The fungus Clonostachys epichloë alters the influence of the Epichloë endophyte on seed germination and the biomass of Puccinellia distans grass.

The fungal grass endophyte Epichloë typhina (Pers.) Tul. & C. Tul. (Ascomycota: Clavicipitaceae) grows intercellulary in aerial plant parts and reproduces asexually by invading host seeds. In this phase, it enhances seed production and germination, which accelerates its vertical spread. This relationship may be distorted by other seed-born fungi, whose spread is not so directly dependent on the success of the grass. Recently, the fungus Clonostachys epichloë Schroers has been observed on Puccinellia distans (Jacq.) Parl seeds originating from grass clumps infested with stromata, sexual structures of Epichloë typhina that are formed in spring on some host culms, preventing flower and seed development ('choke disease'). C. epichloë shows mycoparasitic activity toward Epichloë stromata by reducing the production of ascospores, which are responsible for horizontal transmission of the fungus. The aim of this study was to investigate the effect of seed-borne C. epichloë on seed germination, as well as the size and weight of P. distans seedlings and to examine whether C. epichloë alters the influence of Epichloë in the early developmental stages of P. distans. The results showed that if C. epichloë acts on seeds together with E. typhina endophytes, the seeds were negatively affected due to the elimination of the positive effect of the latter in terms of both seed germination rate and seedling length. At the same time, C. epichloë increased the proportion of E. typhina-untreated germinated seeds. Additionally, only the joint action of the two fungi, E. typhina and C. epichloë, effectively stimulated seedling dry mass; the presence of E. typhina alone was not sufficient to noticeably affect seedling size. Based on the increasing commonality of C. epichloë on Epichloë stromata, as well as its potential to be used in biocontrol of 'choke disease', we should take a closer look at this fungus, not only in terms of its mycoparasitic ability, but also in terms of its cumulative impact on the whole Epichloë-grass system.

Effects of the Clonostachys epichloë fungal hyperparasite on the symbiotic interaction between Botanophila flies and Epichloë fungus.

Botanophila flies are associated with Epichloë fungi in a symbiotic relationship in which the flies benefit from stromata as a food source for both adults and larvae, and the fungus benefits from the transfer of conidia by the flies, resulting in fertilization. Derogations from this pattern indicate that the Epichloë-Botanophila interaction cannot be clearly defined. The situation may be complicated by reports of new elements of the interaction, e.g., Wolbachia bacteria present in Botanophila larvae. The present study investigates the impact of Clonostachys epichloë (Speg.) Schroers, the fungal hyperparasite of Epichloë stromata, on the Botanophila-Epichloë interaction. The interaction between C. epichloë and Botanophila flies associated with Epichloë typhina subsp. clarkii (J.F. White) Leuchtm. & Schardl stromata was studied in the Holcus lanatus L. grass population. C. epichloë was present on 76.5% of stromata, covering on average 44.8 ± 32.1% of its surface and influencing the final perithecial coverage to the same extent as larval feeding. C. epichloë began to appear on stromata much later than the fly eggs and did not affect the preference for Botanophila egg laying. On the other hand, C. epichloë reduced larval hatching success and increased the mortality of the larvae. Clonostachys was responsible for 76.0% of all deaths, overgrowing brood chambers, and its mycelium was present both on and within larvae in all cases. Overall, as a result of the presence of C. epichloë, the number of Botanophila fly offspring decreased by 52.7%. Of the 26 surviving larvae, 10 (38.5%) were affected by C. epichloë, and their weight was significantly lower than that of unaffected larvae. Results show that C. epichloë, a new element of the interaction between E. typhina fungus and Botanophila flies, negatively affects both fungal reproduction and the offspring success of flies. This is the first report on the entomopathogenic activity of C. epichloë against Epichloë-associated 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.