Fungal endosymbionts of plants reduce lifespan of an aphid secondary parasitoid and influence host selection

Complex biotic interactions shape ecological communities of plants, herbivores and their natural enemies. In studies of multi-trophic interactions, the presence of small, invisible micro-organisms associated with plants and those of a fourth above-ground trophic level have often been neglected. Incorporating these neglected factors improves our understanding of the processes within a multi-trophic network. Here, we ask whether the presence of a fungal endosymbiont, which alters plant quality by producing herbivore-toxic substances, trickles up the food chain and affects the performance and host-selection behaviour of aphid secondary parasitoids. We simultaneously offered hosts from endophyte-free and endophyte-infected environments to secondary parasitoids. Older and more experienced parasitoid females discriminated against hosts from the endophyte-infected environment. Developing in lower quality hosts from the endophyte-infected environment reduced the lifespan of secondary parasitoids. This indicates that aphid secondary parasitoids can perceive the disadvantage for their developing offspring in parasitoids from the endophyte environment and can learn to discriminate against them. In the field, this discrimination ability may shift the success of primary parasitoids to endophyte-infected plants, which co-occur with endophyte-free plants. Ultimately, the control of aphids depends on complex interactions between primary and secondary parasitoids and their relative sensitivity to endophytic fungi.     

Apparent competition between two species of aphid via the fungal pathogen Erynia neoaphidis and its interaction with the aphid parasitoid Aphidius ervi

Abstract 1. Motivated by a community study on aphids and their fungal pathogens, three hypotheses were tested experimentally to investigate the influence of the fungal pathogen, Erynia neoaphidis Remaudière and Hennebert, on aphid population and community ecology.
2. Field experiments were performed in 2 years to test whether two susceptible aphid species on different host plants might interact through the shared fungal pathogen. No strong pathogen-mediated indirect interactions (apparent competition) between populations of pea aphid Acyrthosiphon pisum Harris and nettle aphid Microlophium carnosum Buckton were detected.
3. In the first of the field experiments, pea aphids exposed to the fungus showed a weak tendency to produce more winged dispersal morphs than control populations not exposed to the fungus. In a laboratory test, however, no support was found for the hypothesis that the presence of volatiles from fungus-infected cadavers promotes production of winged offspring.
4. The response of the pea aphid parasitoid Aphidius ervi Halliday to colonies containing hosts infected 1 and 3 days previously was assessed. Wasps initiated fewer attacks on 1-day-old infected colonies than on healthy colonies, with the numbers on 3-day-old fungus-infected colonies intermediate.     

Predator-induced morphological shift in the pea aphid

Aphids exhibit a polymorphism whereby individual aphids are either winged or unwinged. The winged dispersal morph is mainly responsible for the colonization of new plants and, in many species, is produced in response to adverse environmental conditions. Aphids are attacked by a wide range of specialized predators and predation has been shown to strongly influence the growth and persistence of aphid colonies. In two experiments, we reared two clones of pea aphid (Acyrthosiphon pisum) in the presence and absence of predatory ladybirds (Coccinella septempunctata or Adalia bipunctata). In both experiments, the presence of a predator enhanced the proportion of winged morphs among the offspring produced by the aphids. The aphid clones differed in their reaction to the presence of a ladybird, suggesting the presence of genetic variation for this trait. A treatment that simulated disturbance caused by predators did not enhance winged offspring production. The experiments indicate that aphids respond to the presence of a predator by producing the dispersal morph which can escape by flight to colonize other plants. In contrast to previous examples of predator-induced defence this shift in prey morphology does not lead to better protection against predator attack, but enables aphids to leave plants when mortality risks are high.     

Connecting plant–microbial interactions above and belowground: a fungal endophyte affects decomposition

Mutualisms can strongly affect the structure of communities, but their influence on ecosystem processes is not well resolved. Here we show that a plant–microbial mutualism affects the rate of leaf litter decomposition using the widespread interaction between tall fescue grass (Lolium arundinaceum) and the fungal endophyte Neotyphodium coenophialum. In grasses, fungal endophytes live symbiotically in the aboveground tissues, where the fungi gain protection and nutrients from their host and often protect host plants from biotic and abiotic stress. In a field experiment, decomposition rate depended on a complex interaction between the litter source (collected from endophyte-infected or endophyte-free plots), the decomposition microenvironment (endophyte-infected or endophyte-free plots), and the presence of mesoinvertebrates (manipulated by the mesh size of litter bags). Over all treatments, decomposition was slower for endophyte-infected fescue litter than for endophyte-free litter. When mesoinvertebrates were excluded using fine mesh and litter was placed in a microenvironment with the endophyte, the difference between endophyte-infected and endophyte-free litter was strongest. In the presence of mesoinvertebrates, endophyte-infected litter decomposed faster in microenvironments with the endophyte than in microenvironments lacking the endophyte, suggesting that plots differ in the detritivore assemblage. Indeed, the presence of the endophyte in plots shifted the composition of Collembola, with more Hypogastruridae in the presence of the endophyte and more Isotomidae in endophyte-free plots. In a separate outdoor pot experiment, we did not find strong effects of the litter source or the soil microbial/microinvertebrate community on decomposition, which may reflect differences between pot and field conditions or other differences in methodology. Our work is among the first to demonstrate an effect of plant–endophyte mutualisms on ecosystem processes under field conditions.     

Endophytic fungus decreases plant virus infections in meadow ryegrass (Lolium pratense)

We studied the effects of fungal endophyte infection of meadow ryegrass (Lolium pratense=Festuca pratensis) on the frequency of the barley yellow dwarf virus (BYDV). The virus is transferred by aphids, which may be deterred by endophyte-origin alkaloids within the plant. In our experiment, we released viruliferous aphid vectors on endophyte-infected and endophyte-free plants in a common garden. The number of aphids and the percentage of BYDV infections were lower in endophyte-infected plants compared to endophyte-free plants, indicating that endophyte infection may protect meadow ryegrass from BYDV infections.     

Effects of Neotyphodium fungi on Lolium multiflorum seed germination in relation to water availability

BACKGROUND AND AIMS: Temperate endophyte-infected (Neotyphodium sp.) grasses have been shown to exhibit an ecological advantage over endophyte-uninfected grasses under abiotic stressful conditions. It is predicted that endophyte-infected plant populations will display higher rates of germination and proportion of germinated seeds under limiting water conditions. METHODS: The hydrotime regression model was used to describe the effect of Neotyphodium endophyte on seed germination of Lolium multiflorum at different water potentials. Additionally, seed mortality after water stress exposure was estimated in endophyte-infected and -uninfected seeds. KEY RESULTS: Endophyte infection inhibited seed germination at all water potentials. The hydrotime model described satisfactorily the germination responses, and revealed that endophyte-free seeds exhibited higher rates of and final percentage germination, probably due to a lower base water potential compared with endophyte-infected seeds. However, Neotyphodium endophyte conferred a higher rate of survival in those seeds that remained ungerminated when exposed to highly water stress conditions. CONCLUSIONS: Changes produced by Neotyphodium endophyte in L. multiflorum seeds might affect fitness in particular ecological scenarios. For example, the presence of the endophyte may curtail seed germination when water is limiting, reducing the risk of seedling death. Conversely, endophyte-free seeds would display an enhanced germination, ensuring a more rapid seedling establishment if later water conditions do not restrict plant growth.     

Bottom–up regulates top–down: the effects of hybridization of grass endophytes on an aphid herbivore and its generalist predator

The ecological consequences of hybridization of microbial symbionts are largely unknown. We tested the hypothesis that hybridization of microbial symbionts of plants can negatively affect performance of herbivores and their natural enemies. In addition, we studied the effects of hybridization of these symbionts on feeding preference of herbivores and their natural enemies. We used Arizona fescue as the host‐plant, Neotyphodium endophytes as symbionts, the bird cherry–oat aphid as the herbivore and the pink spotted ladybird beetle as the predator in controlled experiments. Neither endophyte infection (infected or not infected) nor hybrid status (hybrid and non‐hybrid infection) affected aphid reproduction, proportion of winged forms in the aphid populations, aphid host‐plant preference or body mass of the ladybirds. However, development of ladybird larvae was delayed when fed with aphids grown on hybrid (H+) endophyte infected grasses compared to larvae fed with aphids from non‐hybrid (NH+) infected grasses, non‐hybrid, endophyte‐removed grasses (NH?) and hybrid, endophyte‐removed (H?) grasses. Furthermore, adult beetles were more likely to choose all other types of grasses harboring aphids rather than H+ infected grasses. In addition, development of ladybirds was delayed when fed with aphids from naturally uninfected (E?) grasses compared to ladybirds that were fed with aphids from NH+ and NH? grasses. Our results suggest that hybridization of microbial symbionts may negatively affect generalist predators such as the pink spotted ladybird and protect herbivores like the bird cherry–oat aphids from predation even though the direct effects on herbivores are not evident.     

Competition and dispersal in the pea aphid: clonal variation and correlations across traits

Abstract.  1. The presence of an across-species trade-off between dispersal ability and competitive ability has been proposed as a mechanism that facilitates coexistence. It is not clear if a similar trade-off exists within species. Such a trade-off would constrain the evolution of either trait and, given appropriate selection pressures, promote local adaptation in these traits.
2. This study found substantial levels of heritable variation in competitive ability of the pea aphid, Acyrthosiphon pisum Harris (Homoptera: Aphididae), measured in terms of relative survival when reared with a single clone of the vetch aphid, Megoura viciae Buckton (Homoptera: Aphididae).
3. Pea aphids can move to new patches by either flying (longer distance dispersal) or walking (local dispersal) from plant to plant. There was considerable clonal variation in dispersal ability, measured in terms of the proportion of winged offspring produced, and ability to survive away from their host plant.
4. Winged individuals showed longer off-plant survival times than wingless forms of the same pea aphid clone.
5. There was no evidence of a relationship between clonal competitive ability and either measure of dispersal ability, although the power of the test is limited by the number of pea aphid clones used in the trial.
6. However, there was a positive correlation between clonal fecundity and the proportion of winged offspring produced. Although speculative, it is suggested that clones that are more likely to either overwhelm their host plant or attract higher numbers of natural enemies as a result of having higher fecundity are more likely to produce winged morphs.     

Fungal endophytes of wild barley and their effects on Diuraphis noxia population development

Laboratory experiments were conducted to compare the expression of Diuraphis noxia (Mordvilko) (Homoptera: Aphididae) resistance in four plant introduction (PI) lines of wild barley (Hordeum) infected with different species or strains of endophytic fungi (tribe Balansieae, family Clavicipitaceae, Neotyphodium gen. nov. [formerly Acremonium]). Aphid densities were significantly lower on endophyte-infected plants of PI 314696 (H. bogdanii Wilensky) and PI 440420 (H. brevisubulatum subsp. violaceum (Boissier & Hohenacker)), compared with densities on endophyte-free plants of both PI lines in population growth experiments. This endophyte- associated resistance was the result of antibiosis effects or starvation. In other experiments, endophyte-free plants of PI 269406 and PI 440413 (H. bogdanii) were not superior to endophyte-infected conspecifics as host plants of D. noxia. Our results demonstrate the influence of host plant species/genotype and endophyte species/strain on expression of aphid resistance, provide an explanation of the high levels of D. noxia resistance in PI 314696 and PI 440420 previously reported in the literature, and underscore the potential importance of endophytic fungi in conferring insect resistance in wild barley.     

Natural enemies act faster than endophytic fungi in population control of cereal aphids

1. Fast-growing populations of phytophagous insects can be limited by the presence of natural enemies and by alkaloids that are produced by symbiotic associations of many temperate grass species with endophytic fungi. It is unclear if and how acquired plant defences derived from endophytic fungi interact with natural enemies to affect phytophagous insect populations. 2. To assess the relative importance of endophytic fungi compared to that of natural enemies on the population dynamics of phytophagous insects, we carried out a fully factorial field experiment, in which the presence of natural enemies and the presence of endophytic fungi were manipulated simultaneously. Target colonies of aphids were monitored for 8 weeks starting from their natural appearance in the field to the end of the aphid season. 3. We show that on Lolium perenne increased natural enemy densities reduced the individual numbers of two common cereal aphids, Rhopalosiphum padi and Metopolophium festucae. 4. The presence of the endophytic fungi Neotyphodium lolii reduced the number of M. festucae but did not affect the number of R. padi. The reduction in R. padi numbers by predators and parasitoids was not influenced by the presence of endophytes. For adult M. festucae, however, the negative effects of natural enemies were significant only in the absence of endophytes. 5. Over the duration of the experiment, the effect of natural enemies on aphid colony growth was much stronger than the effect of the endophytic fungi N. lolii, presumably because predator and parasitoid action on aphid colonies is much faster than any effects of endophytes. 6. Our results demonstrate that with simultaneous action of acquired endosymbionts and natural enemies, both factors can control aphid colony growth but they generally act independently of each other.     

Alarm pheromone emission by pea aphid, Acyrthosiphon pisum, clones under predation by lacewing larvae

Genetic variation in anti-predator traits has been shown for a variety of species. Aphid alarm pheromone, ( E )-β-farnesene, is released by attacked aphids and causes a variety of behavioral defense reactions in the signal receivers. In pea aphids, Acyrthosiphon pisum Harris (Homoptera: Aphididae), ( E )-β-farnesene mediates the production of winged offspring in the presence of natural enemies. While variation in the propensity for pea aphids to produce winged offspring is well-documented, little quantitative information is available about clonal differences in ( E )-β-farnesene emission or the amount of alarm pheromone released in aphid colonies. We tested the wing induction response of four clones when attacked by a predatory lacewing larva, Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae), and found that three of the four clones increased the proportion of winged offspring under predator attack. We then investigated the emission of aphid alarm pheromone of these clones of pea aphid under attack. Alarm pheromone emission in aphid colonies of initially 25 adults varied from 81.2 to 10 851.0 ng per aphid colony over 24 h. There were no differences between clones in total emission or in emission per consumption event. These results show that there is substantial variability in alarm pheromone emission within clones and that the propensity to produce winged offspring in some clones is not a simple function of the propensity of alarm pheromone production in these clones.     

Influence of endophyte infection, plant age and harvest interval on Rhopalosiphum padi survival and its relation to quantity of N-formyl and N-acetyl loline in tall fescue

Rhopalosiphum padi L. (Homoptera: Aphididae) is sensitive to loline alkaloids present in tall fescue, Festuca arundinacea Shreb., infected with the endophytic fungus, Acremonium coenophialum Morgan-Jones & Gams. Aphid survival was higher on endophyte-free plants regardless of plant age after germination or age of regrowth tissue after clipping. Survival of aphids on endophyte-infected grass was lower on young tissue but increased as plants aged, although it never reached the same level on endophyte-free plants. Both N-formyl and N-acetyl loline increased as uncut or regrowth tissue aged; however, this was influenced by the age of the plant at the initial cut and the clipping frequency. Although even small amounts of loline cause high aphid mortality, the aphids are able to survive on endophyte-infected plants if the tillers have senescing leaves which contain lower amounts of loline. Preference for senescing leaves may help R. padi avoid plant parts containing high amounts of toxic allelochemicals, thus contributing to higher numbers of aphids on older, endophyte-infected plants.     

Fungal endosymbionts affect aphid population size by reduction of adult life span and fecundity

The symbiosis between grasses and endophytic fungi is a common phenomenon and can affect herbivore performance through acquired, chemical plant defence by fungal alkaloids. In laboratory experiments, two species of common grass aphids, Rhopalosiphum padi and Metopolophium dirhodum were tested, in a population experiment (on four plant cultivars) and individually (on one plant cultivar) for the effects of the endophyte, Neotyphodium lolii, that forms symbiotic associations with perennial ryegrass Lolium perenne. In the population experiment that lasted for four aphid generations both aphid species showed decreased population sizes when feeding on each of the four endophyte-infected cultivars. Individuals of R. padi tested individually showed reduced adult life span and fecundity when feeding on infected plants. Individuals of M. dirhodum showed no response in any of the traits measured. This suggests that R. padi individuals are more sensitive to endophyte infection than M. dirhodum individuals. However, all infected grass cultivars reduced population sizes of both aphid species over four generations. Therefore, fungal endophytes can reduce populations of aphid herbivores independent of plant cultivars.     

Alarm pheromone mediates production of winged dispersal morphs in aphids

The aphid alarm pheromone ( E )- β -farnesene (EBF) is the major example of defence communication in the insect world. Released when aphids are attacked by predators such as ladybirds or lacewing larvae, aphid alarm pheromone causes behavioural reactions such as walking or dropping off the host plant. In this paper, we show that the exposure to alarm pheromone also induces aphids to give birth to winged dispersal morphs that leave their host plants. We first demonstrate that the alarm pheromone is the only volatile compound emitted from aphid colonies under predator attack and that emission is proportional to predator activity. We then show that artificial alarm pheromone induces groups of aphids but not single individuals to produce a higher proportion of winged morphs among their offspring. Furthermore, aphids react more strongly to the frequency of pheromone release than the amount of pheromone delivered. We suggest that EBF leads to a 'pseudo crowding' effect whereby alarm pheromone perception causes increased walking behaviour in aphids resulting in an increase in the number of physical contacts between individuals, similar to what happens when aphids are crowded. As many plants also produce EBF, our finding suggests that aphids could be manipulated by plants into leaving their hosts, but they also show that the context-dependence of EBF-induced wing formation may hinder such an exploitation of intraspecific signalling by plants.     

The interplay between density- and trait-mediated effects in predator-prey interactions: a case study in aphid wing polymorphism

Natural enemies not only influence prey density but they can also cause the modification of traits in their victims. While such non-lethal effects can be very important for the dynamic and structure of prey populations, little is known about their interaction with the density-mediated effects of natural enemies. We investigated the relationship between predation rate, prey density and trait modification in two aphid-aphid predator interactions. Pea aphids (Acyrthosiphon pisum, Harris) have been shown to produce winged dispersal morphs in response to the presence of ladybirds or parasitoid natural enemies. This trait modification influences the ability of aphids to disperse and to colonise new habitats, and hence has a bearing on the population dynamics of the prey. In two experiments we examined wing induction in pea aphids as a function of the rate of predation when hoverfly larvae (Episyrphus balteatus) and lacewing larvae (Chrysoperla carnea) were allowed to forage in pea aphid colonies. Both hoverfly and lacewing larvae caused a significant increase in the percentage of winged morphs among offspring compared to control treatments, emphasising that wing induction in the presence of natural enemies is a general response in pea aphids. The percentage of winged offspring was, however, dependent on the rate of predation, with a small effect of predation on aphid wing induction at very high and very low predation rates, and a strong response of aphids at medium predation rates. Aphid wing induction was influenced by the interplay between predation rate and the resultant prey density. Our results suggests that density-mediated and trait-mediated effects of natural enemies are closely connected to each other and jointly determine the effect of natural enemies on prey population dynamics.     

Influence of the aphid pathogen Pandora neoaphidis on the foraging behaviour of the aphid parasitoid Aphidius ervi

Abstract.  1. The parasitoid Aphidius ervi and the entomopathogenic fungus Pandora neoaphidis both require successful invasion of an aphid host to complete their life cycle. A shorter developmental period allows P. neoaphidis to out-compete A. ervi. Aphidius ervi may reduce this fitness cost by avoiding aphid colonies containing P. neoaphidis . Here the response of A. ervi towards P. neoaphidis was assessed using sequential experiments designed to replicate different stages of parasitoid foraging behaviour.
2. Entry rate experiments showed that A. ervi entered aphid colonies containing P. neoaphidis -sporulating cadavers and that there was no significant difference in the attraction of A. ervi to aphid-damaged Vicia faba plants containing either healthy Acyrthosiphon pisum or P. neoaphidis -sporulating cadavers.
3. Observational behavioural experiments indicated that the presence of P. neoaphidis did not affect the search time or total foraging time of A. ervi on V. faba plants infested with either healthy A. pisum or P. neoaphidis -sporulating cadavers.
4. In Petri dish bioassays using aphids infected with P. neoaphidis over a period of 120 h, A. ervi showed no difference in attack rate against uninfected aphids or living aphids infected with P. neoaphidis for 1, 24, 48, 72, or 96 h. However, sporulating cadavers (120 h infection) were not attacked.
5.  Aphidius ervi appears only able to detect the presence of P. neoaphidis once the host is dead and sporulation has started. The fitness of A. ervi may therefore be severely reduced when foraging in P. neoaphidis -infected aphid colonies.     

Variable effects of endophytic fungus on seedling establishment of fine fescues

Seedborne systemic endophytic fungi of grasses are thought to be plant mutualists, because they have been shown to improve their host’s resistance against biotic and abiotic stresses. The interactions in plant–endophyte associations vary from mutualistic to parasitic with environmental conditions and the genotypes of interacting species. The possible pros and cons of endophytic fungi are expected to be most evident during the seedling establishment, where host fitness is most directly affected. If this holds true, endophytes may play a focal role in local adaptation of hosts to different environments. We examined if endophyte-infected and uninfected seeds and seedlings of two native grass species, Festuca rubra and F. ovina, differ in seed germination and seedling growth rates under greenhouse conditions. The germination of F. rubra seeds was also studied in the field. This is the first time that the effects of Epichloë endophyte on seedling establishment of fine fescues from natural populations have been experimentally evaluated. Mother plant (seed family) had a marked effect on many response variables in both grass species. Length and mean biomass of tillers of endophyte-infected (E+) F. ovina seedlings were lower, but root:shoot ratios were higher than in endophyte-free (E?) seedlings. In F. rubra, the effects of the endophyte were dependent on the habitat where the seeds were collected. The E+ seeds from river banks germinated faster than E+ seeds from meadows, and E+ seedlings from the river banks produced fewer but taller and heavier tillers than the other seedlings. Our data suggest that the effects of the endophyte infection on the seedling stage of fine fescues are dependent the species of grass, host genetic background and mother plant habitat. The germination strategy and growth form of E+ red fescue seedlings from river banks may be beneficial to surviving in the harsh conditions of that habitat.     

The importance of antennae for pea aphid wing induction in the presence of natural enemies

The pea aphid Acyrthosiphon pisum Harris has been shown to produce an increasing proportion of winged morphs among its offspring when exposed to natural enemies, in particular hoverfly larvae, lacewing larvae, adult and larval ladybirds and aphidiid parasitoids. While these results suggest that wing induction in the presence of predators and parasitoids is a general response of the pea aphid, the cues and mechanisms underlying this response are still unclear. Tactile stimuli and the perception of chemical signals as well as visual signals are candidates for suitable cues in the presence of natural enemies. In this paper the hypothesis that the aphids' antennae are crucial for the wing induction in the presence of natural enemies is tested. Antennae of pea aphids were ablated and morph production was scored when aphids were reared either in the presence or the absence of predatory lacewing larvae over a six-day period. Ablation of antennae resulted in a drastic drop in the proportion of winged morphs among the offspring, both in the presence and the absence of a predator whereas predator presence increased wing induction in aphids with intact antennae, as reported in previous experiments. The results show that antennae are necessary for wing induction in the presence of natural enemies. Critical re-examination of early work on the importance of aphid antennae and tactile stimuli for wing induction suggests that a combination of tactile and chemical cues is likely to be involved not only in predator-induced wing formation but also for wing induction in response to factors such as crowding in the aphid colony.     

Multiple Cues for Winged Morph Production in an Aphid Metacommunity

Environmental factors can lead individuals down different developmental pathways giving rise to distinct phenotypes (phenotypic plasticity). The production of winged or unwinged morphs in aphids is an example of two alternative developmental pathways. Dispersal is paramount in aphids that often have a metapopulation structure, where local subpopulations frequently go extinct, such as the specialized aphids on tansy (Tanacetum vulgare). We conducted various experiments to further understand the cues involved in the production of winged dispersal morphs by the two dominant species of the tansy aphid metacommunity, Metopeurum fuscoviride and Macrosiphoniella tanacetaria. We found that the ant-tended M. fuscoviride produced winged individuals predominantly at the beginning of the season while the untended M. tanacetaria produced winged individuals throughout the season. Winged mothers of both species produced winged offspring, although in both species winged offspring were mainly produced by unwinged females. Crowding and the presence of predators, effects already known to influence wing production in other aphid species, increased the percentage of winged offspring in M. tanacetaria, but not in M. fuscoviride. We find there are also other factors (i.e. temporal effects) inducing the production of winged offspring for natural aphid populations. Our results show that the responses of each aphid species are due to multiple wing induction cues.     

Dynamics of Neotyphodium endophyte infection in ageing seed pools: incidence of differential viability loss of endophyte,infected seed and non-infected seed

Symbiotic associations between grasses and vertically transmitted endophytic fungi are widespread in nature. Within grass populations, changes in the frequency of infected plants are driven by influence of the endophyte on the fitness of their hosts and by the efficiency of endophyte transmission from parent plants to their offspring. During the seed stage, the endophyte might influence the fitness of its host by affecting the rate of seed viability loss, whereas the efficiency of endophyte transmission is affected by losses of viability of the fungus within viable seeds. We assessed the viability losses of Lolium multiflorum seeds with high and low level of infection of the endophyte Neotyphodium occultans, as well as the loss of viability of the fungus itself, under accelerated seed ageing and under field conditions. Starting with high endophyte-infected accessions of L. multiflorum, we produced their low endophyte-infected counterparts by treating seeds with a fungicide, and subsequently multiplying seeds in adjacent plots allowing pollen exchange. In our accelerated ageing experiments, which included three accessions, high endophyte-infected seeds lost viability significantly faster than their low endophyte-infected counterpart, for only one accession. High endophyte-infected seeds of this particular accession absorbed more water than low endophyte-infected seeds. In contrast, the endophyte lost viability within live seeds of all three accessions, as the proportions of viable seeds producing infected seedlings decreased over time. In our field experiment, which included only one accession, high endophyte-infected seed lost viability significantly but only slightly faster than low endophyte-infected seed. In contrast, the loss of viability of the endophyte was substantial as the proportions of viable seeds producing infected seedlings decreased greatly over time. Moving the seeds from the air to the soil surface (simulating seed dispersion off the spikes) decreased substantially the rate of seed viability loss, but increased the rate of endophyte viability loss. Our experiments suggest that, in ageing seed pools, endophyte viability loss and differential seed mortality determine decreases in the proportions of endophyte-infected seeds in L. multiflorum. Endophyte viability loss within live seeds contributes substantially more to infection frequency changes than differential viability losses of infected and non-infected seeds.