NZGA: Research and Practice Series

A historical perspective on the International Symposium of Grass Microbial Endophytes

Carolyn Young — 2025-12-04

The emergence of research investigating Epichloë fungal endophytes as ecological keystone species revolutionized our scientific understanding of plant-microbial interactions, illuminating both agricultural challenges (such as livestock toxicosis) and opportunities (enhanced pest resistance, drought tolerance, and sustainable production systems). Pioneering work in the early 20th century, including accounts of vertical endophyte transmission from mother plant to seed by Freeman, Sampson, Neill, and Philipson, laid the foundation for the pivotal 1970s discoveries by Bacon and colleagues in the USA linking endophytes in tall fescue to fescue toxicosis. Shortly after, Fletcher, Mortimer and colleagues in New Zealand linked endophytes to ryegrass staggers and pest resistance. From there, research accelerated to build a comprehensive understanding of how endophytes shape plant productivity, stress tolerance, and ecological resilience. The breeding of cultivars that maintain plant protection while reducing animal toxicity illustrates the field’s ongoing commitment to practical, science-based solutions for pasture-based agricultural systems.

The International Symposium on Fungal Endophytes of Grasses was first held in New Orleans in 1990, arising from a surge of interest in the practical and theoretical implications of endophyte–grass relationships. Originally focused on Epichloë fungi, the symposium has expanded over time to include broader beneficial plant-associated microbes and is now encapsulated as the International Symposium of Grass Microbial Endophytes. Held every 3–4 years, it has become the central international forum for researchers studying endophytic microbes in temperate grasses, drawing participants from around the globe.

Today, the symposium offers not only cutting-edge science—from microbiome interactions to integrated pest management—but also a deep sense of community. For over three decades, it has fostered mentorship, collaboration, and a shared enthusiasm for the intricate biology of grass-endophyte symbioses. As we gather for ISGME 2025 in New Zealand—where many foundational discoveries were made—we celebrate the vibrant, global community that continues to push the boundaries of knowledge in grass microbial endophytes. This historical perspective aims to honour the milestones achieved while embracing the technical sophistication that now allows us to ask—and answer—increasingly complex scientific questions. ISGME 2025 represents not just a continuation, but a reconnection: to place, to progress, and to the people who have shaped this field into what it is today.

Epichloë endophytes – underpinning sustainable food production from pastoral systems

John Roche — 2025-12-04

The world faces an incredible challenge to produce food in an environmentally sustainable manner. In the next 25 years, almost as much protein must be produced as has been produced in the last 2,000 years. Ruminant animal protein sources are key contributors to essential amino acids and human micronutrient needs. This must be achieved while using less land, having a smaller environmental footprint and while providing greater care for production animals; and this must be accomplished in the face of a more volatile climate. Grazing systems are a significant contributor to global food security, with 10-15% dairy products and more than 30% of red meat produced from grazing animals, and there is a growing consumer trend for ‘grass-fed’ or ‘pasture-raised’ animal products. In New Zealand, almost all dairy and red meat are produced from pasture.

The symbiotic relationship between asexual Epichloë endophytes and improved pasture grass species has been critical in developing sustainable animal production systems in many parts of the world, where insect attack would otherwise limit productivity. This need is likely to grow with climate change, as the habitat for these insects grow to regions previously unsuitable, and as consumers demand fewer chemical interventions. Advances in endophyte selection have reduced some of the negative effects on animal health and welfare and further developments will continue to improve their function in grazing systems. Furthermore, advances in genetic technologies may allow the development of endophytes that produce chemicals that confer productivity or sustainability benefits for production systems. With a world in need of more food sustainably produced, advanced genetic manipulation of endophytes with novel traits should be a focus of future research and development.

Epichloë evolution: What ChatGPT won’t tell you

Christopher Schardl — 2025-12-04

Sexual Epichloë species were described by Persoon in 1798, Fries in 1849 and the Tulasne brothers in 1865, whereas strictly seed-borne grass-Epichloë symbioses were first reported in separate 1898 papers by P. Guerín, A. Vogl and A. Nester, then by K. Sampson in 1933 and J.C. Neill in 1941. However, it was the watershed paper by Bacon et al. in 1977 that linked them to livestock toxicoses and sparked the major international research activities that continue today. Amazingly, without having the advantages of well-known model organisms, the Epichloë species have nevertheless provided outstanding examples of numerous evolutionary processes.

That said, I asked ChatGPT to give me a short abstract, which I minimally corrected to give the following paragraph. Epichloë species (fam. Clavicipitaceae) are endophytic fungi that form symbiotic relationships with cool-season grasses (Poaceae subfamily Poöideae), playing a crucial role in the ecology and evolution of grassland ecosystems. Over time, the genus has diversified through both sexual and asexual lineages, with many asexual species arising via hybridization events. These fungi are notable for producing a diverse array of alkaloid compounds—such as ergot alkaloids, lolines, peramine, and indole-diterpenes—that provide chemical defences to host grasses against herbivores. The evolution of these alkaloid pathways is complex, involving gene duplication and neofunctionalization, gene losses and perhaps horizontal gene transfer (HGT), and shaped by selective pressures from plant-herbivore interactions. Comparative genomics has revealed a dynamic and modular organization of alkaloid biosynthetic gene clusters that seemingly facilitates diversification of alkaloid profiles. This chemical diversity contributes to the ecological success of both the fungi and their hosts. Understanding the co-evolution of Epichloë species and their alkaloids sheds light on symbiotic innovation, adaptation, and the evolutionary mechanisms underlying mutualistic interactions in plant-microbe systems.

It is a credit to the current, vibrant research community that the above paragraph requires updating.

One area of new knowledge concerns the diversity of alkaloids and underlying genetics and biosynthetic pathways. Ergot alkaloids of Epichloë species include ergovaline, a complex ergopeptine that is highly toxic to livestock, simpler lysergyl amides that are psychotropic but not as acutely toxic, and chanoclavine, long considered an early intermediate but now recognized as a pathway end-product in several strains, but one that has little or no effect on mammals. Similarly, some Epichloë species or strains produce exo-1-acetamidopyrrolizidine (AcAP) rather than lolines in the 1-aminopyrrolizidine class of alkaloids. Also recently discovered is an array of pyrrolopyrazines, making that class much richer than just peramine, and adding allelic trans-species polymorphism among the evolutionary mechanisms known to enhance chemotypic diversity. Finally, among the highly diverse indole-diterpene class, recent focus has led to newly identified genes for biosynthesis and diversification of the highly complex epoxyjanthitrems, which can help protect against insects.

Host specificity is evident in associations of Epichloë species with particular grass genera or tribes, but underlying mechanisms are only beginning to be revealed (with discovery of afpA), and it is unknown how such specificity is overcome to allow hybrid formation. For example, Epichloë bromicola strains that produce N-formylloline were discovered in Isfahan Province of Iran and are, or are closely related to, the contributors of LOL clusters in ca. half of the known loline-producing hybrid Epichloë species. Those same populations had dramatically broader host ranges for both E. bromicola and Epichloë festucae than previously known. These findings suggest that hybridization of Epichloë species may occur in geographical hotspots where local grasses may have been selected for broaden compatibility with Epichloë species.

Other recent findings indicate rare but important HGTs, including a trichothecene detoxification gene originating from Epichloë that acts as a disease resistance gene in a wild relative of wheat.

Thus, recent findings in the Epichloë evolutionary “model” involve host specificity, polyploidy and hybridization hotspots, and gene dynamics such as duplications, rearrangements, selective losses, trans-species polymorphisms, and even inter-kingdom HGT.

Epichloë endophytes – from science to commercial impact

Linda Johnson — 2025-12-04

Epichloë species are filamentous fungal endophytes within the family Clavicipitaceae that form enduring symbioses with temperate grasses of the Pooideae. These plant-microbe associations are ecologically important constituents of many temperate grassland ecosystems. In New Zealand, selected asexual strains of Epichloë have been incorporated into elite grass cultivars where they predominantly confer protection against invertebrate pests. These novel grass-endophyte associations underpin sustainable pastoral farming practices in managed temperate grasslands in many countries such as in New Zealand, Australia, and the USA. For example, the commercialisation of endophyte strain AR37 in a wide range of ryegrass cultivars has been estimated to contribute NZ$3.6 billion to the New Zealand economy. This has led to greater productivity and persistence of pastures, resulting in increased livestock productivity and improved health and welfare.

In New Zealand, a multidisciplinary science team at AgResearch has, and continues to, underpin the development of selected Epichloë-grass technology with many endophyte products now successfully marketed for pasture persistence and wild-life deterrence. The team’s success has relied on their diverse capability set in areas of science such as agronomy, animal science, entomology, plant biology, fungal genetics, mycology, seed science, toxicology, and natural product chemistry, along with the application of fundamental and applied science to study grass-endophyte symbioses.

In the pursuit of new agriculturally useful grass-endophyte associations, the team at AgResearch has furthered the understanding of the molecular interactions that govern genetic compatibility between endophyte strains and their grass hosts. This new knowledge has led to the development of novel endophyte associations in cereal crops (e.g. rye and wheat). Additionally, the AgResearch team has developed a gene editing platform to precisely custom design previous genetically intractable Epichloë strains to deliver the ideal combination of chemistry and subsequent conferment of bioactivity within the grass host using CRISPR-Cas9 technology. These scientific endeavours aim to broaden the biocontrol capability of Epichloë strains, particularly to mitigate climate related stresses in globally important agriculturally crops.

Prevalence of seedborne symbionts in grass populations: connecting host fitness, endophyte transmission and intergenerational effects

Pedro Gundel — 2025-12-04

Asexual Epichloë fungal endophytes (Clavicipitaceae) are transmitted vertically from mother plants to seeds. These endophytes enhance plant fitness primarily by conferring resistance to herbivores and promoting growth under competitive and stressful conditions. However, variation in symbiosis prevalence among populations likely arises from differences in symbiosis outcome (i.e. relative fitness of symbiotic vs. non-symbiotic plants), and vertical transmission efficiency. Like any other symbiotic interaction, the outcome of plant-Epichloë symbiosis is context-dependent, yet how vertical transmission efficiency aligns with this variability remains unclear. Symbiosis prevalence is generally higher in sites with greater ecosystem productivity, and vertical transmission efficiency is often positively correlated with host fitness. However, exceptions suggest that certain stressors or genetic factors involved in plant-Epichloë compatibility may decouple vertical transmission efficiency from plant fitness. Recent evidence indicates that intergenerational effects induced by environmental factors may reinforce endophyte-mediated benefits, improving plant response to the same stressor (e.g., herbivory or drought). Conversely, other factors that disrupt the symbiosis may have the opposite effects. By linking individual processes to population-level dynamics, we can gain a deeper understanding of the mechanisms driving variation in plant-Epichloë symbiosis prevalence.

Coevolution of Pooideae grasses and their Epichloë endophytes within a hologenome framework

Pilar Catalán — 2025-12-04

Pooideae represents a diverse subfamily of grasses with over 4000 species classified into 12 tribes and 216 genera. Over 67 million years of evolution shaped their dynamic genomes, resulting in their enormous phenotypic and ecological diversity. During this time, many members of the Pooideae have further co-evolved with fungal endophytes of Epichloë (family Clavicipitaceae). Epichloë display long-term associations with their temperate grass hosts and are naturally restricted within the Pooideae to a host genus or closely related grass genera within a tribe. Epichloë grow systemically within the above ground organs of their grass hosts and form a continuum of interactions from vertically transmitted mutualists (asexual species) to horizontally transmitted antagonists (sexual species). While sexual Epichloë species are haploid, most asexual species are alloploids, interspecific hybrids having ancestries involving two or more distinct Epichloë lineages. Comparative genomic and phylogenomic analyses of Pooideae and Epichloë species make these symbioses an excellent model for tracing coevolution through diversification within a hologenomic framework. We hypothesized that the predominant vertical transmission of these endophytes would favour a Pooideae-Epichloë coevolutionary scenario, although horizontal transmission, both sexual and asexual, of endophytes can alter this. We reconstructed the phylogeny of Pooideae host species using single-copy nuclear genes and that of Epichloë species using nuclear barcoding markers and available sequenced genomes. We also filtered and assembled Epichloë genes from host genome skimming data. Phylogenetic reconstructions were performed using maximum likelihood and coalescence-based approaches. Potential coevolutionary trends of Pooideae grasses and their Epichloë fungal endophytes were analysed statistically, based on host-parasite associations, and through concordance scores of host-endophyte topological conflicts. We performed coevolutionary analyses for the main tribal and subtribal representatives of Pooideae and described Epichloë species, and then focused on the more recently evolved Loliinae grasses (e.g., Festuca and Lolium) and their Epichloë symbionts. We found diffuse and lineage-specific coevolutionary trends for some groups of Pooideae and their endophytes, also explored through co-genomic approaches. The more detailed analysis of ~200 Loliinae species confirmed that at least 15% of them associate with Epichloë endophytes. Furthermore, we detected 20 lineage-specific relationships for temperate and tropical mountain fescues and their endophytes. Our analyses revealed significant coevolution for Loliinae and their symbionts at individual scale for two lineages, the fine-leaved F. sect. ovina clade (sheep fescues) with E. festucae strains, and the broad-leaved Schedonorus-Lolium clade (meadow and tall fescues, ryegrasses) with E. typhina, E. hybrida, E. occultans, E. siegelii, and E. uncinata strains. Interestingly, these two groups of Loliinae showed high intraclade hybridization rates, but low interclade rates. Our results highlight the impact of the narrow vs. broad hybridogenous nature of the host plant in the strong vs. weak lineage-specific coevolutionary trends with their respective Epichloë endophytes.

Decomposition potentialities of Epichloë bromicola from Elymus racemifer

Yuki Hatano — 2025-12-04

Fungal endophytes of the genus Epichloë possess weak saprotrophic abilities, but no quantitative studies have been conducted regarding their abilities to decompose grass litter. In the present study, Epichloë bromicola strains were isolated from Elymus racemifer, a common perennial grass in Japan, and assessed for their ability to decompose sterilised grass leaves under pure culture conditions. These abilities were compared with those of two non-systemic endophytic fungi, Phomopsis sp. and Biscogniauxia maritima, sympatrically encountered in the live tissues. Mass loss of leaves caused by 16 strains of E. bromicola on potato dextrose agar at 20 ^oC for 12 weeks ranged from 0.4% to 10.8% of the original leaf mass, with a mean of 4.9%. This value was not significantly different from that of Phomopsis sp. (mean mass loss of 5.6%) but was significantly lower than that of B. maritima (mean mass loss of 20.8%) measured under the same culture conditions. The low decomposing ability of E. bromicola suggests that this fungus is not responsible for significant decomposition of grass litter, which is consistent with the current belief that Epichloë endophytes obtain photosynthetic product from live plant tissues but rarely elongate hyphae on dead tissues.

New Zealand’s pastoral ecosystems: challenges, solutions, and the protective role of Epichloë endophytes

Katrin Gabriela Hewitt — 2025-12-04

New Zealand’s pastoral ecosystems heavily rely on introduced grass species like perennial ryegrass (Lolium perenne) and fescues (Festuca spp.), which can form mutualistic relationships with Epichloë endophytes. These endophytes protect grasses from insect herbivory by producing alkaloids that deter insect pests. However, while they benefit the plant, these alkaloids can also negatively affect grazing livestock. Unlike many other temperate grassland systems, New Zealand’s pastures have low plant diversity, with most sown species originating from Europe. This lack of diversity, combined with a historically limited suite of natural enemies, has contributed to a unique pasture insect pest burden. Pests such as Argentine stem weevil (Listronotus bonariensis), black beetle (Heteronychus arator), and root aphid (Aploneura lentisci) cause significant economic losses, threatening pasture persistence and productivity. To mitigate these challenges, many of New Zealand’s dominant pasture grasses are artificially associated with selected Epichloë endophytes that provide insect protection against key insect pests while minimising impacts on grazing livestock. These endophytes produce alkaloids that act as feeding deterrents and/or reduce the fitness of herbivorous insects, providing a natural bio-protection mechanism. This review explores the unique ecological characteristics of New Zealand’s pasture systems, including the impact of invasive and native insect pests and the role of Epichloë endophytes in pasture protection. Emerging challenges include climate change-driven shifts in pest populations and the need for sustainable pasture management strategies. Integrating ecological principles with advanced endophyte technologies can improve pasture resilience while minimising environmental trade-offs.

Identification of biomarkers associated with the survival of selected Epichloë endophytes while in seed storage

Sushma Prakash — 2025-12-04

Epichloë is a genus of fungal endophytes that form symbiotic associations with cool season grasses in the sub-family Pooideae. In New Zealand, selected strains of asexual Epichloë endophytes have been developed into commercial products for pasture persistence and wild-life deterrence where they confer bioprotective traits to their host grasses. As asexual Epichloë species are exclusively vertically transmitted, their survival in seed is critical for their dissemination to progeny plants. Endophyte survival declines at a much faster rate than seed viability, however the mechanism responsible for this decline is unknown. This study investigated the effects of seed aging on mycelial biomass and selected metabolite concentrations in Epichloë-infected seed of perennial ryegrass (Lolium perenne) with the main objective to identify key metabolites that play a significant role in the viability of endophyte in seed during storage. Fifty-eight seed lines, comprised of three cultivars of perennial ryegrass associated with two economically important endophyte strains (AR1 and AR37), were subjected to an accelerated ageing regime designed to mimic the physical deterioration processes that would occur during natural seed aging. Selected amino acids, neutral sugars, sugar alcohols, phytohormones and Epichloë-derived secondary metabolites were then analysed and their concentrations examined for effect of seed aging, cultivar, and endophyte strain. As predicted, accelerated ageing had little to no effect on the seed germination rate of the selected ryegrass cultivars but negatively impacted the frequency of endophyte viability within seed. Proline and xylose gave the greatest fold changes in concentration across all grass cultivars after seed aging but were not correlated to endophyte infection frequencies and therefore not suitable as biomarkers. Ribitol, a sugar alcohol, exhibited concentration fold changes that correlated with endophyte viability and therefore has potential as a biomarker to trace the viability of endophytes in perennial ryegrass seed during storage.

Using tall fescue symbiotic diversity to improve pasture resilience and sustainability

Alayna Jacobs — 2025-12-04

Tall fescue-dominated pastures cover 14 million ha in the eastern US and support 8.5 million beef cattle, making continued productivity and sustainability a high priority. Most tall fescue stands are infected with the symbiotic fungal endophyte Epichloë coenophiala, which improves grass growth and productivity in hot weather and under heavy grazing. Compounds produced by the most common tall fescue cultivar and strain of the fungus (‘KY 31’ common toxic strain) are harmful to grazing animals and lead to economic losses. Alternative fungal strains that do not produce alkaloid compounds toxic to grazing animals were selected and inoculated into grass cultivars to create novel endophyte-grass associations and these have been commercially available to producers for over two decades. Though novel individual tall fescue-endophyte symbioses have been evaluated for forage production and stand longevity, there is little information about the performance of mixed stands that contain multiple tall fescue cultivar endophyte symbiotic combinations. It was hypothesized that increasing fescue symbiotic diversity in stands would increase pasture resilience and sustainability by promoting fescue dominance and productivity and increasing soil carbon pools. Using custom seed mixes, six levels of increasing symbiotic diversity were established with plant and soil parameters compared after five years. Consistent with the hypothesis, more symbiotically diverse stands produced more fescue biomass (+12%) in the fall and limited the invasion of other weedy grass species, while also increasing soil carbon and nutrient pools. The most symbiotically diverse stand emitted more soil carbon dioxide than the least-diverse stand, perhaps due to greater root growth. These results show that increasing symbiotic diversity represents a new, readily adoptable, and effective strategy for increasing soil fertility and tall fescue productivity.

Mentoring undergraduate research with the Epichloë-tall fescue symbiosis

Tammy Tintjer — 2025-12-04

A science undergraduate research experience can benefit students in a number of ways—students learn first-hand about the process of science, develop critical thinking skills, and begin to identify as a scientist—which can increase retention in science careers. The actual value of an undergraduate research experience depends on a number of factors including the length of the research, the student’s ability to understand the conceptual basis of the project, and the type of mentoring. Faculty-mentored undergraduate research is common at small liberal-arts institutions. These same institutions often have little funding for research, leading to budgetary constraints. The grass fungal endophyte study system between Epichloë and tall fescue will be used to assist students to understand research methodology and processes at a small liberal-arts college where all biology undergraduates complete at least one semester of research. Examples will be provided of student projects to illustrate the utility of this study system to provide undergraduates, who have different levels of preparation and engagement, a successful research experience. The projects have focused on the ecology of the symbiosis and include the use of detection and culturing techniques, feeding assays, field surveys, greenhouse experiments, artificial infections, and the development and application of a novel bioassay. Results of student research, including a recent experiment supporting the ability of a brine shrimp bioassay to detect differences in the mammalian toxicity of wild-type common-toxic and selected commercial Epichloë endophytes of tall fescue will be highlighted. Mentored students often publish and/or present their research—providing the opportunity for them to more fully participate in the process of science. This study system works well for undergraduate research because the projects can be conducted relatively inexpensively, the techniques are accessible, and the conceptual framework to understand the basics of this symbiosis is within reach of undergraduate students.

Combined analysis of population genetic structures in a living-fossil grass Brachyelytrum erectum, and its endophyte Epichloë brachyelytri.

Christopher Schardl — 2025-12-04

Brachyelytrum is the earliest diverging genus known relative to other genera of grass subfamily Poöideae. Brachyelytrum erectum inhabits forest understory in temperate eastern North America and possesses the endophyte Epichloë brachyelytri. A genome sequence of E. brachyelytri included genes for biosynthesis of peramine, the loline alkaloid precursor exo-1-acetamidopyrrolizidine (AcAP), and the ergot alkaloid precursor chanoclavine. In a survey of 24 B. erectum populations in Kentucky and southern Indiana, endophytes were genotyped by PCR for alkaloid genes and mating types. All had ppzA1 for peramine whereas genes for AcAP and chanoclavine exhibited presence/absence polymorphism. Approximate alkaloid levels in leaves were, per g dw, 4–15 µg peramine, 2000–5000 µg AcAP and 175–300 µg chanoclavine when the corresponding gene clusters were present. The sexual state of E. brachyelytri was observed on few plants in only two populations, and the overall ratio of mating types A and B was 1.0:4.3, indicating very limited sexual reproduction for this endophyte. We obtained RNA-seq data from 4–12 symbiotic (E+) plants and 2–7 asymbiotic (E–) plants each in seven populations separated by distances of 10–321 km (average = 171 km), then applied our recently described rna-clique method to infer pairwise genetic distance matrices comparing plants in different populations, plants with different endophyte genotypes, and endophytes with different genotypes. Results indicated geographical structure of B. erectum that distinguished even the closest populations. Within populations we observed partial structure of plant subpopulations based on symbiotic status (E+ vs. E–) or endophyte genotype. The relationship of endophyte genetic structure to endophyte genotypes was consistent with infrequent sexual recombination. Overall, the results indicate local adaptation of B. erectum populations for which its functionally and genetically diverse endophyte species plays a role.

Transgenerational trade-off: the hidden costs of maternal defoliation in grass–endophyte symbiosis

Benjamin Fuchs — 2025-12-04

Epichloë endophytes form systemic, vertically transmitted symbioses with cool-season grasses, providing chemical defence against herbivores through alkaloid production. While often described as a ‘defensive mutualism’, the fitness costs of this relationship for the host plant, particularly across generations, remain poorly understood. This study investigated whether repeated defoliation of Festuca pratensis (meadow fescue) mother plants alters seed production, seed viability, and the endophyte-conferred resistance of progeny plants.

A common-garden experiment was established using both endophyte-symbiotic (E+) and endophyte-free (E–) plants. Over one growing season, half of the plants were subjected to severe, repeated defoliation to simulate grazing stress. Plant biomass, inflorescence number, seed biomass, chlorophyll activity, and alkaloid concentrations in leaves and seeds were quantified. Seed viability and germination rates were tested, followed by a greenhouse experiment where progeny were challenged with the aphid Rhopalosiphum padi to assess resistance conferred by maternally induced alkaloids.

Results revealed a strong resource allocation trade-off. Defoliation reduced leaf alkaloid concentrations in E+ plants but increased alkaloid investment into seeds. This shift enhanced seed protection and provided seedlings with elevated alkaloid levels during the first six weeks of growth. Consequently, progeny of defoliated E+ plants exhibited reduced aphid performance compared with progeny of non-defoliated E+ plants, confirming a transgenerational defensive effect. However, these benefits came at a reproductive cost: seeds from defoliated E+ plants had significantly lower germination rates than seeds from E+ non-defoliated or E– plants. Germination was negatively correlated with seed alkaloid concentrations, suggesting that endophyte-driven defence investment compromised reproductive success.

These findings highlight a context-dependent nature of the grass–endophyte relationship. Under intense foliar stress, the endophyte prioritizes transgenerational defence, allocating resources toward alkaloid-enriched seeds at the expense of seed viability. While this strategy enhances early-stage herbivore resistance in progeny, it reduces the number of viable offspring, suggesting a fitness trade-off between defence and reproduction. From an evolutionary perspective, this dual outcome may help explain the observed variability of endophyte frequencies in natural grass populations. In environments with high herbivory pressure, enhanced seed protection could outweigh reduced germination, whereas under low herbivory, the cost to reproductive output may favour endophyte-free grasses. Overall, the study demonstrates that Epichloë-conferred defensive mutualism does not fully bypass the growth–defence trade-off but instead redistributes it across generations, balancing survival advantages against reproductive costs.

Hidden players in evolution: The impact of mutualistic fungal Epichloë endophytes on niche breadth and diversification across Poa (bluegrass)

Eleanor Hay — 2025-12-04

Understanding what factors promote species’ range expansions and speciation is a central goal in ecology and evolutionary biology. Grasses (Poaceae) are one of the most ecologically and evolutionary successful taxonomic groups and have undergone extensive global radiations. Yet, the underlying processes responsible for their success remain largely unknown. One hypothesis is that symbiotic relationships could be underpinning the radiation of this group, with Epichloë spp. thought to be a hidden player. Infecting up to 20-30% of grass species, Epichloë endophytes can provide a range of advantageous traits to their hosts, including defence from herbivores, drought tolerance, and enhanced nutrient uptake, and are known to expand species’ range limits. However, it is unclear whether these benefits can scale up to have macroevolutionary consequences and influence diversification of clades. In this study, the hypothesis was tested that plant associations with Epichloë expand niche breadth and drive diversification in Poa, a diverse clade of ~600 cool season grass species that are known to form relationships with Epichloë spp. Information on endophyte status was compiled and ecological niche models for over 100 Poa species constructed. Using a range of phylogenetic comparative methods, the evolutionary history of Epichloë and Poa was investigated and the role of endophytes on the evolution of niche breadth and diversification in this group assessed. Epichloë endophytes were found to have played a crucial role in the evolution of Poa, and accelerated niche breadth evolution. Despite this, little evidence was obtained showing that Epichloë spp. significantly influenced diversification dynamics of this plant group. Overall, this study provides insight into how symbiotic interactions can influence the ecological and evolutionary success of species and contributes to understanding of factors underpinning biodiversity across the Tree of Life.

Enhancing plant defence: The role of mutualistic fungi in silicon-mediated pest resistance

Ximena Cibils-Stewart — 2025-12-04

Grasses have primarily coevolved with large grazing herbivores, a relationship that has strongly shaped their defensive architecture. This evolutionary history has led to the development of traits such as basal meristems and prolific tillering, enabling rapid regrowth following grazing. Consequently, grasses typically lack a diverse arsenal of chemical defences and instead rely on alternative strategies, including rapid tissue turnover, physical barriers, and symbiotic associations with microorganisms to deter insect herbivores. Microbial symbionts may provide chemical protection, while structural defences, such as silicon accumulation, play a key role. Silicon serves as a major structural defence against both biotic and abiotic stress, comprising up to 10% of grass dry mass. Many temperate grasses associate with mutualistic fungi, such as Epichloë endophytes and arbuscular mycorrhizal fungi, and these symbiotic plants can exhibit altered host chemistry, subsequently influencing herbivore interactions. However, few studies have investigated how silicon-based physical defences interact with these fungal symbionts and their chemical defence mechanisms. This research addressed this knowledge gap by examining how silicon, Epichloë, and arbuscular mycorrhizal fungi interacted to affect plant resistance to insect herbivores. Two forage grasses (Festuca arundinacea and Lolium perenne) with contrasting leaf dynamics, were studied under factorial combinations of silicon supplementation, fungal inoculation (five novel grass-Epichloë associations [tall fescue: AR584 or common-toxic; perennial ryegrass: AR37, AR1, or common-toxic] or as Epichloë-free controls], and one arbuscular mycorrhizal fungal strain of Rhizophagus irregularis), and herbivore pressure from Helicoverpa armigera (a moth) and Rhopalosiphum padi (an aphid). Experiments showed that all Epichloë strains increased silicon concentrations in tall fescue on average to 31%. Of the five Epichloë strains in perennial ryegrass only strain AR37 had any effect increasing silicon concentrations. Silicon also enhanced Epichloë colonization (fungal mass) in tall fescue, but not in perennial ryegrass. In soil systems, silicon reduced the growth of H. armigera more effectively than any of the Epichloë strains, though both silicon and Epichloë contributed to plant defence in a compatible, non-antagonistic manner. In perennial ryegrass, silicon influenced Epichloë-derived alkaloid production, specifically epoxyjanthitrems. Scanning electron microscopy revealed that silicon and Epichloë could compatibly enhance surface defences and reduce insect feeding efficiency, while also compromising herbivore immunity. In a final experiment using only AR584 in tall grass fescue, tripartite interactions involving arbuscular mycorrhizal fungi, Epichloë, and silicon were explored under aphid attack. While endophytes suppressed aphid performance, arbuscular mycorrhizal fungi counteracted these effects, potentially through reduced alkaloid levels and increased foliar nitrogen. Silicon increased uptake in all symbiotic treatments but did not affect aphid performance. Overall, this research provided novel insights into the complex and context-dependent nature of grass defence systems. Silicon offered stronger protection against folivores, while endophyte-derived alkaloids were more effective against aphids. Interactions between fungal symbionts and silicon varied by plant species, fungal genotype, and herbivore type. These findings underscore the potential of harnessing natural plant–microbe–silicon interactions to enhance grass resilience against a range of biotic stressors, offering valuable insights for the development of more sustainable forage systems.

The pyrrolodiazipic acids: a new class of alkaloids produced during the virulent sexual cycle of Epichloë fungi

Daniel Berry — 2025-12-04

Fungi from the genus Epichloë form systemic and seed-transmissible endophytic infections of grasses in a symbiosis that is considered predominately mutualistic. During asymptomatic growth these Epichloë fungi produce a variety of secondary metabolites that benefit their host by providing protection against herbivory and disease. However, the Epichloë sexual cycle is antagonistic to its host, characterised by the proliferation of hyphae on reproductive tillers to form fungal stromata that encase and sterilise developing host inflorescences. Production of known host-protective Epichloë-derived secondary metabolites is suppressed in these stromata; however, we describe here the pyrrolodiazipic acids, a novel class of fungal alkaloids that are specifically produced in Epichloë stromata.

Transcriptomic analysis identified the five-gene PZA cluster that, unlike other Epichloë biosynthetic gene clusters, is specifically expressed in stromata. Reconstruction of this PZA cluster in a heterologous host enabled the function of the encoded enzymes to be defined, key biosynthetic intermediates to be identified, and the final product characterised – stromatine, a pyrrolodiazepine with carboxylic acid functionality. Analysis of material from many different Epichloë-host associations revealed that stromatine was universally and exclusively found in stroma tissues. Stromatine was also produced in exceptionally large amounts, contributing almost 1% of the total stroma dry mass in some grass-endophyte associations. PZA gene deletion analyses across three different Epichloë species yielded interesting results; stromatine appears to be required for host infection by some Epichloë species, while the accumulation of PZA pathway intermediates appears to be detrimental to symbiosis.

Genomic analyses reveal the PZA cluster is conserved among sexual Epichloë species but is dispensable for asexual strains. Combined with the tissue specificity and high levels of production, this suggests that stromatine plays a critical role in the Epichloë sexual cycle, with potential bioactivities and functions to be dissected further.

Can you see the light? How microbes and polar light environments orchestrate plant adaptations and global biodiversity

Kari Saikkonen — 2025-12-04

Efforts to mitigate biodiversity loss driven by climate change are hindered by an incomplete understanding of the geo-evolutionary processes that shape species’ adaptive capacity. Key factors include polar light regimes characterized by seasonal periods of continuous daylight and darkness ("polar night"), climate change-driven species’ range shifts, microbial interactions, and hybridization. In a recent One Earth Perspective, our team highlighted the role of light-sensitive microbes in plant adaptation and proposed that polar light environments promote circumpolar hybrid zones by synchronizing reproductive phenology, potentially maintaining genetic diversity over geological timescales. To investigate this, Antarctic hair grass (Deschampsia antarctica), one of only two native vascular plant species in Antarctica, was used as a model system. Plants from maritime Antarctica and Chilean Patagonia were (1) examined for the occurrence of plant-associated aerobic anoxygenic phototrophic bacteria (AAPB) with bacteriochlorophyll-based near-infrared fluorescence, and (2) transplanted to two climatically distinct sites in Finland: Turku (60°26′N) and Utsjoki (69°45′N). AAPBs are photoheterotrophs that rely on environmental organic carbon but capture solar energy via anoxygenic photosynthesis.

Overall, AAPBs were detected in 68% of Antarctic plants and 59% of Patagonian plants. The highest proportions were found in the leaf phyllosphere and endosphere. In the phyllosphere, AAPB presence was similar between regions: 85% of Antarctic plants and 84% of Patagonian plants. However, a notable difference was observed in the leaf endosphere, where AAPBs were found in 80% of Antarctic plants, compared to 69% of Patagonian plants. This trend was further highlighted in the root endosphere, where AAPBs were less frequently detected: 37% of Antarctic plants versus only 18% of Patagonian plants. These findings suggest a higher prevalence of AAPBs in the endosphere of Antarctic plants, particularly in the leaf tissues. Despite the shift in hemisphere, the transplanted plants adjusted to the new annual rhythm. After two growing seasons, survival and flowering patterns reflected their latitudinal origin: Patagonian plants performed better in Turku, while Antarctic plants exhibited greater success in Utsjoki. Overall, plant performance was higher in Utsjoki, likely due to more stable winter temperatures and protective snow cover. These findings highlight the role of microbes and geographic adaptation history in shaping phenological responses to extreme light and temperature conditions and suggest that D. antarctica may be capable of adjusting to the increasingly extreme photoperiods at high latitudes under climate change.

Epichloë endophytes mediate transgenerational effects in Achnatherum inebrians by enhancing photosynthetic capacity under drought stress

Chao Xia — 2025-12-04

Transgenerational effects occur when plants exposed to biotic or abiotic stresses influence progeny phenotypes by transferring information and/or resources. As vertically transmitted symbionts, Epichloë endophytes may mediate transgenerational effects, but direct evidence remains limited. Achnatherum inebrians (commonly known as drunken horse grass) is a perennial bunchgrass widespread in arid and semi-arid grasslands of northwest China, where drought stress is common. Most A. inebrians plants in northwest China host the seed-transmitted endophytes Epichloë gansuensis or E. inebrians, making these symbioses ideal systems for studying endophyte-mediated transgenerational effects. This study examined plants of A. inebrians with (E+) and without (E-) Epichloë gansuensis endophyte, derived from the same seed line but originating from either a dry or wet site. Plants were grown under three soil moisture conditions: drought (15% relative soil water content, RSWC), control (45% RSWC), and abundant moisture (60% RSWC). Key photosynthetic parameters were measured to assess the role of Epichloë with respect to transgenerational effects. As expected, E+ performed better than E- under the same treatment. While, under drought, E+ plants from the dry site had 20% higher Mg²⁺ content than E+ plants from the wet site, whereas E- plants from the dry site showed only a 9.7% increase compared to E- plants from the wet site. Similarly, Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase activities within E+ plants from the dry site were significantly higher by 21% and 17% (p<0.05), respectively, than in E+ plants from the wet site, while E- plants showed only 3% and 11% increases in these enzyme activities between dry and wet site. In other words, the Epichloë endophyte amplified the transgenerational effects of host plants, further enhancing the drought resistance of their progeny. These increases in enzymes likely led to higher chlorophyll content and net photosynthetic rates in E+ plants from the dry site, with a greater difference compared to E- plants. Our findings confirm that transgenerational effects occur in A. inebrians and that Epichloë endophytes mediate these effects by enhancing photosynthetic capacity. This may explain why E+ plants have a selective advantage over E- plants in arid grasslands and highlights the potential of Epichloë endophytes in breeding drought-resistant forage germplasm for dry regions.

Similarities and differences of metabolic potential among three Epichloë species by LC-MS

Tian Wang — 2025-12-04

Many Epichloë endophytes are characterized by their mutualistic relationship with Poaceae grasses, which not only enhances the host’s resilience to a range of biotic and abiotic stresses, but also increases its ecological competitiveness. Alkaloids produced by Epichloë spp. play crucial roles in these mutualistic interactions. Numerous studies have systematically characterized four primary classes of Epichloë-derived alkaloids: ergot alkaloids, pyrrolopyrazines (including peramine), indole-diterpenes, and 1-aminopyrrolizidines (including lolines). However, emerging empirical evidence challenges the traditional alkaloid-centric paradigm by demonstrating that the defensive traits mediated by Epichloë in host plants are not exclusively governed by alkaloids. The defensive potential of other metabolites derived from Epichloë remains inadequately understood. Further metabolite profiling could enhance our understanding of the utilization value of Epichloë strains for agricultural benefit. In this study, we employed Liquid Chromatography-Mass Spectrometry (LC-MS) to analyze the metabolites of five Epichloë strains (IB8, QG6, ED11, AD16 and LE7) across three Epichloë species (Epichloë sibirica, E. sinensis, E. bromicola) isolated from five grass species (Achnatherum sibiricum, Psathyrostachys lanuginosa, Festuca sinensis, Elymus cylindricus, and E. dahuricus). Our analysis sought to identify both common and strain-specific metabolites. The findings revealed that 1752 compounds, encompassing 15 distinct classes, were detected across five endophytic fungal strains. The predominant components comprised lipids and lipid-like substances (25.63%), organic acids and their derivatives (24.49%), and organic heterocyclic compounds (12.90%). Although strains with varying alkaloid-producing potential generated similar categories of metabolites, they exhibited quantitative differences. Unique metabolites from strains IB8, AD16, LE7, and ED11 were associated with at least one specific functional pathway each. These pathways corresponded to penicillin and cephalosporin biosynthesis (IB8), folate biosynthesis (AD16), sesquiterpene and triterpenoid biosynthesis (LE7), and the sulfur relay system (ED11). The next phase of this research will focus on elucidating functional linkages between these characterized metabolites and defensive phenotypes in endophyte-symbiotic grasses.

Multi-omics elucidation of the phytohormone biosynthetic pathways in Epichloë sinensis

Yang Luo — 2025-12-04

Epichloë sinensis is an endophyte species that forms a mutualistic symbiosis with Festuca sinensis, which improves host fitness by promoting growth and enhancing stress tolerance. Phytohormones play essential roles in regulating plant–microbial interactions. Previous research has found that E. sinensis is able to produce growth-promoting hormones, including auxin (IAA), cytokinins (CTK), abscisic acid (ABA), and gibberellins (GA). However, the underlying biosynthetic pathways remain uncharacterized. In the present study, E. sinensis strain 57A was used to systematically investigate phytohormone biosynthetic pathways based on genomic and transcriptomic analyses. Genome annotation identified 53 putative phytohormone biosynthesis genes, including key enzymes such as 6 aromatic amino acid aminotransferases, 1 indole-3-pyruvate decarboxylase, 14 aldehyde dehydrogenases, 5 nitrile hydratases, 1 ent-copalyl diphosphate synthase, and 6 cytochrome P450 monooxygenase. Comparative pathway analysis indicated that IAA in E. sinensis is primarily synthesized through the indole-3-pyruvic acid (IPyA) pathway, involving sequential reactions catalysed by aminotransferase, indole-3-pyruvate decarboxylase, and dehydrogenase. Additionally, E. sinensis may synthesize CTK through both the de novo biosynthesis pathway and the tRNA degradation pathway. The former transfers the isopentenyl chain from dimethylallyl pyrophosphate or 4-hydroxy-3-methyl-but-2-enyl pyrophosphate to the N6-amino group of adenosine monophosphate, while the latter generates CTK through the breakdown of prenylated tRNAs. Notably, the canonical GA and ABA biosynthesis pathways in E. sinensis appear incomplete, with absent homologs of alpha-ionylidenethane synthase aba3, ent-kaurene synthase, and ent-kaurenoic acid oxidase. This suggests that E. sinensis may utilize non-canonical or currently unknown pathways to produce GA and ABA, which warrants further investigation. Transcriptome analysis of E. sinensis in vitro showed that the majority of genes involved in IAA and CTK biosynthesis were expressed, which provides molecular evidence supporting our hypothesis regarding the phytohormone biosynthesis pathways in E. sinensis. In conclusion, this study provides the first comprehensive elucidation of phytohormone biosynthesis in E. sinensis and reconstructs the putative IAA and CTK biosynthetic pathways. These findings contribute to a deeper understanding of the E. sinensis endophyte and lay a theoretical foundation for further investigation into the symbiotic mechanisms.

The dynamics of selfish self-splicing introns in mitochondrial genomes of Epichloë endophytes

Jennie Chan — 2025-12-04

The genus Epichloë consists of fungal endophytes that have co-evolved with many Pooideae grass species. Epichloë species display variability in endophytic lifestyle and the propensity to hybridize, and these features make them an interesting group to study the dynamics of selfish elements. Self-splicing group I and II introns, selfish genetic elements that are widely yet patchily distributed across the tree of life, are found in Epichloë species, particularly in their mitochondrial genomes. These introns are considered selfish as they display super-Mendelian inheritance as a result of processes, collectively termed ‘homing’, that allow them to rapidly spread, both within and between populations, into the specific genomic sites they occupy. This study found that the mitochondrial genomes of Epichloë species contain a wide variety of group I and group II self-splicing introns, but these introns show substantial presence-absence polymorphism. Strikingly, the distribution of these introns does not correlate with the known phylogeny of Epichloë, nor does it correlate with Epichloë sexuality/asexuality or hybridity. Moreover, phylogenies made using Epichloë mitochondrial genomes do not correlate with those made nuclear genes. This study showed that the best explanation for the distribution of these self-splicing introns is ongoing invasions and losses over the course of Epichloë evolution. Unexpectedly, however, little evidence was found for the extensive intron degradation that the current self-splicing intron model predicts should occur prior to complete intron loss. Instead, results suggest Epichloë mitochondrial self-splicing introns are rapidly lost following their fixation. However, analysis suggests that additional factors, such as the evolution of homing suppressors, likely contribute to Epichloë self-splicing intron dynamics. Collectively, the results from this study show that self-splicing introns have more diverse evolutionary dynamics than previously appreciated. However, the results also present a conundrum as they suggest there has been relatively frequent exchange of mitochondrial introns between these endophytic Epichloë lineages over the course of genus evolution. The nature of these exchanges and whether they underlie the inconsistency between mitochondrial and nuclear Epichloë phylogenies are unknown and deserve further attention.

CRISPR-Cas9 gene insertion in Epichloë species

Taryn Miller — 2025-12-04

The dairy, meat, and fibre industries in several regions within New Zealand are heavily reliant on selected strains of endophytic fungi, within the genus Epichloë, which confer resistance to a range of insect pests and environmental pressures when in symbiosis with pasture cultivars. Unfortunately, some fungal strains are historically intractable to genetic manipulation, therefore preventing investigation into novel traits. Only recently with the development of CRISPR-Cas systems, a revolutionary gene editing tool, was CRISPR-Cas9 successfully used on one of these intractable strains, Epichloë sp. LpTG-3 strain AR37, to create targeted gene disruptions. This study focused on CRISPR-Cas9 targeted gene insertion capabilities in Epichloë spp. CRISPR-Cas9 was successfully deployed to precisely insert 236 bp of coding sequence from a critical condensation domain of the perA gene, missing in the genetically intractable Epichloë festucae var. lolii strain AR48. CRISPR-Cas9 was also successfully deployed to insert the reporter gene gfp into a precise location within the indole diterpene pathway, a known secondary metabolite pathway in AR37. This research illustrated the ability of CRISPR-Cas9 to repair or insert genes in genetically intractable Epichloë species, with the potential for reconstruction of secondary metabolite pathways for novel compound production and delivery into New Zealand’s pasture-based agricultural system.

Decoding horizontal gene transfer mechanisms in Epichloë–grass symbiosis

Haotian Shi — 2025-12-04

Epichloë endophytes establish mutualistic symbioses with host grasses, significantly enhancing host resistance to diverse biotic and abiotic stresses. Understanding molecular mechanisms of symbiosis and stress response regulatory in Epichloë-host symbionts could provide us effective strategies to overcome the challenges posed by global climate change. Horizontal gene transfer (HGT) is a critical evolutionary mechanism for both fungi and plants to improve their survival, propagation and environmental stress adaptation. A glucanase gene in perennial ryegrass and a disease resistance gene (Fhb7) in wheat are speculated to have originally arrived from Epichloë endophytes based on the nucleotide sequence identity, suggesting that HGT may be a widespread events presented in Epichloë–plant symbionts. However, there is no conclusive evidence for the occurrence of HGT in Epichloë–plant symbionts, and the mechanism by which it occurs as well as its functional implications in symbionts remain unclear. Achnatherum inebrians belongs to the grass tribe Triticeae and is genetically close to the model grass Brachypodium distachyon, the symbiont of A. inebrians with which Epichloë exhibits remarkable stress tolerance. To obtain more definite evidence and reveal the mechanisms of HGT events in Epichloë-plant symbionts, we performed chromosome-level de novo genome assemblies of A. inebrians and E. gansuensis isolated from the symbiont through a combined strategy integrating Illumina HiSeq sequencing, PacBio sequencing and Hi-C technology. The analysis results showed that the genome of A. inebrians is relatively small (~1.47 Gb) with low heterozygosity, which is conductive to the analysis of HGT events. Whole-genome resequencing of the A. inebrians-E. gansuensis symbiont generated over 50 Gb of sequencing data. Through the comparative genome analysis, three possible E. gansuensis-derived HGT reads were detected in the symbiotic A. inebrians genome, with the evidence of each read clearly flanked by definitive A. inebrians genome sequences. This finding strongly support the occurrence of HGT events during the symbiotic interaction of E. gansuensis with A. inebrians. However, only three HGT-derived reads were identified in a quite massive sequencing data, suggesting that these HGT events may not heritable. Intriguingly, functional annotation based on homology comparison revealed that the genes in HGT reads were enriched in biological processes related to DNA recombination and repair, transposon integration, and transcriptional regulation. Our results provide valuable genomic resources and a robust foundation for further dissection of symbiotic regulatory mechanisms mediated by HGT events between Epichloë endophytes and host grasses.

Identification of a domain of unknown function family gene involved in mediating the drought tolerance of Achnatherum inebrians colonised by an Epichloë endophyte

Yali He — 2025-12-04

The increasing frequencies of severe global droughts highlights the importance of cultivating high-yielding, drought-resistant crops. The Epichloë-Achnatherum inebrians symbiosis exhibits remarkable drought tolerance and investigating the molecular mechanism responsible for this trait would provide important information for drought tolerance in graminaceous crops. The transcriptome and proteome of A. inebrians and Epichloë-A. inebrians symbiosis was compared under the conditions of 100% and 40% soil moisture saturation, respectively. The expression of numerous genes exhibited differential patterns even at 100% soil moisture saturation, with the divergence intensifying significantly at 40% saturation. To detect key drought responsive genes, the top 20 genes exhibiting the most significant differences in expression levels were cloned. Utilizing a Agrobacterium-mediated transient expression system in Nicotiana benthamiana and a luciferase (LUC) gene drove by the promoter of a drought response marker gene as a reporter, the functions of these candidate genes in regulating drought response were detected. A DUF (Domain of Unknown Function) family gene, named AiDR6, significantly induced LUC expression under both of the control and PEG6000 treatment conditions. Intriguingly, subcellular localization analysis revealed that AiDR6 is specifically localized to chloroplasts of stomata. Furthermore, compared to the wild type, N. benthamiana overexpressing AiDR6 exhibited a very distinct stomatal closure phenotype. These results suggest that AiDR6 plays a critical role in regulating the Epichloë endophyte mediated A. inebrians drought tolerance. The functional analysis of AiDR6, along with further elucidation of its regulatory mechanisms, will advance the understanding of drought tolerance mechanisms in Epichloë-grass symbionts.

Establishment of an efficient genetic transformation system for the Achnatherum inebrians-Epichloë symbiosis

Zhixia Wang — 2025-12-04

The symbiosis between Achnatherum inebrians and endophytic fungi of the genus Epichloë (e.g., E. gansuensis and E. inebrians) can significantly improve the host plant's ability to tolerate salinity, drought and low nitrogen. A highly efficient genetic transformation system for A. inebrians was established to study mechanisms of the stress tolerance within this plant-fungal symbiosis. Mature seeds were surface disinfected before mechanical wounding of embryos to produce callus. The regeneration step was optimized based on the expression pattern of cell totipotent related genes, such as ARFs, BBM, and WOXs. The regeneration efficiency was around 70%. Agrobacterium tumefaciens strain EHA105 was utilized for stable integration of transgenes into callus-derived genomes through co-cultivation. By vacuum incubating the callus with EHA105 cells (OD₆₀₀=0.2) for 30 mins, the transformation efficiency stabilized at 12%-15%, with a rooting rate of up to 95%. Transformed plants were validated by PCR and sequencing, with a positive rate up to 75%. Our efficient transgenic system for A. inebrians provides a powerful tool for analyzing the molecular mechanism of Epichloë-grass interactions. Combined with gene editing techniques, such as CRISPR-Cas9, the generation of diverse mutant variants of A. inebrians will enable significant acceleration and profound enhancement in both the pace and depth of investigative progression.

Dissection of the epoxyjanthitrem pathway in Epichloë sp. LpTG-3 strain AR37 by CRISPR gene editing to deliver improved SDN-1 type endophytes for grasses

Linda Johnson — 2025-12-04

Epichloë festucae var. lolii and Epichloë sp. LpTG-3 are filamentous fungal endophytes of perennial ryegrass (Lolium perenne) that have a substantial impact on New Zealand’s agricultural economy by conferring biotic advantages to the host grass. Epichloë endophyte strain AR37 provides ryegrass with improved agronomic performance, insect protection and plant persistence. Indole diterpenes are a class of well characterised secondary metabolites produced by Epichloë endophytes, with the AR37 strain expressing epoxyjanthitrems, a class of decorated indole diterpenes. These have been associated with the observed effects of AR37 on livestock and insect pests. Here we used gene inactivation by CRISPR-Cas9 to deconvolute the genetic basis for epoxyjanthitrem biosynthesis and manipulate this secondary metabolite pathway to reduce or remove endophyte-induced mammalian toxicity whilst retaining activity against some important agricultural insect pests. We also show that gene editing of Epichloë can be achieved without off-target events or introduction of foreign DNA (footprint-less) and that these gene edits can be classified as site-directed nuclease 1 (SDN-1), which are not regulated as genetically modified organisms in selected jurisdictions, including Australia. These SDN-1 technology edits have been successfully grown out of containment in the field in Australia for seed multiplication, followed by agronomy trials, which are now being evaluated. This has the potential to provide a step change in the future use of animal safe Epichloë strains in New Zealand pastures which will significantly reduce chemical inputs and increase animal welfare.

Split-marker recombination for fast and efficient targeted deletion of Epichloë genes

Haijuan Zhang — 2025-12-04

Endophytic fungi of the genus Epichloë exist widely in cool season grasses forming a mutualistic symbiotic relationship with their host. Epichloë symbiosis can promote the growth of host plants and improve the hosts resistance to biotic and abiotic stresses. Selected strains of these fungi have therefore been exploited as an important agricultural microbial resource. The establishment of a genetic transformation system in Epichloë is important to understand the molecular mechanisms responsible for host stress resistance. However, due to the slow growth of Epichloë spp. in culture, this has inhibited research in this space. The rate of false positive colonies in gene deletion experiments is often high, typically because gene replacement fails to occur at the targeted locus. Our work therefore introduced a highly efficient protoplast-based transformation method for targeted gene deletion in Epichloë. Cellophane culture was used to disperse mycelium and accelerate its growth, which significantly shortened fungal culture time. High quality protoplasts were subsequently obtained by mixed enzymatic hydrolysis, which reached 4.2×10⁸ protoplasts per ml. In addition, using a split-marker recombination method, knockout mutants of Epichloë were quickly and efficiently obtained increasing rates by 20% over traditional hygromycin methods. This paper discusses a fast and efficient split marker recombination method for knockout and overexpression of slow-growing Epichloë endophytes and its potential use in transforming these fungi for pasture grasses and cereals.

An efficient protoplast transient expression system developed for Achnatherum inebrians

Yiyi Wang — 2025-12-04

With climate change intensifying and extreme weather patterns becoming more prevalent worldwide, there is growing urgency to cultivate crop varieties that demonstrate both high productivity and environmental resilience. Achnatherum inebrians, a cool-season grass, often coexists with the mutualistic endophytic fungi Epichloë gansuensis or Epichloë inebrians. Many studies have shown that the Epichloë symbiont confers excellent resistance to biotic and abiotic stresses over plants that lack this fungal association. A. inebrians is a diploid self-pollinating grass species and its genome was assembled by the College of Pastoral Agriculture Science and Technology (Lanzhou University, China). Polyethylene glycol (PEG)-mediated protoplast transient expression system is a convenient and valid method for supporting investigations into the biological and biochemical functions of plant genes. To better understand the molecular mechanisms of symbiosis and stress resistance in Epichloë-A. inebrians symbiosis, we developed a highly efficient protoplast transient expression system. In detail, as the leaves of A. inebrians seedlings are narrow and contain a high fibre content, we wounded the leaves along veins instead of cutting into segments, which significantly increased the protoplast yield. These 10-15-day old leaves were then subjected to a 5-hour enzymatic treatment using an enzymatic solution containing 1.5% cellulase, 0.4% isolase, and 0.5-0.6 M mannitol, that yielded 2×10⁷ protoplast cells per gram of fresh weight with 85% cell viability. Relative to reported protocols for protoplast isolation for wheat, our method achieved a twofold increase in yield, substantially reducing digestion time, and enhanced cell viability. The transient protein expression system developed here can rapidly evaluate the expression of exogenous genes in A. inebrians by transferring the target genes into A. inebrians protoplasts. This system will be used in future research to support understanding of the functions and interactions of the plant genes. This knowledge will further support investigations into the molecular mechanisms of key biological processes such as endophytic fungal symbiosis and stress tolerance in A. inebrians.

The novel host barley produced a rapid defence response to Epichloë endophyte by elucidating gene regulation

Kamran Malik — 2025-12-04

Epichloë fungal endophytes can have a crucial role in plant growth and development. These fungi are recognized as beneficial microorganisms that form symbiotic relationships with grasses and provide several advantages to the host, such as improving stress resistance and promoting growth. The advantageous traits identified in natural ecosystems can, and have been, exploited in managed ecosystems such as pastoral farming in countries including New Zealand, Australia and the Americas. In the current study, Epichloë bromicola strain WBE1, an endophyte isolated from Hordeum brevisubulatum (wild-barley) was artificially inoculated into endophyte-free plants of H. brevisubulatum and H. vulgare (cultivated-barley, novel host) by researchers at Lanzhou University, China, to study grass-endophyte compatibility. WBE1 successfully infected 54% of the endophyte-free plants (n=107) of wild barley and 10% of the cultivated-barley plants (n=545). WBE1 colonisation enhanced the callus and lignin content in both grass hosts but also led to an increase in plant cell death. Cultivated-barley showed more resistance to endophytic infection as its infection frequency was lower than wild-barley. After 14 days, mycelial growth in both barley hosts was observed with the naked eye at the inoculation sites. The microscopy of endophyte infected leaf sheath tissue of wild barley showed that hyphae were branched and curved. In contrast, hyphae within leaf sheath tissues of cultivated-barley were slightly curved and unbranched. Inoculation of both grass hosts by WBE1 induced the expression of early signalling molecules including MAPK, which increased by 12 and 54% in cultivated barley compared with wild barley, 2- and 4-days post inoculation, respectively. Furthermore, O₂ increased in both wild and cultivated barley after inoculation by 11 and 19%, respectively; H₂O₂increased in both wild and cultivated barley by 67 and 72%, respectively and NO content increased in both wild and cultivated barley by 21 and 18%, respectively. After inoculation, regulated genes related to the synthesis of secondary metabolites involved in phytopathogen detection were expressed earlier in cultivated barley compared with wild barley. NADP+1 oxidoreductase and alcohol dehydrogenase (NADP+) activities were down-regulated in cultivated barley showing that the host was metabolically adjusted to environmental or physiological stress. The expression of signalling molecules and down-regulation of genes related to oxylipin biosynthesis, lipid oxidation, cellular response to environmental stimuli, oxidoreductase activity, and heme binding indicated that inoculation of cultivated barley by WBE1 triggered the host defence responses. This study investigated the gene transcription of an Epichloë endophyte strain across two different host plants, namely the original host and a novel host. The results showed that Epichloë can induce a defence response after inoculation, similar to that exhibited when being invaded by a phytopathogen and was expressed earlier in the novel host compared to the original host. The research results provide new ideas for further revealing the genetic foundations of the symbiotic relationship between Epichloë and their grass hosts.

Receptor-like kinases (RLKs) mediate enhanced immune responses in the Achnatherum inebrians-Epichloë gansuensis symbiotic system against Claviceps pathogen infection

Tao Yang — 2025-12-04

Ergot, is a disease affecting cereals and grasses caused by certain members of the fungal genus Claviceps. Ergot can not only affect the growth and seed yield of the host plant but, when ingested, the fungus can also cause livestock poisoning. Host resistance to Claviceps spp. is rare, but the infection of Achnatherum inebrians by Claviceps purpurea can be significantly reduced if the host plant is colonized by the fungal endophyte Epichloë gansuensis. Therefore, analyzing the disease resistance mechanisms exhibited by this grass-endophyte symbiosis is of great practical significance for developing ergot-resistant germplasm. In this study, we investigated the expression and enrichment analysis of differentially expressed genes within the A. inebrians-Epichloë symbiosis with and without Claviceps purpurea infection. The RNA-seq results indicated that the receptor-like kinase family was highly expressed in A. inebrians-Epichloë symbiosis with C. purpurea infection. Among them, 353 receptor-like kinases were up-regulated (fold changes≥2). Leucine-rich repeat receptor-like kinase, Wall-associated kinase, Lectin receptor-like kinases were found by PFAM (the protein family database, MSA) prediction. Receptor-like kinases are key pattern-recognition receptors for microbe- and plant-derived patterns-triggered immune (patterns-triggered immune, PTI). Thus, further research screened these RLKs through the Nicotiana benthamiana Reactive Oxygen burst system (ROS burst). The results showed that several RLKs consist of WAK domain and Lectin domain had nearly twice ROS burst compared to the mock in response to chitin or Eg- and Cp-derived crude extract. These RLKs are therefore speculated to have important functions in the regulation of PTI in A. inebrians. An understanding of the molecular mechanism of these RLKs will contribute to a deeper understanding of the complex and diverse biological functions of RLKs and provide a theoretical basis for improving ergot resistance in other Poaceae grasses.

Diversity, ecology and applications of Epichloë endophytes from South America

Leopoldo Iannone — 2025-12-04

The presence of Epichloë-infected grasses in South America was inadvertently reported in 1908 when two veterinaries attributed the cause of the poisoning in cattle and livestock to the consumption of Festuca plants infected with a fungus that they wrongly identified and named Endoconidium tembladerae (today Epichloë tembladerae). It was only at the end of the 20th century that systematic studies of endophytes in Argentinian native grasses began, and the host list of new and already known Epichloë endophytes is continually expanding. The team at UBA-FCEyN and CONICET-UBA have, and continue, to work on three central projects:

Toxic and mammalian safe genotypes of Epichloë associated with native fescues.

Festuca fiebrigii and F. argentina associate with Epichloë tembladerae and in populations of F. argentina endophyte incidence is higher than 90%. These endophyte-grass associations are infamous for their toxicity to livestock with preliminary analyses suggesting that mammalian toxicity is due to the production of the alkaloids terpendole C and I. However, extensive surveys in F. fiebrigii populations have now revealed that this grass species also associates with several non-toxic genotypes of E. tembladerae, and these endophytes may contribute to the development of novel forages for Argentina. Recently, we have also detected a new toxic endophyte genetically related to E. aotearoae but more difficult to culture. E. aotearoae is a symbiont of Echinopogon ovatus, a grass endemic to New Zealand.

Endophytes in wild forage grasses.

Bromus auleticus, an important forage grass, is associated with at least three peramine producing Epichloë species, E. pampeana, E. tembladerae and E. platensis. Only the N-formylloline producing E. pampeana is harmful to aphids (Rhopalosiphum padi) while none of these endophytes have effects on the feeding preference of crickets (Gryllus assimilis). However, B. auleticus plants infected with E. platensis are more tolerant to root feeding scarab beetle larvae (Philochloenia bonariensis). Current research aims to further understand the complexity of insect resistance within these grass-endophyte associations. Epichloë endophytes also modulate host physiology in B. auleticus. Germination of endophyte-infected (E+) seeds was prevented under low water activity and salinity and E+ plants, exposed to water deficit, suffered less cellular damage, maintained higher values of stomatal conductance and net photosynthesis than (endophyte-free) E- plants. The Epichloë endophytes also modulate the community of non-systemic foliar endophytes and the community structure of cultivable dark septate endophytes in roots of B. auleticus differed between E+ and E- plants with higher proportions of pathogenic fungi in the E- plants.

Inoculation of Argentinean endophytes in commercial forage grasses.

The wide host range, genetic and chemical diversity, and the advantageous traits conferred upon their original host grasses led to research developing novel grass-endophyte associations. Attempts were made to infect commercial forage grasses and barley (Hordeum vulgare) with E. tembladerae, E. cabralii and E. pampeana. Only Lolium multiflorum inoculated with E. tembladerae formed a stable association. This association remained stable for five generations and these plants did not behave differently from those associated with their original endophyte, E. occultans.

Summarizing, more research is needed to understand the complexity and the potential of endophyte-grass associations in South America.

An Epichloë endophyte associated with the Afromontane grass Festuca simensis

Stuart Card — 2025-12-04

Filamentous fungi of the genus Epichloë form symbiotic associations with Pooideae grasses (family Poaceae) with some estimated at 40 million years old, dating back to the origin of this grass subfamily. Here we provide a report on an Epichloë endophyte from central Africa associating with Festuca simensis, a tetraploid grass species of highland tropical African origin, potentially originated from a cross between a western Mediterranean F. fenas-type maternal parent and a Eurasian Lolium-type paternal parent, followed by genome duplication and likely colonisation of the Afromontane regions that cover the mountains of Africa and the southern Arabian Peninsula during the Pliocene epoch (5.3 to 2.6 million years ago). The endophyte of F. simensis most resembled Epichloë uncinata, a fungus generally associated with F. pratensis (= L. pratense; meadow fescue), with respect to its genetics, plant colonisation patterns and secondary metabolite profile. However, the F. simensis endophyte was slower to emerge from dissected plant tissue, had a significantly slower growth rate in culture and contained an additional copy of the β-tubulin gene, compared with E. uncinata. This paper reviews the preliminary knowledge gained about this intriguing Epichloë-grass symbiosis.

Harnessing rhizosphere Bacillus and Epichloë symbionts to promote the performance of Achnatherum inebrians within arid ecosystems

Jinjin Liang — 2025-12-04

Plants in natural ecosystems form intimate associations with diverse microorganisms, including bacteria and fungi, collectively termed the plant-associated microbiota. Notably, the entire rhizosphere microbial community is widely recognized as the plant's second genome, crucial for host growth and health. Achnatherum inebrians, a perennial grass increasingly dominant in the arid/semiarid grasslands of northwest China, is almost universally colonized by Epichloë fungal endophytes. We isolated bacteria from the rhizosphere soil of Epichloë endophyte-infected (E+) and endophyte-free (E-) plants of A. inebrians. A total of 393 bacterial isolates were identified, primarily belonging to Proteobacteria (195), Actinobacteria (100), and Firmicutes (98) at the phylum level, with dominant genera including Acinetobacter (45 isolates), Pseudomonas (90 isolates), Rhizobium (60 isolates), Bacillus (98 isolates), and Arthrobacter (100 isolates). E+ plants exhibited significantly higher relative abundances of Bacillus and Pseudomonas compared to E- plants. E+ plants showed significantly enhanced germination potential, germination index, seedling length, and fresh weight (P<0.05) compared to E- plants. Inoculation of selected Bacillus isolates onto A. inebrians seedlings demonstrated their growth-promoting ability. All isolates of Bacillus improved germination rate, germination potential, germination index, root length, seedling length, fresh weight, and dry weight (P<0.05). Based on membership function analysis, 80 out of 90 Bacillus isolates positively influenced seed germination of A. inebrians. Both Epichloë endophytes and Bacillus significantly increased tiller number, fresh weight, and dry weight (P<0.05), while their interaction notably affected plant height and reproductive branch number (P<0.05). These findings highlight the synergistic role of Epichloë spp. and rhizosphere bacteria in enhancing host plant performance. Future research will focus on constructing synthetic microbial communities to leverage these beneficial plant-microbial interactions for sustainable agricultural practices.

How Epichloë and arbuscular mycorrhizal fungi interact with wild barley under salt stress

Yufan Pang — 2025-12-04

The symbiotic interactions between plants, endophytic fungi, and arbuscular mycorrhizal fungi (AMF) constitute a critical area of plant-microbe research. In many cool-season grass species, Epichloë endophytes and AMF can simultaneously establish mutualistic relationships with their hosts. The plant provides both habitat and a carbon resource for their fungal partners, while gaining enhanced abiotic and biotic stress tolerance via Epichloë-mediated mechanisms and improved nutrient acquisition and water uptake via AMF symbiosis. Previous studies have primarily focused on nutrient-dependent interactions between Epichloë and AMF, whereas environmental stresses such as salinity remain less explored, limiting our understanding of their synergistic potential in saline ecosystems.

This study investigated the combined effects of Epichloë bromicola, two AMF species (Glomus mosseae and G. claroideum) and a mixed AMF inoculum of both G. mosseae and G. claroideum (known as Gmix) associated with wild barley (Hordeum spontaneum) under salt stress. Epichloë-infected and Epichloë-free seeds of wild barley were inoculated with AMF treatments or maintained as non-mycorrhizal controls, then exposed to three concentrations (0 mM, 100 mM, 300 mM) of salt (NaCl). Plant growth parameters, nutrient content, and AMF colonization rates were evaluated through four harvests conducted at 7-day intervals over 28 days, with qPCR quantification of AMF biomass. The main findings were:

(1) The presence of E. bromicola significantly increased root biomass, root-to-shoot ratio and spikelet number of wild barley plants compared to the control under 300 mM salt stress, but the effect was weakened with time.

(2) Co-infection of wild barley plants with both E. bromicola, and the AMF treatments reduced sodium content, increased the potassium/sodium ratio and reduced salt stress toxicity. G. mosseae increased nitrogen content in aboveground plant organs, while G. claroideum and Gmix increased phosphorus content in the plant root system.

(3) In E. bromicola-infected wild barley plants, colonization by G. mossease and Gmix led to growth reductions in root biomass, root-to-shoot ratio and spikelet numbers indicating an antagonistic interaction between E. bromicola and G. mossease.

Uncovering Alkaloid chemotype diversity in natural Festuca–Epichloë festucae symbioses across Iberian ecosystems

Alba Sotomayor-Alge — 2025-12-04

Epichloë fungal endophytes are characterized by their biotrophic lifestyle and their highly specific and variable interaction with cool-season Pooideae grasses. These endophytes grow systemically in the apoplast of the aerial tissues and can reproduce sexually, asexually or interspersing both lifecycles. Within the cosmopolitan genus Festuca (Poaceae), Epichloë festucae is one of the most prevalent Epichloë endophytes, appearing in up to 30 taxa. This interaction has been thoroughly studied in agricultural species (e.g., Lolium perenne) due to the impact of endophyte-expressed secondary metabolites on farming and livestock production. To date, four alkaloid classes have been identified: ergot alkaloids, 1-aminopyrrolizidines (including lolines), indole-diterpenes and pyrrolopyrazines (including peramine), exhibiting toxicity or deterrence to vertebrates, insects or both.

Our study aimed to characterize the holobionts formed by five fine-leaved Festuca species (F. lambinonii, F. nigrescens, F. rubra subsp. pruinosa, F. yvesii, and F. rothmaleri) and their respective E. festucae strains across diverse Iberian ecosystems. Our specific objectives were: (1) to perform an integrative characterization of both symbionts using molecular, cytogenetic and morphological approaches; and (2) to quantitatively and qualitatively assess the chemotypes and conduct genetic screenings of endophytes in these holobionts.

We recorded a wide variety of chemotypes, finding differences between individuals at intra- and interspecific levels. For pyrrolopyrazines, peramine was detected in some individuals within the species F. yvesii and F. rothmaleri, whereas an alternative compound (pyrrolopyrazine 2a) was detected in the remaining individuals of these species and in all F. rubra subsp. pruinosa and F. lambinonii samples. For ergot alkaloids, only F. rubra subsp. pruinosa showed traces of chanoclavine and quantifiable ergovaline. Indole-diterpenes (e.g., paxilline, terpendole isomers, paspaline and 13-desoxypaxilline) were variably present in F. nigrescens, F. yvesii, and F. rothmaleri, whereas 1-aminopyrrolizidines were absent in all samples. Interestingly, results of the alkaloid analyses were not always consistent with the results of the genetic screens: in some cases, the genes of biosynthetic pathways were present, but the corresponding alkaloids were undetected.

Altogether, we were able to detect the presence of at least two alkaloid classes for all studied species, with exception of F. lambinonii, suggesting that the host species might obtain protection against invertebrates and, for those that produce certain ergovaline or certain indole-diterpenes, also against vertebrates. We believe that these findings evidence the importance of characterizing naturally distributed holobionts to expand our comprehension regarding the secondary metabolism associated with this symbiosis and the plasticity of E. festucae.

Physiological response of Hordeum brevisubulatum-Epichloë bromicola ecotypes to mixed salt stress

Yurun Zhai — 2025-12-04

Soil salinization has become a global agricultural issue, reducing agricultural productivity, threatening livestock production and food security. Wild barley (Hordeum brevisubulatum) is widely distributed in lightly saline meadows in northern China and exhibits high salt tolerance. This tolerance can be further enhanced by the presence of Epichloë bromicola, a mutualistic fungal endophyte and this symbiotic association serves as essential germplasm for developing novel salt-tolerant grass lines. This study evaluated the salt tolerance of 16 wild barley-E. bromicola ecotypes under greenhouse conditions. plants (n=15) from each ecotype were subjected to mixed salt stress (NaCl: Na₂SO₄ = 1: 1) at different concentrations (0 mM, 400 mM, 600 mM, and 800 mM) and their growth responses assessed after 30 days. The results demonstrated that salt stress significantly increased leaf mortality, relative membrane permeability, and Na⁺ content, while reducing plant height, stem diameter, tiller number, biomass, K⁺ content, and the concentration of peramine, an endophyte-derived alkaloid that has insect deterrent activity. Notably, peramine content varied significantly among ecotypes, with DB, B1, B2, B3, and R showing no detectable peramine, whereas D2 exhibited the highest level (424 µg/kg^-¹). Correlation analysis revealed that peramine content was positively correlated with plant chlorophyll content, stem diameter, green leaf rate, and K⁺ content, but negatively correlated with leaf mortality, withering rate, and relative membrane permeability. After 30 days of exposure to 800 mM mixed salt, mortality was detected in a subset of ecotypes. R, DB, LZ E-, D3, and E1 showing mortality rates of 100%, 33%, 33%, 33%, and 27%, respectively. A grey relational analysis ranked the ecotypes' salt tolerance in descending order: D2 > A1 > J > H > G > D1 > K > LZ E+ > B3 > B2 > B1 > E1 > D3 > LZ E- > DB > R. Cluster analysis classified them into five groups: D2 (highest tolerance), A1 (high tolerance), J/ H/ G/ D1/ K/ LZ E+ (moderate tolerance), B3/ B2/ B1 (low tolerance), and E1/ D3/ LZ E-/ DB/ R (lowest tolerance). In conclusion, D2, the most salt-tolerant ecotype, represents a valuable genetic resource for breeding high salt-resistant wild barley-E. bromicola symbiont lines.

Infection frequencies, alkaloid content and characteristics of Epichloë festucae var. lolii in Lolium perenne within German grasslands along a land-use intensity gradient

Jochen Krauss — 2025-12-04

Perennial ryegrass (Lolium perenne) is a dominant cool-season grass species in Germany. It is native in Germany but is also often a component in commercially available seed mixtures on the German market, with numerous grass varieties for turf and pasture. Native and bred varieties can be colonised by the symbiont Epichloë festucae var. lolii, but the infection frequencies and alkaloid concentrations in German grasslands along a land-use intensity gradient are unknown.

In 2014, and again in 2017 we sampled 150 grassland sites in three German regions covering a north to south gradient. Analyses of L. perenne were performed to detect E. festucae var. lolii using different methods as well as alkaloids using HPLC-MS.

The average infection frequencies of the endophyte were between 13 – 15 % of individuals, with no detections in approximately 30 % of L. perenne populations. Concentrations of the vertebrate toxic compound lolitrem B were in 5% of the individual grass samples above published toxicity thresholds for vertebrates (>2 µg/g), while concentrations of the alkaloid ergovaline were below toxicity thresholds of 0.3 µg/g. The insect toxic/deterring alkaloid peramine was in 12 % of individual grass samples above published toxicity thresholds (>3 µg/g). On a population level of L. perenne all grassland sites had alkaloid concentrations clearly below published toxicity thresholds. As the German grasslands were also not monocultures, a toxification of livestock is improbable. Land-use intensity of grasslands had no effect on infection rates or alkaloid content. A characteristic of E. festucae var. lolii in the German grasslands is that the dmaW gene for the ergovaline synthesis is often missing. Another reason, why toxification of livestock in German grasslands is improbable. Depending on climate warming, which could enhance infection rates and alkaloid content and depending on the introduction of L. perenne varieties with genetically different strains of E. festucae var. lolii, the currently low toxification risk might change in the next decades. Regular surveys of infection rates and alkaloid content in grasslands across Germany once per decade seems an appropriate strategy to monitor changes.

Improved methodology for the isolation of Epichloë endophytes from Achnatherum inebrians

Zheng Liang — 2025-12-04

Achnatherum inebrians, a toxic perennial bunchgrass native to northwestern China, establishes a mutualistic symbiotic relationship with two distinct endophyte species, Epichloë gansuensis and Epichloë inebrians. These endophytes produce ergot alkaloids that accumulate in plant tissues and are responsible for frequent poisoning incidents in grazing livestock. The bioaccumulation of these compounds poses significant agricultural risks, necessitating the development of targeted control strategies. Research investigations require the isolation of these fungi from plant material. The current isolation method is internationally accepted but results in a low endophyte isolation frequency.

The conventional protocol involved sequentially treating seeds with 75% ethanol (30 s) followed by 10% sodium hypochlorite (1 min), with 5 consecutive sterile-water rinses performed after each sterilization step. The sterilized seeds were dried on autoclaved filter paper and transferred to potato dextrose agar (PDA) supplemented with antibiotics (50 mg/L benzylpenicillin potassium and 50 mg/L streptomycin sulphate). Following a certain period of time, actively growing mycelia were aseptically selected from the colony periphery for sequential subculturing. Any microbiological contamination was removed as soon as it was detected throughout the experimental process. The methodological enhancements involved two modifications: (1) implementation of a 10 min 50% sulphuric acid pretreatment for seed coat prior to sterilization, and (2) formulation optimization of PDA through supplementation with 0.1% (w/v) yeast extract and 5% (v/v) aqueous foliage extract from A. inebrians. These improvements significantly increased the isolation success rate from 4% to 18% while accelerating fungal growth by 10-20%.

The optimized methodology discussed in this study significantly enhanced the isolation frequency of endophytes from A. inebrians, thereby allowing more effective research studies to be conducted. This method establishes a referenceable technical pathway for the isolation of endophytes from grasses, enhances the practical application potential and advances the technological innovation process in endophyte symbiosis research.

Occurrence and toxicity of Epichloë endophytes in German grasslands

Tabea Lang — 2025-12-04

Epichloë endophytes are symbiotic fungi colonizing cool-season grasses, producing alkaloids potentially toxic to grazing animals. While numerous cases of livestock intoxication have been reported in New Zealand, Australia, and the United States, its prevalence and impact in European pastures remains poorly understood. This study investigates the distribution and toxicity of Epichloë endophytes in German grasslands to estimate possible livestock intoxication risks. Additionally, the research considers the role of Claviceps spp., a closely related fungal genus that can produce similar alkaloids and can cause symptoms potentially mistaken for Epichloë intoxication.

Grass samples were collected from 90 grasslands across two regions of Germany, including cattle pastures, horse pastures, pastures with previous cases of equine laminitis, semi-natural grasslands and football fields. This selection establishes a gradient from non-sown to intensively managed areas. Around 5,000 individuals of 15 grass species were collected between July and September 2024, with particular emphasis on Lolium perenne and Festuca arundinacea, key Epichloë hosts associated with livestock toxicity. Stems and inflorescences were collected for the detection of Epichloë and Claviceps, respectively. Detection of Epichloë was performed using Multiplex-PCR. Subsequently, the positive samples were analyzed to quantify the concentrations of ten alkaloids, including Lolitrem B and Ergovaline, using HPLC-MS and GC-MS.

It is hypothesized that since semi-natural grasslands have never been sown, they likely reflect the natural abundance of Epichloë endophytes in grasses, which might be lower than in sown grasslands. Sown grasslands may contain more Epichloë due to the potential use of endophyte-infected seeds in commercial mixtures. Moreover, the prevalence of Epichloë in turf grass, like sown football fields, could be even higher due to the endophyte's beneficial role in enhancing tolerance to environmental stressors like heat and trampling.

Results are pending, but PCR detection has been successfully established and will be available soon, as well as preliminary analyses on alkaloid contents. This study will provide critical data on Epichloë and Claviceps prevalence and alkaloid profiles in German grasslands. The findings will have significant implications for grassland management and animal health in Germany. The study's relevance is heightened by the potential impact of climate change, which may favour Epichloë-containing grasses and alter future risks to livestock health, potentially making this issue increasingly important in Germany and Europe.

Exploring wild Bromus-Epichloë interactions in Uruguay: genetic diversity, eco-geographic distribution, and antiherbivore effects

Lucia Meneses — 2025-12-04

A comprehensive survey of Epichloë fungal endophytes forming symbioses with the wild C3 forage grass Bromus auleticus across a transitional climate–soil zone in Uruguay was conducted. Among 83 accessions, we detected 70% were infected with Epichloë. Using PCR with 29 genetic markers targeting tefA, mating type and alkaloid biosynthesis genes we identified nine distinct Epichloë genotypes that varied in potential alkaloid production. Of these nine genotypes, 1, 2 and 3 were most commonly observed. All genotypes contained the genes required for the synthesis of pyrrolopyrazines (e.g., peramine, a known insect-feeding deterrent). Two genotypes (designated 1 and 7) contained genes required for the synthesis of pyrrolizidines (e.g. loline alkaloids, known for their broad spectrum insecticidal and insect-deterrent activity), but only genotype 1 was predicted to produce N-formylloline, a compound with strong insecticidal properties. Seven genotypes (2, 3, 4, 5, 6, 8 and 9) contained genes involved in indole diterpene (IDT) synthesis, however, only five genotypes (2, 3, 4, 5, and 6) were predicted to produce early pathway IDTs such as paspaline or terpendoles. Genotypes 8 and 9 lacked essential early pathway genes were not predicted to produce IDTs. Genotypes 3 and 7 contained EAS genes required for ergot alkaloid synthesis; however, these genotypes were only predicted to produce chanoclavine, as genes required for downstream ergovaline synthesis were absent. None of the Epichloë endophytes from this survey contained genes required for the synthesis of the mammalian toxins ergovaline or lolitrem B. As genotypes 1, 2, and 3 were the most prevalent in the collection, six plant accessions, two from each of these genotypes, were selected to evaluate the protective effects of their different Epichloë genotypes against the aphid Rhopalosiphum padi, a generalist insect, in laboratory-controlled bioassays. Notably, only the plant accessions associated with genotype 1, predicted to produce N-formylloline, significantly suppressed R. padi. Endophyte presence and distribution were strongly influenced by environmental factors, particularly water availability, soil organic matter, and temperature extremes. Additionally, plants associated with endophyte genotypes 1, 2 and 3 were grown in two contrasting environments, displayed genotype-specific effects on host performance emerged under different soil fertility conditions. These findings highlight the ecological and agronomic potential of Epichloë endophytes that would lack mammalian toxicity and could provide a foundation for developing sustainable forages with enhanced insect resistance and environmental resilience without compromising livestock health.

Exploring Epichloë endophytes in Scottish wild barley for enhanced crop resilience

Lorena Rangel — 2025-12-04

Barley (Hordeum vulgare) is the most economically important crop in Scotland, yet its yields are increasingly threatened by biotic and abiotic stresses. One promising approach to mitigating these challenges lies in the use of endophytic microorganisms—bacteria and fungi that live within plant tissues and often provide benefits such as pest protection and growth promotion. While the use of selected Epichloë endophytes as biological control agents has been successfully commercialized in countries like New Zealand and the United States, this potential remains largely unexplored in the United Kingdom (UK). Specifically, Epichloë endophytes are known to confer protection against pests and pathogens in grasses. Previous studies have shown that Epichloë strains from grass species within the Triticeae, including wild Hordeum species, cluster according to geographic location rather than host species, suggesting local adaptation is ecologically important to their survival and dissemination.

We hypothesized that locally adapted asexual Epichloë strains in Scottish wild barley may enhance the plants resilience to both abiotic and biotic stresses. To explore this, wild barley seeds were collected from across Scotland and assessed for their bacterial and fungal endophyte communities, with a focus on Epichloë species. Seeds from plants with no choke symptoms were collected to ensure that these endophytes were vertically transmitted within the plant, a key trait for their potential use in sustainable agriculture. Of the 27 sites across Scotland, only one contained Epichloë, identified as E. baconii by ITS sequencing. This isolate was whole genome sequenced to explore its potential secondary metabolite arsenal and other putatively secreted compounds that could be exploited to benefit domesticated barley. We also employed culture-independent methods to map bacterial and fungal endophyte community diversity and their potential associations with specific geographic regions or plant host.

This study fills a significant gap by providing the first inventory of seed-transmitted endophytes in UK wild barley. Future research will explore symbioses between local Epichloë strains and domesticated barley to boost resistance to pests, diseases, and environmental stress. The discovery of specific endophyte-associated compounds may also offer novel avenues for improving barley yield and quality. Ultimately, this research supports the development of more resilient and sustainable agricultural practices, contributing to the long-term health and productivity of barley farming in the UK.

Germplasm survey and collection of Festuca sinensis accessions associated with the endophyte Epichloë sinensis

Pei Tian — 2025-12-04

Festuca sinensis, a native grass in China, is a high-quality forage species in the Qinghai-Tibet Plateau and plays an increasingly important role in restoring degraded grasslands and improving the ecological environment. Epichloë endophytes form a mutualistic symbiosis with F. sinensis, which can be utilized in germplasm innovation and breeding. In order to breed new cultivars with associated beneficial endophytes, our group investigated their distribution in the Qinghai-Tibet Plateau. F. sinensis germplasm was collected across 356 sites, including 46 counties/districts in Qinghai Province, Gansu Province, Sichuan Province, and Xizang Autonomous Region, which suggested that F. sinensis was widely distributed in the Qinghai-Tibet Plateau. During the investigation process, we noticed that F. sinensis normally grew in moist and shady areas, along streams, under forested areas and/or at the edges of farmland at altitudes ranging from 2,300 m to 4,700 m. It is normally a companion species of other grasses including Elymus nutans and Poa annua in natural grasslands. F. sinensis plants completed their growth stages and produced mature seeds between August 20^th to September 30^th, depending on their location. F. sinensis was scattered in the community with a height range of 30 cm to 80 cm, and a tiller number range of 1 to 4 in most survey sites. The frequency of endophyte within F. sinensis populations was also investigated by assessing seed. One hundred and sixty-six lines from 173 lines were infected by endophyte, with an infection frequency between 10% to 100%. The infection frequency of 122 lines was above 80%. This research clarified that the vast majority of F. sinensis on the Qinghai-Tibet Plateau was infected by endophyte and had a high infection frequency (>80%). Based on these collected resources, the first germplasm resource nursery of F. sinensis can be established to meet diverse breeding needs.

Fungal endophytes for disease control in cereals

David Collinge — 2025-12-04

Plant diseases and pests are responsible for significant crop losses worldwide both in terms of yield and quality. Conservative estimates lie at 20-40%. These losses are caused not only by direct inhibition of plant growth but also by insidious factors such as toxic specialised metabolites, termed mycotoxins, which result in reduced quality and safety of the consumable products. Although diseases are managed through a combination of technologies, ranging from farmer skills, the use of pesticides, disease resistance and biological control, there are many diseases for which effective methods for management are yet to be developed. The use of biological control as a means of controlling disease, and particularly the potential of endophytic microorganisms for control of diseases will be explored. Our studies have focussed on cereal diseases, especially Fusarium head blight and Septoria tritici blotch. Both diseases cause losses in cereals, with Fusarium head blight further resulting in the accumulation of several mycotoxins in the grain.

An ecological approach was used for identifying and isolating endophytic fungi in cereal crops, specifically in plants subject to disease pressure that do not show disease symptoms. The chief rationale is that an organism isolated from a particular environment is likely to be a successful competitor with other microorganisms – including pathogens – in that environment. Therefore, it may represent an efficient means of identifying fungi which can be used as biological control agents (BCAs). A reason for concentrating on endophytic fungi is that, once they have successfully colonised the host tissues, they are less affected by external environmental conditions than phyllosphere or rhizosphere microbes and this may lead to increased stability in changing environments. A focus of biological control of Fusarium head blight is to determine whether this can lead to a reduction in mycotoxin levels in the harvested grain. We have studied this in barley, wheat and oats, primarily with an effective rhizosphere BCA, Clonostachys rosea IK732, and have shown that this organism can stimulate detoxification of trichothecene mycotoxins such as deoxynivalenol. A commercially successful biological control strategy is associated with the ability of the BCA to colonise the host tissue and outcompete pathogens. In the case of Fusarium diseases in cereals, several microbes have individually been shown to control disease under different conditions. Whether more effective control can be achieved by combining several microorganisms will be explored.

Breeding turfgrasses with Epichloë endophytes for abiotic and biotic stress tolerance

Steve Reid — 2025-12-04

Breeding objectives for turfgrass species include continued improvement of aesthetics and persistence while minimizing inputs such as water, fertilizers, and fungicides. In order to achieve the objectives, breeders must evaluate large amounts of germplasm for multiple traits in multiple environments to develop resilient grasses suitable for sport, lawn, and landscape uses. Fungal endophytes of the genus Epichloë develop a mutualistic relationship with many turfgrass species, including ryegrass (Lolium spp.) and fescues (Festuca spp.), imparting a high tolerance to biotic and abiotic stresses. The development of improved turfgrasses with endophyte is of great value to reduce maintenance costs and conserve resources. It has been reported that red thread disease (caused by the pathogen Laetisaria fuciformis) and dollar spot disease (caused by Sclerotinia homoeocarpa) can be suppressed in fine fescues that are infected with endophytes. Grasses colonised by Epichloë endophytes have shown high tolerance to foliar feeding insects such as billbugs (Sphenophorus spp.). Several studies have also demonstrated that endophytes confer protection to perennial ryegrass and tall fescue from water stress by increasing the availability of primary metabolites, e.g. concentrations of glucose and fructose. Breeders can use established immunological tests to determine if Epichloë endophytes are present in plant seed, but there is no guarantee that the endophyte is viable. To determine viability, the same immunological tests (commonly known as growouts) can be performed on grass seedlings to determine viable endophyte infection frequencies across grass populations. To deliver grass cultivars with high (>70%) endophyte infection frequencies, these cultivars must be developed with endophyte infection frequencies near 100%. Many grass breeders in the United States maintain high endophyte frequencies in the live plant material to ensure the mutualistic relationship between the plant genotype and endophyte genotype is maintained throughout the breeding cycle. However, viable endophyte infection frequencies can decrease over time depending on the environment and cultural practices deployed. Maintaining breeder seed of a cultivar with a high endophyte infection frequency requires cold storage with low humidity in addition to an established endophyte assessment protocol. However, delivering this quality endophyte-seed product for commercial production requires a quality assurance program that is not currently available on an industry wide scale in the United States.

AR128 - a new Epichloë fungal endophyte product for ryegrass

John Caradus — 2025-12-04

Perennial ryegrass (Lolium perenne) persistence and yield in many parts of New Zealand is critically reliant on its association with mutualistic fungal endophytes of the genus Epichloë. Epichloë spp. can protect their host plants from insect pests and improve tolerance to drought. Over the last 25 years several novel endophyte strains have been commercialised. While ensuring effective persistence of ryegrass, they have also minimised the negative impacts on livestock of ryegrass staggers, heat stress and productivity losses associated with the standard endophyte strain. A new endophyte product is becoming available – AR128. This Epichloë sp. LpTG-3 strain originated from Italy and has a similar known chemical profile to AR37, through expression of epoxyjanthitrems. AR128 protects the host ryegrass plant against the same insect pests as AR37. It also exhibits similar animal safety to that of AR37. However, AR128 transmits through seed production cycles at a greater frequency than AR37 and it also stores for a longer duration in seed at ambient temperature and humidity conditions. These features ensure that the end-user farmer obtains a high quality and effective product, which should ensure improved perennial ryegrass persistence and production compared to endophyte-free grasses and possibly other endophyte strains which are less amenable to transmission and storage. The human consumer of products derived from animals feeding on ryegrasses with AR128 can be assured that there are no food safety issues associated with this technology based on mouse feeding studies.

Impact of selected Epichloë endophyte on perennial ryegrass yield performance across regions in New Zealand

Justine Ferguson — 2025-12-04

Over the past couple of decades selected Epichloë endophytes have been vital to farmers and the economy in New Zealand, with AR1 and AR37 being two of the frontier and foundational ryegrass endophyte strains. These endophytes produce different alkaloid profiles which defines the scope of pest resistance afforded by them to the host grass and suitability for use in different areas of New Zealand. Data collected by the New Zealand Plant Breeding and Research Association as a part of the National Forage Variety Trials provide information to understand the yield advantages that endophyte can have in regions with different insect and environmental limitations. The analysis focused on the impact of AR37, AR1 and without endophyte in a diploid perennial ryegrass cultivar (cv. Ceres ONE⁵⁰) evaluated in 20 trials throughout New Zealand.

There were significant regional and seasonal endophyte effects, with the greatest benefits being consistent with when insect and abiotic pressures were expected to be the greatest. AR37 had the broadest level of increased yield while AR1 still offered yield advantages over no endophyte in many cases. Dry matter yield did not differ between AR37 and AR1 at a national level except for autumn, or regionally except for the Upper North Island. The benefit of AR37 was seen to the greatest extent in the Upper North Island, with a 33% and 51% yield advantage over AR1 and without endophyte respectively. For the Upper South Island both AR37 and AR1 gave a significant increase in DM yield over without endophyte for annual dry matter yield.

There were individual trials in which AR1 and without endophyte gave inadequate protection, resulting in pasture failure, others in which insect pests or other stressors are likely to have impacted yield without causing pasture failure, and some where there was little differentiation between selected endophyte and without endophyte. This highlights the wide variation that can be observed on individual farms across New Zealand and the role that selected endophyte plays in creating resilient pastures.

Effects of fungicide treatment on transmission of Epichloë endophyte in rye (Secale cereale)

David Edward Hume — 2025-12-04

Seed-borne asexual species of Epichloë fungal endophyte have proven to be of critical importance in temperate grasses, particularly those grasses sown in pastures in the USA, New Zealand and Australia. These obligate mutualistic fungal endophytes impart meaningful benefits to the host plant through enhancing the plant’s ability to counter various abiotic and biotic stresses. Although there may be detrimental effects on livestock, selected endophytes have been identified and used in commerce to address animal health and productivity issues, while still providing protection to the host grass from stresses.

The use of selected endophytes has been explored for cultivated cereals such as rye and wheat (Triticum spp.), with the goal of reducing usage of fungicides and insecticides, and improving the tolerance of the plant to abiotic stresses. A complicating factor is that fungicides may still be necessary for crop management which in turn could be detrimental to the Epichloë endophyte. While this issue has been studied for pasture and amenity grasses, no similar studies have been performed with endophyte-infected cereals.

Fungicide treatments, representing a total of five chemical families, were applied either to seed prior to sowing or to foliage of established crops of rye infected with selected strains of Epichloë bromicola (AR3002) or Epichloë bromicola x amarillans (AR3068). In a field trial, two spring applications of five foliar-applied fungicidal products had no detrimental effect on transmission of endophyte to grain (seed-borne endophyte), but one combination did result in lower viable endophyte in the grain. In a greenhouse trial, three seed-applied fungicidal products had no detrimental effect on transmission of endophyte to seedlings. Similar results have been obtained with ryegrass (Lolium perenne). These initial results are promising in demonstrating that while the Epichloë endophyte will reduce the need for fungicides to control plant pathogens, if there are times when fungicides are needed, then options are available that will not adversely affect the endophyte. In addition to further work on rye, the effect of fungicides on Epichloë-infected wheat requires investigation.

Prospects of Epichloë endophytes on fall armyworm growth and survival in tall fescue

Kendall Lee — 2026-03-20

Fall armyworm (Spodoptera frugiperda) larvae are detrimental pests to many crops in the Southeastern USA, including tall fescue (Schedonorus arundinaceus (Schreb.) Dumort.), grown for turf and forage. In most natural and managed ecosystems, tall fescue plants harbour an Epichloë endophyte that imparts protection to the grass from abiotic and biotic stresses, including insect deterrence. Enhancing tall fescue resistance to fall armyworms may be achieved by identifying Epichloë endophyte strains that exhibit antibiosis or antixenosis. In this study, we inoculated 22 Epichloë strains (16 nontoxic and 6 toxic), isolated from tall fescue collections of various origins, into an endophyte-free continental-type tall fescue cultivar and a Mediterranean-type tall fescue cultivar. Leaves from each strain × host combination were used in feeding studies with fall armyworm larvae. We investigated the effect of different strain × host combinations on fall armyworm development by measuring larval weight gain, days to pupation, and survival in the laboratory. Complementary ratings of defoliation were conducted in a field infested with fall armyworm to assess pest damage under natural conditions. The Mediterranean-type tall fescue, in combination with most strains, showed detrimental effects on larval development. Two endophyte strains (one capable of producing mammalian-toxic ergot alkaloids and the other not capable of producing ergot alkaloids) significantly reduced fall armyworm larval development and minimised defoliation in the field. These will be characterised further for use in integrated pest management.

Physiological responses and ergot alkaloid kinetics in steers rotating grazing toxic endophyte-infected tall fescue

Ignacio Llada — 2025-12-04

Fescue toxicosis is a mycotoxin-related disease caused by ingestion of tall fescue infected with the ergot alkaloid-producing endophyte Epichloë coenophiala. Weight gain and physiological parameters related to thermoregulation are affected. Although rotational grazing might be a mitigation approach, the relationship between ergot alkaloid levels and clinical signs under such conditions remains unclear. Thus, we sought to explore how quickly clinical signs appear and resolve after exposure to toxic pastures, identify residual effects post-removal, assess environmental conditions that impair thermoregulation, and examine ergot alkaloid dynamics in biological matrices relative to onset and recovery. Steers grazed non-toxic, toxic, or endophyte-free fescue pastures. After 14 days, groups switched diets (toxic to non-toxic and vice versa). Physiological parameters and biological samples were collected. Wireless sensors monitored skin temperature and environmental conditions. Fourteen days were sufficient to induce clinical changes; rumen and urinary alkaloid dynamics preceded or aligned with their onset and recovery. While most physiological parameters recovered quickly after removal from toxic pastures, a residual effect on skin temperature, exacerbated by rain and mild heat, but not cold, persisted. Steers on toxic pastures could not dissipate heat below 18–20°C; above this, heat loss was unaffected. Rotational fescue grazing is a promising management tool, as long as thermoregulation and environmental conditions are considered.

Epichloë is not associated with the pollen of infected ryegrass plants

Natasha Forester — 2025-12-04

Observations taken over decades of seed production, and examination of anther tissues from Epichloë infected grasses suggest that Epichloë endophytes are vertically transmitted through seed and are not horizontally transferred by pollen to new host plants. However, no study has quantified the potential for Epichloë transmission via pollen. We studied the incidence of Epichloë DNA detection in pooled pollen samples collected from subsets of 1680 Lolium perenne plants undergoing seed multiplications in a containment glasshouse. Most plants were infected with one of several Epichloë strains, representing different species of Epichloë. These data were compared with the incidence of Epichloë DNA in anthers, pseudostem and seed with known infection status. Sensitive qualitative and quantification assays, high resolution melting analysis and digital droplet PCR were employed to detect and/or quantify either Epichloë or Lolium DNA within plant-host tissues. Epichloë DNA sequences were not detected in any pollen samples tested. Epichloë is therefore unlikely to be spread between plants via pollen. In countries such as New Zealand, where genetically modified organisms are regulated, risks of potential spread into the environment associated with maternal compared to paternal transmission may affect regulatory decisions regarding the use of such Epichloë-grass symbioses outside of physical containment facilities.

Epichloë endophyte survival in rye (Secale cereale) and perennial ryegrass (Lolium perenne) seeds under different storage conditions

Anouck de Bonth — 2025-12-04

Epichloë fungal endophytes are an important symbiont of some temperate forage grasses, providing the plant host with protection from biotic and abiotic stresses. Recent research has focussed on applying the potential benefits of the asexual form of this endophyte to cultivated cereal crops. Cereals are not known to naturally host Epichloë endophytes but can be artificially inoculated with selected fungal strains isolated from Elymus and Hordeum species. A critical aspect of a successful association is the ability of these obligate mutualistic endophytes to transmit through the seed and colonise the resulting offspring. Within agricultural and cropping systems, seed can be stored for varying lengths of time before being sown. The ability of these fungal endophytes to survive in stored seed and colonise the next generation of plants is an important consideration.

Optimal storage conditions required for Epichloë survival in seed of perennial ryegrass (Lolium perenne L.) and tall fescue (Festuca arundinacea Schreb.) are well documented, but little is known about endophyte survival within stored cereal grain, nor the conditions that are required for maximising its survival. Rye (Secale cereale L.) grain colonised with endophyte strain AR3002 and perennial ryegrass seed colonised with AR1 endophyte were stored under different temperature and humidity conditions, with the survival of both host and endophyte evaluated over a 2-year period.

Under controlled conditions of 0°C and a relative humidity of 24%, the endophyte and host maintained stable levels of viability for both species for the duration of the trial. In environments where both the temperature and relative humidity were not controlled and the seed was exposed to ambient conditions, the endophyte generally declined rapidly within 12-18 months, although in most cases the seed itself remained viable, for both rye and perennial ryegrass seed. The endophyte associated with rye responded in a similar way to endophyte associated with perennial ryegrass in these storage environments. This understanding provides guidance on seed storage conditions required to ensure sustained endophyte viability but also identifies timeframes in which endophytic grain will need to be resown if held at ambient conditions. Further work will be required to determine if similar limitations are applicable to Epichloë endophytes in wheat.

Meadow fescue and its Epichloë endophytes in New Zealand

Alan Stewart — 2025-12-04

In New Zealand, meadow fescue (Festuca pratensis) was introduced from Europe, along with many other grasses, as Europeans settled in the 1800s. By the 1950s it had fallen completely out of favour and ceased to be used due to its poor agronomic performance. The discovery of an ecotype in Northland in the 1990s, with Epichloë uncinata endophyte, stimulated research on this grass and the effect of its endophyte on insect pests. In environments with little insect pressure, trials with meadow fescue generally exhibit pasture yields lower than perennial ryegrass (Lolium perenne) and tall fescue (Festuca arundinacea). However, in environments with high insect pressure endophyte free meadow fescue can fail in the first year particularly if conditions are dry, while endophyte-infected meadow fescue exhibits strong tolerance to grass grub (Costelytra zealandica), black beetle (Heteronychus arator) and other insect pests. In these conditions it can perform better than both perennial ryegrass with AR37 endophyte and tall fescue with AR584 endophyte. Today the use of mixtures of meadow fescue and meadow fescue type Festulolium, with their E. uncinata endophyte, has found a niche commercial role on farms in New Zealand due to the protection it provides pastures from a number of insect pests. In addition, mixing meadow fescue with tall fescue has been found to improve palatability over tall fescue alone, with animals leaving lower residuals after grazing, leading to improved quality at subsequent grazings. This provides a solution to the challenge of managing tall fescue on its own. This paper reviews the knowledge about this grass/endophyte combination.

The impact of selected Epichloë endophytes on invertebrate biodiversity in New Zealand ryegrass sheep pastures

Mark McNeill — 2025-12-04

This study investigated the impact of six novel perennial ryegrass-Epichloë-associations in New Zealand sheep pasture on the above-ground and subterranean invertebrate fauna. The perennial ryegrass-endophyte treatments included cv. GA66 without endophyte, and infected with the selected Epichloë strains AR1, AR37 and the common-toxic endophyte. Additionally, two further perennial ryegrass lines, cv. GPD and cv. GPT, both infected with Epichloë strain AR37, were included to examine a cultivar effect. Invertebrates from above ground and subterranean environments were collected from pastures sown with each ryegrass-endophyte treatment at six intervals during 2021. Analysis of above-ground invertebrates found significant differences in density across ryegrass-endophyte treatments for Aranae and Opiliones, Acari, Psocoptera, Neuroptera, and Listronotus bonariensis (Argentine stem weevil), but overall differences for other taxa were not significant. Analysis of the Richness Index for all above-ground invertebrate taxa found significant differences amongst treatments, but not for Shannon’s diversity index H. Overall, all taxa showed a decline in density following sheep grazing, but the effect was taxa specific, with Acari, Coleoptera (beetles) and Hymenoptera (wasps, bees and ants) showing the most significant impact across all ryegrass-endophyte treatments.

The mostabundant soil invertebrates were Annelida (earthworms) and larvae of Costelytra giveni (New Zealand grass grub), with peak densities of both taxa observed in June (winter). Earthworm densities were significantly higher under all treatments following sheep grazing compared to pre-grazing densities, but the response varied for endogeic, epigeic and anecic earthworm taxa. Population fluctuations reflected grazing by sheep and the presence of dung introduced to the soil, soil moisture and season. There appearedto be minimal ryegrass-endophyte treatment effects on earthworm abundance or diversity, whereas for C. giveni larvae there were significant ryegrass-endophyte effects, attributed to colonisation flights in summer. Overall, large herbivore grazing had a significant impact on above-ground and subterranean invertebrate communities, irrespective of ryegrass-endophyte associations and invertebrates biology associated with seasonal changes, with the scale of impact being taxa dependent.

Performance of Sheep Grazing Tall Fescue Cultivars Containing Non-Ergovaline Producing Endophytes

James Thompson — 2025-12-04

Fescue toxicosis is one of the costliest animal disorders facing the livestock industry in the eastern United States, affecting over 8.5 million cattle and costing the United States beef industry nearly US $2 billion annually in lost revenue due to reduced reproductive and growth rates in cattle herds. An study was conducted to determine the effects of tall fescue (Festuca arundinacea Schreb.) containing wild-type ergovaline producing endophyte (Kentucky 31 E+), cultivars containing non-ergovaline producing endophyte (Martin 2 PROTEK, Martin 2 ISO 207, Duramax Gold), and tall fescues containing no endophyte (Martin 2, Au Triumph). Three Polypay yearling ewes were assigned to one of 6 different pasture treatments containing either ergovaline producing endophyte, non-ergovaline producing endophyte or non-endophyte cultivars in 0.1 hectare replicated pasture treatments. The 36 ewes were evaluated for changes in body weight, serum prolactin, and rectal temperatures during three grazing periods over three years (2009 to 2011) to determine animal performance and evaluate fescue toxicosis. The nutritional quality of endophyte-infected tall fescue was shown to be comparable to other tall fescues that did not contain the Epichloë endophyte. Ergovaline was only detected in the Kentucky 31 E+ forage, when sampled during each year of each grazing period (139 ppm). Pre-treatment sheep body weight did not differ (P<0.9948) among treatments. Mean total weight gain (P=0.0039) and mean average daily gain (P=0.0026) was lower for Kentucky 31 E+ compared to all other treatments. Pre-treatment serum prolactin concentration did not differ (P=0.5526) among treatments. Post-treatment prolactin level showed a highly significant effect in the 3-year mean (P<0.0001). Serum prolactin concentrations in sheep consuming wild-type endophyte fescue were lower than those sheep consuming endophyte fescues that did not produce the ergovaline at detectable levels. No differences in rectal temperatures were observed, either; pre-treatment (P=0.8404), treatment (P=0.7180), or post-treatment (P=0.5905). However, rectal temperature measurements can be subjected to confounding variables, such as sheep handling stress, and ambient air temperature. Sheep grazing tall fescue cultivars inoculated with endophytes not producing ergovaline displayed total weight gains similar to sheep consuming cultivars not containing endophyte. Lowered prolactin concentration and depressed weight gain of sheep grazing Kentucky E+ tall fescue indicates that the use of non-toxic endophyte tall fescue cultivars is a viable management strategy for minimizing the effects of tall fescue toxicosis.

Exploring ways to improve wheat productivity through the use of beneficial Epichloë endophytes

Lisa Wood — 2025-12-04

A fifth of the calories consumed by humans come from wheat. In 2023, 789 million tonnes of wheat were produced worldwide. With an increasing human population, demand for wheat is expected to increase. With rising production costs and increased environmental instability, novel solutions are required to enhance productivity. Beneficial fungal endophytes belonging to the Epichloë genus may provide a solution. These endophytes are used as protection against insect herbivory in commercial pasture grasses throughout the world via animal safe secondary metabolites. Other benefits from Epichloë include resistance to fungal pathogens and increased resistance to abiotic stress such as drought. Asexual Epichloë, which are used in commercial forage grasses, grow intercellularly and are vertically transmitted (maternally inherited). Modern wheat could benefit from this technology by reducing the synthetic chemistry requirements for control of biotic stress. However, to date there are no Epichloë found naturally in symbiosis with modern wheat cultivars and establishing symbioses has been a challenge. Early work successfully involved Chinese spring-based wheat lines that contained alien chromosomes from wild grasses known to host Epichloë endophytes. One of the more successful associations was with TACBOW0011, which has a Leymus racemosus H chromosome substitution. However, there were several negative phenotypes associated with endophyte colonisation, which included stunting, delayed flowering, shrivelled seed, low germination and low endophyte transmission. Through traditional breeding methods, improvements in plant phenotypes compatible with Epichloë endophytes will be described. Progress includes improved plant height, reduced flowering delay, increased seed size and improved germination and endophyte transmission.

Impact of respiration and CO2 on the survival of Epichloë festucae var. lolii strain AR37 in a cultivar of perennial ryegrass during short-term seed storage

Maria Duter — 2025-12-04

During storage, ryegrass seeds undergo cellular respiration to stay alive. When seeds respire, they take in oxygen (O₂) while releasing an equal amount of carbon dioxide (CO₂). Oxygen reduces seed longevity due to the oxidation of cells and membranes, even at ambient temperatures, but the effect of increased CO₂ concentration on seed and Epichloë endophyte viability is unknown. Two experiments were conducted to identify (i) whether seed respiration rate was affected by storage temperature and time, and whether this affected the viability and survival of the endophyte in seed and (ii) to determine whether a reduction in atmospheric O₂ concentration, and therefore increased CO₂ concentration, would affect seed and endophyte survival during storage. The first experiment was set up using a split-plot design with seeds in airtight containers. There were two endophyte treatments, with endophyte AR37 (E+), and endophyte-free (E-) three storage temperatures (5°C, 20°C, and 30°C), and six storage sampling periods (1, 2, 4, 8, 16, and 32 days). The hypotheses were that high CO₂ concentration resulting from the respiration of seeds would (i) have a negative host-endophyte effect, decreasing the survival of AR37 in an airtight container, (ii) E+ seeds would have a higher respiration rate than E- seeds, and that (iii) increasing storage temperature and storage time would decrease endophyte viability. The second experiment, designed as a completely randomised block, used ascorbic acid dust (ascorbic acid -AA) to create anoxic conditions, elevating relative carbon dioxide concentrations. The experiment was conducted in airtight glass vials with E+ and E- seeds at 20°C only, for the same storage periods as experiment one. Unsealed vials were used as control treatments. The hypotheses were that (i) high concentrations of CO₂ (greater than 400ppm) and increasing storage time would have a negative host-endophyte effect, (ii) anoxic conditions would not affect seed survival, and (iii) germination would differ between E+ and E- seeds. The endophyte viability and seed germination were assessed using an established tissue-print immunoblot procedure, and top of paper method respectively. Endophyte survival after storage was significantly reduced at all three storage temperatures, dropping from 86% at the beginning of the experiment to a grand mean of 77%; the loss in viability was lower at 5°C (-2.6%) than the other two temperatures (-11%). Seed respiration rate (mean=1.83 LogCO₂mg g^-1 h^-1) increased significantly with storage time at all three temperatures. E+ seeds had a significantly higher respiration rate (1.923 LogCO₂mg g^-1h^-1) than E- seeds (1.748 LogCO₂mg g^-1 h^-1). Within a storage temperature, the respiration rate did not differ between E+ or E- seeds. When stored under anoxic and high CO₂ concentration conditions, AR37 survival fell significantly to 72% after 32 days at 20°C. However, germination was not affected for either E+ or E- seeds. While seed germination was not affected by the modified storage conditions used a suitable explanation for the loss of AR37 viability remains to be determined. Further studies on the biochemical changes in the seed resulting from exposure to high concentrations of CO₂ are required.

Alliance for Grassland Renewal’s impact on novel Epichloë endophyte tall fescue adoption in the US

Nicholas Hill — 2025-12-04

Novel Epichloë endophyte infected tall fescue was commercially introduced to US livestock producers in 2001. Initial acceptance of the technology was poor with sales amounting to a small fraction of market studies. Acceptance of novel technology was attributed to mixed messaging from livestock professionals and companies with novel products. University, industry, and seed companies coalesced in the fall 2012 to develop the Alliance for Grassland Renewal to provide uniform messaging to livestock producers, operating under the assumption that educating producers would increase novel endophyte technology adoption. Methods: A minimum of four ‘producer’ schools were conducted by the Alliance each year beginning in 2013 until present. School topics included: differentiating the endophyte types in tall fescue, common falsehood remedies to fescue toxicosis, methods to renovate toxic tall fescue, pasture management, seed testing for quality control and assurance of novel products, and economics of pasture renovation. The ‘Alliance’ selected Agrinostics Ltd. Co. as the standard laboratory to monitor seed lots for quality (viable infection frequencies, mammalian toxic off types). Agrinostics monitored the number of seed lots tested over time to determine the impact of the Alliance on novel endophyte adoption. Findings: There was a lag in novel endophyte technology acceptance by producers from 2013 through 2016. However, beginning in 2017 there has been a linear increase in the number of seed lots tested. We conclude that novel endophyte technology is gaining acceptance by livestock producers because of the educational efforts provided by the Alliance. However, producer resistance to pasture renovation remains. Resistance to renovation is related to a) current livestock market conditions and b) a poor understanding when renovation is the best strategic option.

Temperature-driven variations in Epichloë endophyte-mediated porina resistance in perennial ryegrass

Katrin Hewitt — 2025-12-04

New Zealand’s pastoral systems rely heavily on perennial ryegrass (Lolium perenne), which are intentionally associated with selected Epichloë fungal endophytes that form a mutualistic symbiosis that protects the host against herbivorous insects through the production of alkaloids. The commercially available endophyte Epichloë LpTG-3 strain AR37 is a perennial ryegrass endophyte that confers resistance against the larvae of porina (Wiseana spp.), a significant pest in cooler regions of New Zealand. To expand pest management options, a newly identified strain of Epichloë, designated AR128 has been identified. This study investigated how temperature influences alkaloid concentrations in AR37 and AR128-infected perennial ryegrass plants and the subsequent effects on porina fitness, plant damage, and plant size.

Perennial ryegrass plants, both with and without AR37 or AR128, were grown at three temperature regimes: 8°C, 13°C, and 18°C. After 4 weeks of growth, one porina larva was added to each plant, and left to feed for 8 weeks. Following this feeding period, the tillers were assessed for feeding damage using a scoring system from 0-5. Porina survival and weight gain were also measured. Generalised linear mixed models were used to analyse the effect of perennial ryegrass and/or temperature on porina fitness, plant damage, and plant growth. There was no significant interaction between temperature and endophyte strain; however, both factors significantly affected the proportion of severely damaged tillers. Endophyte infection with AR37 or AR128 significantly reduced the proportion of severely damaged tillers (score 5) by 1.5% and 1.3%, compared to endophyte-free plants with 4.4%. The highest proportion of severely damaged tillers was observed at 8°C, while the lowest occurred at 18°C. The reduction in tiller damage at 18°C was associated with lower porina survival. Although there was no significant difference in porina survival between AR37 (41%) and AR128 (29%), both strains significantly reduced porina survival compared to endophyte-free plants (73%). Across all treatments, temperature influenced porina survival, which was lowest at 18°C with 21%. However, temperature had no significant effect on the weight gain of surviving larvae. The temperature-dependent trend in damage corresponded with epoxyjanthitrem concentrations in endophyte-infected plants, an alkaloid compound highly deterrent/toxic to porina. Although there was no significant difference in total epoxyjanthitrem levels between AR37 and AR128, concentrations were lowest at 8°C (5.9 µg/mL) and increased with temperature, reaching 29.4 µg/mL at 18°C.

Across all treatments, this research demonstrated that porina causes greater plant damage at lower temperatures. Although endophyte infection with AR37 or AR128 consistently reduced plant damage, their efficacy declined at lower temperatures, which was associated with a reduction in alkaloid concentrations. These findings highlight the importance of temperature in modulating endophyte-derived insect protection and suggests that the efficacy of this endophyte technology may be compromised under cooler growing conditions.

Impact of AR37, a selected Epichloë endophyte, on Italian ryegrass yield performance across regions in New Zealand

Sarah McKenzie — 2025-12-04

The selected Epichloë endophyte strain AR37 first became commercially available in 2007 in New Zealand cultivars of perennial ryegrass (Lolium perenne), before being extended into shorter-term ryegrass cultivars. In 2012, the cultivar Asset was the first commercially available Italian ryegrass (L. multiflorum) with the options of AR37 and endophyte-free. Over the past 10 years, the use of Epichloë endophytes in shorter-term ryegrass cultivars has expanded, with more than 10 hybrid (L. boucheanum, syn. L. hybridum) or Italian ryegrass cultivars with a selected Epichloë endophyte option now available.

The National Forage Variety Trials (NFVT) evaluates commercial and pre-commercial ryegrass cultivars within New Zealand through the New Zealand Plant Breeding and Research Association. The diploid Italian ryegrass cultivar Asset has been evaluated in 15 of these trials with AR37 and without endophyte, varying in duration from 12 to 18 months. To understand the effect in production that Epichloë endophyte AR37 provides in Asset Italian ryegrass, these trials were analysed for seasonal and total dry matter yield nationally and by region.

Asset with AR37 endophyte demonstrated increased production over no endophyte, with the combined multisite analysis showing an 11% increase in dry matter yield in the first summer post sowing, and a 14% increase in dry matter yield in the second autumn (P<0.05). The four trials located in the Upper North Island displayed the greatest benefits of AR37, with a 9% mean increase in dry matter yield over the period from sowing to the end of the first summer. In three of these four trials, AR37 had a significantly greater dry matter yield in the summer and second autumn.

Of the 11 trials located in the Lower North Island and Upper South Island, there were no seasons that differed significantly in the combined seasonal analysis within each region. In an analysis of the individual trials, two of the four Lower North Island trials had significantly greater dry matter production with AR37 in summer and the second autumn than without endophyte. In the Upper South Island, three of the seven trials had significantly greater dry matter yield for AR37 in both the summer and in total production over an 18-month period.

The results confirm that AR37 endophyte in Asset Italian ryegrass provided equal or greater total dry matter yield over without endophyte across all trials within 12 to 18 months, giving confidence that endophyte can have a positive impact on Italian ryegrass dry matter yield. While this work did not investigate the specific factors contributing to the benefits of endophyte, future work would help validate how Epichloë endophyte supports the resilience of shorter-term ryegrasses under varied environments within regions and seasons.

Resistance to dollar spot disease in fine fescues is linked to the presence of the endophyte Epichloë festucae

Ruying Wang — 2025-12-04

Dollar spot is a major disease in many turfgrass species. Resistance to dollar spot is critical for fine fescues to be used on golf courses or as low-input turfgrass lawns. Many fine fescue species, including Festuca rubra and its subspecies, have coevolved with Epichloë festucae, a filamentous fungal endophyte with which they form long-term, symbiotic associations. These endophytes confer several benefits to their host, including pest and disease resistance. In fine fescues, different sources of resistance to dollar spot linked to genotypes of E. festucae could exist. Dollar spot resistance in strong creeping red fescue (F. rubra subsp. rubra) has been attributed to an antifungal protein gene, Efe-afpA. In hard fescue (F. brevipila), a diallel cross between three resistant endophyte-containing and three susceptible endophyte-free parents was performed to determine the inheritance of dollar spot disease resistance. The highly significant maternal effect demonstrated that dollar spot disease resistance is maternally inherited in hard fescue and linked with the maternal inheritance of E. festucae. However, Efe-afpA is absent in endophyte strains that associate with hard fescue. The mechanism of endophyte-mediated disease resistance in hard fescue is therefore unknown. It is possible that small effector proteins from E. festucae strains associated with hard fescue could function similarly to Efe-AfpA and/or trigger plant defence responses against fungal pathogens. Regardless of the source of resistance, E. festucae strains that confirmed dollar spot resistance can be transferred within or between turfgrass species. Our research suggested that artificially inoculating turfgrass species with Epichloë endophytes can generate novel dollar spot resistant germplasm, creating more opportunities for turfgrass breeding and cultivar development.

A novel Lolium multiflorum-Epichloë tembladerae association: Evaluation of plant growth, seed production and vertical endophyte transmission under drought and waterlogging conditions

Daniel Ruiz Mínguez — 2025-12-04

Endophytes of the genus Epichloë are key allies for many cool-season grasses, providing them with significant advantages such as increased resistance to herbivores and tolerance to adverse environmental conditions. Epichloë tembladerae is a widely distributed endophyte in Argentina, associated with more than 30 grass species. Recently, a strain of E. tembladerae was inoculated into Lolium multiflorum and formed a compatible association. This study investigated the impact of the novel symbiotum E. tembladerae-Lolium multiflorum on grass host performance and its stability under waterlogging and drought conditions. Plant growth, biomass, seed production, and vertical transmission of the endophyte to plant progeny were evaluated.

Seeds of L. multiflorum of three symbiotic statuses: 1) endophyte-free, 2) colonised with E. occultans and 3) colonised with E. tembladerae were germinated on filter paper at 15°C. Ninety seedlings per symbiotic status were sown in potting mix and grown at a soil water content of 80% field capacity for four months. All plants were trimmed 5 cm above the crown, and their aerial biomass dried and measured. Thirty plants from each symbiotic status were then subjected to one of three water treatments: 1) drought (40% field capacity), 2) waterlogging (2 cm of water above the soil surface), and 3) control (80% field capacity). After 20 days of treatment all the plants were cut to evaluate regrowth and half of the plants were harvested to measure root and shoot biomass. The remaining plants were allowed a 15-day resting period before resuming the water stress treatments, which were maintained until seed harvest. At the end of the experiment, seed yield and endophyte transmission frequencies were evaluated. No significant differences in regrowth, shoot and root biomass production, and seed yield among the three symbiotic statuses were detected. However, the drought treatment significantly reduced plant regrowth, biomass production and seed yield compared to the control and waterlogging conditions. Transmission efficiency of E. occultans and E. tembladerae to seeds exceeded 90% in both cases, and under all water treatments.

These results suggest that water deficiency plays a crucial role in the productivity of L. multiflorum and that the association with Epichloë has no effect in drought tolerance. Interestingly, the novel association between L. multiflorum and E. tembladerae resulted in compatible associations even under unfavourable environmental conditions and no negative effects on the plants were observed. Our findings expand the knowledge about the specificity between Epichloë and their host plants. Considering endophytes in forage and crop breeding, our findings encourage future research to study the effect of E. tembladerae on novel grass hosts under different stresses and environmental conditions.

Festulolium–Epichloë symbiosis: suppressing stubby root nematodes and delivering nematotoxic plant metabolites

Nyambura Mwangi — 2025-12-04

This study investigated the potential of utilising cool season grasses naturally associated with Epichloë endophytic fungi for the management of plant-parasitic stubby root nematodes (Trichodorus spp. and Paratrichodorus spp.). Stubby root nematodes are economically important in East England where they cause docking disorder in sugar beet which causes up to 50% root yield reduction. The lack of chemical nematicides for the management of stubby root nematodes due to environmental concerns warrants the development of more eco-friendly measures. Epichloë fungal endophytes are known to confer protection from herbivory to their cool-season grass hosts, via the production of alkaloidal compounds. Several Epichloë species are known to produce loline alkaloids, a group of compounds known for their insecticidal and insect-deterrent properties. Lolines have also been reported to inhibit nematode mobility, egg hatching and mortality. In this study, Festulolium loliaceum and its associated endophyte Epichloë uncinata, known for its production of loline alkaloids, were investigated for their efficacy in suppressing stubby root nematodes. In a field experiment in Docking, Norfolk, England, endophyte-infected grasses significantly reduced the reproduction of stubby root nematodes by seven times compared to endophyte-free grass associations. Laboratory experiments using extracts from shoots and roots of F. loliaceum plants showed that both endophyte-infected and endophyte-free plants could immobilize nematodes. Age, source of extract and extract concentration had a significant effect on the nematocidal activity. Extracts from younger grasses were more potent compared to older grasses, where shoot extracts from 8-week-old endophyte-infected grass plants had six times lower LD50 values compared to 20-week-old grass plants. A contrasting effect was found for grass root extracts where roots from older plants were more potent than extracts from roots from younger plants, and had lower potency compared to shoot extracts. Further laboratory experiments showed that artificial wounding of endophyte-infected F. loliaceum plants elevated the loline alkaloids in the regrowth tissue within the first 11 days. Nematicidal activity of extracts from this regrowth tissue was significantly greater as compared to extracts from unwounded grass tissue. Overall, the results from this study suggest that endophyte-infected grasses could serve as a potential cultural management strategy for stubby root nematodes in a sugar beet crop rotation system. It is also evident that stubby root nematodes are sensitive to loline alkaloids produced from the grass-endophyte symbiosis used in this study, and this system could be optimised in the future to support nematode suppression under field conditions.

Epichloë endophytes from Elymus dahuricus enhance the growth and nutritional quality of Poa pratensis

Mingxiang Du — 2025-12-04

In natural ecosystems, symbiotic Epichloë endophytes can contribute significantly to the growth and development of cool season grasses in the Pooideae subfamily with which they form host specific, long-term associations. Selected Epichloë strains can also be transferred to alternate grass hosts, such as elite grass germplasm used as forage, where they can also confer advantageous traits. Most of these ‘novel’ endophyte-grass associations have been developed in fescue and ryegrasses that are agriculturally important to farm systems in many countries including New Zealand and the USA. Limited reports exist regarding the symbiotic relationship between Epichloë and Poa pratensis within natural ecosystems, and no studies have documented the application of this Epichloë species in managed pastoral systems. In this study, three Epichloë strains (LE1, LE3, and WBE1) isolated from Elymus dahuricus, determined as non-toxic to grazing livestock, were inoculated into modern cultivars of P. pratensis to investigate their colonization ability, compatibility and potential effects on agronomic traits and nutritional quality of the novel symbioses. The colonization rates of strains LE1, LE3, and WBE1, reached 4.1% (n=369), 4.4% (n=341), and 4.1% (n=320), respectively. All three strains significantly enhanced key agronomic traits, including plant height, leaf length, potassium (K) accumulation, and soluble sugar content. PCA revealed that LE3 exhibited the most pronounced synergistic effects on host nutritional growth, as evidenced by its superior comprehensive evaluation score. Furthermore, endophytic fungal colonization induced significant structural alterations in the phyllosphere bacterial community, characterized by shifts in dominant taxa and functional guilds, thereby establishing a novel plant-microbe functional network. These findings underscore the potential of Epichloë-mediated microbiome remodelling as a driver of phenotypic optimization in grass-endophyte symbioses. This study confirms that novel symbiotic associations between Epichloë endophytes and Poa pratensis can be achieved to confer advantageous traits to the host grass. These findings contribute valuable insights to the field of grass-endophyte symbiosis research and practical breeding applications.

Perennial ryegrass microbiomes: manipulating microbial communities for enhanced pasture performance

Shengjing Shi — 2025-12-04

It is well recognised that the plant microbiome plays a critical role in plant health, productivity, and resilience to environmental stresses. Recent research has suggested the two disciplines of plant breeding and microbiome engineering should be integrated at an early stage of crop development and this will enhance plant performance via the promotion of beneficial plant-microbe interactions. Achieving this goal requires a greater understanding of key factors that shape the plant microbiome. Perennial ryegrass (Lolium perenne), the most common pastoral forage species utilised in New Zealand, was used as a model system. The microbiome associated with different plant zones (including the rhizosphere, root and shoot) was assessed from plants collected from multiple field trials across the North and South Islands of New Zealand. Results indicate that plant zone, geographic location and plant growth stage were the strongest drivers of ryegrass microbiome structure as analysed by amplicon sequencing of 16S and ITS genes for bacteria and fungi, respectively. In contrast, ryegrass cultivar and the presence of agriculturally beneficial strains of Epichloë endophyte had minor impacts on the ryegrass microbiome but significantly shaped the ryegrass seed microbiome. Current research is focused on investigating the influence of diverse ryegrass genetics (>400 different families) on the plant microbiome to uncover host genetic contributions to microbial community assembly. This knowledge will provide a critical foundation for the development of novel forage crops with beneficial microbiome traits to enhance plant performance and promote sustainability.

Effects of Epichloë endophytes on the microbial communities and nutrient concentrations of Achnatherum inebrians roots

Xiumei Nie — 2025-12-04

This study investigated the impact of Epichloë gansuensis, a fungal endophyte that forms a long-term symbiosis with the grass Achnatherum inebrians, on fungal and bacterial communities, nutrient concentrations, and microorganismal-nutrient relationships associated with host roots. Both fungal and bacterial communities associated with the host root endosphere were characterized for plants infected with E. gansuensis (symbiotic) and for those that lacked a symbiosis with E. gansuensis (non-symbiotic) using high-throughput sequencing. Concentrations of multiple nutrients were measured in the plant roots, i.e., total carbon, total nitrogen, total phosphorus, total potassium, and organic carbon. In the roots of symbiotic and non-symbiotic plants, fungal communities were dominated by members within Ascomycota, Basidiomycota, and Mortierellomycota, while bacterial communities were predominantly composed by members within Pseudomonadota, Acidobacteriota, and Actinomycetota. The presence of E. gansuensis in plants increased the root concentrations of total carbon, total nitrogen, and organic carbon, but decreased the concentration of total potassium. There were 11 positive (3 fungi and 3 bacteria) and 2 negative (0 fungi and 2 bacteria) significant correlations between microorganismal taxa abundance and nutrient concentrations. The abundance of the fungal phyla Aphelidiomycota, Glomeromycota, and Olpidiomycota were positively correlated with the concentrations of total carbon and organic carbon. The abundance of the bacterial phylum Methylomirabilota was positively correlated with the concentrations of total carbon, total nitrogen, and organic carbon, while negatively correlated with total potassium concentration. Furthermore, the abundance of bacterial phyla Actinomycetota and Nitrospirota were positively correlated with the concentrations of total potassium and organic carbon, respectively, while Pseudomonadota abundance was negatively correlated with the concentration of total potassium. Our findings emphasize the effect of a fungal endophyte, E. gansuensis, that is exclusively found in the aboveground organs of its host plants and absent from belowground plant organs on the concentrations of nutrients within plant roots and the existence of putative functional relationships between root-associated fungal/bacterial communities and root nutrients.

Using niche occupancy from microbiome data to reveal potential fungal pathogens of established perennial ryegrass roots

Nigel Bell — 2025-12-04

Perennial ryegrass is the most widely sown grass in Aotearoa New Zealand grazed pastures. Poor persistence of perennial ryegrass pastures in the northern part of the country is an on-going issue for farmers. Both above- and below-ground fungal plant diseases are likely to be part of the persistence issue, and a better understanding of the range of pathogens present across the country will help in the design of new approaches to overcome their effects. Microbiome data gives a culture-independent measure of potential pathogen, and beneficial, microbiota presence and relative abundance, including in different niches. This may be a useful tool to improve our knowledge of potentially pathogenic and beneficial microbiota for further study. Forty plots of three-year-old perennial ryegrass plants were sampled from each of four dairying regions across New Zealand. Samples were taken in autumn and separated into soil, rhizosphere soil, roots, shoots and phyllosphere niches, giving a total of 800 niche samples. Fungal communities were determined by extracting DNA and analysing using ITS MiSeq amplicon sequencing. Percentage of reads of individual amplicon sequence variants (ASVs) amongst the five niches was used to identify any that were preferentially present and abundant in particular niches, as an indication of potential pathogen status. For roots there were ca. 100 fungal ASVs across all sites which were almost exclusively associated with this niche (>80% of reads). This high niche occupancy rate was particularly striking for 10 closely related ASVs whose sequences matched Slopeiomyces or Gaeumannomyces spp., with many of these species known as grass pathogens. For shoots, only ca. 20 ASVs across all sites were as closely associated with this niche. This included species such as Neoascochyta sp., known for causing disease in grasses along with Epichloë sp., known in New Zealand for their mutualistic interaction with fescues and ryegrasses. These patterns of occurrence may change temporally but do show that potential pathogens and beneficials could be identified in microbiome data by their niche occurrence pattern. Clearly, isolation and plant growth testing of the most prevalent of these fungi is now needed to understand their impact on ryegrass growth and therefore what part they may be playing in poor plant persistence.

Wild progenitor species of maize (teosintes) have greater seedborne microbial diversity than domesticated maize

Sandeep Kumar — 2025-12-04

Maize (Zea mays) is a globally vital cereal crop that sustains billions of people as a food staple while supporting livestock, biofuel, and industrial applications. Maize was domesticated from teosintes, a group of Zea mays subspecies that originated in ancient Mesoamerica. Plant domestication and intensive agriculture may have reduced beneficial endophytic diversity in maize, underscoring the need to characterise microbial roles in plant performance. Investigating seed and seedling microbiomes is critical for unravelling how microbial communities influence plant ecology and function. The study tested the hypothesis that the seedborne microbial communities of teosintes were more diverse than those of domesticated maize. Seedborne microbial endophytic communities were surveyed from 85 geographical diverse Zea accessions, 65 cultivars and 20 teosintes accessions, originating from 28 countries sourced from international genebanks. The fungal ITS2 region and bacterial 16S rRNA genes were sequenced using the Illumina MiSeq platform to analyse the microbiomes associated with the shoot, root and kernel from five seedlings of each accession. The teosintes accessions, on average, had a significantly higher bacterial and fungal Shannon diversity index and observed taxon indices than the domesticated maize accessions. Pseudomonadota and Actinomycetota were the most abundant and prevalent bacterial phyla across all accessions of teosintes, comprising an average of 80% and 17% of the total reads, respectively. In contrast, Pseudomonadota was extremely abundant in domesticated maize, comprising an average of 98% of the bacterial reads, compared to 71% in teosintes. Sarocladium and Fusarium were the most abundant fungal genera across the accessions averaging 90% of the total reads. This study revealed significant differences in seed and seedling microbiomes between domesticated maize and teosintes, with accessions of the latter harbouring greater microbial diversity. The dominance of Pseudomonadota in domesticated maize and the reduced microbial richness suggest that domestication has resulted in specialised microbial associations, potentially impacting plant resilience. These findings highlight the importance of preserving and restoring microbial diversity in modern crops that have the potential to enhance stress tolerance, nutrient uptake, and disease resistance.

Arbuscular mycorrhizal fungi coupled with Epichloë endophytes reshaped the rhizosphere microbiome structure and induced synergistic resistance to leaf spot disease in perennial ryegrass

Youlei Shen — 2025-12-04

Perennial ryegrass (Lolium perenne) frequently forms simultaneous associations with arbuscular mycorrhizal (AM) fungi and Epichloë endophytes, residing belowground and aboveground of host plants, respectively. These beneficial symbionts enhance plant stress resistance by regulating nutrient absorption, increasing the activity of defensive enzymes and hormone levels, and upregulating stress-related gene expression. The rhizosphere microbiome, often referred to as the plant's ‘second genome’, plays a crucial role in influencing productivity, and resilience to stress. While AM fungi and Epichloë endophytes are known to independently alter rhizosphere microbial communities, their interactive effects on rhizosphere microbiome, particularly under pathogen stress, remain poorly understood and underexplored.

A greenhouse experiment was conducted to investigate the individual and interactive influences of the AM fungus (Acaulospora delicata) and Epichloë sp. LpTG-3 strain AR37 on defence physiology (enzymes and hormones), and rhizosphere microbiome-metabolite profiles related to resistance against Bipolaris sorokiniana, responsible for leaf spot disease.

When applied alone, A. delicata reduced disease incidence by 38% and disease index by 57%, while Epichloë strain AR37 alone decreased disease incidence by 26% and disease index by 44%. Co-colonization of A. delicata plus AR37 synergistically amplified these effects, achieving a 54% reduction in disease incidence and a 76% reduction in disease index, with A. delicata playing a predominant role in pathogen suppression. The tripartite interaction significantly enhanced defensive enzyme activities (superoxide dismutase, peroxidase, catalase), which scavenge pathogen-induced ROS, while reducing malondialdehyde (MDA) content by 29%, indicating mitigated lipid peroxidation. Notably, the A. delicata-Epichloë AR37 interaction increased ethylene levels by 11%, a critical shift linked to defence priming. A. delicata also increased the alpha diversity of both the rhizosphere bacterial and fungal communities, in contrast to the minimal microbiome alterations observed from AR37 or pathogen alone.

In the presence of the phytopathogen B. sorokiniana, the interaction between A. delicata and AR37 enriched fungal taxa within the division Basidiomycota and the bacterial phyla within the Acidobacteria, Actinobacteria, and Candidatus Sumerlaeota, while simultaneously suppressing the dominance of the fungi within the division Ascomycota.

Further analysis revealed that the relative abundance of Basidiomycota, Acidobacteria, Actinobacteria, and Candidatus Sumerlaeota was negatively correlated with the disease index and positively correlated with MDA and ethylene levels in ryegrass. Additionally, rhizosphere metabolomics identified four pathogen-inhibitory compounds that significantly accumulated in the multisymbiont systems: (benzo(b)thiophene-6-yl) acetic acid, erucic acid, acetamide, and dodecanoic acid. The concentrations of these metabolites were negatively correlated with the disease index and positively correlated with the relative abundance of some taxa. In vitro experiments indicated that acetamide exhibited a pathogen inhibition rate of 52%.

Our findings demonstrate that the synergistic effects of A. delicata and Epichloë sp. strain AR37 stimulated the production of rhizosphere metabolites, such as (benzo(b)thiophene-6-yl) acetic acid, acetamide, which may have recruited a beneficial microbiome to the host rhizosphere to strengthen anti-disease defences.

Diversity of endophytic fungi in live and dead leaf sheaths of Brachypodium sylvaticum infected with stroma-forming and asymptomatic Epichloë endosymbionts

Yuki Hatono — 2025-12-04

The fungal endosymbionts of the genus Epichloë (Clavicipitaceae, Ascomycota) systemically infect the aerial parts of certain cool-season grasses in the sub-family Pooideae. Epichloë is a monophyletic genus that includes anamorphic (asexual) and teleomorphic (sexual) species. Asexual Epichloë species transmit vertically via host seed while sexual species can transmit horizontally between hosts, and some species can simultaneously transmit both vertically and horizontally. With respect to the sexual reproductive lifecycle, Epichloë form external stromata that prevent the development of inflorescences and seed production of the host. Recent studies reported that the presence of Epichloë in both live and dead grass leaves can affect the leaves composition of non-systemic endophytes. However, the effects of the sexual reproductive cycle, particularly the presence of Epichloë stromata on the diversity and functional groups of non-systemic endophytes are yet to be investigated. We assessed the fungal diversity in live and dead leaf sheaths of Brachypodium sylvaticum plants that were endophyte-free or colonised by Epichloë sylvatica, a species that can be genetically differentiated into sexual and asexual subpopulations by DNA metabarcoding. The species richness of non-systemic endophytes and those that were functionally assigned by the FungalTraits database to litter saprotrophs and plant pathogens was greater in live plants than dead plants. In live grass plants, the species richness of non-systemic endophytes, and those that were functionally assigned to litter saprotrophs and plant pathogens, was significantly greater in individuals exhibiting stroma than in asymptomatic plants colonised by Epichloë and those that were endophyte-free. The composition of non-systemic endophytes that were functionally assigned to litter saprotrophs was significantly different between stroma-forming plants vs asymptomatic and endophyte-free plants, whereas such differences were not significant in the composition of non-systemic endophytes that were functionally assigned to plant pathogens. These results suggest that the presence of Epichloë stroma on B. sylvaticum affects the diversity of saprotrophic fungi and these could potentially alter ecosystem processes such as decomposition of dead plant tissues in soil.

Bacterial symbionts of Epichloë fungal endophytes of grasses

Daniel Bastías — 2025-12-04

Bacteria often form symbiotic associations with plant beneficial fungi, and they can increase the fitness of either their fungal and/or plant hosts. We characterised the bacterial microbiota associated with Epichloë fungal endophytes of perennial ryegrass (Lolium perenne) and investigated the effects of the bacterial symbionts on the performance of Epichloë or L. perenne (e.g., growth, Epichloë-derived alkaloid concentrations). We hypothesised that the bacterial microbiota associated with Epichloë would not be detrimental towards the fungal or plant host and instead promote plant growth without affecting either fungal growth or production of Epichloë-derived alkaloids. The bacterial microbiota associated with Epichloë was dominated by members of the genus Paenibacillus, although bacteria within genera Delftia, Micrococcus and Bradyrhizobium were also present. Two bacterial strains, Paenibacillus E222 and Micrococcus E226, were isolated from Epichloë mycelia and characterised further. Both E222 and E226 formed ectosymbiotic relationships with the fungal endophyte (i.e., bacterial cells were located on the hyphal surface) and possessed plant-growth promoting traits identified by whole genome analysis. Further experiments, enriching the abundance of E222 and E226 by inoculating these bacteria onto perennial ryegrass seed colonised by Epichloë, confirmed their plant growth promoting abilities in planta. E226 did not affect the growth of Epichloë or the concentration of Epichloë-derived alkaloids while E222 did the opposite. The mechanism of plant growth promotion by E222 and E226 is likely related to the production of growth promoting hormones/volatiles, abilities to solubilise phosphorus, and/or synthesise vitamins/metabolic cofactors.

Effects of a fungal volatile organic compound on microbiomes associated with perennial ryegrass and white clover under drought stress

Sereyboth Soth — 2025-12-04

Drought can significantly reduce pasture growth in many parts of New Zealand with subsequent negative effects on the economy. Fungal volatile organic compounds (FVOCs) can facilitate plant-microbe interactions including host-microbiome recognition, assembly, and maintenance via chemical signalling. This study investigated the impact of coating seeds with a FVOC on plant biomass and plant associated microbiomes of drought stressed pasture plants (perennial ryegrass, white clover, and a mixture of both) in a glasshouse study. Plant dry matter and soil microbial samples were collected when plants had recovered from three consecutive drought events. The ITS3/ITS4 and 16S V5/V7 regions were sequenced using the DNA nanoball sequencing platform, with sequence denoising using the DADA2 pipelines, and microbiome analyses conducted via MicrobiomeAnalyst. FVOC treatment increased plant dry weight after the three drought events by 9%, 8% and 33% respectively in the absence of drought and by 15%, 31%, 48% under drought conditions for grass, clover, and the mixture, respectively. Bacterial amplicon sequence variants (ASVs) of Rhizobiaceae, Bacillaceae, and Xanthobacteraceae from FVOC treatments were 11%, 5%, and 22% higher than controls, whereas fungal ASVs of Nectriaceae, Chaetomiaceae, Mortierellaceae, Filobasidiaceae, and Hoehnelomycetaceae were reduced by 9%, 6%, 10%, 3%, and 38%, respectively. Bacterial families that incorporate known mutualistic species were associated with plant growth and stress responses, while fungal families that showed a reduction in ASVs were primarily those that include species capable of causing plant disease. These results suggest that FVOC treatment can enhance plant biomass and modulate microbiomes to improve drought resilience, offering a potential strategy for pasture management under water-limited conditions.