epichloe australiensis, while the endophyte of P. matthewsii is a new species named here E. novae-zelandiae. The new species is a hybrid derived from E. amarillans, E. bromicola and E. typhina subsp. poae. Alkaloid analyses in planta suggested that E. novae-zelandiae can produce small amounts of peramine, early pathway indole-diterpenes and ergot alkaloids, but no lolines or lolitrems. Target specific primers suggested the presence of genes for ergot alkaloids and peramine, but genes of only early pathway steps for the other alkaloids. Furthermore, genes for both mating type idiomorphs (MTA and MTB) were present, a single copy of MTA and two copies of MTB. Endophytes of native grasses may provide a genetic resource that could be exploited for developing pasture grass cultivars with improved performance.