Does the Degree of Mutualism between Epichloë Fungi and Botanophila Flies Depend upon the Reproductive Mode of the Fungi?

Epichloë (Ascomycota: Clavicipitaceae) fungi can form an intriguing interaction with Botanophila flies. The fungi live within above-ground shoots of grasses. Some species (type I) only reproduce sexually by forming stromata on all host culms (choke disease). Stromata produce haploid spores (spermatia) that fertilize stromata of opposite mating type to form dikaryotic cells. A second category of Epichloë species (type II) produces stromata on only some of the host culms; culms without choke produce flowers and seeds. These Epichloë can reproduce asexually by invading host seed, as well as sexually. Female Botanophila flies visit stromata for feeding and oviposition. Spermatia pass through the gut of Botanophila intact and viable. Flies can cross-fertilize the fungus during defecation after egg laying. Hence, we described the interaction as a mutualism similar to pollination. Yet, subsequent work by others and ourselves showed that visitation by Botanophila flies was not necessary for cross fertilization of Epichloë. We believe these contradictory results can be reconciled from an evolutionary perspective, if one takes into account the reproductive mode of the fungus. We explore a novel hypothesis to reconcile this contradiction, its predictions and discuss ways in which to test them.

Long-term shifts in the functional diversity of abandoned wet meadows: Impacts of historical disturbance and successional pathways.

Investigating the direction of changes in functional diversity involving successional pathways and historical disturbances may be a promising tool for predictions of the effectiveness of the seminatural meadows conservation, with great emphasis on formulation of more cost-effective restoration strategies. The goal of this research was to assess the differences in long-term shifts in the functional diversity of plant species in seminatural wet meadows unmanaged for the last 40 years, under the influence of different successional pathways and historical disturbances. Using ordination techniques, linear mixed-effect models, a set of plant functional traits and parameters of functional diversity, we assessed the importance of habitat filtering, competition, and niche partitioning in shaping community assembly changes over time. The most dramatic shifts in functional diversity were found in the Carex acutiformis successional pathway after topsoil removal, where colonization by successional inhibitors was the main driver causing decreases in functional dispersion and divergence. This was expressed as a decrease in the importance of habitat filtering and replacement of specialized species by competitors with heavier seeds and higher specific leaf area. Regarding the C. cespitosa and Salix cinerea pathways, the magnitudes of shifts in functional diversity were milder and differed less between the historical topsoil removal and mowing treatments, thereby maintaining a large role for niche partitioning in shaping the vegetation structure. The results of our study highlight the importance of tussock sedges and shrubs as effective buffers against the functional homogenization of meadows driven by the decreases in functional diversity of plant species, even from a long-term perspective.

Cooling effect of fungal stromata in the Dactylis-Epichloë-Botanophila symbiosis.

The stromata of Epichloë fungi are structures covering part of the stem of grasses. Under the fungal layer, still green tissues of the plant survive, although the development of the new leaves is inhibited. Stromata are the places where conidia and ascospores develop. Also, here Botanophila flies dine on mycelium, lay the eggs, defecate, and the larvae develop. The interaction of the three symbionts was analyzed concerning the organisms' adaptation to understand the differences in physiology and ecology of this microenvironment that support stable symbiosis spreading presently in Europe since the beginning of the XXI century. For analysis of the infrared radiation emitted by stromata, a high-resolution infrared camera FLIR E50 was used. The visualization of stromata temperature profiles was shown in the form of pseudo-colored (false) infrared images. The 13C discrimination was used to characterize photosynthesis of the plant tissue enclosed within the stromata. The stromata had a substantially lower temperature than the green plant tissues. The difference reached ~5.6°C during midday hours, whereas it was smaller in the evening, reaching only ~3.6°C. The mycelium of Epichloë cultivated on agar showed about 2°C lower temperature in comparison to the surrounding. The plant tissues enclosed within the stroma were photosynthetically active, although this activity was of phosphoenolpyruvate carboxylase (PEPC) type and less involved in heat dissipation during the day. The stromata, built by fungal hyphae, on which fungal reproductive structures develop, form a cool shelter. This shelter provides a place for the larvae of Botanophila flies.

Botanophila flies, vectors of Epichloë fungal spores, are infected by Wolbachia.

Epichloë fungi are endophytes within grasses that can form stromata on culms of their hosts. Botanophila flies visit the stromata for egg laying and in the process can vector spermatial spores, thereby cross fertilising the fungus. Following egg hatch, larval flies consume fungal tissue and spores. Thus, Epichloë individuals with traits that limit larval consumption could be at a selective advantage. We assessed Botanophila fly larvae from sites within the United States and Europe for infection by the bacterial sexual parasite Wolbachia through amplification of the Wolbachia surface protein gene (wsp). Nearly 70% of fly larvae in our samples were infected by Wolbachia. This is the first record of infection by Wolbachia within Botanophila and could have far reaching effects on not only the fly host, but also the Epichloë fungi upon which Botanophila feeds as well as the grass host within which the fungi live. For example, infection by Wolbachia could limit consumption of Epichloë spores by Botanophila larvae if the bacteria promoted premature larval death.

Incidence, Identification, and Mycoparasitic Activity of Clonostachys epichloë, a Hyperparasite of the Fungal Endophyte Epichloë typhina.

Fungi of the genus Epichloë (Clavicipitaceae, Ascomycota) cause choke disease in many grass species. The disease manifests itself as fungal stromata that form around developing inflorescences, thereby suppressing their maturation. Economic losses in agricultural production due to choke have long been known in the U.S.A. and France, but attempts to control choke disease have not been successful. The interaction between Epichloë typhina (Pers.) Tul. & C. Tul. and its naturally occurring fungal hyperparasite, Clonostachys epichloë (Speg.) Schroers (sexual morph Bionectria epichloë) was investigated in populations of the grass Puccinellia distans (L.) Parl. Fungal hyperparasites occur widely in nature, and many are successfully used commercially as biological control agents against plant pathogenic fungi. Microscopy of Epichloë stromata infected with C. epichloë revealed a lack of asci with ascospores in perithecia and damage to mycelia at sites colonized by C. epichloë. The ability of C. epichloë to colonize E. typhina was confirmed via two in vitro experiments. The percent inhibition of growth of E. typhina strains by C. epichloë varied from 18.40 to 46.50%, and the mycoparasite colonized up to 100% of Epichloë mycelia in a precolonization experiment. We discuss the possibility of using C. epichloë to control choke disease caused by E. typhina in grass populations.

Change in abundance of three phytophagous mite species (Acari: Eriophyidae, Tetranychidae) on quackgrass in the presence of choke disease.

Phytophagous mites and endophytic fungi may interact when sharing a host plant, potentially influencing one another's growth or population dynamics; however, interactions between them are poorly known and remain largely unexplored. In this study, quantitative associations between three species of phytophagous mites and the endophytic fungus Epichloë bromicola Leuchtm. & Schardl (Clavicipitaceae, Ascomycotina) on quackgrass, Elymus repens (L.) Gould are reported. The mites' abundance was assessed on field-collected grass shoots that were either exhibiting choke disease symptoms or without the fungus. Overall, the abundance of Tetranychus urticae and Aculodes mckenziei was significantly lower on quackgrass plants infected by E. bromicola compared to plants without the fungus. Conversely, populations of Abacarus hystrix were significantly larger on plants colonised by the fungus than on uninfected plants. Thus, the presence of this endophytic fungus may have divergent effects on different phytophagous mite species although the basis of these effects is not yet known.

Age-specific response of the grass Puccinellia distans to the presence of a fungal endophyte.

Asexual fungal endophytes, which do not produce reproductive structures, spread in host populations only vertically via the propagules of their hosts. With such a close relationship between the fitness of the asexual endophyte and that of the host, the relationship is traditionally thought to be mutualistic. Here we present data showing that the positive effect of asexual endophytes may concern only a short period of the host's life and that its lifetime reproductive success may be reduced. We followed 180 individuals of a perennial grass, Puccinellia distans, for the first 3 years of their growth both in the field and in a common garden experiment. In the first 2 years, infected individuals produced more generative shoots with longer inflorescences. Three-year-old individuals produced significantly fewer generative shoots with shorter inflorescences when infected with the fungus Epichloë typhina. Moreover, the dry mass of above- and belowground parts after three seasons was significantly lower in infected individuals. We suggest that if the endophyte retains control over plant reproduction and if shorter generation time is more crucial for the fungus than for the perennial host, then the fungus should stimulate plant reproduction early in life even though infection incurs a future cost. This reasoning suggests that discovering an endophyte's beneficial effect on its perennial host in one season provides insufficient grounds for concluding that a mutualistic relationship exists.