Adrian Leuchtmann

Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae reveals dynamics of alkaloid loci

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.

Nomenclatural realignment of Neotyphodium species with genus Epichloë

Nomenclatural rule changes in the International Code of Nomenclature for algae, fungi and plants, adopted at the 18th International Botanical Congress in Melbourne, Australia, in 2011, provide for a single name to be used for each fungal species. The anamorphs of Epichloë species have been classified in genus Neotyphodium, the form genus that also includes most asexual Epichloë descendants. A nomenclatural realignment of this monophyletic group into one genus would enhance a broader understanding of the relationships and common features of these grass endophytes. Based on the principle of priority of publication we propose to classify all members of this clade in the genus Epichloë. We have reexamined classification of several described Epichloë and Neotyphodium species and varieties and propose new combinations and states. In this treatment we have accepted 43 unique taxa in Epichloë, including distinct species, subspecies, and varieties. We exclude from Epichloë the two taxa Neotyphodium starrii, as nomen dubium, and Neotyphodium chilense, as an unrelated taxon.

Taxonomy of Acremonium endophytes of tall fescue (Festuca arundinacea), meadow fescue (F. pratensis) and perennial ryegrass (Lolium perenne)

Isozyme analysis, morphological characters, sensitivity to benomyl in vitro, and the production of alkaloids in the fungal-host-grass associations were used to examine the relationships between Acremonium endophytes of Festuca arundinacea, F. pratensis and Lolium perenne. Isozyme analysis identified six taxonomic groupings, with each grouping comprising isolates from just one of the three grass species. Three taxonomic groupings occurred in F. arundinacea, two in L. perenne, and one in F. pratensis. Alkaloid production in the natural host grass-Acremonium associations proved a useful taxonomic criterion, with the profile of alkaloids being consistent in the host plants for all isolates within a single isozyme phenotype and for most isolates within a taxonomic grouping. On the basis of morphological, cultural and secondary product criteria, only one of the three taxonomic groupings identified amongst the F. arundinacea endophytes fitted the definition of A. coenophialum, and only one grouping of L. perenne endophytes could be accommodated within A. lolii. All isolates from F. pratensis could be accommodated within A. uncinatum even though the conidia differed between isolates of the two isozyme phenotypes identified during this study.

Sexual compatibility and taxonomy of a new species of Epichloe symbiotic with fine fescue grasses

Mating tests among stroma-forming and nonstroma-forming strains of Epichloe from several fine fescue species, including Festuca rubra subsp. rubra and F. rubra subsp. commutata, indicated a distinc...

Different Levels of Protective Alkaloids in Grasses with Stroma-forming and Seed-transmitted Epichloë/Neotyphodium Endophytes

The three alkaloid groups—lolines, ergopeptides, and peramine— are typically associated with endophyte infection of grasses, with the main function to protect hosts against herbivores. We determined levels of N-formylloline, N-acetylloline, ergovaline, and peramine in 18 European grasses naturally infected with seed-transmitted Neotyphodium endophytes or sexual Epichloë species. Peramine was the most common alkaloid, whereas lolines and ergovaline were only detected in Festuca hosts infected with E. festucae, N. coenophialum, or N. uncinatum. Only ten of the grass species analyzed contained detectable amounts of one or more of these alkaloids. There was a clear tendency for plants associated with stroma-forming Epichloë species to be free of alkaloids, and those that did produce alkaloids contained only small levels of peramine. In contrast, plants infected with seed-transmitted Neotyphodium endophytes often contained extremely high levels of lolines. Lolines enhance host survival through increased protection from herbivores and, thus, may be particularly favored in asexual endophytes that depend on host seed-production for their dispersal.

Currencies of Mutualisms: Sources of Alkaloid Genes in Vertically Transmitted Epichloae

The epichloae (Epichloë and Neotyphodium species), a monophyletic group of fungi in the family Clavicipitaceae, are systemic symbionts of cool-season grasses (Poaceae subfamily Poöideae). Most epichloae are vertically transmitted in seeds (endophytes), and most produce alkaloids that attack nervous systems of potential herbivores. These protective metabolites include ergot alkaloids and indole-diterpenes (tremorgens), which are active in vertebrate systems, and lolines and peramine, which are more specific against invertebrates. Several Epichloë species have been described which are sexual and capable of horizontal transmission, and most are vertically transmissible also. Asexual epichloae are mainly or exclusively vertically transmitted, and many are interspecific hybrids with genomic contributions from two or three ancestral Epichloë species. Here we employ genome-scale analyses to investigate the origins of biosynthesis gene clusters for ergot alkaloids (EAS), indole-diterpenes (IDT), and lolines (LOL) in 12 hybrid species. In each hybrid, the alkaloid-gene and housekeeping-gene relationships were congruent. Interestingly, hybrids frequently had alkaloid clusters that were rare in their sexual ancestors. Also, in those hybrids that had multiple EAS, IDT or LOL clusters, one cluster lacked some genes, usually for late pathway steps. Possible implications of these findings for the alkaloid profiles and endophyte ecology are discussed.

Evolution of 'pollinator'-attracting signals in fungi

Fungi produce a plethora of secondary metabolites yet their biological significance is often little understood. Some compounds show well-known antibiotic properties, others may serve as volatile signals for the attraction of insects that act as vectors of spores or gametes. Our investigations in an outcrossing, self-incompatible fungus show that a fungus-produced volatile compound with fungitoxic activities is also responsible for the attraction of specific insects that transfer gametes. We argue that insect attraction using this compound is likely to have evolved from its primary function of defence—as has been suggested for floral scent in the angiosperms. We, thus, propose that similar yet convergent evolutionary pathways have lead to interspecific communication signals in both fungi and plants.

Isozyme relationships of Acremonium endophytes from twelve Festuca species

The relationships of 159 isolates of Acremonium endophytes from twelve Festuca host species, representing five subgenera, were investigated using starch gel electrophoresis of 11 enzymes. Cluster analysis of isozyme genotypes identified six distinct groupings with genetic identities ( I ) of 0·5 or less among groupings. Three groupings were previously described taxa associated with a single Festuca host species, A. coenophialum from F. arundinacea, A. uncinatum from F. pratensis , and A. starrii from F. obtusa . On F. arundinacea a second distinct grouping (FaTG-2) was recognized. All genotypes associated with species of fine fescue (subgenus Festuca ) or with F. gigantea and F. pulchella formed another distinct grouping with identical or very similar genotypes. The endophyte from F. altissima appeared to be most divergent from all other isolates studied and may represent a separate species. Most of the gene diversity within the total isolate sample was due to differentiation among subgroups from different hosts (H T = 0·484; G ST = 0·801). In addition, morphological and cultural characteristics of representative isolates from each host were studied and found to be distinctive for all major isozyme groupings. The possibility of coevolution of endophytes along host subgenera of Festuca is discussed based on genetic relationships of endophytes and subgeneric classification of their hosts.

Periglandula, a new fungal genus within the Clavicipitaceae and its association with Convolvulaceae

We describe two newly discovered fungi living on the adaxial leaf surface of plants belonging to the Convolvulaceae, Ipomoea asarifolia and Turbina corymbosa. The fungi apparently are epibionts because hyphae were never observed to penetrate epidermal cells or stomata of their respective host plants, and most remarkably are intimately associated with secretory glands on the leaf surface. Hyphae and structures resembling chlamydospores and synnemata (but lacking conidia), formed by both fungal species are phenotypically nearly indistinguishable after in vitro growth or when examined in vivo on the leaf surface. Phylogenetic trees based on aligned DNA sequences from nuclear genes for β-tubulin (tubB) and RNA Polymerase II subunit 1 (rpbA), and the mitochondrial gene for ATP synthase F0 subunit A (atp6), grouped the fungal species in a clade within the Clavicipitaceae. Clavicipitaceous fungi isolated from the two different plant species could be distinguished by their atp6 and rpbA sequences, and nuclear genes for γ-actin (actG), translation elongation factor 1–α (tefA), and 4–(γ,γ-dimethylallyl)tryptophan synthase (dmaW), the determinant step in ergoline (i.e. ergot) alkaloid biosynthesis. Based on these findings we propose a new fungal genus, Periglandula, gen. nov., and describe two new species, Periglandula ipomoeae sp. nov., from host plant I. asarifolia, and Periglandula turbinae sp. nov., from T. corymbosa.

Isozyme subgroups in Trichoderma section Longibrachiatum

Seventy-eight representatives of Trichoder? ma sect. Longibrachiatum, Hypocrea jecorina, and H. schweinitzii were compared using ten enzyme systems. The results essentially supported Bissetts morpholog? ically based taxonomy of Trichoderma sect. Longibra? chiatum, in that the species T longibrachiatum, T ci- trinoviride, T parceramosum, and T pseudokoningii could be distinguished. Trichoderma reesei was closely related to H. jecorina and more distantly linked to T longibrachiatum, thus the synonymy of T reesei with T longibrachiatum was not supported. The possibility that T. reesei is a clonal derivative of H. jecorina is discussed. Strains of Trichoderma citrinoviridewere in?