Neotyphodium endophytes in grasses: deterrents or promoters of herbivory by leaf-cutting ants?

Endophytic fungi, particularly in the genus Neotyphodium, are thought to interact mutualistically with host grasses primarily by deterring herbivores and pathogens via production of alkaloidal mycotoxins. Little is known, however, about how these endophytes interact with host plants and herbivores outside the realm of agronomic forage grasses, such as tall fescue, and their livestock grazers or invertebrate pest herbivores. We tested the effects of Neotyphodium inhabiting introduced tall fescue and native Arizona fescue on preference, survival, and performance of the leaf-cutting ant, Acromyrmex versicolor, an important generalist herbivore in the southwestern United States. In a choice experiment, we determined preferences of foraging queens and workers for infected and uninfected tall fescue and Arizona fescue. In a no-choice experiment, we determined queen survival, worker production, and size of fungal gardens for foundress queens reared on diets of infected and uninfected tall fescue and Arizona fescue. Foraging workers and queens did not significantly prefer either uninfected tall fescue or Arizona fescue relative to infected grasses, although ants tended to harvest more uninfected than infected tall fescue and more infected than uninfected Arizona fescue. Queen survivorship and length of survival was greater on uninfected tall fescue, uninfected Arizona fescue, and infected Arizona fescue than on infected tall fescue or the standard diet of palo verde and mesquite leaves. No queens survived beyond 6 weeks of the study when fed the infected tall fescue diet, in contrast to the effects of the other diets. Likewise, worker production was much lower and fungal garden size much smaller on infected tall fescue than in all other treatments, including the standard diet. In general, ant colonies survived and performed better on uninfected tall fescue and infected and uninfected Arizona fescue than standard diets of palo verde and mesquite leaves. The interaction of Neotyphodium with its host grasses is highly variable and these endophytes may increase, not alter, or even decrease resistance to herbivores. The direction of the interaction depends on host and fungal genotype, herbivore species, and environmental factors. The presence of endophytes in most, if not all, host plants suggests that endophytes may alter foraging patterns, performance, and survival of herbivores, such as leaf-cutting ants, but not always in ways that increase host plant fitness. Received: 27 October 1998 / Accepted: 19 October 1998     

FUNGAL ENDOPHYTES OF CYPERUS AND THEIR EFFECT ON AN INSECT HERBIVORE

Populations of the sedges Cyperus virens Michx. and C. pseudovegetus Steud. (Cyperaceae) in Louisiana often contain individuals infected by the systemic fungal endophyte Balansia cyperi Edg. (Clavicipitaceae, Ascomycetes). Related fungal endophytes infecting grasses are known to have detrimental effects on insect and mammalian herbivores consuming infected plants. In this study herbivory of infected and uninfected sedges was compared in two laboratory experiments. Newly hatched larvae of the fall armyworm (Spodoptera frugiperda [J. E. Smith], Noctuidae, Lepidoptera) were reared on leaves from either infected or uninfected plants of C. virens and C. pseudovegetus grown in the greenhouse. Survival, growth, and development of each insect were monitored. For both sedges larval survival and rate of weight gain were reduced, and length of the larval period was increased for larvae reared on leaves from infected plants compared to larvae reared on leaves from uninfected plants. The results of this study parallel results obtained from grasses, suggesting that the endophyte may defend its host against herbivory in natural populations.     

Influence of temperature on alkaloid levels and fall armyworm performance in endophytic tall fescue and perennial ryegrass

The symbiotic relationships between Neotyphodium endophytes (Clavicipitacea) and certain cool‐season (C3) grasses result in the synthesis of several alkaloids that defend the plant against herbivory. Over a 3 month period we evaluated the effects of temperature on the expression of these alkaloids in tall fescue, Festuca arundinacea Schreb, and perennial ryegrass, Lolium perenne L. (Poaceae). Response surface regression analysis indicated that month, temperature, and their interaction had an impact on the alkaloid levels in both grasses. We aimed to identify the alkaloids most closely associated with enhanced resistance to the fall armyworm, Spodoptera frugiperda JE Smith (Lepidoptera: Noctuidae), and clarify the role of temperature in governing the expression of these alkaloids. The dry weights and survival of fall armyworms feeding on endophyte‐infected tall fescue or perennial ryegrass were significantly lower than for those feeding on uninfected grass, whereas endophyte infection had no significant influence on survival. For tall fescue, a four‐alkaloid model consisting of a plant alkaloid, perloline, and the fungal alkaloids ergonovine chanoclavine, and ergocryptine, explained 47% of the variation in fall armyworm dry weight, whereas a three‐alkaloid model consisting of the plant alkaloid perloline methyl ether and the fungal alkaloids ergonovine and ergocryptine explained 70% of the variation in fall armyworm dry weight on perennial ryegrass. Although temperature had a significant influence on overall alkaloid expression in both grasses, the influence of temperature on individual alkaloids varied over time. The levels of those alkaloids most closely linked to armyworm performance increased linearly or curvilinearly with increasing temperature during the last 2 months of the study. We conclude that the growth temperature of grasses can influence the performance of fall armyworm, and that this effect may be mediated through a set of plant‐ and endophyte‐related alkaloids.     

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.     

Effects of fungal endophytes on the seed and seedling biology of Lolium perenne and Festuca arundinacea

Summary Many grasses are infected by endophytic fungi that grow intercellularly in leaves, stems, and flowers and are transmitted maternally by hyphal growth into ovules and seeds. The seed biology and seedling growth of endophyte-infected and uninfected perennial ryegrass (Lolium perenne) and tall fescue (Festuca arundinacea) were investigated under controlled environmental conditions. The percentage of filled seeds produced by infected tall fescue was over twice of uninfected tall fescue; infected and uninfected perennial reegrass had similar percentages. Weights of seeds from infected and uninfected plants were similar in both species. Seeds from infected plants of both species exhibited a higher rate of germination than seeds from uninfected plants. Shoot growth in the greenhouse was compared by making three sequential harvests of above-ground plant parts from infected and uninfected plants of both species. Infected perennial ryegrass plants produced significantly more biomass and tillers than uninfected plants after 6 and 10 weeks of growth and significantly more biomass after 14 weeks of growth. Infected tall fescue plants produced significantly more biomass and tillers than uninfected plants after 10 and 14 weeks of growth. The physiological mechanism of enhancement of growth is not known. The results of this study suggest that infected plants may have a selective advantage in populations with uninfected members.     

Grass species and endophyte effects on survival and development of fall armyworm (Lepidoptera: Noctuidae)

Grass selections including 10 zoysiagrasses, 18 paspalums, 34 Bermuda grasses, tall fescue, creeping red fescue, and perennial ryegrasses with and without endophyte were evaluated for potential resistance to fall armyworm, Spodoptera frugiperda (J. E. Smith), larvae. Laboratory evaluations assessed the degree of antibiosis among >70 grass lines to first-instar fall armvworms. When all parameters measured were considered, the trend in resistance to fall armyworm among endophyte-infected (E+) and endophyte-free (E-) cool season grasses from greatest to least was: 'Dawson' E+ > APR 1234 > 'Dawson' E- > 'Rosalin' E+ > Lp 5425, 'Rosalin' E-, ATF 480 > 'Tulsa' or: E+ slender creeping red fescue > E+ turf- type perennial ryegrass > E- slender creeping red fescue > E+ forage-type perennial ryegrass > E- forage-type perennial ryegrasses, and E+ tall fescue > E- turf-type tall fescue. Among warm season grasses larval weight gain was reduced on all zoysiagrasses. Larval weight gain also was lower on the Bermuda grasses 'Tifsport', 'Tifgreen', 97-4, 97-14, 97-22, 97-28, 97-39, 97-40,97-54, 98-15, 98-30, and 98-45 than when larvae were fed 'Tulsa' tall fescue or the diet control. Only APR1234 and 'Dawson' creeping red fescue reduced larval survival to the same extent that was observed for zoysiagrasses. Survival on Bermuda grasses was least on 97-8. Seashore paspalums were only rarely less susceptible to fall armyworm than tall fescue, although pupal weights were consistently lower on 'Temple 1' and 'Sea Isle 1' paspalums than that on 'Tulsa' tall fescue. Genetic resistance to key grass pests can reduce insecticide use and simplify management of these cultivars.     

Effects of CO2 enrichment,nutrient addition,and fungal endophyte-infection on the growth of two grasses

Summary Increasing atmospheric carbon dioxide (CO₂) concentration is expected to increase plant productivity and alter plant/plant interactions, but little is known about its effects on symbiotic interactions with microorganisms. Interactions between perennial ryegrass, Lolium perenne (a C3 plant), and purpletop grass, Tridens flavus (a C4 plant), and their clavicipitaceous fungal endophytes (Acremonium lolii and Balansia epichloe, respectively) were investigated by growing the grasses under 350 and 650 l l^– 1 CO₂ at two nutrient levels. Infected and uninfected perennial ryegrass responded with increased growth to both CO₂ enrichment and nutrient addition. Biomass and leaf area of infected and uninfected plants responded similarly to CO₂ enrichment. When growth analysis parameters were calculated, there were significant increases in relative growth rate and net assimilation rate of infected plants compared to uninfected plants, although the differences remained constant across CO₂ and nutrient treatments. Growth of purpletop grass did not increase with CO₂ enrichment or nutrient addition and there were no significant differences between infected and uninfected plants. CO₂ enrichment did not alter the interactions between these two host grasses and their endophytic-fungal symbionts.     

Interactions among fungal endophytes,grasses and herbivores

The interaction between two species often depends on the presence or absence of a third species. One widespread three-species interaction involves fungal endophytes infecting grasses and the herbivores that feed upon them. The endophytes are allied with the fungal family Clavicipitaceae and grow systemically in intercellular spaces in above-ground plant tissues including seeds. Like relatedClaviceps species, the endophytes produce a variety of alkaloids that make the host plants toxic or distasteful to herbivores. A large number of grass species are infected, especially cool-season grasses in temperate areas. Field and laboratory studies have shown that herbivores avoid infected plants in choice trials and suffer increased mortality and decreased growth on infected grasses in feeding experiments. Resistance to herbivores may provide a selective advantage to infected plants in competitive interactions with noninfected plants. Recent studies have shown that differential herbivory can reverse competitive hierarchies among plant species. Both endophyte-infected and noninfected tall fescue grass (Festuca arundinacea) are outcompeted by orchardgrass (Dactylis glomerata) in the absence of insect herbivory. However, when herbivores are present infected tall fescue outcompetes orchardgrass. These results suggest that the frequency of infection in grass species and grassland communities will increase over time. Several studies are reviewed illustrating increases in infection frequency within grass populations subject to herbivore pressure. Endophytic fungi may be important regulators of plant-herbivore interactions and so indirectly affect the structure and dynamics of plant communities.     

Influence of genetic variation in the fungal endophyte of a grass on an herbivore and its parasitoid

Neotyphodium coenophialum (Glenn, Bacon, Price & Hanlin) (Ascomycota: Clavicipitaceae) is an endophytic fungus that lives symbiotically within grasses and produces alkaloids that can help protect its hosts from some insect pests. We used laboratory‐based experiments to investigate whether fungal genotype influences an herbivore and its parasitoid. We tested whether variation in novel isolates, plus a control lacking fungal infection, affected preference by fall armyworm, Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae: Amphipyrini), and growth and survival of Euplectrus comstockii Howard (Hymenoptera: Eulophidae: Euplectrini), a parasitoid of fall armyworm. Caterpillars preferred leaf blades in choice experiments from uninfected tall fescue [Lolium arundinaceum (Schreb.) Darbysh., cultivar Jesup (Poaceae)] and tended to avoid blades from plants containing fungal isolates AR502, AR542, or the most common strain from pastures in Georgia, USA, in tall fescue. However, caterpillars fed as much on leaf blades from plants containing isolate AR502 as from those lacking infection. Parasitoid pupal mass was not influenced by fungal isolate, yet fungal isolate did influence parasitoid survival. Survival was higher than expected for parasitoids reared from hosts fed plants lacking fungal infection, but was lower than expected for those reared from hosts fed plants infected with the common strain or AR542 isolates. In contrast, parasitoids reared from hosts fed plants infected with isolate AR502 did not experience higher mortality than expected by chance. Our results show that N. coenophialum can modify bottom‐up trophic cascades through direct effects on herbivores, as well as indirect effects on a natural enemy of the herbivores and that the fungus may influence the tritrophic interaction in ways that counterbalance herbivore protection provided by the symbiont. Our work also shows that these effects are influenced by fungal genotype. As attempts are made to produce forage cultivars with strains of fungal endophyte that lack negative influences on livestock, it will be prudent for investigators to assess the multi‐trophic effects of these novel associations within agroecosystems.     

Interaction between fungal endophytes and environmental stressors influences plant resistance to insects

Neotyphodium coenophialum, an endophytic fungus that infects shoots of tall fescue (Festuca arundinacea), may protect its host from herbivory through production of alkaloids. Yet, the fungus can also modify plant resource allocation, regrowth dynamics, and drought tolerance, and these changes may also influence herbivores. We tested if N. coenophialum infection interacted with stress (drought or simulated herbivory) to modify plant resistance to insects. We assigned greenhouse plants to one of four treatments: 1) clipping at 3 cm above the soil surface, 2) drought stress during insect bioassays, 3) drought stress prior to insect bioassays, or 4) daily watering. Treatments were crossed with presence or absence of endophyte to give eight treatment combinations, and we assessed the performance of bird cherry‐oat aphid (Rhopalosiphum padi) and fall armyworm (Spodoptera frugiperda) feeding on plants in two separate experiments from each of the eight treatments. Aphids were placed into clip bags on leaf blades and allowed to reproduce parthenogenetically. Plant tissue was fed to third instar fall armyworm caterpillars until they molted into the fifth instar. Developmental time was recorded and larval growth was obtained gravimetrically. We also assessed total protein nitrogen (N) and loline alkaloids in plants.
Total protein N was unaffected by endophyte infection. In contrast, stress influenced total protein N, but its effect varied with endophyte infection. Uninfected plants that were clipped had higher total protein N; this trend was absent in infected plants. Plants in drought stress had lower N, but only if they were infected. Lolines were nearly absent from uninfected plants. In infected plants they tended to be higher in clipped plants. The effect of endophyte infection differed between the two insects: aphid reproduction was reduced by the endophyte, but endophyte infection enhanced caterpillar performance. Both insects were affected by interactions between the endophyte and stress. Aphids were negatively affected by drought stress, but only when feeding on uninfected plants, while caterpillars showed the opposite response, displaying lower performance on drought stressed plants only if they were infected. Aphids reproduced faster on regrowth tissue (following damage by clipping) of uninfected plants, but endophyte infection cancelled this effect. In contrast, performance of caterpillars was not influenced by an interaction between damage and infection. We conclude that N. coenophialum does not provide universal resistance to insects. Endophyte‐mediated resistance varies with insect species and will be a complex function of environmental stress, including drought and prior damage.     

Arthropod abundance in tall fescue,Lolium arundinaceum,pastures containing novel ‘safe’ endophytes

Poor livestock performance on tall fescue is linked to infection by a fungal endophyte that enhances grass resistance to stress, including herbivory, while producing ergot alkaloids toxic to vertebrate grazers. Novel ‘safe’ endophyte/grass associations produce no ergot alkaloids yet retain stand persistence, but they could be more susceptible to insect outbreaks. We tested the hypothesis that grass‐feeding insects are more abundant in novel endophyte pastures compared with those containing common endophyte. Above‐ and below‐ground herbivores were sampled across two growing seasons in pastures containing common strain endophyte (KY31), novel endophytes (MaxQ or AR584) or endophyte free. We also sampled natural enemies as an indicator of possible tritrophic effects. With a few exceptions, numbers of predatory, chewing (grasshoppers, crickets, caterpillars) or sucking insects (leafhoppers, planthoppers) were similar in common and novel endophyte pastures. Contrary to our hypothesis, Aphrodes spp. leafhoppers were more abundant in KY31 than other pasture types in 2008; their nymphs also were more abundant in KY31 than in MaxQ in 2009. Adults (but not total numbers or nymphs) of another leafhopper, Psammotettix striatus, were less abundant in KY31 than other pasture types in 2009. Popillia japonica and Cyclocephala spp. grubs did not differ in density, weight or instar among the endophyte‐containing associations. In feeding assays, armyworm (Mythimna unipuncta) survival was higher on MaxQ and NE9301, and weight gain was higher on NE9301, suggesting armyworm outbreaks could be more severe on such grasses, but that result may not be attributable solely to alkaloids because common strain and endophyte‐free grasses did not differ in either parameter. Caterpillar abundance did not differ among pasture types in the field. Our results suggest that re‐seeding common strain endophytic pastures with livestock‐safe novel endophyte/grass associations to alleviate fescue toxicosis is unlikely to promote markedly higher populations of plant‐feeding insects.     

Loline alkaloid production by fungal endophytes of Fescue species select for particular epiphytic bacterial microflora

The leaves of fescue grasses are protected from herbivores by the production of loline alkaloids by the mutualist fungal endophytes Neotyphodium sp. or Epichloë sp. Most bacteria that reside on the leaf surface of such grasses can consume these defensive chemicals. Loline-consuming bacteria are rare on the leaves of other plant species. Several bacterial species including Burkholderia ambifaria recovered from tall fescue could use N-formyl loline as a sole carbon and nitrogen source in culture and achieved population sizes that were about eightfold higher when inoculated onto plants harboring loline-producing fungal endophytes than on plants lacking such endophytes or which were colonized by fungal variants incapable of loline production. In contrast, mutants of B. ambifaria and other bacterial species incapable of loline catabolism achieved similarly low population sizes on tall fescue colonized by loline-producing Neotyphodium sp. and on plants lacking this endophytic fungus. Lolines that are released onto the surface of plants benefiting from a fungal mutualism thus appear to be a major resource that can be exploited by epiphytic bacteria, thereby driving the establishment of a characteristic bacterial community on such plants.     

Modification of esterified cell wall phenolics increases vulnerability of tall fescue to herbivory by the fall armyworm

Feruloylation of arabinoxylan in grass cell walls leads to cross-linked xylans. Such cross-linking appears to play a role in plant resistance to pathogens and insect herbivores. In this study, we investigated the effect of ferulate cross-linking on resistance to herbivory by fall armyworm (Spodoptera frugiperda) making use of genetically modified tall fescue [Schedonorus arundinaceus (Festuca arundinacea)] expressing a ferulic acid esterase gene. Mature leaves of these plants have significant reduced levels of cell wall ferulates and diferulates but no change in acid detergent lignin. These reduced levels of esterified cell wall ferulates in transgenic plants had a positive effect on all measures of armyworm larval performance examined. More larvae survived (89 vs. 57?%) and grew faster (pupated 2.1?days sooner) when fed transgenic leaves with reduced levels of cell wall ferulates, than when fed control tall fescue leaves where levels of cell wall ferulates were not altered. Overall, mortality, growth and food utilization were negatively associated with level of esterified cell wall ferulates and diferulates in leaves they were fed. This study is the first to use transgenic plants with modified level of cell wall esterified ferulates to test the role of feruloylation in plant resistance to insects. It is concluded that the accumulation of ferulates and the cross-linking of arabinoxylans via diferulate esters in the leaves of tall fescue underlies the physical barrier to insect herbivory. Reducing ferulate cross-linking in grass cell walls could increase susceptibility of these plants to insect folivores.     

Endophyte-grass-herbivore interactions: the case of Neotyphodium endophytes in Arizona fescue populations

Neotyphodium endophytes in introduced agronomic grasses are well known to increase resistance to herbivores, but little is known of interactions between Neotyphodium endophytes and herbivores in native grass populations. We investigated whether endophytes mediate plant-herbivore interactions in a native grass species, Festuca arizonica in the southwestern United States, in two ways. First, to test the prediction that the presence and frequency of endophyte-infected (E+) plants should increase with increasing herbivory, we determined endophyte frequencies over a 4-year period in six natural Arizona fescue populations. We compared Neotyphodium frequency among plants growing inside and outside long-term vertebrate grazing exclosures. Second, we experimentally tested the effects of Neotyphodium infection, plant clone, and soil nutrients on plant resistance to the native grasshopper Xanthippus corallipes. Contrary to predictions based upon the hypothesis that endophytes increase herbivore resistance, levels of infection did not increase in plants subjected to grazing outside of exclosures relative to ungrazed plants within exclosures. Instead, endophyte frequencies tended to be greater inside the exclosures, where long-term vertebrate grazing was reduced. The grasshopper bioassay experiment corroborated these long-term patterns. Survival of grasshoppers did not differ between infected (E+) and uninfected (E–) plants. Instead, mean relative growth rate of grasshoppers was higher on E+ grasses than on E– ones. Growth performance of newly hatched grasshopper nymphs varied among host plant clones, although two of six clones accounted for most of this variation. Our results suggest that Neotyphodium-grass-herbivore interactions may be much more variable in natural communities than predicted by studies of agronomically important Neotyphodium-grass associations, and herbivory is not always the driving selective force in endophyte-grass ecology and evolution. Thus, alternative hypotheses are necessary to explain the wide distribution and variable frequencies of endophytes in natural plant populations. Received: 15 February 1999 / Accepted: 19 July 1999     

Insect oviposition preference between Epichloë‐symbiotic and Epichloë‐free grasses does not necessarily reflect larval performance

Variation in plant communities is likely to modulate the feeding and oviposition behavior of herbivorous insects, and plant‐associated microbes are largely ignored in this context. Here, we take into account that insects feeding on grasses commonly encounter systemic and vertically transmitted (via seeds) fungal Epichloë endophytes, which are regarded as defensive grass mutualists. Defensive mutualism is primarily attributable to alkaloids of fungal origin. To study the effects of Epichloë on insect behavior and performance, we selected wild tall fescue (Festuca arundinacea) and red fescue (Festuca rubra) as grass–endophyte models. The plants used either harbored the systemic endophyte (E+) or were endophyte‐free (E?). As a model herbivore, we selected the Coenonympha hero butterfly feeding on grasses as larvae. We examined both oviposition and feeding preferences of the herbivore as well as larval performance in relation to the presence of Epichloë endophytes in the plants. Our findings did not clearly support the female's oviposition preference to reflect the performance of her offspring. First, the preference responses depended greatly on the grass–endophyte symbiotum. In F. arundinacea, C. hero females preferred E+ individuals in oviposition‐choice tests, whereas in F. rubra, the endophytes may decrease exploitation, as both C. hero adults and larvae preferred E? grasses. Second, the endophytes had no effect on larval performance. Overall, F. arundinacea was an inferior host for C. hero larvae. However, the attraction of C. hero females to E+ may not be maladaptive if these plants constitute a favorable oviposition substrate for reasons other than the plants' nutritional quality. For example, rougher surface of E+ plant may physically facilitate the attachment of eggs, or the plants offer greater protection from natural enemies. Our results highlight the importance of considering the preference of herbivorous insects in studies involving the endophyte‐symbiotic grasses as host plants.     

Fungal endophytes of native grasses decrease insect herbivore preference and performance

Endophytic fungal symbionts of grasses are well known for their protective benefit of herbivory reduction. However, the majority of studies on endophyte–grass symbioses have been conducted on economically important, agricultural species—particularly tall fescue (Lolium arundinaceum) and perennial ryegrass (Lolium perenne)—raising the hypothesis that strong benefits are the product of artificial selection. We examined whether fungal endophytes found in natural populations of native grass species deterred insect herbivores. By testing several native grass–endophyte symbiota, we examined phylogenetic signals in the effects of endophytes on insects and compared the relative importance of herbivore and symbiotum identity in the outcome of the interactions. Preference was assessed using three herbivore species [Spodoptera frugiperda (Lepidoptera), Schistocerca americana (Orthoptera), Rhopalosiphum padi (Hemiptera)] and ten native symbiota, which spanned seven grass genera. We also assessed herbivore performance in a no choice experiment for five native symbiota against S. frugiperda. We compared greenhouse and laboratory trials with natural levels of herbivory measured in experimental field populations. In all cases, we included the agronomic grass species, L. arundinaceum, to compare with results from the native grasses. Both in the field and in experimental trials, herbivores showed a significant preference for endophyte-free plant material for the majority of native grasses, with up to three times lower herbivory for endophyte-symbiotic plants; however, the degree of response depended on the identity of the herbivore species. Endophyte presence also significantly reduced performance of S. frugiperda for the majority of grass species. In contrast, the endophyte in L. arundinaceum had few significant anti-herbivore effects, except for a reduction in herbivory at one of two field sites. Our results demonstrate that the mechanisms by which native symbionts deter herbivores are at least as potent as those in model agricultural systems, despite the absence of artificial selection.     

Sesquiterpene lactones of Vernonia — influence of glaucolide-A on the growth rate and survival of Lepidopterous larvae

Summary Sesquiterpene lactone glaucolide-A from Vernonia, incorporated in the rearing diets of five species of Lepidoptera, significantly reduced the rate of growth of larvae of the southern armyworm, Spodoptera eridania; fall armyworm, S. frugiperda; and yellowstriped armyworm, S. ornithogalli. Quantitative feeding tests demonstrated that decreased feeding levels and reduced growth resulted from ingestion of a sesquiterpene lactone. Ingestion of glaucolide-A increased the number of days to pupation in four of the species. In the southern armyworm, it significantly reduced pupal weight. Glaucolide-A decidedly reduced percentage of survival of southern and fall armyworms. Yellow woollybear, Diacrisia virginica, and cabbage looper, Trichoplusia ni, larvae were essentially uneffected by the ingestion of the sesquiterpene lactone. Sesquiterpene lactones adversely affect growth rate and survival of certain insects that feed upon plants containing them. They apparently function as defensive products, screening out a portion of the potential herbivores.Vernonieae: Compositae     

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.     

Fungal loline alkaloids in grass–endophyte associations confer resistance to the rice leaf bug, Trigonotylus caelestialium

Plant symbiotic fungi (endophytes) of the genus Neotyphodium [anamorphs, asexual derivatives of Epichloë (Ascomycota: Clavicipitaceae)] often associate with grass species of the subfamily Pooideae, which includes important forage and turf species. These endophytes are known to produce a range of alkaloids that enhance their host's resistance to insects or are toxic to grazing animals. Among the alkaloids, loline alkaloids (saturated 1‐aminopyrrolizidines) are generally observed in the highest concentrations in many Neotyphodium–grass symbiotic associations, and are known to be toxic to insects but not to mammals. Some Neotyphodium‐infected grasses have enhanced resistance to rice leaf bug, Trigonotylus caelestialium (Kirkaldy) (Heteroptera: Miridae), one of the major pests for rice production in Japan. Our laboratory experiments quantified the effects of purified loline (N‐formylloline) and in planta synthesis of loline alkaloids by meadow fescue [Lolium pratense (Huds.) S.J. Darbyshire (syn. Festuca pratensis Huds.)]–Neotyphodium uncinatum (Gams, Petrini & Schmidt) Glenn, Bacon & Hanlin and Italian ryegrass (Lolium multiflorum Lam.)–Neotyphodium occultans Moon, Scott & Christensen associations on the development and survival of T. caelestialium. No‐choice feeding assays with laboratory populations of the insect revealed that their growth was significantly decreased by the infected grasses, and the effect was greater for N. uncinatum than for N. occultans, in keeping with differences in N‐formylloline concentrations in the plants. Artificial feeding of N‐formylloline through feeding sachets indicated that the chemical has an adverse effect on survival of larvae, even at the lowest concentration tested (50 µg/g), which was considerably lower than the typical concentrations in many Neotyphodium–grass associations. The results confirmed the ability of Neotyphodium‐infected forage grasses to control T. caelestialium propagation in meadows, which may cause damages to nearby rice paddies.     

Plant-endophyte-herbivore interactions: More than just alkaloids?

A recent paper by Rasmussen et al., (New Phytol 2007; 173:787–97) describes the interactions between Lolium perenne cultivars with contrasting carbohydrate content and the symbiotic fungal endophyte Neotyphodium lolii at different levels of nitrogen supply. In a subsequent study undertaken by Rasmussen et al., (Plant Physiol 2008; 146:1440–53) 66 metabolic variables were analysed in the same material, revealing widespread effects of endophyte infection, N supply and cultivar carbohydrate content on both primary and secondary metabolites. Here, we link insect numerical responses to these metabolic responses using multiple regression analysis.Key words: Neotyphodium lolii, Lolium perenne, high sugar grasses, metabolomics, insect herbivoresPasture grasses are often infected with symbiotic fungal endophytes and benefits for host plants arising out of these associations are generally ascribed to endophyte produced anti-herbivorous alkaloids. We tested the effects of (i) infection with three strains of endophytes differing in their alkaloid profiles, (ii) high vs. low nitrogen (N) supply, and (iii) ryegrass cultivars with high vs. control levels of water soluble carbohydrates (WSCs) on numerical insect responses (aphids, thrips, mites). A difference in WSC content between the cultivars had no significant effect on insect numbers, whereas high N compared to low N supply increased mites, thrips and alate Rhopalosiphum spp., but decreased apterous Rhopalosiphum spp. The effect of endophyte infection was strain dependant and differed for the different insects.A total of 66 metabolic variables of the same plants analysed prior to insect treatment were linked to insect responses using multiple regression analysis. One of the major conclusions to be drawn is that alkaloids are not always the most important factor influencing numerical insect responses which will also be determined by other metabolites, clearly indicating the importance of metabolomics type studies to point the way toward a mechanistic explanation of grass-endophyte-herbivore interactions.Grass species are often hosts of symbiotic clavicipitaceous endophytic fungi¹ residing in the apoplastic spaces of above ground plant parts and usually not causing any visible symptoms of infection.^2–4 These fungal symbionts confer protection from insect herbivory to their host plants through alkaloids,^5–8 some of which (ergovaline, lolitrem B) are also toxic to grazing mammals.^9,10 Natural endophyte strains lacking these mammalian toxins, but still retaining at least some of their insect deterring features, have been commercialized and are now widely used in ryegrass and tall fescue based pastures.^11,12