Endophytic fungi and drought tolerance: ecophysiological adjustment in shoot and root of an annual mesophytic host grass.

Epichloid endophytic fungi, vertically transmitted symbionts of grasses, can increase plant tolerance to biotic and abiotic stress. Our aim was to identify ecophysiological mechanisms by which the endophyte Epichloë occultans confers drought tolerance to the annual grass Lolium multiflorum Lam. Endophyte-associated or endophyte-free plants were either well-watered or subjected to water deficit. We evaluated plant biomass, root length and nitrogen concentration, and we assessed intrinsic water use efficiency (iWUE) and its components net photosynthesis and stomatal conductance, by carbon and oxygen isotope analysis of shoot tissues. Endophyte-free plants produced more biomass than endophyte-associated ones at field capacity, while water deficit strongly reduced endophyte-free plants biomass. As a result, both types of plants produced similar biomass under water restriction. Based on oxygen isotope composition of plant cellulose, stomatal conductance decreased with water deficit in both endophyte-associated and endophyte-free plants. Meanwhile, carbon isotope composition indicated that iWUE increased with water deficit only in endophyte-associated plants. Thus, the isotope data indicated that net photosynthesis decreased more strongly in endophyte-free plants under water deficit. Additionally, endophyte presence reduced root length but increased its hydraulic conductivity. In conclusion, endophytic fungi confer drought tolerance to the host grass by adjusting shoot and root physiology.

Glyphosate decreases mycorrhizal colonization and affects plant-soil feedback.

Our aim was to study the effects of glyphosate, tilling practice and cultivation history on mycorrhizal colonization and growth of target (weeds) and non-target (crops) plants. Glyphosate, the world's most widely used pesticide, inhibits an enzyme found in plants but also in microbes. We examined the effects of glyphosate treatment applied in the preceding fall on growth of a perennial weed, Elymus repens (target plant) and a forage grass, Festuca pratensis (non-target plant) and their arbuscular mycorrhizal fungal (AMF) root colonization in a field pot experiment. Non-target plants were sown in the following spring. Furthermore, we tested if glyphosate effects depend on tillage or soil properties modulated by long cultivation history of endophyte symbiotic grass (E+ grass). AMF root colonization, plant establishment and growth, glyphosate residues in plants, and soil chemistry were measured. Glyphosate reduced the mycorrhizal colonization and growth of both target and non-target grasses. The magnitude of reduction depended on tillage and soil properties due to cultivation history of E+ grass. We detected glyphosate residues in weeds and crop plants in the growing season following the glyphosate treatment. Residues were higher in plants growing in no-till pots compared to conspecifics in tilled pots. These results demonstrate negative effects of glyphosate on non-target organisms in agricultural environments and grassland ecosystems.

Testing the roles of vertical transmission and drought stress in the prevalence of heritable fungal endophytes in annual grass populations.

Beneficial inherited symbionts are expected to reach high prevalence in host populations, yet many are observed at intermediate prevalence. Theory predicts that a balance of fitness benefits and efficiency of vertical transmission may interact to stabilize intermediate prevalence. We established populations of grass hosts (Lolium multiflorum) that varied in prevalence of a heritable fungal endophyte (Epichloё occultans), allowing us to infer long-term equilibria by tracking change in prevalence over one generation. We manipulated an environmental stressor (elevated precipitation), which we hypothesized would reduce the fitness benefits of symbiosis, and altered the efficiency of vertical transmission by replacing endophyte-positive seeds with endophyte-free seeds. Endophytes and elevated precipitation both increased host fitness, but symbiont effects were not stronger in the drier treatment, suggesting that benefits of symbiosis were unrelated to drought tolerance. Reduced transmission suppressed the inferred equilibrium prevalence from 42.6% to 11.7%. However, elevated precipitation did not modify prevalence, consistent with the result that it did not modify fitness benefits. Our results demonstrate that failed transmission can influence the prevalence of heritable microbes and that intermediate prevalence can be a stable equilibrium due to forces that allow symbionts to increase (fitness benefits) but prevent them from reaching fixation (failed transmission).

Mutualism effectiveness and vertical transmission of symbiotic fungal endophytes in response to host genetic background.

Certain species of the Pooideae subfamily develop stress tolerance and herbivory resistance through symbiosis with vertically transmitted, asexual fungi. This symbiosis is specific, and genetic factors modulate the compatibility between partners. Although gene flow is clearly a fitness trait in allogamous grasses, because it injects hybrid vigor and raw material for evolution, it could reduce compatibility and thus mutualism effectiveness. To explore the importance of host genetic background in modulating the performance of symbiosis, Lolium multiflorum plants, infected and noninfected with Neotyphodium occultans, were crossed with genetically distant plants of isolines (susceptible and resistant to diclofop-methyl herbicide) bred from two cultivars and exposed to stress. The endophyte improved seedling survival in genotypes susceptible to herbicide, while it had a negative effect on one of the genetically resistant crosses. Mutualism provided resistance to herbivory independently of the host genotype, but this effect vanished under stress. While no endophyte effect was observed on host reproductive success, it was increased by interpopulation plant crosses. Neither gene flow nor herbicide had an important impact on endophyte transmission. Host fitness improvements attributable to gene flow do not appear to result in direct conflict with mutualism while this seems to be an important mechanism for the ecological and contemporary evolution of the symbiotum.

Inherited fungal symbionts enhance establishment of an invasive annual grass across successional habitats.

Plants infected with vertically transmitted fungal endophytes carry their microbial symbionts with them during dispersal into new areas. Yet, whether seed-borne endophytes enhance the host plant's ability to overcome colonisation barriers and to regenerate within invaded sites remains poorly understood. We examined how symbiosis with asexual endophytic fungi (Neotyphodium) affected establishment and seed loss to predators in the invasive annual grass Lolium multiflorum (Italian ryegrass) across contrasting successional plots. Italian ryegrass seeds with high and low endophyte incidence were sown into three communities: a 1-year-old fallow field, a 15-year-old grassland, and a 24-year-old forest, which conformed to an old-field chronosequence in the eastern Inland Pampa, Argentina. We found that endophyte infection consistently increased host population recruitment and reproductive output. Endophyte presence also enhanced aerial biomass production of ryegrass in a low recruitment year but not in a high recruitment year, suggesting that symbiotic effects on growth performance are density dependent. Endophyte presence reduced seed removal by rodents, although differential predation may not account for the increased success of infected grass populations. Overall, there was no statistical evidence for an endophyte-by-site interaction, indicating that the fungal endosymbiont benefitted host establishment regardless of large differences in biotic and abiotic environment among communities. Our results imply that hereditary endophytes may increase the chances for host grass species to pass various ecological filters associated with invasion resistance across a broad range of successional habitats.

Searching for evidence against the mutualistic nature of hereditary symbioses: a comment on Faeth.

Abstract: Associations between fungal endophytes and grasses have served as particularly useful systems for exploring the nature and significance of hereditary symbiosis. Here, we propose alternative explanations for recent work by Faeth, in which endophytes were proposed to function as reproductive parasites. Faeth argued that his data demonstrated a symbiosis-induced shift to earlier host reproduction that could generate parasitism through life-history trade-offs with growth/survival. We contend that identifying a symbiosis as mutualistic or parasitic requires studies that incorporate both correlations among demographic pathways and rates of symbiont transmission; such work can advance understanding of the ecology and evolution of symbiosis.

The interplay between the effectiveness of the grass-endophyte mutualism and the genetic variability of the host plant.

Neotyphodium endophytic fungi, the asexual state of Epichloë species, protect cool-season grasses against stresses. The outcomes of Neotyphodium-grass symbioses are agronomically relevant as they may affect the productivity of pastures. It has been suggested that the mutualism is characteristic of agronomic grasses and that differential rates of gene flow between both partners' populations are expected to disrupt the specificity of the association and, thus, the mutualism in wild grasses. We propose that compatibility is necessary but not sufficient to explain the outcomes of Neotyphodium-grass symbiosis, and advance a model that links genetic compatibility, mutualism effectiveness, and endophyte transmission efficiency. For endophytes that reproduce clonally and depend on allogamous hosts for reproduction and dissemination, we propose that this symbiosis works as an integrated entity where gene flow promotes its fitness and evolution. Compatibility between the host plant and the fungal endophyte would be high in genetically close parents; however, mutualism effectiveness and transmission efficiency would be low in fitness depressed host plants. Increasing the genetic distance of mating parents would increase mutualism effectiveness and transmission efficiency. This tendency would be broken when the genetic distance between parents is high (out-breeding depression). Our model allows for testable hypotheses that would contribute to understand the coevolutionary origin and future of the endophyte-grass mutualism.

Imperfect vertical transmission of the endophyte Neotyphodium in exotic grasses in grasslands of the flooding pampa.

Cool-season grasses establish symbioses with vertically transmitted Neotyphodium endophytes widespread in nature. The frequency of endophyte-infected plants in closed populations (i.e., without migrations) depends on both the differential fitness between infected and non-infected plants, and the endophyte-transmission efficiency. Most studies have been focused on the first mechanism ignoring the second. Infection frequency and endophyte transmission from vegetative tissues to seeds were surveyed in two grasses growing in vegetation units that differ in flood and grazing regimes, and soil salinity. Transmission efficiency and infection frequency for tall fescue did not vary significantly and were 0.98 and 1.00, respectively. For Italian ryegrass, transmission efficiency and infection frequency were 0.88 and 0.57 in humid prairies, and 0.96 and 0.96 in the other vegetation units. Only in humid mesophytic meadows, the observed pattern was irrespective of the presence or absence of grazers. Our results suggest that selection forces for endophyte infection are different for both species. Imperfect transmission was only compensated in tall fescue through an increased fitness of infected plants. Interpreting variations of infection frequency only in terms of differential fitness can be misleading, considering that endophyte transmission can be imperfect and variable in nature. Therefore, this study highlights the importance of measuring transmission efficiency.

Neotyphodium endophyte infection frequency in annual grass populations: relative importance of mutualism and transmission efficiency.

Persistence and ubiquity of vertically transmitted Neotyphodium endophytes in grass populations is puzzling because infected plants do not consistently exhibit increased fitness. Using an annual grass population model, we show that the problems for matching endophyte infection and mutualism are likely to arise from difficulties in detecting small mutualistic effects, variability in endophyte transmission efficiency and an apparent prevalence of non-equilibrium in the dynamics of infection. Although endophytes would ultimately persist only if the infection confers some fitness increase to the host plants, such an increase can be very small, as long as the transmission efficiency is sufficiently high. In addition, imperfect transmission limits effectively the equilibrium infection level if the infected plants exhibit small or large reproductive advantage. Under frequent natural conditions, the equilibrium infection level is very sensitive to small changes in transmission efficiency and host reproductive advantage, while convergence to such an equilibrium is slow. As a consequence, seed immigration and environmental fluctuation are likely to keep local infection levels away from equilibrium. Transient dynamics analysis suggests that, when driven by environmental fluctuation, infection frequency increases would often be larger than decreases. By contrast, when due to immigration, overrepresentation of infected individuals tends to vanish faster than equivalent overrepresentation of non-infected individuals.