Broad-scale geographic patterns in the distribution of vertically-transmitted, asexual endophytes in four naturally-occurring grasses in Sweden

The presence and distribution of vertically-transmitted, asexual Neotyphodium/Epichloë endophytes in four species of widely distributed grasses, all members of the sub-family Pooideae, were determined at 57 spatially-distinct sites across Sweden in naturally-occurring grass populations. While these endophytes are well-studied in agricultural systems, much less is known about their abundance and distribution in non-agricultural systems at larger scales across landscapes. We used two screening methods to detect endophytes: 1) visual inspection of fixed, stained grass shoots, and 2) immunoblot cards with monoclonal antibodies specific for Neotyphodium / Epichloë endophytes. Infection frequency (i.e. the proportion of individuals) varied widely among species and among sites, a finding common to most studies performed in non-agricultural ecosystems. Festuca rubra had the highest overall infection frequency (23% visual screening, 27% immunoblot screening), while Deschampsia flexuosa (4%) and Poa trivialis (4%) had low frequencies of infection. None of the sampled Agrostis capillaris populations were infected. In F. rubra , the within-site infection frequency varied from 0 to 100%. A significant altitudinal gradient was found for F. rubra , with the infection frequency of the vertically-transmitted endophyte Epichloë festucae decreasing with increasing altitude above sea level. The high variation across and within grass species at different geographical locations that ours and others' studies reveal, agree with suggestions that agronomic model systems cannot be readily extrapolated to understand the ecological role of endophytes in native grasses in the wild. Further investigation into the underlying cause of the observed altitudinal gradient is required to understand broader-scale patterns in the distribution of endophyte-infected grasses.     

禾草内生真菌的遗传多样性及其共生关系

 近百年的禾草内生真菌研究历经了由浅入深的过程,从最初的家畜中毒事件认识到是一种共生内生真菌存在的缘故,到如今利用分子生物学技术揭示其共生机制,人类发现这类植物内生真菌并非想象中的对生态系统无足轻重。Epichloë及其无性型Neotyphodium与禾本科植物是系统发生的互利共生关系,尤其是Neotyphodium可提高宿主抵抗环境胁迫的能力和抵御动物的取食,增强植物的竞争力。禾草内生真菌有3种生活史：有性生活史、无性生活史和兼性生活史,后者表明真菌在不同的宿主及环境下既能营有性生殖也可营无性繁殖,是一种更灵活而有效的生活史对策。对内生真菌分子系统学、生活史以及与宿主禾草协同进化的研究发现,Neotyphodium起源于禾草致病真菌Epichloë的某些种,或是Epichloë与Neotyphodium的种间杂交后代。植物和内生真菌各异的生活史策略,真菌的种间杂交,两者的协同进化亦或种群间基因流的差异,都促成了共生体多样化的基因组合（Genetic combination ）,也是其共生关系多样化的根源。内生真菌对宿主的有益作用只在特定基因型真菌、宿主和一定环境条件下才起作用,自然生态系统的共生关系要比农业系统复杂得多,是一个从互利共生至寄生关系的连续系统。未来对于更多共生体的遗传背景和基因与环境相互作用的阐明将有助于对禾草内生真菌共生关系本质更加深入的认识。     

Symbiosis between grasses and asexual fungal endophytes

The symbiosis between vertically transmitted asexual endophytic fungi and grasses is common and generally considered to be mutualistic. Recent studies have accumulated evidence of negative effects of endophytes on plant fitness, prompting a debate on the true nature of the symbiosis. Genetic factors in each of the two partners show high variability and have a range of effects (from positive to negative) on plant fitness. In addition, interacting environmental factors might modify the nature of the symbiosis. Finally, competition and multitrophic interactions among grass consumers are influenced by endophytes, and the effects of plant neighbours or consumers could feedback to affect plant fitness. We propose a mutualism-parasitism continuum for the symbiosis between asexual endophytes and grasses, which is similar to the associations between plants and mycorrhizal fungi.     

Endophytes of native grasses from South America: Biodiversity and ecology

We review and present preliminary results of studies on cool-season grass endophytes native to South America. These fungi have been studied only in Argentina, where they have been detected in 36 native grass species. The hybrid Neotyphodium tembladerae is present in an extremely wide host range found in diverse environmental conditions, but some other endophytes seem to be strictly associated with one host species in a particular environment. In host species that inhabit different environments, the incidence of endophytes is highly variable among populations and in most of the cases is clearly associated with environmental conditions. In these native grasses, Neotyphodium presents a mutualistic behaviour, conferring enhanced growth, promoting the symbiosis with arbuscular mycorrhizal fungi, and inhibiting growth of pathogenic fungi. In native forage grasses, preliminary analyses indicate that some Argentinian endophytes can produce lolines but are unlikely to produce lolitrem B or ergot alkaloids.     

Direct classification of related species of fungal endophytes (Epichloë spp.) using visible and near-infrared spectroscopy and multivariate analysis

The aim of this work was to investigate the potential of visible and near-infrared (Vis-NIR) reflectance spectroscopy for the classification of three morphologically similar species of fungal endophytes of grasses. Vis-NIR spectra (400–2498 nm) from 34 isolates of Epichloë sylvatica , 32 of Epichloë typhina and 38 of Epichloë festucae were recorded directly from fresh mycelium growing in potato dextrose agar plates. Multivariate procedures applied to the spectral data were discriminant modified partial least squares regression, soft independent modelling of class analogy and discriminant partial least squares regressions (PLS1, PLS2). Several types of data pretreatments were tested to develop the classification models. The best predictive models were achieved with PLS2 analysis; with this method, 90% of E. typhina and 100% of E. festucae and E. sylvatica external validation samples were successfully classified. These results show the potential of Vis-NIR spectroscopy combined with multivariate analysis as a rapid method for classifying morphologically similar species of filamentous fungi.     

Stroma-forming endophyte Epichloë glyceriae provides wound-inducible herbivore resistance to its grass host

Fungal endophyte-grass associations are diverse and complex. Some endophytes (e.g. Neotyphodium spp.) reproduce asexually by growing vegetatively into host seeds and many of these vertically-transmitted endophytes form mutualisms with their hosts by providing high levels of alkaloids, such as lolines, that reduce herbivore performance. Additionally, Neotyphodium coenophialum provides wound-inducible herbivore resistance through increased production of lolines. Neotyphodium likely evolved from Epichloë spp. which are sexually reproducing endophytes that are transmitted horizontally to the next host generation through production of stromata (fruiting bodies), which sterilize host grasses. We asked if wound-inducible resistance like that in N . coenophialum also occurs in the ancestral, sexually reproducing Epichloë glyceriae , which infects Glyceria striata . Host grasses were damaged by fall armyworm caterpillars, artificially cut, or left undamaged. An aphid bioassay tested the plant's toxicity to herbivores, expression of lolc (a gene in the loline biosynthesis pathway) was quantified using real-time RT-PCR, and loline concentration was quantified using gas chromatography and mass spectrometry. Artificially-damaged plants supported fewer live aphids, had greater lolc mRNA expression, and greater loline concentration than undamaged plants. Herbivore-damaged plants supported intermediate performance by aphids, low lolc mRNA expression, and minimal loline concentration. Our study is the first to demonstrate sexual endophytes can produce lolines following wounding. This suggests wound-induced responses are ancestral within the Epichloë / Neotyphodium clade and reveals a trait of grass endophytes that may have predisposed them for the evolution of defensive mutualisms with their hosts.     

Potential analysis of grass endophytes Neotyphodium as biocontrol agents

Many grasses in the subfamily Pooideae develop symbioses with Neotyphodium fungal endophytes, which exist widely in nature. The stably symbiotic relationship not only ensures accessible nutrients required by Neotyphodium fungal endophytes, but also significantly increases the resistance of host grasses to biological stresses through the production of secondary metabolites. Previous studies show that infected grasses with endophytic fungi have prominently enhanced resistance to pests, plant diseases, companion plants and other biological stresses. Grass endophytic fungi show remarkable resistant to at least 79 species of pests from three classes; arachnida, nematode and insecta, and to at least 22 species of pathogenic fungi. Although the biotechnological application of endophytic fungi in grass breeding for variety selection and quality improvement has progressed well, opportunities remain for further exploring the use of fungal endophytes among different host grasses coupled with the examination of genetic stability of Neotyphodium in novel host grasses. In the future application of endophytic fungi as a bio-control method, researchers should not only consider specificities of host grasses, but also need to have comprehensive analysis and knowledge about the mutual relationships among grasses, endophytic fungi and ecological environments, which will help use endophytic fungi to better serve humanity.     

Can seed-borne endophytes promote grass invasion by reducing host dependence on mycorrhizas?

Symbiotic interactions between plants and microorganisms have recently become the focus of research on biological invasions. However, the interaction between different symbionts and their consequences in host-plant invasion have been seldom explored. Here, we propose that vertically transmitted fungal endophytes could reduce the dependency of invasive grasses on mycorrhizal fungi allowing host establishment in those environments where the specific mutualist may be not present. Through analyzing published studies on nine grass species, we evaluated the effect of seed-borne Epichloë endophytes on the relationship of invasive and non-invasive grasses with arbuscular mycorrhizal fungi (AMF), a symbiosis known to be fundamental for plant fitness and invasion success. The endophyte effect on AMF colonization differed between invasive and non-invasive grasses, reducing mycorrhization only on invasive species but with no impact on their biomass. These results allowed us to propose that Epichloë endophytes could reduce the dependency of host plants on the mutualism with AMF, promoting host grass establishment and subsequent invasion. Simultaneous interactions with different types of mutualists may have profound effects on the host-plant fitness facilitating its range expansion. Our findings suggest that some specific mutualistic fungi such as epichloid endophytes facilitate host invasion by reducing the requirements of the benefits derived from other mutualisms.     

禾草内生真菌作为生防因子的潜力分析

早熟禾亚科多种禾草可与Neotyphodium内生真菌形成禾草-内生真菌共生体, 这种植物-微生物共生体性状较为稳定, 且在自然界中广泛存在。禾草-内生真菌共生体稳定的互利共生关系不但保证了内生真菌所需的全部营养物质, 而且共生体产生的次生代谢物又可显著提高宿主禾草对生物胁迫的抗逆性。众多研究表明, 内生真菌的侵染可显著提高宿主禾草对虫害、病害及伴生植物等多种生物胁迫的抗性。据不完全统计, 禾草内生真菌对蛛形纲、线虫纲、昆虫纲3个纲至少79个种的害虫表现出较明显的抗性, 对至少22个种的病原真菌表现出明显的抗性。尽管利用内生真菌进行禾草品种选育及其品质改良的技术日趋成熟, 但是内生真菌在不同宿主禾草之间高效的替代转化技术, 及其在宿主体内遗传的稳定性仍有待于进一步深入探索。研究者把禾草内生真菌作为生防手段, 在未来的应用过程中不应只考虑其与宿主禾草之间的共生特异性, 而应更全面地分析禾草-内生真菌-生态环境之间的相互关系, 让内生真菌更好地为人类服务。     

Endophytic fungus-vascular plant-insect interactions

Insect association with fungi has a long history. Theories dealing with the evolution of insect herbivory indicate that insects used microbes including fungi as their principal food materials before flowering plants evolved. Subtlety and the level of intricacy in the interactions between insects and fungi indicate symbiosis as the predominant ecological pattern. The nature of the symbiotic interaction that occurs between two organisms (the insect and the fungus), may be either mutualistic or parasitic, or between these two extremes. However, the triangular relationship involving three organisms, viz., an insect, a fungus, and a vascular plant is a relationship that is more complicated than what can be described as either mutualism or parasitism, and may represent facets of both. Recent research has revealed such a complex relationship in the vertically transmitted type-I endophytes living within agriculturally important grasses and the pestiferous insects that attack them. The intricacy of the association depends on the endophytic fungus-grass association and the insect present. Secondary compounds produced in the endophytic fungus-grass association can provide grasses with resistance to herbivores resulting in mutualistic relationship between the fungus and the plant that has negative consequences for herbivorous insects. The horizontally transmitted nongrass type-II endophytes are far less well studied and as such their ecological roles are not fully understood. This forum article explores the intricacy of dependence in such complex triangular relationships drawing from well-established examples from the fungi that live as endophytes in vascular plants and how they impact on the biology and evolution of free-living as well as concealed (e.g., gall-inducing, gall-inhabiting) insects. Recent developments with the inoculation of strains of type-I fungal endophytes into grasses and their commercialization are discussed, along with the possible roles the endophytic fungi play in the galls induced by the Cecidomyiidae (Diptera).     

内生真菌与禾本科植物之间的关系及其生态学意义

1　前　言内生真菌是一类生长在植物体内的真菌 ,至今已在许多高等植物中发现了内生真菌的存在 ,但以禾本科植物中尤为常见。这些真菌包括子囊菌纲、麦角菌科 (Ｃｌａｖｉｃｉｐｉｔａｃｅａｅ)、瘤座菌族 (Ｂａｌａｎ ｓｉｅａｅ)的许多种类 ,也包括与瘤座菌族关系密切的一些半知菌类 ,如Ａｃｒｅｍｏｎｉｕｍ[1 5] 。与菌根真菌的一个显著不同是 ,它们仅存在于植物地上部分的组织内而不是根的组织中。至少有 80个属、几百种禾本科植物被确认含内生真菌[1 6] ,但到目前为止 ,大量的研究都集中在两个有重要经济意义的植物种 ,即高羊茅 (Ｆｅ…     

Do the costs and benefits of fungal endophyte symbiosis vary with light availability?

? Here, we examined whether fungal endophytes modulated host plant responses to light availability. First, we conducted a literature review to evaluate whether natural frequencies of endophyte symbiosis in grasses from shaded habitats were higher than frequencies in grasses occupying more diverse light environments. Then, in a glasshouse experiment, we assessed how four levels of light and the presence of endophyte symbioses affected the growth of six grass species. ? In our literature survey, endophytes were more commonly present in grasses restricted to shaded habitats than in grasses from diverse light environments. ? In the glasshouse, endophyte symbioses did not mediate plant growth in response to light availability. However, in the host grass, Agrostis perennans, symbiotic plants produced 53% more inflorescences than nonsymbiotic plants at the highest level of shade. In addition, under high shade, symbiotic Poa autumnalis invested more in specific leaf area than symbiont-free plants. Finally, shade increased the density of the endophyte in leaf tissues across all six grass species. ? Our results highlight the potential for symbiosis to alter the plasticity of host physiological traits, demonstrate a novel benefit of endophyte symbiosis under shade stress for one host species, and show a positive association between shade-restricted grass species and fungal endophytes.     

Host selection by a mycophagous fly and its impact on fly survival

Epichloë fungi interact with Botanophila flies in a co-evolved mutualism that appears to be analogous to angiosperm pollinator-parasite systems. Female flies act as vectors of fungal spores as they lay eggs on the fungi which grow within and on grass stems. To understand selective pressures operating on choice of host fungi by flies, we monitored the interaction of two species of Epichloë with Botanophila at two sites in southern England. At one site (Ashurst), the hosts Epichloë clarkii and Epichloë baconii co-occurred, while at a second site (Farm Gate), only E. clarkii was present. Flies ovipositing on E. baconii showed preferences for large, somewhat isolated fungi occurring within small clumps of grass. Flies ovipositing on E. clarkii at the Ashurst site did not select hosts based on any physical features of fungi that we measured. However, flies ovipositing on E. clarkii at the Farm Gate site tended to prefer large fungi that occurred within small clumps of grass. Fungal characteristics preferred by the flies showed no correlation with egg/larval survivorship. Insect survival on E. clarkii varied between sites and was related to differences in egg dispersion among fungi at the two sites. The absence of an alternate host ( E. baconii ) was associated with greater aggregation of eggs on E. clarkii at the Farm Gate site and these insects experienced higher levels of mortality at this compared to the other site.     

Intraspecific competition of endophyte infected vs uninfected plants of two woodland grass species

Grass endophytes (Clavicipitaceae, Ascomycota) are generally considered to be mutualists which increase the host's fitness. Infected plants are often more persistent and competitive than uninfected plants, influencing population dynamics and plant community diversity. However, most of this empirical evidence is based on studies focusing on agronomically important grass species such as tall fescue or perennial ryegrass and their implications for livestock and man-made habitats. Recent studies indicate that endophyte-plant associations may be more variable, ranging from parasitic to mutualistic. In the present study, we investigated the influence of endophyte infection on two wild woodland grasses, which are naturally infected with distinct fungal endophytes: Brachypodium sylvaticum with Epichloë sylvatica and Bromus benekenii with Epichloë bromicola . An intraspecific competition experiment was conducted over two growing seasons in the greenhouse and in an experimental garden. At first harvest (after 12 weeks growing), endophyte infection had a significant negative effect on above ground dry matter yield (DMY) of B. sylvaticum , but a significant positive effect on DMY of Br. benekenii under competition. The same differential effects on DMY and on total seed number were also observed at final harvest (after 62 weeks growing). Results from Br. benekenii were consistent with our hypothesis of increased competitive abilities of infected plants in nature which could explain the high infection rate observed in natural populations. In contrast, this explanation does not hold true for B. sylvaticum , and other factors such as increased herbivore and pathogen resistance together with frequent horizontal transmission may be responsible for the very high incidence of this association in nature. Our results confirm previous predictions that beneficial effects of endophyte infection in wild grasses can vary for different grass species, even in comparable habitats.     

Chromosome-level genomes provide insights into genome evolution,organization and size in Epichloe fungi

Epichloe fungi are endophytes of cool season grasses, both wild species and commercial cultivars, where they may exhibit mutualistic or pathogenic lifestyles. The Epichloe-grass symbiosis is of great interest to agricultural research for the fungal bioprotective properties conferred to host grasses but also serves as an ideal system to study the evolution of fungal plant-pathogens in natural environments. Here, we assembled and annotated gapless chromosome-level genomes of two pathogenic Epichloe sibling species. Both genomes have a bipartite genome organization, with blocks of highly syntenic gene-rich regions separated by blocks of AT-rich DNA. The AT-rich regions show an extensive signature of RIP (repeat-induced point mutation) and the expansion of this compartment accounts for the large difference in genome size between the two species. This study reveals how the rapid evolution of repeat structure can drive divergence between closely related taxa and highlights the evolutionary role of dynamic compartments in fungal genomes.     

Conspicuous epiphytic growth of an interspecific hybrid Neotyphodium sp. endophyte on distorted host inflorescences

Selected Neotyphodium sp. endophytes are now commonly used to enhance pasture persistence and livestock productivity, with seed of perennial ryegrass and tall fescue cultivars with these selected endophytes being commercially available. In a large population of perennial ryegrass plants infected with a Neotyphodium sp. endophyte that was being grown for seed production a small percentage of inflorescences were distorted and covered with a conspicuous white mycelial growth. Within individual plants only a small number of inflorescences were affected and the amount of distortion differed between affected inflorescences. This Neotyphodium sp. is an interspecific hybrid of Epichloë typhina and Neotyphodium. lolii and like nearly all other Neotyphodium spp is symptomless in host grasses. The fungus isolated from distorted inflorescences had colonies that were identical to those isolated from symptomless inflorescences and these were characteristic of this Neotyphodium sp. This is the first report of distorted inflorescences covered with epiphytic hyphal growth on host grasses infected with an interspecific hybrid Neotyphodium sp.     

A test of host specialization by insect vectors as a mechanism for reproductive isolation among entomophilous fungal species

Epichloë species are self incompatible (heterothallic) fungi that must be fertilized by spermatia from individuals of opposite mating type for successful sexual reproduction to occur. Female flies of the genus Botanophila act as vectors of the fungi by ingesting and defecating spermatia (gametes) onto fungal stromata (fruiting bodies) after oviposition. Larvae feed and develop on the stromata and thus maintain a symbiotic relationship with Epichloë fungi. We hypothesized that sole dependence on fertilized stromata as a food source would promote specialization by flies to single compatible host species and that this specialization would promote reproductive isolation among Epichloë species. Analysis of progeny of ascospores from experimental field plots in Zurich, Switzerland, indicated prevalence of specific matings between stromata of the same host, and thus was consistent with the hypothesis that flies are species-specific in their visitation behaviour. Genetic analyses of spermatia contained in the faeces of individual flies also gave some support for this hypothesis. We recovered spermatia of 4 different Epichloë species from fly faeces. Comparison of spermatia found in fly faeces to those available from stromata showed flies avoided Epichloë clarkii and may have preferred Epichloë typhina . Interestingly, these are the only two Epichloë species known to be interfertile with one another. Individual flies tended to carry spermatia predominantly from one fungal species. Thus, flies may adopt a type of "majoring" and "minoring" behaviour when visiting fungi. Yet, Botanophila flies are not monolectic and often visited all hosts that were available within screened cages. In addition to any reproductive isolation flies may provide to some fungal species, differences in competitiveness among spermatia of different species deposited on the same stroma may favor intraspecific matings.     

Mollusc herbivory influenced by endophytic clavicipitaceous fungal infections in grasses

Clavicipitaceous fungi of the genus Neotyphodium occur widely as mutualistic, systemic, seed‐borne infections in festucoid grasses. Grass infection by these fungi is associated with the presence of a range of secondary metabolites (SM), several of which have been demonstrated to confer to the plant resistance against herbivorous vertebrates and insects. An initial experiment demonstrated that endophytic infection by Neotyphodium can influence the utilisation of grasses by Deroceras, with feeding preferences and impact on plant yields affected differentially by endophytes with different SM profiles. The role of Neotyphodium SM in feeding preferences of Deroceras slugs were then evaluated in artificial diets. Among the indole diterpenoids tested, lolitrem B was demonstrated to reduce feeding, while diets containing paxilline, lolitriol, α‐paxitriol and β‐paxitriol tended to be preferred over that of untreated diet. The pyrrolopyrazine alkaloid peramine had no effect. Among the ergopeptine alkaloids tested in the diets, ergotamine and ergovaline were demonstrated to be phagostimulatory. These results with artificial diets were generally consistent with Deroceras reticulatum preferences among plants of known Neotyphodium endophyte strain and SM profile. Deroceras slugs obtained from sites containing contrasting frequencies of Neotyphodium‐infected grasses, exhibited differential responses to Neotyphodium SM incorporated into artificial diet. This study demonstrates that infection of grasses by different isolates of Neotyphodium endophytes differentially influence herbivory by molluscs, reflecting their SM profile. These results offer an explanation for variable acceptability of grasses to molluscs and their importance in the diet of molluscs in the field reported in previous studies in both natural and agricultural systems. Neotyphodium endophytes potentially offer novel approaches to management of mollusc pests in agricultural gramineous crops.     

Fungal Symbionts Alter Plant Drought Response

Grassland productivity is often primarily limited by water availability, and therefore, grasslands may be especially sensitive to climate change. Fungal symbionts can mediate plant drought response by enhancing drought tolerance and avoidance, but these effects have not been quantified across grass species. We performed a factorial meta-analysis of previously published studies to determine how arbuscular mycorrhizal (AM) fungi and endophytic fungal symbionts affect growth of grasses under drought. We then examined how the effect of fungal symbionts on plant growth was influenced by biotic (plant photosynthetic pathway) and abiotic (level of drought) factors. We also measured the phylogenetic signal of fungal symbionts on grass growth under control and drought conditions. Under drought conditions, grasses colonized by AM fungi grew larger than those without mycorrhizal symbionts. The increased growth of grasses conferred from fungal symbionts was greatest at the lowest soil moisture levels. Furthermore, under both drought and control conditions, C3 grasses colonized by AM fungi grew larger than C3 grasses without symbionts, but the biomass of C4 grasses was not affected by AM fungi. Endophytes did not increase plant biomass overall under any treatment. However, there was a phylogenetically conserved increase in plant biomass in grasses colonized by endophytes. Grasses and their fungal symbionts seem to interact within a context-dependent symbiosis, varying with biotic and abiotic conditions. Because plant–fungal symbioses significantly alter plant drought response, including these responses could improve our ability to predict grassland functioning under global change.     

Endophytic fungi: resource for gibberellins and crop abiotic stress resistance

The beneficial effects of endophytes on plant growth are important for agricultural ecosystems because they reduce the need for fertilizers and decrease soil and water pollution while compensating for environmental perturbations. Endophytic fungi are a novel source of bioactive secondary metabolites; moreover, recently they have been found to produce physiologically active gibberellins as well. The symbiosis of gibberellins producing endophytic fungi with crops can be a promising strategy to overcome the adverse effects of abiotic stresses. The association of such endophytes has not only increased plant biomass but also ameliorated plant-growth during extreme environmental conditions. Endophytic fungi represent a trove of unexplored biodiversity and a frequently overlooked component of crop ecology. The present review describes the role of gibberellins producing endophytic fungi, suggests putative mechanisms involved in plant endophyte stress interactions and discusses future prospects in this field.