Endophyte-mediated Resistance Research Articles

Whole-Genome Sequence Resource of Fusarium oxysporum Strain TH15, a Plant Growth Promoting Endophytic Fungus Isolated from Tetrastigma hemsleyanum

Whole-Genome Sequence Resource of <i>Fusarium oxysporum</i> Strain TH15, a Plant Growth Promoting Endophytic Fungus Isolated from <i>Tetrastigma hemsleyanum</i>

Pattern-triggered immunity restricts host colonization by endophytic fusaria, but does not affect endophyte-mediated resistance.

Fusarium oxysporum (Fo) is best known as a host‐specific vascular pathogen causing major crop losses. Most Fo strains, however, are root endophytes potentially conferring endophyte‐mediated resistance (EMR). EMR is a mechanistically poorly understood root‐specific induced resistance response induced by endophytic or nonhost pathogenic Fo strains. Like other types of induced immunity, such as systemic acquired resistance or induced systemic resistance, EMR has been proposed to rely on the activation of the pattern‐triggered immunity (PTI) system of the plant. PTI is activated upon recognition of conserved microbe‐associated molecular patterns (MAMPs) of invading microbes. Here, we investigated the role of PTI in controlling host colonization by Fo endophytes and their ability to induce EMR to the tomato pathogen Fo f. sp. lycopersici (Fol). Transgenic tomato and Arabidopsis plants expressing the Fo effector gene Avr2 are hypersusceptible to bacterial and fungal infection. Here we show that these plants are PTI‐compromised and are nonresponsive to bacterial‐ (flg22) and fungal‐ (chitosan) MAMPs. We challenged the PTI‐compromised tomato mutants with the EMR‐conferring Fo endophyte Fo47, the nonhost pathogen Fom (a melon pathogen), and with Fol. Compared to wild‐type plants, Avr2‐tomato plants became hypercolonized by Fo47 and Fom. Surprisingly, however, EMR towards Fol, induced by either Fo47 or Fom, was unaffected in these plants. These data show that EMR‐based disease resistance is independent from the conventional defence pathways triggered by PTI, but that PTI is involved in restricting host colonization by nonpathogenic Fo isolates.

Maternal Exposure to Ozone Modulates the Endophyte-Conferred Resistance to Aphids in Lolium multiflorum Plants.

Simple SummaryGlobal change is driving the incidence of novel stress factors such as the tropospheric pollutant ozone. Plants can overcome the environmental challenges by adjusting their phenotypes that eventually, can be transmitted to the progeny. Plants also establish symbiotic interactions with beneficial fungal endophytes, some of which can be transmitted to the progeny through the seeds. We worked with the grass Lolium multiflorum and its common endophyte, Epichloë occultans that benefits the host by coffering resistance to herbivores. Specifically, we studied the effect of exposing endophyte-symbiotic and non-symbiotic plants to ozone on the level of resistance to herbivorous aphids in the progeny. The ozone history impaired the endophyte-conferred resistance to aphids in progeny. This was evident at individual weight level of aphids but not so at population level. Defensive compounds were higher in endophyte-symbiotic seeds but depressed by mother plants exposure to ozone. Despite the negative effect of maternal ozone on the resistance level of plants, symbiotic plants showed a superior biomass compared to endophyte-free plants. Our work illustrates how the environment explored by parent plants can persist transgenerationally and, depending on the symbiotic state, will affect the fate of the progeny.Plants are challenged by biotic and abiotic stress factors and the incidence of one can increase or decrease resistance to another. These relations can also occur transgenerationally. For instance, progeny plants whose mothers experienced herbivory can be more resistant to herbivores. Certain fungal endophytes that are vertically transmitted endow plants with alkaloids and resistance to herbivores. However, endophyte-symbiotic plants exposed to the oxidative agent ozone became susceptible to aphids. Here, we explored whether this effect persists transgenerationally. We exposed Lolium multiflorum plants with and without fungal endophyte Epichloë occultans to ozone (120 or 0 ppb), and then, challenged the progeny with aphids (Rhopalosiphum padi). The endophyte was the main factor determining the resistance to aphids, but its importance diminished in plants with ozone history. This negative ozone effect on the endophyte-mediated resistance was apparent on aphid individual weights. Phenolic compounds in seeds were increased by the symbiosis and diminished by the ozone. The endophyte effect on phenolics vanished in progeny plants while the negative ozone effect persisted. Independently of ozone, the symbiosis increased the plant biomass (≈24%). Although ozone can diminish the importance of endophyte symbiosis for plant resistance to herbivores, it would be compensated by host growth stimulation.

Diminished Pathogen and Enhanced Endophyte Colonization upon CoInoculation of Endophytic and Pathogenic Fusarium Strains.

Root colonization by Fusarium oxysporum (Fo) endophytes reduces wilt disease symptoms caused by pathogenic Fo strains. The endophytic strain Fo47, isolated from wilt suppressive soils, reduces Fusarium wilt in various crop species such as tomato, flax, and asparagus. How endophyte-mediated resistance (EMR) against Fusarium wilt is achieved is unclear. Here, nonpathogenic colonization by Fo47 and pathogenic colonization by Fo f.sp. lycopersici (Fol) strains were assessed in tomato roots and stems when inoculated separately or coinoculated. It is shown that Fo47 reduces Fol colonization in stems of both noncultivated and cultivated tomato species. Conversely, Fo47 colonization of coinoculated tomato stems was increased compared to single inoculated plants. Quantitative PCR of fungal colonization of roots (co)inoculated with Fo47 and/or Fol showed that pathogen colonization was drastically reduced when coinoculated with Fo47, compared with single inoculated roots. Endophytic colonization of tomato roots remained unchanged upon coinoculation with Fol. In conclusion, EMR against Fusarium wilt is correlated with a reduction of root and stem colonization by the pathogen. In addition, the endophyte may take advantage of the pathogen-induced suppression of plant defences as it colonizes tomato stems more extensively.

Biocontrol by Fusarium oxysporum Using Endophyte-Mediated Resistance.

Interactions between plants and the root-colonizing fungus Fusarium oxysporum (Fo) can be neutral, beneficial, or detrimental for the host. Fo is infamous for its ability to cause wilt, root-, and foot-rot in many plant species, including many agronomically important crops. However, Fo also has another face; as a root endophyte, it can reduce disease caused by vascular pathogens such as Verticillium dahliae and pathogenic Fo strains. Fo also confers protection to root pathogens like Pythium ultimum, but typically not to pathogens attacking above-ground tissues such as Botrytis cinerea or Phytophthora capsici. Endophytes confer biocontrol either directly by interacting with pathogens via mycoparasitism, antibiosis, or by competition for nutrients or root niches, or indirectly by inducing resistance mechanisms in the host. Fo endophytes such as Fo47 and CS-20 differ from Fo pathogens in their effector gene content, host colonization mechanism, location in the plant, and induced host-responses. Whereas endophytic strains trigger localized cell death in the root cortex, and transiently induce immune signaling and papilla formation, these responses are largely suppressed by pathogenic Fo strains. The ability of pathogenic strains to compromise immune signaling and cell death is likely attributable to their host-specific effector repertoire. The lower number of effector genes in endophytes as compared to pathogens provides a means to distinguish them from each other. Co-inoculation of a biocontrol-conferring Fo and a pathogenic Fo strain on tomato reduces disease, and although the pathogen still colonizes the xylem vessels this has surprisingly little effect on the xylem sap proteome composition. In this tripartite interaction the accumulation of just two PR proteins, NP24 (a PR-5) and a β-glucanase, was affected. The Fo-induced resistance response in tomato appears to be distinct from induced systemic resistance (ISR) or systemic acquired resistance (SAR), as the phytohormones jasmonate, ethylene, and salicylic acid are not required. In this review, we summarize our molecular understanding of Fo-induced resistance in a model and identify caveats in our knowledge.

Endophyte-Mediated Resistance in Tomato to Fusarium oxysporum Is Independent of ET, JA, and SA.

Root endophytes can confer resistance against plant pathogens by direct antagonism or via the host by triggering induced resistance. The latter response typically relies on jasmonic acid (JA)/ethylene (ET)-depended signaling pathways, but can also be triggered via salicylic acid (SA)-dependent signaling pathways. Here, we set out to determine if endophyte-mediated resistance (EMR), conferred by the Fusarium endophyte Fo47, against Fusarium wilt disease in tomato is mediated via SA, ET or JA. To test the contribution of SA, ET, and JA in EMR we performed bioassays with Fo47 and Fusarium oxysporum f. sp. lycopersici in tomato plants impaired in SA accumulation (NahG), JA biosynthesis (def1) or ET-production (ACD) and -sensing (Nr). We observed that the colonization pattern of Fo47 in stems of wildtype plants was indistinguishable from that of the hormone mutants. Surprisingly, EMR was not compromised in the lines affected in JA, ET, or SA signaling. The independence of EMR on SA, JA, and ET implies that this induced resistance-response against Fusarium wilt disease is distinct from the classical Induced Systemic Resistance (ISR) response, providing exciting possibilities for control of wilt diseases independent of conventional defense pathways.

Xylem Sap Proteomics Reveals Distinct Differences Between R Gene- and Endophyte-Mediated Resistance Against Fusarium Wilt Disease in Tomato.

Resistance (R) genes and endophytic organisms can both protect plants against pathogens. Although the outcome of both processes is the same, little is known about the commonalities and differences between both immune responses. Here we set out to phenotypically characterize both responses in the tomato-Fusarium pathosystem, and to identify markers to distinguish these responses at the molecular level. As endophyte Fusarium oxysporum (Fo) strain Fo47 was employed, which confers protection against various pathogens, including the vascular wilt fungus F. oxysporum f.sp. lycopersici (Fol). As R-gene conferring Fol resistance, the I-2 gene of tomato (Solanum lycopersicum) was used. Fol colonizes the xylem vessels of susceptible and I-2 resistant tomato plants, but only causes disease in the former. Fol was found to colonize the vasculature of endophyte-colonized plants, and could be isolated from stems of non-diseased plants co-inoculated with Fo47 and Fol. Because the xylem vessels form the main interface between plant and pathogen, the xylem sap proteomes during R gene- and Endophyte-Mediated Resistance (RMR and EMR) were compared using label-free quantitative nLC-MS/MS. Surprisingly, both proteomes were remarkably similar to the mock, revealing only one or two differentially accumulated proteins in the respective resistant interactions. Whereas in I-2 plants the accumulation of the pathogenesis-related protein PR-5x was strongly induced by Fol, the endophyte triggered induction of both NP24, another PR-5 isoform, and of a β-glucanase in the presence of Fol. Notably, over 54% of the identified xylem sap proteins have a predicted intracellular localization, which implies that these might be present in exosomes. In conclusion, whereas both resistance mechanisms permit the pathogen to colonize the vasculature, this does not result in disease and this resistance coincides with specific induction of two distinct PR-5 isoforms and a β-glucanase.

When host-plant resistance to a pest leads to higher plant damage

The effects of the association between grasses and fungal endophytes on orthopterans are very poorly studied although they are important grassland pests. Here, the endemic New Zealand weta, Hemiandrus sp. ‘promontorius’, and Festulolium loliaceum infected with Epichloe uncinata, were used to study the effect of endophyte-mediated resistance in grasses on this large orthopteran insect in the laboratory, and the effects of this interaction on the grass. The insect was presented with F. loliaceum with and without E. uncinata infection in no-choice and paired choice experiments. Other controls were Epichloe festucae-infected Festuca rubra and endophyte-free Lolium perenne. In no-choice experiments, persistent attempts by the insect to graze the endophyte-infected grasses (but promptly abandoning them) resulted in a significantly higher number of plants lost due to excision at their stems after the first bite (P = 0.004). The inability of affected grasses to compensate for the lost biomass resulted in a lack of significant difference between the dry biomass of endophyte-infected and endophyte-free controls (P = 0.206). However, in choice experiments, there was a preference for the endophyte-free controls when they were paired with the endophyte-infected grasses (P < 0.05). The current work shows that endophyte-infected grasses can sustain high plant losses when attacked by an orthopteran insect in the absence of an alternative food source. This contrasts other endophyte/herbivory experiments in which high herbivory occurs because chemical plant defences are at a low concentration or the endophytes have other non-toxin roles in the plant.

An alpha-glucosidase inhibitor from an endophytic Cladosporium sp. with potential as a biocontrol agent.

This study highlights the importance of alpha-glucosidase inhibitors as mechanisms for endophyte-mediated resistance to insect pests. One of the major benefits which endophytes confer on plants is providing resistance against insect pests. This built-in defense mechanism of the plant can be used for exploring ecofriendly strategies for pest control. In the present study, 34 endophytic fungi were isolated from Tinospora cordifolia and screened for their ability to produce alpha-glucosidase inhibitors. Maximum inhibitory activity was observed in an isolate from T. cordifolia (TN-9S), identified to be Cladosporium sp. The inhibitor was purified using chromatographic techniques. The insecticidal activity of the purified inhibitor was evaluated against Spodoptera litura. The inhibitor induced a significant mortality in the larvae of S. litura and adversely affected its survival and development. It also inhibited the activity of α-glycosidases in vivo in the gut of the larvae. The purified inhibitor was determined to be a phenolic compound with amine groups, demonstrating a noncompetitive type of inhibition in vitro. The production of the inhibitor was optimized. Response surface methodology (RSM) analysis revealed a significant interaction between dextrose and malt extract, with first-order effect of pH.

An endophytic fungus reduces herbivory in its recently colonised grass host: a food-choice experiment on common voles, weeping alkaligrass and Epichloë typhina

The interactions of endophytes with plants are believed to evolve over time from parasitic to mutualistic, and in the seed-transmitted fungal endophytes, these interactions are conventionally treated as mutualistic. The weeping alkaligrass Puccinellia distans has recently dispersed to new habitats in Europe, where it was colonised by the seed-transmitted fungus Epichloe typhina. The E. typhina-infected weeping alkaligrass has a fertility advantage over -uninfected plants, but this advantage is held only over shorter time scales. We assess the antiherbivore value of E. typhina in mature weeping alkaligrass against common voles Microtus arvalis, a herbivore naturally co-occurring with weeping alkaligrass. In two consecutive food-choice tests conducted in a lab, we examined feeding by naive voles on E. typhina-infected (E+) and -uninfected grass (E−) originating from Central Europe. In the first test, all voles consumed comparable amounts of E+ biomass and E− biomass. In the subsequent test, the voles reduced their feeding rates by 57 %, but E+ food was avoided twice as frequently as E− food (75 vs. 33 % reduction). This result suggests that toxins produced by E. typhina repel herbivores soon after the first contact. We show that in addition to its direct fitness consequences, endophyte-mediated resistance to herbivory increases the fertility advantage of E. typhina-infected weeping alkaligrass. This effect can increase the ability of weeping alkaligrass to invade habitats with intense herbivory.

Evaluating the fungal endophyte Neotyphodium occultans for resistance to the rice leaf bug, Trigonotylus caelestialium, in Italian ryegrass, Lolium multiflorum

Fungal endophytes of the genus Neotyphodium (Ascomycota: Clavicipitaceae) form symbiotic associations with many grass species of the subfamily Pooideae, including some important forage and turf grasses from the genus Lolium. Some Neotyphodium-infected grasses have enhanced resistance to the rice leaf bug, Trigonotylus caelestialium (Kirkaldy) (Heteroptera: Miridae), one of the major pests in rice production in Japan. The insects can grow on Lolium grasses and thus cause a conflict of interest between production of forage and rice. Endophyte-mediated resistance to T. caelestialium is likely to be conferred by N-formylloline, one of the major loline alkaloids (saturated 1-aminopyrrolizidines) observed in some grass–Neotyphodium associations. We checked the feasibility of using Neotyphodium occultans, the native endophyte of Italian ryegrass (Lolium multiflorum Lam.), to reduce T. caelestialium, as this endophyte produces N-formylloline, but no known mammalian toxins. To this end, we conducted choice and no-choice feeding assays using T. caelestialium first instars and 35 N. occultans-infected Italian ryegrass plants with different in planta N-formylloline concentration. Some N. occultans-infected ryegrass showed significant resistance to T. caelestialium. The preference and the survival of this insect were considerably lower on infected plants, and the higher the in planta N-formylloline concentration, the greater the adverse effects on the insect. The incremental risk of death of the insect was assumed to be due to intake of the chemical, and in addition, to the decreased amount of feeding caused by its insect-deterrent effect. These results indicated that the use of the N. occultans–grass associations has potential to reduce T. caelestialium propagation in ryegrass crops. The breeding of N. occultans-infected Italian ryegrass cultivars with high N-formylloline concentrations should be an effective way to develop pasture grasses with a greater protective effect against T. caelestialium.

Endophyte-Mediated Resistance to Black Cutworm as a Function of Plant Cultivar and Endophyte Strain in Tall Fescue

To improve Neotyphodium endophyte-mediated resistance to black cutworm Agrotis ipsilon (Hufnagel) (BCW), a series of experiments was conducted by using several different cultivars of tall fescue, Schedonorus arundinaceus (Schreb.) Dumort. in combination with several different haplotypes of the endophyte Neotyphodium coenophialum (Morgan-Jones & Gams) (plant cultivar × endophyte haplotype = plant line), each producing unique alkaloid profiles. BCW settling response, survival at 5 and 10 d, and larval biomass varied significantly among plant lines. In general, greater variation BCW performance was observed within a single plant cultivar infected with different endophyte haplotypes than among different plant cultivars infected with the same endophyte haplotype, but comparisons among the former were far more numerous. Although five endophyte-mediated alkaloids representing three alkaloid classes were quantified in the plants, the pyrrolizidine alkaloid N-acetyl norloline was consistently the single best predictor of BCW performance. BCW settling response, 5-d survival, and 10-d survival decreased as levels of the alkaloid N-acetyl norloline increased. The same three response variables also decreased with increasing levels of peramine, but increased with increasing levels of ergovaline. Minor variation in endophyte infection levels occurring among infected plant lines had no significant influence on BCW performance. Results indicate a potentially important role for N-acetyl norloline and peramine in providing resistance to black cutworm whereas ergovaline appears to be much less important. Therefore, endophyte haplotypes expressing high levels of N-acetyl norloline and peramine may be of particular importance for developing 'friendly' endophyte-enhanced turf and pasture grasses that resist challenging lepidopteran pests, although remaining safe for wildlife and grazing mammals.

Endophyte-Mediated Resistance to Herbivores Depends on Herbivore Identity in the Wild GrassFestuca subverticillata

Understanding factors that affect the context dependency of species interactions has been identified as a critical research area in ecology. The presence of symbionts in host plants can be an important factor influencing the outcome of plant-insect interactions. Similarly, herbivore identity can alter the outcome of plant-symbiont interactions. Symbiotic foliar fungal endophytes confer resistance to herbivores in economically important agronomic grasses, in part through the production of alkaloids. Although endophytes are common in nature, relatively little is known about their effects on herbivores of native, wild grass species, and a recent meta-analysis suggested that endophytes are only beneficial in agronomic settings. In this study, we performed choice trials for five insect species and a greenhouse experiment with one species to assess effects of the fungal endophyte Neotyphodium sp. on herbivores of the wild grass Festuca subverticillata. In feeding trials, endophyte presence altered the preference of all five insect species tested. However, the magnitude and direction of preference varied among species, with Pterophylla camellifolia (F.), Spodoptera frugiperda (J. E. Smith), and Rhopalosiphum padi L. preferring endophyte-disinfected plants and Encoptolophus costalis (Scudder) and Romalea guttata (Houttuyn) preferring endophyte-symbiotic plants. Despite reducing insect preference, the endophyte had no significant effect on S. frugiperda performance in a no-choice greenhouse experiment and did not increase plant growth in response to this herbivore. Our results show that endophyte-mediated resistance to herbivory depends strongly on herbivore identity and suggest that the fitness consequences of endophyte symbiosis for host plants will be context dependent on the local composition of insect herbivores.

Endophyte-mediated resistance against white pine blister rust in Pinus monticola

Induced resistance responses, including fungal endophyte-mediated resistance, have been well studied in both agricultural crops and grass systems. Yet, the effect of these processes and symbionts in forest trees is poorly known. Fungal endophytes have been found in all conifer forest systems examined to date and have been hypothesised to be involved in resistance-mediated responses. However, in the absence of functional studies the influence of these endophytes on the extended phenotype of the host plant is unclear. In this study we demonstrate that fungal endophytes from Pinus monticola were effective at increasing survival in host plants against the exotic pathogen Cronartium ribicola, which is responsible for the devastating disease called white pine blister rust. Seedlings previously inoculated with fungal endophytes lived longer than endophyte-free seedlings and also showed some reduction in white pine blister rust disease severity. This endophyte-mediated resistance was found to be effective over time, indicating persistence, and is hypothesised to be a form of induced resistance. Overall, this suggests fungal endophytes may play a determinative role in the structure of biological communities and could provide a useful alternative or ancillary management tool for combating pests and diseases.

Importance of Host Plant Species, &lt;I&gt;Neotyphodium&lt;/I&gt; Endophyte Isolate, and Alkaloids on Feeding by &lt;I&gt;Spodoptera frugiperda&lt;/I&gt; (Lepidoptera: Noctuidae) Larvae

Three grass host species--tall fescue, Festuca arundinacea Schreber; meadow fescue, Festuca pratensis Hudson; and perennial ryegrass, Lolium perenne L.--each infected with a number of different Neotyphodium endophyte isolates, were investigated for their effects on fall armyworm, Spodoptera frugiperda (J.E. Smith). Alkaloid profiles varied among associations. Choice and no-choice tests comparing feeding and early development of S. frugiperda larvae on endophyte-infected and endophyte-free leaf blade material were performed. Endophyte-mediated resistance to S. frugiperda was greatest in meadow fescue and weakest in tall fescue. Some endophyte isolates, particularly in perennial ryegrass and meadow fescue, had a major effect on feeding and development of S. frugiperda, whereas others had no effect or were only weakly efficacious. In tall fescue, some associations deterred S. frugiperda from feeding in choice tests but had no effect on development, whereas larvae reared on other associations weighed significantly more than control larvae fed endophyte-free grass. It was concluded that the deleterious consequences of endophyte infection were easily masked by other factors in tall fescue. Relative leaf age had no effect on feeding preferences in the three host species. Chemical analysis of herbage from the plants used, and results from a no-choice study using spiked artificial diets, failed to individually implicate any of the major known alkaloids (peramine, lolitrem B, ergovaline, and lolines) in the observed effects on S. frugiperda. Hypotheses explaining these observations, and their impact on creating desirable grass-endophyte associations for use in pastures, are discussed.

Overseeding endophytic perennial ryegrass into stands of Kentucky bluegrass to manage bluegrass billbug (Coleoptera: Curculionidae).

Studies were conducted to determine the utility of overseeding endophyte enhanced varieties of perennial ryegrass, Lolium perenne L., into existing stands of Kentucky bluegrass, Poa pratensis L., to manage bluegrass billbug, Sphenophorus parvulus Gyllenhal, larval populations and their damage. Two commercial varieties of endophyte enhanced perennial ryegrass (Repell II and Triple Play) were overseeded at two different rates in the field. S. parvulus larval populations, their damage, and turfgrass composition were monitored over the course of 3 yr. Significantly fewer larvae were found in plots overseeded with Repell II, whereas Triple Play had no significant effect on larval population density. Endophyte infection was confirmed in approximately 58% of all Repell II shoots, whereas only approximately 31% of Triple Play shoots were infected. Regression analysis, using a segmented model, demonstrated that larval populations declined as the proportion of endophyte-infected perennial ryegrass increased. However, no additional reductions in larval population density were achieved in stands containing >40% endophyte-infected perennial ryegrass. Visual damage varied with cultivar, seeding rate, and year, but generally decreased as the proportion of endophyte-infected perennial ryegrass increased. Although seeding rate had an initial effect on turfgrass composition, these differences were not detected after the first year of the study. Results indicate overseeding existing stands of Kentucky bluegrass with endophytic varieties of perennial ryegrass can reduce S. parvulus larval populations and their damage. However, plant genetic characteristics (i.e., different cultivars) may be overriding determinants of many aspects of plant performance including establishment, tolerance to insect feeding, and expression of endophyte-mediated resistance.

Leaf Age and Related Factors Affecting Endophyte-mediated Resistance to Fall Armyworm (Lepidoptera: Noctuidae) in Tall Fescue

Differences in fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith), larval preference for and development on fungal endophyte-infected (high incidence of infection) and uninfected (low incidence of infection) tall fescue, Festuca arundinacea Schreb., at three relative leaf ages were examined. Neonate larvae significantly preferred older, uninfected leaves to equivalent age infected leaves and fed more upon them in paired choice tests. Fourth-instar preference and consumption were not affected by infection status of leaves of equivalent age in 6- or 16-h choice tests. Eight-day larval weights were significantly higher for larvae reared on infected older leaves. Percent wet weight of fescue leaves was significantly higher in infected older leaves. Water-soluble carbohydrate and crude protein content did not differ between infected and uninfected leaves at any leaf age, although crude protein was significantly higher in young leaves. Loline alkaloid content was significantly higher in infected leaves and decreased as leaf age increased. The data suggest that both larval age and relative leaf age affect FAW response to endophyte-infected tall fescue.