The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.

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The aim of this study was to investigate whether Neoptyphodium spp. endophytes, fungal symbionts of cool‐season grasses, can be selected to improve plant growth and seed yield of perennial ryegrass (Lolium perenne L.). Endophyte‐infected (EI) and endophyte‐free (EF) clones of 13 L. perenne genotypes, collected from native habitats with stressful environmental conditions, were evaluated in a field experiment for symbiotic effects on plant growth (herbage yield, reproductive tiller number), seed production, and seed quality parameters [1000‐seed weight (TSW), germination] over 3 yr in an area with low rainfall (Halle, Germany). The results revealed high variability in endophyte effects on the investigated parameters. In seven genotypes, endophyte presence improved plant growth and seed production during the first harvest year. However, in four of these genotypes, endophyte effects were reversed in the following harvests. In the remaining six genotypes, endophyte infection reduced plant performance. The impact of the fungus on TSW was inconsistent, while seed germination was either improved or not affected by the endophyte. A positive endophyte effect was detectable for genotypes with low performance of EF clones, whereas genotypes with high‐yield EF clones were not, or negatively, affected by the endophyte. Genotypes originating from sites exposed to flooding and periodic drought showed consistent negative endophyte effects on herbage yield and seed production. Since reduced shoot growth is a drought avoidance mechanism, further studies are needed to determine whether endophytes in these genotypes improve adaptation to drought, or represent a “metabolic cost.” Our results indicate that some Neotyphodium endophytes have the potential to improve plant performance of L. perenne, but the selection of strains with consistent beneficial effects on plant growth is difficult.

Interspecific hybridization is a common evolutionary process for the many epichloid fungi that consequently possess multiple gene copies accumulated from their parental strains. Serial gene manipulations in such strains are impeded by the limited availability of selectable resistance marker genes. Therefore, we developed a method for marker elimination suitable for a range of filamentous fungi that allows the reuse of the same marker for successive manipulations, and can also generate gene knockout mutants free of any foreign genes. For epichloae, the complete elimination of the marker gene from the genome would mitigate public concerns and regulatory hurdles to the use of such fungal strains in field experiments.

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